PubMed Health⌕ Search

Biomedical subjects

T Imoto

Publications and source records attributed to T Imoto.

At least 55 records · Page 3Linked to original sources
PubMed1999

Construction of the single-chain Fv from 196-14 antibody toward ovarian cancer-associated antigen CA125.

The variable regions of heavy- and light-chains of mouse monoclonal antibody 196-14 toward ovarian cancer-associated antigen CA125 were linked with a peptide linker (GSTSGSGKSSEGKG) and a histidine tag was attached at the carboxyl terminal. This single-chain Fv (scFv) with a histidine tag was expressed in Escherichia coli as an inclusion body. The inclusion body was solubilized with guanidium chloride, followed by purification on nickel nitrilotriacetic acid agarose column and refolding into the active form. The scFv thus obtained bound to the Siso cells, which express CA125, and may recognize the same epitope as the parental 196-14 antibody IgG does.

Animals↗
PubMed1999

Resolution and characterization of tryptophyl fluorescence of hen egg-white lysozyme by quenching- and time-resolved spectroscopy.

The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region.

Acetylglucosamine↗
PubMed1998

Investigation of the structural basis for thermostability of DNA-binding protein HU from Bacillus stearothermophilus.

Site-directed mutagenesis was used to identify amino acid residues essential for the thermostability of the DNA-binding protein HU from the thermophile Bacillus stearothermophilus (BstHU). Two mutants, BstHU-A27S and BstHU-V42I, in which Ala27 and Val42 in BstHU were replaced by the corresponding amino acids Ser27 and Ile42, respectively, in the homologue from a mesophile B. subtilis (BsuHU), were less stable than the wild-type BstHU (63.9 degreesC), showing Tm values of 58.4 degreesC and 60.1 degreesC, respectively, as estimated by circular dichroism (CD) analysis at pH 7.0. The denaturation of two mutants was further characterized using differential scanning calorimetry; the Tm values obtained by calorimetric analysis were in good agreement with those estimated by CD analysis. The results suggest that Ala27 and Val42 are partly responsible for enhancing the thermostability of BstHU. When considered together with previous results, it is revealed that Gly15, Ala27, Glu34, Lys38, and Val42 are essential for the thermostability of thermophilic protein BstHU. Moreover, five thermostabilizing mutations were simultaneously introduced into BsuHU, which resulted in a quintuple mutant with a Tm value of 71.3 degreesC, which is higher than that of BstHU, and also resulted in insusceptibility to proteinase digestion.

Bacterial Proteins↗
PubMed1998

Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues.

The internal motion of lysozyme was described by the steady-state and time-resolved fluorescence anisotropy of its tryptophan residues. The fluorescence of mutant lysozymes W62Y- and W108Y-lysozyme, in which Trp62 or Trp108 of hen egg white lysozyme was replaced with a tyrosine residue, could be respectively assigned to Trp108 or Trp62 at the longer wavelength region of the total fluorescence spectrum. The segmental motion of Trp62 as shown by its fluorescence anisotropy decay was described with two components originating from the fluctuational rotation of an indole moiety about the Calpha-Cbeta bond and rotational wobble of the peptide segment adjacent to Trp62. Although Trp62 showed a high degree of motional freedom, its motion was significantly suppressed by the interaction of the mutant protein with a trimer of N-acetyl-D-glucosamine. By contrast, the segmental motion of Trp108 is hindered by the local cage structure at temperatures below 30 degreesC, but Relief from restricted motion occurred on the formation of ligand complex or by thermal agitation. Because of overlaps of the fluorescence spectrum, it is difficult to assign the segmental motion of Trp28 or Trp111, the other two tryptophan residues in lysozyme. However, a careful analysis of the fluorescence anisotropy decay of W62Y- and W108Y-lysozyme showed that the fluctuation of the hydrophobic matrix box was greater than that expected from lysozyme's crystal structure, although it was suppressed by the binding of the ligand to the active site of lysozyme.

Amino Acid Substitution↗
PubMed1998

Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype.

A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Smr) gene and streptomycin-dependent (Smd) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Smr gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Smr gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Smr gene of pHA394 was replaced by the fusion gene, followed by transformation of Smd E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.

Anti-Bacterial Agents↗
PubMed1998

High pressure NMR study of a small protein, gurmarin.

The effect of pressure on the structure of gurmarin, a globular, 35-residue protein from Gymnema sylvestre, was studied in aqueous environment (95% 1H2O/5% 2H2O, pH 2.0) with an on-line variable pressure NMR system operating at 750 MHz. Two-dimensional TOCSY and NOESY spectra were measured as functions of pressure between 1 and 2000 bar at 40 degrees C. Practically all the proton signals of gurmarin underwent some shifts with pressure, showing that the entire protein structure responds to, and is altered by, pressure. Most amide protons showed different degrees of low field shifts with pressure, namely 0-0.2 ppm with an average of 0.051 ppm at 2000 bar, showing that they are involved in hydrogen bonding and that these hydrogen bonds are shortened by pressure by different degrees. The tendency was also confirmed that the chemical shifts of the amide protons exposed to the solvent (water) are more sensitive to pressure than those internally hydrogen bonded with carbonyls. The pressure-induced shifts of the H alpha signals of the residues in the beta-sheet showed a negative correlation with the 'folding' shifts (difference between the shift at 1 bar and that of a random coil), suggesting that the main-chain torsion angles of the beta-sheet are slightly altered by pressure. Significant pressure-induced shifts were also observed for the side-chain protons (but no larger than 10% of the 'folding' shifts), demonstrating that the tertiary structure of gurmarin is also affected by pressure. Finally, the linearity of the pressure-induced shifts suggest that the compressibility of gurmarin is invariant in the pressure range between 1 and 2000 bar.

Amino Acid Sequence↗
PubMed1998

Tolerogenic activity of polyethylene glycol-conjugated lysozyme distinct from that of the native counterpart.

Conjugation of proteins with polyethylene glycol (PEG) has been reported to make the proteins tolerogenic. Native proteins are also potentially tolerogenic when given without adjuvants. We compared immunotolerogenic activities between PEG-conjugated and native hen egg-white lysozyme (HEL). BALB/c mice were given consecutive weekly intraperitoneal administrations of PEG-conjugated HEL, unmodified HEL or phosphate-buffered saline (PBS), for 3 weeks, then challenged with HEL in Freund's complete adjuvant. The pretreatment with PEG-HEL tolerized both T-cell and humoral responses, while native HEL tolerized only the T-cell response. Immunoglobulin G1 (IgG1) antibody was already elevated in HEL-pretreated mice prior to challenge immunization, followed by suppressed IgG2a and IgG2b, but spared IgG1 production after the antigen challenge, whereas PEG-HEL-pretreated mice produced no antibody in all IgG subclasses prior and subsequent to the antigen-challenge. Production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by lymphoid cells in response to HEL in vitro was markedly suppressed in both the antigen-pretreated groups, while suppression of IL-4 production was evident only in PEG-HEL-, not in HEL-pretreated animals. Involvement of suppressor cells in these tolerance states was found to be unlikely, and the immunological property of PEG-HEL differed from deaggregated HEL that was similar to the original HEL. These results suggest a unique immunotolerogenic activity of PEG-conjugated proteins to suppress both T-helper type-1 (Th1)- and Th2-type responses, the result being extensive inhibition of all IgG subclass responses, while tolerance induction by unconjugated soluble proteins tends to be targeted on Th1-, but spares Th2-type responses.

Adjuvants, Immunologic↗
PubMed1998

Reduction of the suppressive effects of gurmarin on sweet taste responses by addition of beta-cyclodextrin.

Cyclodextrins (CDs) have the remarkable ability to form inclusion complexes with a wide variety of guest molecules. In the present study, possible influences of CDs on gurmarin inhibition of the chorda tympani responses to sucrose were examined in C57BL mice. Responses to sucrose were suppressed to approximately 50% of control by treatment of the tongue with 30 micrograms/ml (approximately 7.1 microM) gurmarin. Rinsing the tongue with 15 mM beta-CD after gurmarin gave rapid recovery of the suppressed sucrose responses to approximately 85% of control, whereas 15 mM alpha- or gamma-CD did not. When gurmarin was mixed with beta-CD, the suppressive effects of gurmarin on sucrose responses were largely reduced. No such reduction was observed for mixtures with alpha- and gamma-CD. Gurmarin includes tyrosine and tryptophan residues whose aromatic rings are directed outward and can probably form inclusion complexes with beta-CD. Therefore, the observed reduction of the effects of gurmarin may be due to steric hindrances in inclusion complexes of gurmarin with beta-CD that may interfere with gurmarin binding to sweet taste receptors.

Animals↗
PubMed1998

Detection of a local interaction of hen lysozyme under highly denaturing conditions using chemically 13C-enriched methionine resonance.

Using hen lysozyme in which the epsilon-carbons of two methionine residues are enriched with 13C nuclei, we found that there is a subtle difference in the chemical shift of the epsilon-carbon resonances between Met 12 and Met 105 in thermally denatured lysozyme without any reduction of disulfide bonds at pD 3.8, and also in reduced S-alkylated lysozyme at pD 3.8 and 35 degrees C. The difference in the chemical shift was abolished on digestion with TPCK-trypsin and the chemical shifts of both resonances converged to that of Met 12, whose chemical shift is identical to that in the randomly coiled state. Therefore, it is suggested that the chemical shift in the epsilon-carbon resonance of Met 105 is different from that in the randomly coiled state due to an interaction involving Met 105. In order to locate the interaction involving Met 105, fragmentation of the reduced S-alkylated lysozyme into the peptides was carried out by means of chemical cleavage or specific endoprotease digestion. As a result, the local interaction of Met 105 or the residues around Met 105 with eleven residues at the C-terminus of lysozyme is suggested to occur.

Alkylation↗
PubMed1998

Kinetic measurement of the interaction between a lysozyme and its immobilized substrate analogue by means of surface plasmon resonance.

A method for evaluating the association and dissociation rate constants of interaction between a lysozyme and its substrate analogue, an immobilized p-aminophenyl-tri-N-acetyl-beta-chitotrioside, by means of surface plasmon resonance has been developed. Site-specific immobilization of p-aminophenyl-tri-N-acetyl-beta-chitotrioside, which is a product of p-nitrophenyl-tri-N-acetyl-beta-chitotrioside, on carboxymethyldextran linked to the surface of the cuvette of the instrument, IAsys, was carried out by catalysis with EDC/NHS. The kinetic parameters of the interaction between hen or human lysozyme and the immobilized substrate analogue indicated that a larger dissociation constant of the human lysozyme-immobilized substrate analogue complex depended on a smaller association rate constant. The kinetic parameters of the interaction between the immobilized substrate analogue and a mutant hen lysozyme, in which Arg14 and His15 are deleted, with higher activity than the wild type hen lysozyme were measured. It was suggested that the higher activity of the mutant lysozyme was due to faster removal of the substrate from the active site cleft and/or the formation of a stabler and better complex as to hydrolysis.

Animals↗
PubMed1998

Enhanced responses of the chorda tympani nerve to nonsugar sweeteners in the diabetic db/db mouse.

Genetically diabetic db/db mice show greater neural and behavioral responses to sugars than lean control mice. The present study examined chorda tympani responses of db/db mice to nonsugar sweeteners and their inhibition by a sweet response inhibitor, gurmarin. The results showed that responses to sucrose, saccharin, glycine, L-alanine, and D-tryptophan, but not to D-phenylalanine, were approximately 1.5 times greater in db/db mice than in control mice. Treatment of the tongue with gurmarin suppressed responses to these sweeteners in db/db and control mice, but the extent of suppression was considerably smaller in db/db mice. The magnitudes of gurmarin-sensitive components of the response to sweeteners in db/db mice were not significantly different from those in control mice, whereas the magnitudes of gurmarin-insensitive components in db/db mice were about twice as large as those in control mice. These results suggest that the enhancement of chorda tympani responses in db/db mice to sucrose and other nonsugar sweeteners may occur through gurmarin-insensitive membrane components.

Animals↗
PubMed1997

Depression of T-cell epitope generation by stabilizing hen lysozyme.

Conformational stability of proteins is an important factor that determines their resistance/susceptibility to proteolytic digestion. Intracellular proteolysis is the key step in antigen presentation events for protein antigens; hence, it is likely that increasing protein stability reduces the antigenicity of proteins. We prepared three hen egg white lysozyme derivatives possessing different stabilities by chemical modification to clarify the relationship between conformational stability and the antigenicity of the protein. One of the derivatives was conformationally unstabilized by removing one intramolecular disulfide bond, whereas the two others were stabilized by the addition of an intramolecular cross-link. The antigenicity of these derivatives was evaluated using hen egg white lysozyme-specific T-cell hybridoma cells and a B-lymphoma cell line, A20, as antigen-presenting cells. With an increase in conformational stability, the T-cell response decreased. However, the reduction was not derived from the inefficiency of internalization to A20 cells nor the alteration of antigenicity by chemical modifications. Moreover, from analyses of their susceptibility to proteolysis and the kinetics of presentation of the T-cell epitope, it was confirmed that increasing protein stability led to the depression of T-cell epitope generation by increasing resistance to proteolysis. These results have an important implication in devising a new strategy for manipulating T-cell response by the stability of protein antigen.

Animals↗
PubMed1997

A covalent enzyme-substrate adduct in a mutant hen egg white lysozyme (D52E).

A mutant hen egg white lysozyme, D52E, was designed to correspond to the structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H., and Matthews B. W. (1993) Science 262, 2030-2033) to investigate the role of the catalytic residue on the alpha-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of the N-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position of the catalytic residue on the beta-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme, and T4 phage lysozyme, the adaptability of the side chain on the alpha-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.

Animals↗
PubMed1997

Quaternary structure-dependent idiotope and antigen binding of a monoclonal antibody specific for conformational epitope on type II collagen.

We previously generated a monoclonal antibody (mAb) against a putative pathogenic epitope on native type II collagen (CII) for the induction of collagen-induced arthritis in mice (mAb1), and an anti-idiotypic mAb which appears to possess the internal image of the CII epitope (mAb2). In the present study, the structural basis of the antigen/mAb1 and mAb1/mAb2 interactions was examined. When partially SH-reduced mAb1 was analysed on Western blots, only fragments containing both heavy (H) and light (L) chains were recognized by mAb2. When mAb2 was partially SH-reduced, only fragments containing both H and L chains were recognized by mAb1. H and L chains were separated from mAb1 in a reduced, denatured condition, and each chain and a mixture of the two were refolded. mAb2 reacted specifically to the renatured whole IgG molecule of mAb1, but not to the refolded L or to H chains. Recombinant single chain Fv (scFv) generated from mAb1 and mAb2 had properties of the original mAbs, whereas genetically constructed chimeric scFvs, consisting of VH from mAb1 and an irrelevant VL. or VL of mAb1 and an irrelevant VH. did not react either to CII or to mAb2. Thus, interactions among CII, mAb1 and mAb2 appear to depend on quaternary structures containing different protein subunits. These observations support the internal image property of the mAb2. In addition, this dependency on quaternary structure for recognition of proteins may also be relevant to other protein-protein interactions.

Amino Acid Sequence↗
PubMed1997

Stabilization of protein.

The stabilization of proteins is discussed from the theoretical and practical points of view. Methods are described for kinetic stabilization and protection from deterioration, as well as the thermodynamic stabilization of proteins.

Animals↗
PubMed1997

Favourable interaction between heavy and light chains arrests the undesirable oligomerization of heavy chains in the refolding of denatured and reduced immunoglobulin G.

Recently we developed a slow dialysis method that effectively refolds denatured and reduced immunoglobulin G (IgG) [Maeda, Ueda and Imoto (1996) Prot. Engng 9: 95-100]. This method allows both individual and simultaneous refolding of denatured and reduced H and L chains. Analysis by SDS-polyacrylamide gel electrophoresis revealed that some oligomers were formed through disulfide bonds when H chains were refolded individually. It was also shown that the extent of IgG obtained by rejoining the mixture of refolded H and L chains which had been refolded individually was similar to that obtained by refolding denatured and reduced whole IgG. The results indicated that a favourable interaction between H and L chains prevented formation of H-chain oligomers to yield intact IgG. The present results suggest a mechanism whereby individually folded chains might associate to form IgG molecules in vivo.

Animals↗
PubMed1997

Prevention of collagen-induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one.

Administration of a soluble protein into animals prior to challenge immunization induces immunological tolerance which is specific for the protein. In addition, chemical modification of proteins with polyethylene glycol (PEG) has been reported to convert the immunogenic proteins to become tolerogenic. However, differences in tolerogenic properties between PEG-modified proteins and the native counterparts have never been analysed. The ability of PEG-conjugated type II collagen (PEG-CII) to attenuate CIA, an animal model for rheumatoid arthritis, was compared with the native unconjugated CII. Groups of DBA/1 J mice were treated weekly with i.p. injections with PEG-CII, native CII, or vehicle alone for 3 weeks, before they were challenged with CII in adjuvants. The induction of tolerance was confirmed in both PEG-CII- and CII-pretreated mice when suppression of lymph node T cell proliferation in response to CII was noted. The degrees of suppression of T cell proliferation were comparable between the two pretreated groups. However, induction of arthritis and production of IgG anti-CII antibody were more markedly suppressed in PEG-CII-pretreated mice than in native CII-pretreated mice, although the severity of arthritis and antibody levels in the latter group were also lower than in control mice. IgG2a and IgG2b antibody levels were equally suppressed in the two pretreated groups, whereas the IgG1 level was significantly lower in the PEG-CII-pretreated group than in the native CII-pretreated group. The results provide the first evidence that attachment of PEG to CII renders the protein more tolerogenic.

Animals↗