Biomedical subjects

I Shimada

Publications and source records attributed to I Shimada.

At least 73 records · Page 4Linked to original sources

PubMed1995

Three-dimensional structures of the Fab fragment of murine N1G9 antibody from the primary immune response and of its complex with (4-hydroxy-3-nitrophenyl)acetate.

The three-dimensional structures of the Fab fragment, in its unliganded and liganded crystals, of mouse anti-(4-hydroxy-3-nitrophenyl)acetate (NP) antibody N1G9 have been determined by the molecular replacement method. The unliganded and NP-liganded structures were refined at 2.4 A resolution to crystallographic R-factors of 0.194 and 0.196, respectively. Antibody N1G9 bears lambda light chains, and is one of the primary immune response antibodies. Fab N1G9 exhibits an elbow angle of 197 degrees in both structures. This large angle is ascribed to the VL-CL interface formed by lambda-chain residues. A hydrophobic pocket surrounded by the complementarity-determining regions except L2 is identified as a hapten-binding site. Between the liganded and unliganded structures, root-mean-square (r.m.s.) positional deviations are 0.42 A for the main-chain atoms, and 0.74 A for all the protein atoms. The major structural differences between these structures are localized in the hapten-binding site, and yield an r.m.s. deviation of 1.03 A for the side-chain atoms. The soaked NP ligand is in van der Waals contact with the aromatic side-chains of Tyr32L and Trp91L of the light chain, and Trp33H and Tyr97H of the heavy chain, and is hydrogen-bonded to the side-chains of Trp96L, His35H, Arg50H, Tyr95H, and Ser100aH. The side-chain of Lys58H is salt-bridged to the NP hydroxyl group. The side-chains of Arg50H, Trp33H, and Tyr97H are shifted toward the NP carboxyl group. The side-chain of Trp33H, whose replacement to Leu increases affinity by tenfold, is sandwiched between the Arg50H and Tyr97H side-chains, and is in cramped contact both with the ligand and with these side-chains. Affinity increases in the maturation of the anti-NP antibodies are ascribable to conformational relief of these cramped contacts through the replacement of Trp33H or through suitable structural alterations in the H3 region.

Animals↗

PubMed1995

The affinity maturation of anti-4-hydroxy-3-nitrophenylacetyl mouse monoclonal antibody. A calorimetric study of the antigen-antibody interaction.

To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.

Animals↗

PubMed1995

Three-dimensional solution structure of the calcium channel antagonist omega-agatoxin IVA: consensus molecular folding of calcium channel blockers.

The three-dimensional solution structure of omega-agatoxin IVA, which is a specific blocker of the P-type calcium channel isolated from funnel web spider venom and has a molecular mass of 5.2 kDa, was determined by two dimensional 1H NMR spectroscopy, combined with simulated annealing calculations. On the basis of 563 experimental constraints, including 516 distance constraints obtained from the nuclear Overhauser effect, 21 torsion angle (phi, chi 1) constraints, and 26 constraints associated with hydrogen bonds and disulfide bonds, a total of 14 converged structures were obtained. The atomic root mean square difference for the 14 converged structures with respect to the mean coordinates is 0.42 (+/- 0.07) A for the backbone atoms (N, C alpha, C) and 0.95 (+/- 0.15) A for all heavy atoms of the central part (residues 4 to 38) constrained by four disulfide bonds. The N- and C-terminal segments (residues 1 to 3 and 39 to 48, respectively) have a disordered structure in aqueous solution. The molecular structure of omega-agatoxin IVA is composed of a short triple-stranded antiparallel beta-sheet, three loops, and the disordered N- and C-terminal segments. The overall beta-sheet topology is +2x, -1, which is the same as that reported for omega-conotoxin GVIA, an N-type calcium channel blocker. Irrespective of differences in the number of disulfide bonds and low primary sequence homology, these two peptide toxins show a significant structural similarity in three dimensions. The whole-cell voltage-clamp recording using rat cerebellar slices suggests that the hydrophobic C-terminal segment of omega-agatoxin IVA, which does not exist in omega-conotoxin GVIA, plays a crucial role in the blocking action of omega-agatoxin IVA on the P-type calcium channel in rat cerebellar Purkinje cells. The present study provides a molecular basis for the toxin-channel interaction, and thereby provides insight into the discrimination of different subtypes of calcium channels.

Amino Acid Sequence↗

PubMed1995

Proteolytic fragmentation with high specificity of mouse immunoglobulin G. Mapping of proteolytic cleavage sites in the hinge region.

We report the results of fragmentation of mouse IgG by clostripain, lysyl endopeptidase, metalloendopeptidase, and V8 protease that have a narrower substrate specificity than papain and pepsin. A panel of mouse monoclonal switch variant antibodies with IgG1, IgG2a, and IgG2b subclasses were examined. Cleavage sites by these proteases were mapped on the hinge region of each of the IgG subclasses. It was demonstrated that lysyl endopeptidase can cleave the core hinge portion of IgG2a and IgG2b without perturbing the inter-chain disulfide bridges. Digestion products were successfully isolated by a combined use of protein A affinity chromatography and HPLC techniques. This is a first successful attempt of obtaining the F(ab')2 fragment of IgG2b by proteolytic digestion.

Alkylation↗

PubMed1995

Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.

The distribution of neutral and sialyl N-linked oligosaccharides from human serum glycoproteins has been studied using three kinds of HPLC columns as described below. N-linked oligosaccharides were released from chymotrypsin- and trypsin-digested glycopeptides of human serum by means of glycoamidase (from almond) digestion. The reducing ends of the oligosaccharides were derivatized with the fluorescent reagent 2-aminopyridine (PA). The mixture of PA-oligosaccharides was separated by high-performance liquid chromatography on a diethylaminoethyl column according to the sialic acid content. Each fraction separated was then applied on an octadecylsilyl (ODS) column, and the elution volume of each peak was recorded as a glucose unit on the X-axis. Then, each of the separated oligosaccharides was applied to the amide column. Each peak's elution volume was recorded as a glucose unit on the Y-axis. The elution volumes from these two columns (ODS and amide) provide a unique set of coordinates. The structure of each sialyl oligosaccharide fraction was analyzed by the same two-dimensional sugar-mapping technique as previously developed for neutral oligosaccharides. This was combined with exoglycosidase digestion and high-resolution proton NMR measurement. Fourteen neutral, eight mon-sialyl, four di-sialyl, and five tri-sialyl oligosaccharides were isolated from human serum and their structures were characterized.

Amidohydrolases↗

PubMed1995

Comparative thermodynamic analyses of the Fv, Fab* and Fab fragments of anti-dansyl mouse monoclonal antibody.

In order to investigate the role of the constant domains on the antigen-binding property of the variable domains, we have carried out a comparative thermodynamic study of the anti-dansyl Fv, Fab* and Fab fragments that possess the identical amino acid sequence of the variable domains. The thermodynamic analyses have shown that binding constants, enthalpy changes and entropy changes are similar for the three antigen-binding fragments, whereas the thermal stability of Fab is much higher than that of Fv and Fab*. We have concluded that (i) the variable domains of the three antigen-binding fragments possess identical intrinsic capability for antigen binding and (ii) the two constant domains serve to improve the stability of the variable domains.

Animals↗

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

Elevation of ratio of urinary N-acetylneuraminlactose to free sialic acid in some advanced cancer patients.

We estimated the levels of free sialic acid and sialylated oligosaccharides excreted in the urine of normal donors (n = 10) and patients with gastric cancer (n = 6) and colorectal cancer (n = 4). The total sialic acid level in cancer patients was similar to that in normal donors. However, the ratios of glycosidically bound sialic acids to free sialic acid were higher in some advanced cancer patients than in the normal donors. A major component of sialylated oligosaccharides was N-acetylneuraminyl alpha (2-->3) lactose. The elevation of the urinary ratio of this sialylated oligosaccharide to free sialic acid observed in some advanced cancer patients in this study may reflect the elevation of sialyltransferase activity in tumor tissues.

Adult↗

PubMed1995

[An analysis of decision-making process in help-seeking behavior].

The purpose of this study was to analyze decision-making process in help-seeking behavior with the information monitoring method. Four simulated situations were prepared in which help became necessary. Eight categories of further situational information were explained, and 67 subjects were asked to indicate which categories of information they would like to see before actually seeking others' help in each of the four situations. The number and categories of information requested, together with their order, were recorded. Also, questions were asked concerning the difficulty, hesitation, and confidence they had felt during the task, and ratings were made regarding how adequate the eight information categories and how real the four situations were. Results suggested that (1) only two steps were taken before a help-seeking decision was made in every situation, and (2) it was made in a fairly fixed pattern in term of the help's possible cost and probable attributions of why it became necessary.

Adult↗

PubMed1995

[Mitral and aortic annular enlargement for small mitral annulus after mitral annuloplasty].

Mitral and Aortic valve rings were enlarged in a 70-year-old female with tiny mitral annulus after mitral annuloplasty before. She had undergone aortic valve replacement with No. 21 SJM valve and Kay's annuloplasty 10 years previously. The reoperation was needed because of mitral stenosis and tricuspid regurgitation. We applied the Manouguian technique for the enlargement of the mitral annulus because it was too small to implant a prosthetic valve, even though the Aortic prosthetic valve was functioning well. As too tight a mitral annuloplasty may make the possibility of mitral stenosis real, we should make the annulus remain wide enough.

Aged↗

PubMed1995

[A case report of tracheal stenosis due to true aortic arch aneurysm of retroesophageal right aortic arch associated with so called vascular ring-facial syndrome].

A 63-year-old man admitted to our hospital because of dyspnea and inspiratory stridor. The X-ray computed tomography and angiogram revealed tracheal stenosis due to compression by aortic arch aneurysm of retroesophageal right aortic arch. His face was congenitally asymmetrical, and he also showed anotia, and meatal atresia. In the operation, we approached the aneurysm via median sternotomy and left thoracotomy by the 3rd intercostal space, and found atretic left aortic arch in front of trachea. So, operative diagnosis was Edwards Ib complete vascular ring associated with right aortic arch aneurysm. The aneurysm was incised and the arch and its branches were reconstructed with vascular prosthesis under ECC using selective cerebral perfusion. Postoperatively, until 5th postoperative day his condition was uneventful, and he was neurologically almost normal. But on the 5th postoperative day, his hemodynamics suddenly deteriorated because of severe pneumonia and septicemia. On the 6th postoperative day, he died in spite of earnest resuscitation. We could not find any previous reports about this rare combination of diseases.

Abnormalities, Multiple↗

PubMed1995

[A successful case of emergency CABG using PCPS due to acute myocardial infarction].

A successful case of emergency CABG (Coronary Artery bypass Grafting) by using PCPS (Percutaneous Cardio Pulmonary Bypass) for a 60-year-old with shock due to acute myocardial infarction was reported. He developed shock due to acute myocardial infarction before arrival at our hospital. He was resuscitated by PCPS. Coronary angiography revealed that LMT was 99% stenosed and LAD just proximal was totally occluded. Emergency CABG was performed to LAD and CX using saphenous vein grafts. Post-operative course was uneventful and the patient was discharged at hospital two months after the operation. PCPS is useful for bridge until emergency CABG.

Cardiopulmonary Bypass↗

PubMed1995

[The hazards of coronary revascularization under hypothermic ventricular fibrillation].

Since January 1992 till June 1994, we experienced 22 cases of coronary revascularization (CABG) under hypothermic ventricular fibrillation (Vf) in patients with unclampable ascending aorta (Group 2). We compared them with patients undergoing conventional CABG with cardioplegic cardiac arrest (Group 1). All these 362 cases were primary isolated CABG. Comparing preoperative patient profile, patients of Group 2 were older (Group 1: 64.3 +/- 8.5, Group 2: 68.2 +/- 6.7 year old, p < 0.05), had more unstable angina (Group 1: 20.3%, Group 2: 54.5%, p < 0.001), and had more severe NYHA classification (Group 1: 2.22 +/- 0.55, Group 2: 2.73 +/- 0.46, p < 0.05) than Group 1. There were no significant difference between both groups about other factors. Comparing post operative complication, low output syndrome (Group 1: 2.6%, Group 2: 18.2%, p < 0.005) perioperative myocardial infarction (PMI) (Group 1: 4.4%, Group 2: 36.4%, p < 0.0001) ventricular tachycardia (VT) (Group 1: 1.4%, Group 2: 18.2%, p < 0.0001), respiratory failure (Group 1: 3.8%, Group 2: 18.2%, p < 0.005), and post operative hospital death (Group 1: 2.9%, Group 2: 18.2%, p < 0.0005) occurred more frequent in Group 2 than Group 1. Other complication (renal failure, wound infection, cerebrovascular accident, rethoracotomy for bleeding) equally occurred in both groups. Long Vf time (mean 92.8 minutes) and low perfusion pressure during Vf (mean 60.0 mmHg) were suspected to be major cause of high incidence of PMI and VT in Group 2. But there were no correlation between Vf time, perfusion pressure and occurrence of PMI and/or VT.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1995

[Risk factors of wound infection following cardiac surgery and antimicrobial prophylaxis].

To make investigation about risk of wound infection following cardiac surgery, we analyzed cases of primary aorto-coronary bypass surgery and/or primary valvular surgery. Cases required respirator for longer than 2 days, and those with drainage tube for longer than 5 days were excluded from this study, because these cases had antimicrobial agent postoperatively as therapeutic use rather than prophylactic use. Those received preoperative antimicrobial agent for infectious endocarditis and so on were also excluded for the same reason. 523 cases were entered this study. These cases received cefazolin (CEZ) postoperatively as prophylaxis. Sternal wound and leg/groin wound were separately analyzed. Risk factors (age, sex, diabetes mellitus, unstable angina, use of intraaortic balloon, internal thoracic artery harvest, re-exploration, previous myocardial infarction, NYHA classification, emergent operation, operation time, extracorporeal circulation time, blood product use, preoperative blood hemoglobin concentration, preoperative serum albumin, preoperative creatinine clearance (Ccr), duration of drainage tube insertion, body surface area, daily dose of CEZ, duration of prophylaxis) were examined using univariate (Chi square test was used for contingency table analysis, unpaired t test was used to compare averages) and multiple regression analysis. For sternal wound infection, only Ccr showed significant (P = 0.027) correlation. For leg/groin wound infection, 2 factors (smaller daily dose of CEZ: P = 0.009, more severe NYHA class: P = 0.03) showed significant correlation. To investigate appropriate duration of CEZ prophylaxis, cases were divided into 2 groups, those had CEZ within 48 hours postoperatively (group S) and those had CEZ for longer than 48 hours (group L).(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed1995

[A case of tongue cancer effectively treated with tegafur].

We administered a slow-release formulation of tegafur (SF-SP) to a patient with cancer of the tongue. We would like to report the findings of this case which showed clear improvement. SF-SP was administered at 800 mg/day to a 58 year-old male for cancer of the tongue (T4N1). By the 17th day after the start of SF-SP administration, the tumor had shrunk, and by the 37th day the submandibular lymph nodes could not be located by touch. At the completion of the test on the 60th day, no cancer cells were detected. Following the completion of the test, there have been no symptoms of recurrence and a complete response was observed. At present, we are continuing administration of SF-SP and making observations.

Adenocarcinoma↗

PubMed1995

[A case report of successful surgical treatment of acute postinfarction left ventricular free wall rupture using the percutaneous cardiopulmonary support system].

The percutaneous cardiopulmonary support system (PCPS) was used in a 75-year-old female with a blow out type heart rupture of the posterior wall complicating acute myocardial infarction. The patient was transported to the operating room with the PCPS and IABP in place, and had the ventricular rupture repaired as well as CABG. Although the postoperative cardiac function was satisfactory she sustained ischemic cerebral stroke and went on to die of aspiration pneumonia 166 day later. PCPS buys time to enable surgical repair of the heart, but how to protect the brain remains as a problem to be solved.

Aged↗

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↗