Biomedical subjects

T Okayama

Publications and source records attributed to T Okayama.

At least 19 recordsLinked to original sources

PubMed2000

4-Alkoxy-2-hydroxybenzaldehyde (AHB): a versatile aldehyde linker for solid-phase synthesis of C-terminal modified peptides and peptidomimetics.

[reaction: see text] A new and versatile 4-alkoxy-2-hydroxybenzaldehyde (AHB) linker for solid-phase syntheses is described. Acylation of the polymer-bound secondary amine obtained from reductive amination of the aldehyde in the AHB linker showed good reactivity. Following acylation of the phenolic hydroxyl group, the resulting carboxamide resin was stable to treatment with 95% TFA. The O-acyl functional group was removed with 20% piperidine and the desired compound was cleaved from the resin by TFA treatment.

Biochemistry↗

PubMed1999

Exploring the structure-activity relationships of [1-(4-tert-butyl-3'-hydroxy)benzhydryl-4-benzylpiperazine] (SL-3111), a high-affinity and selective delta-opioid receptor nonpeptide agonist ligand.

SL-3111 [1-(4-tert-butyl-3'-hydroxy)benzhydryl-4-benzylpiperazine] is a de novo designed, high-affinity and selective nonpeptide peptidomimetic agonist of the delta-opioid receptor. In a previous report we had described the unique biological characteristics of this ligand and also a need for further structural evaluation.(6) To pursue this, we have introduced a completely different heterocyclic template (2 and 3), which, based on molecular modeling studies, may present the required structural features to properly orient the pharmacophore groups. We also have made more subtle changes to the original piperazine scaffold (5 and 11). The biological activities of these compounds revealed an important participation of the scaffold in the ligand-receptor interaction. To further explore functional diversity on the scaffold, we have maintained the original piperazine ring and introduced four different functionalities at position 2 of the heterocyclic ring (15a-d; a = CH(2)-O-CH(2)-Ph; b = Me; c = CH(2)Ph; d = CH(2)OH). The biological activities observed for these compounds showed a very interesting trend in terms of the steric effects of the groups introduced at this position. A decrease of almost 2000-fold in affinity and potency at the delta-receptor was observed for 15c compared with 15b. This difference may be explained if we postulate that the bioactive conformation of these peptidomimetics is close to the minimal energy conformations calculated in our study. On the basis of these findings we have realized the importance of this position to further explore and simplify the structure of future generations of peptidomimetic ligands.

Animals↗

PubMed1999

A low rate of nucleotide changes in Escherichia coli K-12 estimated from a comparison of the genome sequences between two different substrains.

Two genome sequences of Escherichia coli K-12 substrains, one partial W3110 and one complete MG1655, have been determined by Japanese and American genome projects, respectively. In order to estimate the rate of nucleotide changes, we directly compared 2 Mb of the nucleotide sequences from these closely-related E. coli substrains. Given that the two substrains separated about 40 years ago, the rate of nucleotide changes was estimated to be less than 10(-7) per site per year. This rate was supported by a further comparison between partial genome sequences of E. coli and Shigella flexneri.

Escherichia coli↗

PubMed1999

Topology selection in unrooted molecular phylogenetic tree by minimum model-based complexity method.

In reconstruction of phylogenetic trees from molecular data, it has been pointed out that multifurcate phylogenetic trees are difficult to be correctly reconstructed by the conventional methods like maximum likelihood method(ML). In order to resolve this problem, we have been engaged in developing a new phylogenetic tree reconstruction method, based on the minimum complexity principle widely used in the inductive inference. Our method, which we call "minimum model-based complexity (MBC) method", has been proved so far to be efficient in estimating multifurcate branching when the tree is described in the form of rooted one. In this study, we make further investigations about the efficiency of MBC method in estimating the multifurcation in unrooted phylogenetic trees. To do so, we conduct computer simulation in which the estimations by MBC method are compared with those by ML, AIC and statistical test approach. The results show that MBC method also provides good estimations even in the case of multifurcate unrooted trees and suggest that it could be generally used for reconstruction of phylogenetic tree having arbitrary multifurcations.

Biological Evolution↗

PubMed1999

Identification of a member of the serralysin family isolated from a psychrotrophic bacterium, Pseudomonas fluorescens 114.

An extracellular metalloprotease named No. 114 protease is one of the major secretions of a psychrotrophic bacterium, Pseudomonas fluorescens 114, the cold-adaptation mechanism of which has not been identified. In this study, we purified and cloned No. 114 protease, which is a single polypeptide having a molecular mass of 47 kDa. This protease contains a zinc-binding motif (HEXXHXUGUXH: X, arbitrary amino acid; U, bulky hydrophobic amino acid), glycine-rich repeats (GGXGXD) and no cysteine residue, which are the features specifically found in serralysin subfamily. No. 114 protease has its maximum activity at the temperature of 35-40 degrees C, which is about 20 degrees C lower than that of a serralysin from a mesophilic bacterium, Pseudomonas aeruginosa. All these results imply that No. 114 protease from this psychrophilic bacterium is a unique member of the serralysin group characterized by a low optimal temperature.

Adaptation, Physiological↗

PubMed1999

Interaction of paratropomyosin with beta-connectin and its 400-kilodalton fragment from chicken skeletal muscle as influenced by the calcium ion concentration.

The binding of paratropomyosin to beta-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of beta-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10(-7) M to 10(-5) M and markedly suppressed above 10(-4) M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of beta-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.

Animals↗

PubMed1998

Formal design and implementation of an improved DDBJ DNA database with a new schema and object-oriented library.

MOTIVATION: The DNA Data Bank of Japan (DDBJ) has developed a new DNA database system with a new schema design to accommodate rapid change and growth of requirements on the system. RESULTS: The new schema and systems were created using an object-oriented design approach. The design was accomplished in accordance with ANSI/SPARC three-level schema architecture. First, the conceptual schema was designed using a functional model named AIS (associative information structure) and was visualized in extended diagram format. The model is a natural extension of an ER (entity relationship) model and describes real-world objects in binary associations between entities with the concept of order. Second, the schema was mapped on a relational database as a physical schema. All details are concentrated in this schema and the layer lying above enjoys physical independence. Finally, as another layer, external modeling was introduced for the database applications interface. It provides set-at-a-time basis operations and was implemented as a C++ object-oriented library. On this common framework of a new schema, a new annotator's workbench named Yamato II and a World Wide Web (WWW) submission system named Sakura have been successfully developed to improve drastically daily transactions in the DDBJ. AVAILABILITY: Sakura is available at the following address: http://sakura.ddbj.nig.ac.jp. CONTACT: hsugawar@genes.nig.ac.jp

Base Sequence↗

PubMed1998

Binding of paratropomyosin to beta-connectin from chicken skeletal muscle.

We found that paratropomyosin bound to beta-connectin, in examining binding of paratropomyosin at the junction of A- and I-bands of sarcomeres. The turbidity of a mixture of beta-connectin and paratropomyosin was greater with more paratropomyosin added, but high concentrations of Ca2+ suppress this increase. These results suggest that paratropomyosin is released from connectin filaments at the A-I junction region by increased concentrations of calcium ions in postmortem skeletal muscles.

Animals↗

PubMed1998

The application of model-based complexity inference method to molecular evolution analysis.

In this study, a new method based on the concept of complexity in inductive inference is proposed for reconstructing molecular phylogenetic tree. This method describes the complexity of molecular phylogenetic tree by three terms, which are related to tree topology, the branch lengths and fitness between the model and data measured by likelihood function. The computer simulation is used to investigate the efficiency of this method. The results suggest that this method is superior to the traditional methods because it avoids excess-complexity in the tree model estimation available from DNA sequence.

Base Sequence↗

PubMed1997

Antithrombotic effects of NE-6505, a novel anion-binding exosite inhibitor.

NF-6505, a bi-O-Tyr-sulfated decapeptide, which specifically interacts with the anion-binding exosite of the thrombin molecule, was chemically synthesized and assessed for its antithrombotic effects in vitro and in vivo. The IC50 value of this peptide on fibrin-clot formation in vitro was about 0.05 microgram/ml, which indicated a potency similar to that of a recombinant hirudin. NF-6505 caused a 2-fold prolongation of activated partial thromboplastin time when intravenously administered at 1 mg/kg in rats. In a rat venous thrombosis model, a bolus intravenous administration of this peptide dose-dependently inhibited the thrombus formation with an ED50 value of 0.03 mg/kg, a value smaller than that of recombinant hirudin (ED50 = 0.1 mg/kg) or of argatroban (ED50 = 0.2 mg/kg). These results suggest that NF-6505 is a highly potent and safe agent for the clinical treatment of venous thrombosis diseases.

Amino Acid Sequence↗

PubMed1997

Antithrombotic effects of the novel inhibitor of thrombin-induced offtelet aggregation and thrombus formation, 3-[2-[1,1':2',1"]-terphenyl-4'-yl)ethyl]phenoxyacetic acid.

The new compound 3-[2-([1,1':2,1"]-terphenyl-4'yl)ethyl]phenoxyacetic acid (F1070) was synthesized and its effects on platelet aggregation induced by thrombin, thrombin receptor agonist peptide (TRAP), ADP and collagen were evaluated in humans, guinea pigs and rats, and were compared with the effects of he thrombin antagonists argipidine and (D)Phe-Pro-Arg-CH2Cl (FPR). F1070 inhibited the platelet aggregation induced by these agonists and was highly selective in its inhibition of thrombin. F1070 inhibited fibrin formation induced by thrombin, but far less effectively than argipidine. In a guinea pig model of extracorporeal circulation thrombosis, F1070 (10 mg/kg p.o.) significantly inhibited the development of a thrombus. F1070 is thus a key compound that should facilitate the development of new orally active antithrombotic drugs that are specific for thrombin.

Animals↗

PubMed1997

Inference of molecular phylogenetic tree based on minimum model-based complexity method.

In this study, starting with a newly introduced concept of data complexity ("empirical data complexity"), we specify the concept of complexity more concretely in relation to mathematical modeling and introduce "model-based complexity (MBC)". Inductive inference based on the minimum model-based complexity method is then applied to the reconstruction of molecular evolutionary tree from DNA sequences. We find that minimum MBC method has good asymptotic property when DNA sequence lengths approach to infinite and compensates the bias of maximum likelihood method due to the difference of tree topology complexity. The efficiency of minimum MBC method for reconstruction of molecular tree is studied by computer simulation, and results suggest that this method is superior to the traditional maximum likelihood method or its modification by Akaike's AIC.

Animals↗

PubMed1997

Anticoagulant activity of the novel thrombin inhibitor 1-butyl-3-(6,7-dimethoxy-2-naphthylsulfonyl) amino-3-(3-guanidinopropyl)-2-pyrrolidinone hydrochloride.

1-Butyl-3-(6,7-dimethoxy-2-naphthylsulfonyl)amino-3-(3-guanidin opropyl)-2-pyrrolidinone hydrochloride (CAS 173440-64-7, SPI-501), which has highly selective thrombin-inhibitory activity, caused a concentration-dependent increase in the time taken for coagulation induced by thrombin in rabbit plasma. The IC50 of SPI-501 was 1.7 mumol/l. Argipidine also prolonged coagulation time and its activity was one order of magnitude greater than that of SPI-501. SPI-501 and argipidine caused dose-dependent increases in the activated partial prothrombin time (APTT) and prothrombin time (PT) of rat plasma. When APTT and PT were measured, IC50 values of SPI-501 were 38.0 and 18.5 mumol/l and those of argipidine, 1.4 and 1.9 mumol/l, respectively. Intravenous administration of SPI-501 (10 and 30 mumol/kg) and argipidine (1 and 3 mumol/kg) prolonged both APTT and PT in rats. While SPI-501 was less potent than argipidine, the durations of the effects of both were the same.

Animals↗

PubMed1996

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues.

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO3H)YLQ, -PYY(SO3H)YLQ, -PYYY(SO3H)LQ and -PYY(SO3H)Y(SO3H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N,N'-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO3H)Y(SO3H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO3H). The inhibitory activity by substitution with Tyr(SO3H) at position 63 was greater than that obtained by the substitution at position 62.

Amino Acid Sequence↗

PubMed1996

Anticoagulant peptides; synthesis, stability and antithrombin activity of hirudin C-terminal-related peptides and their disulfated analog.

We designed a unique anticoagulant decapeptide, which possesses two O-sulfated tyrosine residues, based on the structure of hirudin's C-terminal functional domain. We first prepared a series of octa-, nona- and decapeptides with no sulfation, Suc-Phe-Glu-Pro-Ile-Pro-Glu-Tyr-Tyr-X-OH [X = bond, Leu or Leu-Gln], by a solution phase method and measured their thrombin times (TT) using human thrombin and rabbit plasma. The shortest octapeptide (3a) showed full antithrombin activity comparable to that of the lead compound hirudin (54-65), and the longest decapeptide (3c) prolonged TT most potently with an IC50 value of 5.8 microM. We consequently converted 3c to a disulfated decapeptide (NF-22) with SO3.pyridine complex and compared its antithrombin activity with that of known hirudin-related peptides: hirugen, MDL28050 and hirulog-1. NF-22 showed potent antithrombin activity with an IC50 value of 0.3 microM, being more potent than hirugen and MDL28050 (IC50 values of 4.0 microM and 1.1 microM, respectively). NF-22 was as potent as hirulog-1. NF-22 showed no change in activity in aqueous solution for 10 d at 60 degrees C, and remained about 90% unchanged in rat plasma on incubation for 24h at 37 degrees C, whereas the corresponding unsulfated peptide (3c) was completely digested under the same condition. NF-22 appears to be one of the most potent and stable peptide anticoagulants among the hirudin analogs.

Amino Acid Sequence↗

PubMed1995

Lactam-conformationally restricted analogs of N alpha-arylsulfonyl arginine amide: design, synthesis and inhibitory activity toward thrombin and related enzymes.

Three new lactam-conformationally restricted arginine derivatives, 1-butyl-3-(6,7-dimethoxy-2-naphthylsulfonyl)-3-(3-guanidinoprop yl)-substituted gamma-, delta-, and epsilon-lactams (2-4), were synthesized on the basis of backbone modification of the lead structure, 6,7-dimethoxy-2-naphthylsulfonylarginine n-butylmethylamide (1). We tested these compounds for inhibitory activity toward thrombin and other trypsin-like enzymes (trypsin, factor Xa, plasmin, and kallikrein). All the compounds synthesized (1-4) potently inhibited thrombin with IC50 values of 0.75, 0.70, 0.92, and 3.2 microM, respectively; they inhibited thrombin over 40-fold more effectively than the other enzymes tested. The gamma-lactam (2) with the most profound inhibitory activity toward thrombin was a reversible inhibitor with a Ki of 0.26 microM. Compound 2 also showed better thrombin selectivity than the lead compound (1). The lactam-conformational restriction of arylsulfonylarginine amides, especially gamma-lactam, has thus proved to be a useful device for the improvement of antithrombotic activity.

Arginine↗

PubMed1994

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↗

PubMed1994

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↗