[Novel approach for drug design by using NMR].
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Combinatorial Chemistry Techniques↗
Biomedical subjects
I Shimada
Publications and source records attributed to I Shimada.
The pH-dependent structural variation of complementarity-determining region H3 in the crystal structures of the Fv fragment from an anti-dansyl monoclonal antibody.
The Fv fragment from an anti-dansyl antibody was optimally crystallized into two crystal forms having slightly different lattice dimensions at pH 5.25 and 6.75. The two crystal structures were determined and refined at high resolution at 112 K (at 1.45 A for the crystal at pH 5.25 and at 1.55 A for that at pH 6.75). In the two crystal structures, marked differences were identified in the first half of CDRH3 s having an amino acid sequence of Ile95H-Tyr96H-Tyr97H-His98H-Tyr99H-Pro1 00H-Trp100aH-Phe100bH-Ala101H- Tyr102H. NMR pH titration experiments revealed the p Kavalues of four histidine residues (His27dL, His93L, His55H and His98H) exposed to solvent. Only His98H (p Ka=6.3) completely changed its protonation state between the two crystallization conditions. In addition, the environmental structures including hydration water molecules around the four histidine residues were carefully compared. While the hydration structures around His27dL, His93L and His55H were almost invariant between the two crystal structures, those around His98Hs showed great difference in spite of the small conformational difference of His98H between the two crystal structures. These spectroscopic and crystallographic findings suggested that the change in the protonation state in His98H was responsible for the structural differences between pH 5.25 and 6.75. In addition, the most plausible binding site of the dansyl group was mapped into the present structural models with our previous NMR experimental results. The complementarity-determining regions H1, H3 and the N-terminal region in the VH domain formed the site. The side-chain of Tyr96H occupied the site and interacted with Phe27H of H1, giving a clue for the binding mode of the dansyl group in the site.
Antibodies, Monoclonal↗
Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.
The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.
Amino Acid Sequence↗
A multinuclear NMR study of the active site of an endoglucanase from a strain of Bacillus. Use of Trp residues as structural probes.
In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity; (b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pKa values to 5.5 for both amino acid residues; and (c) the pKa values of Glu130 and Asp191 would lead to an unusually narrow pH-activity profile of the endoglucanase K.
Amino Acid Sequence↗
Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis.
We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.
Alanine↗
Plastic surgical reconstruction of left main coronary artery.
OBJECTIVE(S): To report the early and mid-term results of surgical plasty of the left main coronary artery in 12 patients operated upon between 1993 and 1997. METHODS: The anterior approach was used in all patients. Saphenous vein (n = 4) of glutaraldehyde treated autologous pericardium (n = 8) were used as patch material. Additional coronary artery bypass grafting was performed in 7 patients, the first 3 as a safety back up, and for coexisting stenosis of other coronary branches not revascularized by the plastic procedure in the remaining 4. RESULTS: Pathologic specimens of the left main coronary artery in 5 revealed atheroma in 3 and myxomatous intimal thickening in 2. The left main coronary artery was widely patent angiographically in all patients prior to discharge. Six patients consented to angiographic restudy 5-40 months after the procedure and revealed excellent results in 5. One patient upon whom autologous pericardial patch had been used underwent percutaneous coronary angioplasty for restenosis of the left main coronary artery and a new lesion of the proximal left anterior descending branch 5 months after the operation. There were no late deaths nor other cardiac events. All patients were in CCS class 1 at their last follow-up. CONCLUSIONS: Surgical angioplasty of the left main coronary artery could be used to revascularize the left heart safely in patients with a discrete localized lesion of the left main coronary artery and is particularly useful in the face of unavailability of other conduits.
Aged↗
Prediction of thoracic aortic aneurysm expansion: validation of formulae describing growth.
BACKGROUND: The expansion rate of thoracic aortic aneurysms may be an important and clinically relevant index of the risk of rupture. The aims of this study were to assess the validity of three published exponential equations that predict expansion rate in a separate sample population, and to calculate an expansion rate formula for this cohort of patients. METHODS: We studied 88 consecutive patients undergoing serial computed tomographic or magnetic resonance imaging scanning to monitor thoracic aortic aneurysm progression. In interval scans of at least 6 months, we measured minimum coronal aortic diameter at seven set levels and maximal diameter, yielding 780 segment-intervals. RESULTS: The linear expansion rate (mean 2.6 mm/year) increased with incremental aortic diameter (aortic diameter < 40 mm: 2.0; 40-49 mm: 2.3; 50-59 mm: 3.6; > or = 60 mm: 5.6 mm/year; p < 0.01). Regression analysis showed close correlation between predicted and sample data, but there were significant differences between observed and expected measurements. The Yale formula underestimated growth by 0.8 mm, while Mt. Sinai and Osaka formulae overestimated actual change by 1.5 and 0.2 mm, respectively. The expansion rate derived from our population was: last diameter = initial diameter x e(0.00367 x time) (r = 0.617). CONCLUSIONS: Although formulae derived from one thoracic aortic aneurysm sample population may not extrapolate exactly to others, there is close concordance of results for patient populations in three different continents.
Aortic Aneurysm, Thoracic↗
Multiple receptor sites for nucleotide reception in the labellar taste receptor cells of the fleshfly Boettcherisca peregrina.
Nucleotides applied to the labellar chemosensory hair of the fleshfly, Boettcherisca peregrina, stimulated the taste receptor cells. Adenosine 5'-diphosphate (ADP) evoked a large response of the sugar receptor cell (sugar response) and guanosine 5'-diphosphate (GDP) evoked a large response of the salt receptor cell (salt response), but the salt response to ADP and the sugar response to GDP were relatively small. While the sugar response to ADP was independent over a wide range, pH5-9, the salt responses to GDP and ADP were inhibited at neutral and alkaline pH's, even though they elicited a marked salt response between pH's of about 5 and 6. Only adenine nucleotides (ADP, AMP, ATP) could stimulate the sugar receptor cell, with an order of stimulating effectiveness of ADP>>AMP>/=ATP. However, the salt receptor cell could respond significantly not only to GDP but various nucleoside 5'-diphosphates, nucleoside 5'-monophosphates, cyclic nucleotides and thiamine diphosphate. These results clearly suggest that the specificity of the receptor site reacting with nucleotide in the sugar receptor cell is very different from that in the salt receptor cell.
Journal Article↗
Reproducibility of thoracic aortic diameter measurement using computed tomographic scans.
OBJECTIVES: Decisions to recommend elective surgical repair of thoracic aortic aneurysms (TAA) may be based on size or expansion rate, which are used as indices of the risk of rupture. Measurement error may thus affect clinical decision-making. In order to evaluate the reproducibility of aortic diameter measurements of TAA, we assessed departmental inter- and intra-observer variability of measurement of pre-selected computed tomographic scan images of aneurysmal segments of the thoracic aorta. METHODS: We compared measurements of minimum aortic diameter made by four observers in 50 pre-selected scans and at different times by two observers using a calliper method and a measurement tool within the scan. Differences in measured dimension were analysed using Wilcoxon's signed ranks test and the repeatability assessed using the method of Bland and Altman. RESULTS: There were no significant inter-observer differences among three observers but there were significant differences between another observer and two other observers (P < 0.05). No significant intra-observer differences existed. The best intra-observer repeatability was 2.25 while the worst inter-observer repeatability was 4.37. The mean and maximum difference in measurement were +/-0.88 mm and +/-8.0 mm, respectively. Variability of measurement increased with aortic diameter. CONCLUSIONS: Calliper measurement of TAA is an acceptable measurement method for surveillance of TAA but appears most accurate with a single observer. Increasing error is seen with increasing diameter which may compound error in estimation of expansion rate. Standardisation of technique is advisable for multiple observers and aortic units should adopt quality assurance protocols to minimise error.
Aortic Aneurysm, Thoracic↗
DNA polymorphisms in the 5'-flanking sequence of human ABO blood group genes and their association with the alleles for the common ABO phenotypes.
Previously we found that minisatellite located on the 5'-flanking sequence far from the transcription start site of the human ABO blood group genes and containing four tandem repeats of a 43 base pair (bp) consensus sequence was an enhancer element for the transcription of the ABO genes. Concerning the number of tandem repeats in the minisatellite, there are at least three alleles: ABOU1*1, ABOU1*3, and ABOU1*4. Besides the variability of the minisatellite, another location of the 5'-flanking sequence has two alleles associated with the insertion of the 35 bp DNA segment: ABOU2*0 allele with an uneventful sequence and ABOU2*1 allele with the 35-bp insertion, respectively. Both of the DNA polymorphisms are closely associated with the common alleles of blood group ABO phenotypes. Namely, the 5'-flanking configuration consisting of the alleles ABOU1*1 and ABOU2*0 links up with the common A1 allele, whereas, the other 5'-flanking configuration consisting of the alleles ABOU1*4 and ABOU2*1 links up with common B and O alleles (abbreviated as 1-0-A1, 4-1-B and 4-1-O). In these linkages between three loci of alleles of 102 unrelated healthy individuals, two are not consistent with the rules described above. One is 3-1-O and the other is 4-1-A2. These findings suggest that the 3 or 4 times repeats of a 43 bp consensus sequence at ABOU1 locus and the 35 bp-insertion at ABOU2 locus found in 5'-flanking sequence of the B and O genes are incidental to the ABO gene evolution, subject to the ancestral A1 gene.
Journal Article↗
N-glycan structures of murine hippocampus serine protease, neuropsin, produced in Trichoplusia ni cells.
N-glycans of neuropsin (serine protease in the murine hippocampus) expressed in Trichoplusia ni cells were released from the glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a two dimensional high-performance liquid chromatography (HPLC) mapping technique on two kinds of HPLC columns. Fourteen different major N-glycan structures were identified, of which 6 were high-mannose type (9.1%), and the remaining 8 were paucimannosidic type. The presence of insect specific N-glycan structures containing both alpha1,3- and alpha1,6- di-fucosylated innermost N-acetylglucosamine residue (23.3%), as below, was also confirmed by 600 MHz 1H-NMR spectroscopy.
Animals↗
Chemical synthesis and properties of (6-4) photoproduct and its analogs.
We report the preparation of a deoxyribooligonucleotide containing a new thymine (6-4) photoproduct analog. The (6-4) photoproduct is one of the major forms of DNA lesions, and leads to mutation in DNA. An antibody (64M5) that binds the (6-4) photoproduct has been described. To investigate the interaction of the photoproduct with the 64M5 antibody, we prepared a (6-4) photoproduct analog in which the two thymines were connected with a formacetal linkage. With UV-irradiation, the thymine dimer with the formacetal linkage reacted to the (6-4) photoproduct faster than the phosphodiesterified dimer, and the yields of the analog was higher than those of the natural thymine dimer. The 64M5 antibody exhibited sufficient binding to a tetranucleotide containing the (6-4) photoproduct analog with a formacetal linkage, although the association constant was slightly lower than that for the natural lesion. This (6-4) photoproduct analog may be useful for investigation of other proteins that recognize the (6-4) photoproduct.
Antibodies↗
[Structural biology of the immune response on cell surface].
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Animals↗
[Efficacy of mupirocin in eradicating methicillin-resistant Staphylococcus aureus from nasal discharge in carrying cardiovascular surgical patients].
Methicillin-resistant Staphylococcus aureus from nasal discharge was identified in 37 (2.5%) cardiovascular patients operated between 1995 and 1997; 25 male and 12 female, ranging from 1 to 83 years (mean 63); 2 were excluded because of Arbekacin or Isodine-gel treatment. The first 17 were treated with Vancomycin inhalation (V group) and eradication was considered to have been achieved when 3 consecutive negative cultures were obtained; the subsequent 18 were treated with Mupirocin (M group) and eradication was determined by 1 negative culture. In post-eradication electively operated 13 V and 15 M, postoperative MRSA infection was observed in one M (wound infection); the interval from the first nasal culture to the operation was 68 +/- 58 in V and 32 +/- 12 days in M, respectively (p < 0.05). In the remaining 7 who had to undergo emergency surgery while waiting for eradication because of progression of symptoms (2 V) or prior to instituting treatment (2 V, and 3 M), postoperative MRSA infection was observed in 2 M (both pneumonia). No deaths from infection were observed. Though the time required for conversion of the nasal culture was longer in V (13 +/- 20) than in M (7 +/- 1 days) differences were not significant. Mupirocin is easier to use, eradication can be achieved generally within a week.
Administration, Intranasal↗
Complete and rapid peptide and glycopeptide mapping of mouse monoclonal antibody by LC/MS/MS using ion trap mass spectrometry.
Complete and rapid peptide and glycopeptide mapping of a mouse monoclonal immunoglobulin (IgG2b) were carried out by liquid chromatography/electrospray ionization ion trap-mass spectrometry/mass spectrometry (LC/ ESI IT-MS/MS). It was possible to obtain spectra of a minor glycopeptide at a quantity as low as 1.8 pmol. Reduced and carboxymethylated mouse antidansyl monoclonal IgG2b (RCM-IgG2b) was digested with lysyl-endopeptidase. Proteolytic peptides were subjected to capillary HPLC separation followed by analysis with an ion trap mass spectrometer. The complete amino acid sequence of the IgG2b was characterized by using LC/ ESI IT-MS/MS. The structures of 12 different types of O-linked oligosaccharides attached to Thr-221AH in the hinge region and those of three major types of N-linked oligosaccharides attached to Asn-297H have been characterized.
Amino Acid Sequence↗
31P NMR study of the interactions between oligodeoxynucleotides containing (6-4) photoproduct and Fab fragments of monoclonal antibodies specific for (6-4) photoproduct.
A 31P nuclear magnetic resonance (NMR) study of the interactions between oligonucleotides containing the (6-4) photoproduct and the Fab fragments of monoclonal antibodies (64M3 and 64M5) recognizing the (6-4) photoproduct is reported. The 31P chemical shift data indicate that backbone conformation of (64) adduct is affected by the presence of flanking oligodeoxynucleotides, and (6-4) adducts with different backbone conformations are accommodated in the antigen binding sites of these antibodies. It was also revealed that epitopes for these antibodies consist of not only the (6-4) adduct but the flanking di- or tri-deoxynucleotides on both the 5' and 3' sides as well.
Antibodies, Monoclonal↗
NMR study of the interaction between the B domain of staphylococcal protein A and the Fc portion of immunoglobulin G.
The solution structure of the B domain of staphylococcal protein A (FB) complexed with the Fc fragment of immunoglobulin G (IgG) is reported. A previous NMR analysis has shown that in solution FB is composed of a bundle of three alpha-helices, helix I, helix II, and helix III [Gouda, H., Torigoe, H., Saito, A., Sato, M., Arata, Y., and Shimada, I. (1992) Biochemistry 31, 9665-9672]. In contrast, the crystal structure of FB in the FB-Fc complex lacks helix III. Uniformly 15N- and 15N/13C-labeled FB were prepared, and the backbone 13C resonances were assigned. The spectral data obtained in the present study indicated that in solution all three helices including helix III are preserved in the FB-Fc complex. The mode of interaction of FB with the Fc fragment was discussed on the basis of the combined data of hydrogen-deuterium exchange experiments and 1H-15N correlation spectroscopy. It was concluded that a contiguous surface shaped by F14, Y15, E16, L18, and H19 in helix I, and N29, Q33, L35, and K36 in helix II is responsible for the binding.
Amino Acid Sequence↗
N-glycan structures of a recombinant mouse soluble Fcgamma receptor II.
N-glycans of a recombinant mouse soluble Fcgamma receptor II (sFcgammaRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226:139-46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was alpha-(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and alpha-(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry. Carbohydrate sequence [see text]
Animals↗