Biomedical subjects

T Yomo

Publications and source records attributed to T Yomo.

At least 37 records · Page 2Linked to original sources

PubMed1997

Insertion of foreign random sequences of 120 amino acid residues into an active enzyme.

Random sequences of 120-130 amino acid residues were inserted into a surface loop region of Escherichia coli RNase HI. This library was screened and about 10% of the clones were found to retain RNase H activity. Subsequent random mutagenesis led to an increase in RNase H activity and solubility of the protein. The inserted regions were found not to contribute to the secondary structure of the mutant protein. The high frequency of insertion of flexible sequences and the increase in the protein's function by further mutagenesis simulate one of the events in protein evolution.

Amino Acid Sequence↗

PubMed1997

Isologous diversification: a theory of cell differentiation.

An isologous diversification theory for cell differentiation is processed, based on simulations of interacting cells with biochemical networks and the cell division process following consumption of some chemicals. According to the simulations of the interaction-based dynamical systems model, the following scenario of the cell differentiation is proposed. (1) Up to some threshold number, divisions bring about almost identical cells with synchronized biochemical oscillations. (2) As the number is increased, the oscillations lose synchrony, leading to groups of cells with different phases of oscillations. (3) Amplitudes of oscillation and averaged chemical compositions start to differ by groups of cells. The differentiated behavior of states is transmitted to daughter cells. (4) Recursivity is formed so that the daughter cells keep the identical chemical character. This "memory" is made possible through the transfer of initial conditions. (5) Successive differentiation proceeds. The mechanism of tumor cell formation, origin of stem cells, anomalous differentiation by transplantations, apoptosis and other features of cell differentiation process are also discussed, with some novel predictions.

Animals↗

PubMed1996

Application of N-terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) to DNA sequencing and polymerase chain reactions: comparative study of delta Tth and wild-type Tth polymerases.

N-Terminally truncated DNA polymerase from Thermus thermophilus (delta Tth polymerase) lacking 5'-3' exonuclease activity was used for DNA sequencing and polymerase chain reaction (PCR). In contrast to the high background of the sequencing ladder observed with the wild-type Tth polymerase, delta Tth polymerase gave readable sequencing patterns which extend up to more than 500 bases from the primer site on cycle sequencing and automated sequencing. The delta Tth polymerase was used for the standard and mutagenic PCR, and net amplification of the DNA and the mutations accumulated during PCR were analyzed. Under mutagenic PCR, the mutation rates were 7.0 x 10(-4) (Tth) and 8.3 x 10(-4) (delta Tth) per nucleotide per cycle of amplification, which were 4-9 times higher than the rates under standard PCR.

Automation↗

PubMed1996

Solubility of artificial proteins with random sequences.

A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3-42 and RP3-45 and insoluble RP3-70 were purified. The solubility of the purified form is the same as that in the cell lysate.

Amino Acid Sequence↗

PubMed1995

A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2.

The entire nucleotide sequence of nylon oligomer degradative plasmid pOAD2 from Flavobacterium sp. KI723T1 was determined. pOAD2 comprises 45519 bp, with a 66.6 mol% G+C content. The precise loci of the four nylon oligomer degradation genes, namely nylA (6-aminohexanoate-cyclic-dimer hydrolase gene), nylB (6-aminohexanoate-dimer hydrolase), nylB' (a gene having 88% homology to nylB) and nylC (endo-type 6-aminohexanoate oligomer hydrolase), and five IS6100 elements were identified on this plasmid. Comparison of the sequence of pOAD2 with those in the GenBank and EMBL databases revealed that the deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (oligopeptide permeases), ftsX (filamentation temperature sensitive), penDE (isopenicillin N-acyltransferase) and rep (plasmid incompatibility). A functional map of pOAD2 is presented.

Amidohydrolases↗

PubMed1995

Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution.

Through selective cultivation with 6-aminohexanoate linear dimer, a by-product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains.

Amidohydrolases↗

PubMed1995

[Production of medicinal plants by soilless culture system. I. Studies of morphological characteristics and saikosaponins content in Bupleurum falcatum cultivated by Ebb & Flood system].

We studied that the morphological and histological characteristics, and the content (%DW) of saikosaponins on the root of Bupleurum falcatum cultivated in an Ebb & Flood system (E & F), a kind of soilless culture system, by both the direct sowing and the transplanting methods, and that effects of pinching on the root growth and the content (%DW) of saikosaponins in each part of root. Yield of root and content (%DW) of saikosaponins in each part of root, 8-months-old, cultivated in E & F by both methods were at the same level as that cultivated for the same period in soil condition by generally standard procedures. Morphological characteristics of the root cultivated by the direct sowing method were the same appearance as that by soil condition, but by the transplanting method main root branched off in all direction and the lateral root were more developed than by the direct sowing method. By pinching lignification in xylem on the main root were inhibited, but the dried weight of total root part and content (%DW) of saikosaponins in each part of the root were not shown to be significantly changed.

Botany↗

PubMed1994

A frame-specific symmetry of complementary strands of DNA suggests the existence of genes on the antisense strand.

The bacterial DNA sequence in GenBank database were divided into coding and noncoding regions and examined for the base-trimer distribution in every triplet frame on the sense and antisense strands. The results revealed that for the noncoding region, both strands have very similar base-trimer distributions and have no frame specificity; that is, DNA is symmetric in the noncoding region. For the coding region, on the other hand, the symmetry is broken only in the triplet framework, and we found a special triplet-frame-specific symmetry which appears when the two complementary strands of the coding region are read from their 5' ends. In addition, the following frame specificity was also observed in the distribution of stop codons on the antisense strand of the coding region. When the antisense sequences of the open reading frames (ORFs) in the database are read in the three reading frames, the same reading frame as the corresponding ORF contains a significantly larger amount of long open frames without stop codons (i.e., nonstop frames [NSFs]) than expected, while the number of NSFs in the other two reading frames is similar to that of the expected one. That is, NSFs as well as ORFs are maintained in a frame-specific manner, and in this sense, DNA becomes symmetrical even in the coding region. These two kinds of frame-specific symmetries indicate that only an ORF and its complementary triplets are specifically recognized and maintained in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Antisense↗

PubMed1994

Insertion sequence IS6100 on plasmid pOAD2, which degrades nylon oligomers.

The nucleotide sequence of repeated sequence I, which appears in five regions on nylon oligomer-degrading plasmid pOAD2, harbored in Flavobacterium sp. strain K172, was determined. The five regions of repeated sequence I had 880 bp of identical sequence, and the sequence was identical to that of IS6100, an insertion sequence classified in the IS6 family, initially found in Mycobacterium fortuitum. Sequences homologous to that of IS6100 were found for another nylon oligomer-degrading plasmid, pNAD2, harbored in Pseudomonas sp. strain NK87, by Southern hybridization experiments.

Amino Acid Sequence↗

PubMed1994

[Study on development of linear-traction type skeletal muscle powered pump].

We successfully developed Linear-Traction Type Skeletal Muscle Powered Pump (LSMPP). Unlike the Insertion-Type SMPP (ISMPP), previously used by us, in which a polyurethane chamber is inserted between skeletal muscle and chest wall, this pump can utilize the traction of muscle fiber in a linear direction. This pump also enabled us to obtain a sufficient flow with a low preload by the use of a spring for the filling of the pump. Comparison was made between the measurement of flow using the origin side of Latissimus Dorsi Muscle (o-group) (n = 6) and that using the upper arm side (i-group) (n = 6), both measured with the Linear-Traction Type equipment, and the result obtained with the ISMPP (s-group) (n = 6). Under a rate of 60 beat/min and a preload of 40 mmHg, the flows (ml/min) of o-group, i-group and s-group were 668.0 +/- 108.0, 1202 +/- 478.6 and 620.0 +/- 188.0, respectively. In i-group (n = 6), different rate, preload and spring constant were given to examine how the flow would vary with them. Mean flow (Mean +/- Std ml/min) was 815.0 +/- 378.0 at 60 beat/min, significantly different in comparison with that of 80 beat/min and that of 100 beat/min. For 40, 30 and 20 mmHg of preloads, mean flows were 877.0 +/- 366.0, 625.0 +/- 311.0 and 422.0 +/- 270.0, respectively. This showed that, as far as the rate remains low, even low preload can provide some 30% of the cardiac output of a dog as compensate cardiac flow. In contrast, there were no significant differences in mean flow with spring constants such as 0, 80, 160, 240 and 320 g/cm.

Animals↗

PubMed1993

Stabilization of xylanase by random mutagenesis.

Four heat-resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser-26 to Trp, Gly-38 to Asp and Thr-126 to Ser; N102, Gly-38 to Asp; N104, Gly-38 to Ser and Arg-48 to Lys; F1, Ser-12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Endo-1,4-beta Xylanases↗

PubMed1992

No stop codons in the antisense strands of the genes for nylon oligomer degradation.

Genes for nylon oligomer-degrading enzymes are unique in the sense that the enzymes encoded by them are found not to have any appropriate substrates during most of the period of their evolution. Furthermore, these nylB genes form a family not related to any other known gene families. The base sequences of these genes were examined and a common characteristic was found: a long stretch of sequence without chain-terminating base triplets, defined as a nonstop frame (NSF), is being maintained on the antisense strand. Moreover, a certain coding frame is open for both the sense and the antisense sequences, while the other frames have many stop codons. The probability of the presence of these NSFs on the antisense strand of a gene is very small (0.0001-0.0018). In addition, another gene for nylon oligomer degradation was found to have a NSF on its antisense strand, and this gene is phylogenically independent of the nylB genes. Therefore, the presence of these NSFs is very rare and improbable. Even if the common ancestral gene of the nylB family was originally endowed with a NSF on its antisense strand, the probability of this original NSF persisting in one of its descendants of today is only 0.007. Unless an unknown force was maintaining the NSF, it would have quickly disappeared by random emergences of chain terminators. Therefore, the presence of such rare NSFs on all three antisense strands of the nylB gene family suggests that there is some special mechanism for protecting these NSFs from mutations that generate the stop codons. Such a mechanism may enable NSFs to evolve into new functional genes and hence seems to be a basic mechanism for the birth of new enzymes.

Amidohydrolases↗

PubMed1992

Preparation and kinetic properties of 5-ethylphenazine-lactate-dehydrogenase-NAD+ conjugate, a semisynthetic lactate oxidase showing a hide-and-seek effect.

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP(+)-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 microM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.

Animals↗

PubMed1992

Principles for designing enzyme-like catalysts based on the rate-acceleration mechanisms of semisynthetic oxidases.

Combinations of substrate-binding sites and catalytic groups constitute various kinds of enzyme-like catalysts. The design of such catalysts can be evaluated by the enhancement of the overall catalytic activity by combining these parts into one catalyst. For a catalyst having one substrate-binding site and one catalytic group, an equation was obtained which shows the relationship between the rate-acceleration due to the combination, the affinity of the site (1/Kd), intrinsic effective concentration (kin/kex) and substrate concentration ([S]). The intrinsic effective concentration is the ratio of the first-order rate constant (kin) of the intramolecular reaction between the catalytic group and the bound substrate and the second-order rate constant (kex) of the intermolecular reaction between the catalytic group and the free substrate; the value depends on the method of linking the catalytic group and the binding site. This equation provides the following principles for designing catalysts of this type with a considerable grade of rate-acceleration: [S] less than or equal to kin/kex and (1/10)[S] less than or equal to Kd less than or equal to kin/kex. To increase kin/kex, the structure of the binding site is required not to reduce the reactivity of the bound substrate, and the linker connecting the binding site and the catalytic group is required to be flexible and to have an appropriate length. A subunit structure is also found to be effective to improve the catalytic activity: the activity of an n-mer is at most n2 times as high as that of the monomer. As for the substrate-binding sites, the sites of natural enzymes and antibodies are good candidates because various kinds of binding sites with high affinity and specificity to the corresponding substrates are available. In addition, the equation relating the rate-acceleration with Kd, kin/kex, and [S] is used for explaining the catalytic efficiency of enzymes energetically. The principle for designing a multifunctional catalyst having several kinds of binding sites for its substrates and intermediates and several kinds of catalytic groups was then investigated. In this case, the diffusion of the intermediates strongly affects the activity of the multifunctional catalyst, and such a diffusion process was also analyzed. On the basis of these analyses, the following principles were obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗

PubMed1992

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Amino Acid Sequence↗

PubMed1992

Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030.

Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase. Two of them, gdhI and gdhII, were cloned from B. megaterium IAM1030 in our previous work (T. Mitamura, R. V. Evora, T. Nakai, Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Ferment. Bioeng. 70:363-369, 1990). In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified. Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids. Thus, a total of four glucose dehydrogenase genes have been cloned from B. megaterium IAM1030. In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis. The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively. These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues. Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II. These glucose dehydrogenases were stabilized in the presence of 2 M NaCl. The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme. Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP. The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

Amino Acid Sequence↗

PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-glucose-dehydrogenase-NAD+ conjugate, a semisynthetic glucose oxidase.

5-Ethylphenazine-glucose-dehydrogenase-NAD+ conjugate (EP(+)-GlcDH-NAD+) was prepared by linking both poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to glucose dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.8, and that of the NAD+ moieties was 1.2. This conjugate is a semisynthetic enzyme having glucose oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following values of the kinetic constants: for the system with oxygen, the turnover number per subunit is 0.40 s-1 and Km for oxygen is 1.57 mM; and for the system with MTT, the turnover number is 0.11 s-1 and Km for MTT is 0.072 mM. The catalytic cycle of the semisynthetic oxidase has two catalytic steps: reduction of the NAD+ moiety by the active site of the glucose dehydrogenase moiety and oxidation of the NADH moiety by another catalytic site of the ethylphenazine moiety. The apparent intramolecular rate constants of these steps were estimated, and the values are as follows: 0.39 s-1 for the reductions of the NAD+ moiety, 2.2 s-1 and 0.12 s-1 for the oxidation of the NADH moiety in the systems with oxygen and with MTT, respectively, and 3.2 s-1 and 0.18 s-1 for the reduction of the ethylphenazine moiety in the systems with oxygen and with MTT, respectively. On the bases of these results, the following three rate-acceleration mechanisms of the semisynthetic glucose oxidase are discussed: high effective concentration, intramolecular coupling of successive catalytic reactions, and multiple connection between the two kinds of the catalytic sites.

Binding Sites↗

PubMed1991

Preparation and kinetic properties of 5-ethylphenazine-poly(ethylene-glycol)-glutamate-dehydrogenase conjugate. A semisynthetic NADH oxidase.

5-Ethylphenazine-poly(ethylene glycol)-glutamate dehydrogenase conjugate (EP(+)-PEG-GluDH) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme-binding site of glutamate dehydrogenase works as a substrate-binding site. The effects of the presence of the substrate-binding site near the catalytic group were studied by using EP(+)-PEG-GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme-binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP(+)-PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1-(3-carboxypropyloxy)-5- ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate-binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP(+)-PEG-GluDH show that the apparent turnover number of the active site is 0.38 s-1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate-binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate-accelerating effect of the binding site.

Binding Sites↗