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Biomedical subjects

Y Arata

Publications and source records attributed to Y Arata.

At least 73 records · Page 4Linked to original sources
PubMed1995

Effect of the tertiary structure alteration by ligation on the interface contacts between subunits of hemoglobin.

In order to obtain knowledge of molecular mechanism underlying the cooperative ligand binding to hemoglobin (Hb), atomic contacts between subunits in the hypothetical intermediate quaternary structures have been investigated using the atomic coordinates of O2-liganded subunits' within the T-state. The atomic coordinates are obtained by energy minimization of hypothetical atomic coordinate sets in which an O2 molecule is placed near the iron atom of a deoxy subunit. In an 'O2-liganded beta-subunit', drastic atomic displacements occur at the distal side amino-acid residues E11Val and E12Leu in order to produce sufficient space for the O2 molecule, while this kind of atomic displacement cannot be seen in an 'O2-liganded alpha-subunit'. Therefore the features of structural alterations at heme surroundings by O2 ligation are markedly different between alpha- and beta-subunits. When an alpha-subunit in deoxy Hb is replaced by the corresponding 'O2-liganded subunit', tight inter-subunit contacts between alpha 1FG4(92)Arg and the two residues beta 2C3(37)Trp and beta 2C6(40)Arg strengthen greatly, while, when a beta-subunit is replaced by a corresponding O2-liganded one, serious steric hindrances occur between beta 2FG4(97)His and alpha 1CD2(44)Pro. The characteristics of structural alteration confirm that the effect of O2-ligation at alpha-subunit is transmitted to the other subunit through the alpha FG4Arg. However, the role of FG4His in beta-subunit is not as clear as that of the alpha FG4Arg.

Binding Sites↗
PubMed1995

Model for the complex between protein G and an antibody Fc fragment in solution.

BACKGROUND: Streptococcal protein G and staphylococcal protein A are bacterial antibody-binding proteins, widely used as immunological tools, whose antibody-binding domains are structurally quite different. The binding of protein G to Fc fragments is competitive with respect to protein A, suggesting that the binding sites for protein A and protein G on Fc overlap, notwithstanding the fact that they lack sequence or structural similarity. RESULTS: To resolve this issue, the residues involved in the interaction between an IgG-binding domain of protein G (domain II) and the Fc fragment of mouse IgG2a have been identified by use of 13C and 15N NMR. Binding of protein G domain II selectively perturbed resonances from residues between the CH2 and CH3 domains of Fc, whereas in domain II the residues affected are primarily those on the alpha-helix and the third strand of the beta-sheet. This information was used, together with the structures of the two uncomplexed proteins, to construct a model of the complex, using Monte Carlo minimization techniques. In this model, the alpha-helix of protein G lies in the same position as helix 1 of protein A in the crystal structure of the protein A:Fc complex, but its orientation differs from the latter by 180 degrees. CONCLUSIONS: The interactions of the bacterial antibody-binding proteins with their 'target' immunoglobulins involve a very versatile set of protein-protein interactions. First, the IgG-binding domains of protein A and protein G have quite different three-dimensional structures, but bind to sites on the Fc fragment that overlap extensively. Secondly, protein G employs two quite different regions of its surface to bind to the Fab and Fc regions of IgG.

Antigens, Bacterial↗
PubMed1995

3'-Deoxy-3'-fluoroinosine as a potent antileishmanial agent. The metabolism and selective cytotoxic effect of 3'-deoxy-3'-fluoroinosine against Leishmania tropica and L. donovani in vitro and in vivo.

We studied the antileishmanial activity of 3'-deoxy-3'-fluoroinosine (3'-FI) against Leishmania tropica and L. donovani. In in vitro cultivation, the EC50 values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3'-FI against the promastigotes of L. tropica and L. donovani were 2.3 x 10(-7) and 1.0 x 10(-6) M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC50 value was 1.9 x 10(-4) M. Leishmania promastigote metabolized 3'-FI to 3'-deoxy-3'-fluoroadenosine 5'-triphosphate (3'-FATP) but FM3A cells did not. 3'-FI was effective against L. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3'-FI (50 mg/kg, given i.v.) showed a cytotoxic effect against the amastigotes of L. donovani in mice.

Animals↗
PubMed1995

Impaired colour discrimination among workers exposed to styrene: relevance of a urinary metabolite.

OBJECTIVES: To survey the loss of colour vision among Japanese workers who have been exposed to styrene concentrations currently considered low (about 20 ppm). Also to assess the effects of styrene by examination of the nature of the relation between disorder of colour vision and age, alcohol consumption, and other variables. METHODS: Colour discrimination was examined in 64 male workers exposed to styrene (mean age; 38.0, mean exposed years; 7.0) and in 69 controls (mean age; 38.0). A standardised questionnaire was adopted to collect work history, occupational or non-occupational solvent exposure, alcohol consumption, and drug use. Colour vision was evaluated by the Lanthony desaturated panel D-15 test. The results of the test were expressed as the colour confusion index (CCI). RESULTS: The mean atmospheric styrene concentration was about 20 ppm. The mean urinary concentration of mandelic acid was 0.22 g/l. There was a significant difference in CCI between exposed workers and age matched controls. Colour vision of workers whose concentration of urinary mandelic acid was > or = 0.42 g/l was significantly impaired when compared with workers whose concentration was < 0.42 g/l. Multiple linear regression analysis that controlled confounding variables such as age, alcohol consumption, smoking, and educational attainment showed that the CCI was significantly related to the concentration of urinary mandelic acid. In both exposed workers and controls, the types of defects were mostly blue-yellow loss, although a few subjects showed complex loss. No one showed only red-green loss. CONCLUSIONS: These findings suggest that exposure to moderate styrene concentrations can lead to impairment of colour vision, and that there is a significant correlation with the urinary metabolite of styrene.

Adult↗
PubMed1994

A stable isotope-aided NMR study of the active site of an endoglucanase from a strain of Bacillus.

Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.

Amino Acid Sequence↗
PubMed1994

Role of the domain-domain interaction in the construction of the antigen combining site. A comparative study by 1H-15N shift correlation NMR spectroscopy of the Fv and Fab fragments of anti-dansyl mouse monoclonal antibody.

A comparative NMR structural study of anti-dansyl Fv and Fab fragments is reported. Both of these antigen binding fragments have been prepared using antibodies that originate from the identical anti-dansyl switch variant cell lines. It has been confirmed that the Fv and Fab fragments possess the identical binding property. The antigen binding fragment analogs labeled with 15N of the main chain amide group of the aromatic residues (His, Phe, Trp, and Tyr) were used. The chemical shift and hydrogen-deuterium exchange rate of the amide protons are compared for the Fv and Fab fragments. On the basis of the NMR data obtained, we have concluded that (1) the structural change induced in the VH domain upon antigen binding significantly affects the dynamical structure of the VL domain and (2) the existence of the constant regions affects the fluctuation of the VL domain, increasing the thermal stability of the variable region.

Amides↗
PubMed1994

Application of 13C NMR spectroscopy to paratope mapping for larger antigen-Fab complexes.

For the purpose of engineering the antibody combining site, mapping residues that are involved in antigen binding provide us with valuable information. By use of 13C NMR spectroscopy with selectively 13C-labeled Fv fragments, we have established a general strategy to identify the residues that are perturbed upon binding of small antigen (hapten) molecules [(1990) Biochemistry 30, 6604-6610]. In the present paper, we demonstrate that this strategy can be extended to molecular structural analyses of the complexes of an Fab fragment and a larger antigen molecule such as Pseudomonas aeruginosa exotoxin A with a molecular mass of 67 kDa.

ADP Ribose Transferases↗
PubMed1994

O-glycosylation in hinge region of mouse immunoglobulin G2b.

Mouse monoclonal immunoglobulin G2b (IgG2b) antibodies are known to contain two forms of the heavy chain that are different in susceptibility to the protease attack. In the present study, by use of an affinity column containing sialic acid-binding lectins from Maackia amurensis seeds, a mouse monoclonal IgG2b was successfully separated into three phenotypes, which are different in the degree of sialylation in the heavy chain. In the N-linked oligosaccharides from all of the IgG2b phenotypes, virtually no sialylation was detected. Elution profiles of the lysyl endopeptidase digestion products were compared for the three phenotypes. The peptides eluted at different retention times were subjected to fast atom bombardment-mass spectrometry and amino acid sequence analyses. It was revealed that approximately 40% of the heavy chain of the mouse IgG2b are O-glycosylated at Thr-221A in the hinge region, predominantly with a tetrasaccharide composed of GalNAc, Gal, and two N-glycolylneuraminic acid residues. We suggest that the O-glycosylation renders the hinge region resistant against the proteolyses of the heavy chain. A therapeutic significance of the O-glycosylation of IgG2b is briefly discussed.

Amino Acid Sequence↗
PubMed1994

Synthesis and characterization of sapecin and sapecin B.

Two insect defencins, sapecin and sapecin B, were chemically synthesized to confirm their structure and antibacterial activity and also to examine the possibility that these peptides bind to the same site on the large conductance calcium-activated potassium channel as charybdotoxin. Both synthetic peptides showed the same antibacterial activity as native sapecins, indicating that the synthetic peptides folded correctly in the chemical synthesis. Synthetic sapecins did not show an inhibitory effect on [125I]charybdotoxin binding to rat brain synaptic membranes, suggesting that sapecin B recognizes a different binding site from that of charybdotoxin despite the similar structural motif.

Amino Acid Sequence↗
PubMed1994

Dynamical structure of the hinge region of immunoglobulin G as studied by 13C nuclear magnetic resonance spectroscopy.

The segmental flexibility of the hinge region of immunoglobulin G (IgG) molecules has been considered to play important roles in the mediation between antigen recognition and effector functions. Here we report the result of a 13C nuclear magnetic resonance study of the dynamical structure of the hinge region of a mouse monoclonal IgG2a antibody. In the present work, a variety of IgG2a analogs selectively labeled with 13C at the carbonyl carbon atom were prepared. The flexibility of the hinge region was discussed on the basis of the linewidth data of 13C resonances obtained using a Carr-Purcell-Meiboom-Gill pulse train. It was concluded that the hinge region of the mouse IgG2a molecule has a mosaic structure with heterogeneous nature of flexibility with the rigid core part flanked by upper and lower hinges, which are highly flexible peptide segments. Cleavage of the inter-chain disulfide bridges led to homogeneous flexibility of the hinge region, except for the N-terminal side of the upper hinge. It was suggested that the rigidity in the N-terminal side of the upper hinge is due to the interaction with the CH1 domain. Biological significance of the mosaic structure of the hinge region was briefly discussed.

Amino Acid Sequence↗
PubMed1994

Naive T cells can mediate delayed-type hypersensitivity response in T cell receptor transgenic mice.

We produced transgenic mice expressing T cell receptor-alpha beta chain genes, derived from the chicken ovalbumin (OVA)-specific I-Ad-restricted CD4+CD8- T helper cell clone 7-3-7. In transgenic mice with H-2d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-gamma, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.

Animals↗
PubMed1993

A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies: comparison of antibody combining sites of primary response antibody N1G9 and secondary response antibody 3B62.

On the basis of multinuclear NMR data, the structures of the antibody combining sites of anti-4-hydroxy-3-nitrophenylacetyl (NP) antibodies were compared for N1G9, which is one of the primary response antibodies with low affinity for NP, and 3B62, which is one of the secondary response antibodies with high affinity for NP. It has been concluded, on the basis of the results of antibody engineering, that in most secondary response antibodies a Trp-->Leu exchange at position 33 of the heavy chain is primarily responsible for the increased affinity for NP [Allen, D., Simon, T., Sablitzky, F., Rajewsky, K., & Cumano, A. (1988) EMBO J. 7, 1995-2001]. Although 3B62 exhibits one of the highest affinities for NP, it lacks the Trp-->Leu exchange at position 33 of the heavy chain. A variety of stable isotope-labeled Fab analogues of N1G9 and 3B62 have been prepared. Chain-specific resonance assignments were made by recombination of the heavy chains and light chains of the Fab fragments. Binding experiments of a spin-labeled hapten and NOESY experiments have demonstrated that, compared with the environment of the antibody combining site of N1G9, the combination of mutations (including one codon deletion) and the particular D-JH rearrangement in the heavy chain of 3B62 affords a more hydrophobic environment, which is formed by one Tyr residue originating from the light chain and two Tyr residues originating from the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Solution conformation of an antibacterial peptide, sarcotoxin IA, as determined by 1H-NMR.

The solution conformation of sarcotoxin IA, which is an antibacterial peptide isolated from Sarcophaga peregrina with a molecular mass of 4 kDa, was determined by NMR spectroscopy and hybrid distance geometry/dynamical simulated annealing calculations. On the basis of 227 experimental constraints, including 185 distance constraints obtained from NOE and 42 constraints associated with 21 hydrogen bonds, a total of 18 converged structures of sarcotoxin IA were obtained. The final 18 converged structures exhibit backbone-atomic root-mean-square differences about the averaged coordinate positions of 0.070 +/- 0.027 nm for residues 3-23 and 0.040 +/- 0.017 nm for residues 28-38. It has been indicated that sarcotoxin IA consists of two amphiphilic alpha-helical regions, i.e. helix I (Leu3-Gln23) and helix II (Ala28-Ala38), with a hinge region (Gly24-Ile27), which connects helix I and helix II. We conclude that these two amphiphilic helical segments of sarcotoxin IA are of importance for the expression of the antibacterial activity.

Amino Acid Sequence↗
PubMed1993

13C NMR study of the mode of interaction in solution of the B fragment of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G.

The mode of interaction of the B domain (FB) of staphylococcal protein A and the Fc fragments of mouse immunoglobulin G (IgG) has been investigated by 13C NMR spectroscopy. Mouse IgG1, IgG2a, and IgG2b proteins have been selectively labeled with 13C at the carbonyl carbon of His, Met, Trp or Tyr residue and used to prepare the corresponding Fc fragments by limited proteolysis. Site-specific resonance assignments have been made for each of these Fc analogues. FB was reported to form two contacts (contact 1 and contact 2) with human Fc in the crystal [Biochemistry 20 (1981) 2361-2370]. Comparisons of the chemical shift data of the Fc fragments observed in the absence and presence of FB have led us to conclude that in solution contact 1 is responsible for the formation of the Fc-FB complexes.

Animals↗
PubMed1993

Structural study of the oligosaccharide moieties of sphingolipid activator proteins, saposins A, C and D obtained from the spleen of a Gaucher patient.

We have determined and compared the structures of the oligosaccharide moieties of saposin A, C and D purified from the spleen of a patient with Gaucher disease. These saposins, together with saposin B, are small glycoproteins, derived from separate domains of a single precursor, prosaposin, and are required for the lysosomal hydrolysis of various sphingolipids. The characteristic features of the oligosaccharide moieties of saposin A are (a) the predominance of a fucosylated trimannosyl core structure and (b) the occurrence of several different oligomannose-type and N-acetyllactosamine-type oligosaccharides. Saposin C contains (a) a predominance of oligomannose-type oligosaccharides and monoantennary oligosaccharides and (b) the presence of four different oligosaccharides having bisecting N-acetylglucosamine residues (found only in this saposin). Saposin D is distinguished by the occurrence of oligomannose-type oligosaccharides, which comprise nearly 90% of its total oligosaccharides. The possible reasons for the unique glycosylation of each saposin is discussed.

Carbohydrate Sequence↗
PubMed1993

Structural elucidation of a variety of GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase.

Fifteen different structures of terminal GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase have been identified. These N-linked oligosaccharides were mostly neutral, because sialic acid content was lower than 0.13 mol of sialic acid/mol of sugar chain, and sulfate was not detected. The oligosaccharides were released from pepsin-digested protein by glycoamidase A (from almond) digestion. The reducing ends of the oligosaccharide chains were aminated with a fluorescent reagent, 2-aminopyridine. The resulting mixture of pyridylamino derivatives of the oligosaccharides were separated by high performance liquid chromatography on an ODS-silica column, and 15 oligosaccharides were isolated. The structure of each oligosaccharide fraction was analyzed by two-dimensional sugar mapping, component sugar analysis, high resolution proton nuclear magnetic resonance and methylation analysis. It was found that each N-linked oligosaccharide associated with human urinary kallidinogenase contains unsubstituted GalNAc residues at the nonreducing terminal. These 15 oligosaccharides include 5 biantennary, 7 triantennary, and 3 tetraantennary oligosaccharides.

Acetylgalactosamine↗