Amounts of tPA and PAI-1 in the euglobulin fraction obtained at different pH: their relation to the euglobulin clot lysis time.
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Adult↗
Biomedical subjects
Y Takada
Publications and source records attributed to Y Takada.
Changing ligand specificities of alphavbeta1 and alphavbeta3 integrins by swapping a short diverse sequence of the beta subunit.
Integrins mediate signal transduction through interaction with multiple cellular or extracellular matrix ligands. Integrin alphavbeta3 recognizes fibrinogen, von Willebrand factor, and vitronectin, while alphavbeta1 does not. We studied the mechanisms for defining ligand specificity of these integrins by swapping the highly diverse sequences in the I domain-like structure of the beta1 and beta3 subunits. When the sequence CTSEQNC (residues 187-193) of beta1 is replaced with the corresponding CYDMKTTC sequence of beta3, the ligand specificity of alphavbeta1 is altered. The mutant (alphavbeta1-3-1), like alphavbeta3, recognizes fibrinogen, von Willebrand factor, and vitronectin (a gain-of-function effect). The alphavbeta1-3-1 mutant is recruited to focal contacts on fibrinogen and vitronectin, suggesting that the mutant transduces intracellular signals on adhesion. The reciprocal beta3-1-3 mutation blocks binding of alphavbeta3 to these multiple ligands and to LM609, a function-blocking anti-alphavbeta3 antibody. These results suggest that the highly divergent sequence is a key determinant of integrin ligand specificity. Also, the data support a recent hypothetical model of the I domain of beta, in which the sequence is located in the ligand binding site.
Amino Acid Sequence↗
Defining the topology of integrin alpha5beta1-fibronectin interactions using inhibitory anti-alpha5 and anti-beta1 monoclonal antibodies. Evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the alpha5 subunit.
The high affinity interaction of integrin alpha5beta1 with the central cell binding domain (CCBD) of fibronectin requires both the Arg-Gly-Asp (RGD) sequence (in the 10th type III repeat) and a second site (in the adjacent 9th type III repeat) which synergizes with RGD. We have attempted to map the fibronectin binding interface on alpha5beta1 using monoclonal antibodies (mAbs) that inhibit ligand recognition. The binding of two anti-alpha5 mAbs (P1D6 and JBS5) to alpha5beta1 was strongly inhibited by a tryptic CCBD fragment of fibronectin (containing both synergy sequence and RGD) but not by GRGDS peptide. Using recombinant wild type and mutated fragments of the CCBD, we show that the synergy region of the 9th type III repeat is involved in blocking the binding of P1D6 and JBS5 to alpha5beta1. In contrast, binding of the anti-beta1 mAb P4C10 to alpha5beta1 was inhibited to a similar extent by GRGDS peptide, the tryptic CCBD fragment, or recombinant proteins lacking the synergy region, indicating that the RGD sequence is involved in blocking P4C10 binding. P1D6 inhibited the interaction of a wild type CCBD fragment with alpha5beta1 but had no effect on the binding of a mutant fragment that lacked the synergy region. The epitopes of P1D6 and JBS5 mapped to the NH2-terminal repeats of the alpha5 subunit. Our results indicate that the synergy region is recognized primarily by the alpha5 subunit (in particular by its NH2-terminal repeats) but that the beta1 subunit plays the major role in binding of the RGD sequence. These findings provide new insights into the mechanisms, specificity, and topology of integrin-ligand interactions.
Animals↗
Multiple loop structures critical for ligand binding of the integrin alpha4 subunit in the upper face of the beta-propeller mode 1.
A non-I-domain integrin, alpha4beta1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of alpha4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of alpha4 (which spans repeats 2-5 of the seven N-terminal repeats) with the corresponding regions of alpha5. Swapping residues 112-131 in repeat 2, or residues 237-247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40-52 in repeat 1, residues 151-164 in repeat 3, or residues 282-288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112-131, 151-164, and 186-191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published beta-propeller folding model of the integrin alpha4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65-72], in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetric axis. The regions of alpha4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the beta-propeller model, although they are not adjacent in the primary structure.
Amino Acid Sequence↗
Remnant-like particles stimulate platelet aggregation in whole blood.
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Adult↗
Propolypeptide of von Willebrand factor is a novel ligand for very late antigen-4 integrin.
We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) promotes melanoma cell adhesion in a beta1 integrin-dependent manner. In this report, we identified the alpha subunit of the cell adhesion receptor for pp-vWF as alpha4. Human leukemia cell lines that express alpha4beta1 integrin (very late antigen-4, VLA-4), but not cell lines which lack VLA-4, attached well to pp-vWF substrate and these adhesions were completely inhibited by anti-alpha4 integrin monoclonal antibody HP2/1. Adhesion of mouse melanoma expressing alpha4 integrin was also inhibited by anti-mouse alpha4 mAb PS/2. Furthermore, transfection of human alpha4 cDNA into alpha4(-) Chinese hamster ovary cells resulted in an acquisition of adhesive activity to pp-vWF, indicating that pp-vWF is a ligand for VLA-4 integrin. Using a recombinant fragment of pp-vWF, the cell attachment site was shown to be located within amino acid residues 376-455 of pp-vWF. A series of synthetic peptides covering this region were tested for the ability to promote cell attachment and a 15-residue peptide designated T2-15 (DCQDHSFSIVIETVQ, residues numbered 395-409) promoted VLA-4 dependent cell adhesion. The peptide was also capable of inhibiting cell adhesion to pp-vWF, suggesting that this sequence represents the cell attachment site. By affinity chromatography using peptide T2-15-Sepharose, it was found that alpha4beta1 integrin complex from extracts of surface iodinated B16 cells specifically bound to the peptide. These results strongly suggest that pp-vWF is a novel physiological ligand for VLA-4.
Animals↗
Hepatic allograft procurement from non-heart-beating donors: limits of warm ischemia in porcine liver transplantation.
To investigate the tolerance to warm ischemia of liver grafts from non-heart-beating donors, porcine orthotopic liver transplantation was performed using grafts obtained at various periods after cardiac arrest. Graft viability was investigated in relation to changes in hepatic adenine nucleotide metabolism. In donors, livers were divided into four groups according to warm ischemic time after cardiac arrest (group 1: 0 min, n=3; group 2: 30 min, n=3; group 3: 60 min, n=5; group 4: 90 min, n=4). Thereafter, the livers were flushed and preserved for 4 hr using 4 degrees C Euro-Collins solution. After surgery, all of the recipients in groups 1, 2, and 3 survived more than 4 days, except for one pig in group 3 that died of bleeding from an arterial catheter on day 2. By contrast, all of the recipients in group 4 died within 12 hr. The serum glutamic oxaloacetic transaminase concentration at 4 hr after reperfusion of the graft was significantly higher in group 4 (mean+/-SE, 2563+/-556 IU/L) than in groups 1, 2, and 3 (298+/-29 IU/L, 1226+/-222 IU/L, and 1181+/-174 IU/L, respectively). The adenylate energy charge of the liver graft recovered at 1 hr after reperfusion of the graft to 0.852+/-0.013, 0.845+/-0.003, and 0.842+/-0.003 in groups 1, 2, and 3, respectively. The recovery was significantly suppressed in group 4 (0.796+/-0.011). The hepatic adenosine triphosphate concentration also was significantly lower in group 4 compared with the other groups. The present study suggests that liver allografts can be used from non-heart-beating donors subjected to warm ischemia for less than 60 min. Postoperative survival is associated with prompt recovery of the adenylate energy charge of the liver graft.
Adenine Nucleotides↗
Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP).
Multidrug resistance-associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP-dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate-induced trapping of 8-azido-ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti-cancer drugs VP-16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8-azido-[alpha-32P]ATP by the vanadate-trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP x MgADP x Vi, an analog of the MRP x MgADP x Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti-cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.
ATP-Binding Cassette Transporters↗
Intestinal Behçet's disease--pathognomonic changes in intramucosal lymphoid tissues and effect of a "rest cure" on intestinal lesions.
To clarify the pathognomonic changes of intestinal lesions of Behçet's disease and to determine effective therapeutic measures, we recruited 13 patients with the intestinal form of this disease for study. We performed pathology studies on the resected specimens of 7 patients and treated 5 of the other 6 patients with a low-residue diet. Pathology examination revealed that 6 of 7 had inflammatory ulcerations in the ileocecal region. The ileal ulcers were mainly on the antimesenteric side. We observed remnants of Peyer's patches at the margins of the major ulcerative lesions in 2 of 2 patients examined. There were aggregations of lymphocytes resembling destroyed lymph follicles in the superficial layer at the mouths of small fissuring lesions, and ulcer scars were also noted in Peyer's patches in 4 of 5 other patients. X-ray and endoscopic examinations revealed the disappearance of intestinal lesions in 5 patients within 1 month during, or following the low-residue diet treatment. We found the intestinal lesions of Behçet's disease at sites coinciding with intramucosal lymphoid tissue. The "rest cure" for the affected bowel was effective, i.e., there was significant alleviation of gastrointestinal symptoms and the intestinal lesions disappeared. We speculated that acute exudative inflammation, abscess formation, and consequent ulceration may occur in these tissues by the same mechanisms as those that operate in the positive needle-prick reactions seen in patients with Behçet's disease.
Adult↗
Neutrophil elastase activity in hepatic ischemia reperfusion injury.
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Alanine Transaminase↗
Long-term intelligence outcome of post-encephalo-duro-arterio-synangiosis childhood moyamoya patients.
Long term post-encephalo-duro-arterio-synangiosis (EDAS) intelligence quotient (IQ) evaluated by Wechsler tests stays around the normal range (mean full intelligence quotient (FIQ), verbal intelligence quotient (VIQ) and performance intelligence quotient (PIQ) at 100.0 +/- 155, 100.0 +/- 15.7 and 100.2 +/- 163, respectively) at more than 9.5 years after the operation, provided that the patient has FIQ above 70 pre-operatively. This fact encourages us to employ operative treatment for this difficult disease in which intelligence deterioration has been reported to be a common occurrence.
Cerebral Revascularization↗
Structural basis of integrin-mediated signal transduction.
Integrins are a family of alpha/beta heterodimers of cell adhesion receptors that mediate cell-extracellular matrix and cell-cell interactions. Both alpha and beta subunits have a large extracellular domain and a short cytoplasmic domain. The alpha subunit has seven sequence repeats of 60-70 residues in its N-terminal region. The beta-propeller model, in which seven four-stranded beta-sheets are arranged in a torus around a pseudosymmetry axis, has been proposed as a structural model of these seven repeats. Several predicted loops critical for ligand binding have been identified in the upper face of the proposed beta-propeller model. Several alpha subunits (e.g., alpha 2, alpha L and alpha M) have I-domains of about 200 residues inserted between their second and third repeats. These I-domains adopt a Rossman-fold structure and have major ligand and cation binding sites (the MIDAS site) on their surfaces. The beta subunit has an I-domain-like structure in its N-terminal region. This structure includes multiple sequences/conserved oxygenated residues critical for ligand binding (e.g., Asp-119 in beta 3), and non-conserved residues critical for ligand specificities. Several "activation-dependent" epitopes have been identified in the Cys-rich (stalk) region of beta 1. It has yet to be determined how these multiple ligand binding sites in the alpha and beta subunits are involved in ligand binding, and how conformational changes on activation/ligand occupancy relate to signal transduction.
Animals↗
Structural interlock between ligand-binding site and stalk-like region of beta1 integrin revealed by a monoclonal antibody recognizing conformation-dependent epitope.
Integrin activation and sebsequent ligand binding to it are regulated by intracellular mechanisms called inside-out signaling, which are not fully understood and are accompanied by dynamic structural changes of the integrin molecule itself. A monoclonal antibody recognizing a conformation-dependent epitope on human beta1 integrin was produced and characterized in detail. This antibody, AG89, reacted with human integrin beta1 chain regardless of the alpha subunit. AG89 can recognize resting state beta1 integrin on the cells, but the reactivity is increased approximately 2-fold upon integrin activation by activating anti-beta1 antibodies and approximately 3-fold by Mn2+. Furthermore, occupation of the ligand-binding pocket by a soluble ligand (RGD peptide for alpha(v)beta1 and CS-1 peptide for alpha4beta1) resulted in maximum binding of AG89, indicating that the epitope for AG89 is exposed during the conformational changes of beta1 integrin upon activation/ligation. Epitope mapping by using interspecies chimeric beta1 revealed that the epitope for AG89 lies within residues 426-587, which corresponds to the cysteine-rich repeat structure located in the middle of the beta1 chain. The fact that binding of AG89 itself could activate the resting beta1 integrin indicates that exposure of the AG89 epitope in the membrane-proximal stalk-like domain and "opening" of the ligand-binding pocket at the outermost domain are physically linked. We propose that the integrin "signaling" is mediated by this direct physical transduction of conformational information along the integrin molecule.
Antibodies, Monoclonal↗
Negative transcriptional regulation of the VCAM-1 gene by fluid shear stress in murine endothelial cells.
To explore the mechanism of shear stress-induced downregulation of vascular cell adhesion molecule 1 (VCAM-1) expression in murine endothelial cells (ECs), we examined the effect of shear stress on VCAM-1 gene transcription and assessed the cis-acting elements involved in this phenomenon. VCAM-1 mRNA expression was downregulated at the transcriptional level as defined by nuclear run-on assay and transient transfection of VCAM-1 promoter-luciferase gene constructs. The luciferase assay on the VCAM-1 deletion mutants revealed that the cis-acting element is contained between -694 and -329 bp upstream from the transcription initiation site. Gel shift assay using overlapping oligonucleotide probes of this region showed that oligonucleotides containing a double AP-1 consensus sequence (TGACTCA) formed distinct complexes with nuclear proteins extracted from shear-stressed cells. Mutation of either one or both of two AP-1 consensus sequences completely abolished the ability of the promoter to respond to shear stress. These results suggest that fluid shear stress downregulates the transcription of the VCAM-1 gene via an upstream cis-element, a double AP-1 consensus sequence, in murine lymph node venule ECs.
Animals↗
IORT combined with resection for stage IV gallbladder carcinoma.
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Adult↗
Comparison of resting beta-methyl-iodophenyl pentadecanoic acid (BMIPP) and thallium-201 tomography using quantitative polar maps in patients with unstable angina.
We compared resting beta-methyl-iodophenyl pentadecanoic acid (BMIPP) tomography with resting thallium-201 tomography in 28 patients with unstable angina. Tracer distribution was displayed on a polar map and compared with a normal standard deviation map obtained from a group of 12 normal subjects. The extent scores and severity scores obtained by BMIPP were significantly greater than those obtained by thallium-201. Confirmation by coronary angiography revealed the sensitivity of the methods in identifying patients to be 89% for BMIPP and 54% for thallium-201. There were significant differences between BMIPP and thallium-201 in the sensitivities of detecting postischemic jeopardized myocardium in the area supplied by the right coronary artery (RCA; 53% vs 18%, p < 0.05), left circumflex artery (LCX; 78% vs 39%, p < 0.025) and all 3 vessels combined (71% vs 35%, p < 0.001) but no significant differences in specificity (RCA: 82% vs 64%; LCX: 70% vs 90%; and total 3 vessels combined: 75% vs 79%). In conclusion, resting BMIPP tomography is more sensitive than resting thallium-201 tomography in detecting postischemic myocardial damage in patients with unstable angina.
Angina, Unstable↗
Fibrinolytic parameters as an admission prognostic marker of head injury in patients who talk and deteriorate.
Because it has been difficult to predict the outcome of patients with head injury in the early period after admission, a clinical study was undertaken to evaluate if the fibrinolytic parameters could be reliable indicators of outcome. Seventy patients presenting with head injury without obvious trauma in other regions were studied, and plasma levels of alpha2-plasmin inhibitor-plasmin complex (PIC) and D-dimer on admission (within 2 hours postinjury) were assessed. Plasma levels of both PIC and D-dimer were elevated and correlated well to patient outcomes. When plasma PIC levels were higher than 15 microg/ml or D-dimer levels were higher than 5 microg/ml, 92% of patients died regardless of their consciousness level on admission, whereas all patients made good recoveries when their PIC levels were less than 2 microg/ml or D-dimer levels were less than 1 microg/ml. Therefore, plasma levels of both PIC and D-dimer on admission were revealed to be reliable prognostic markers of head injury. Using these markers, patients with poor outcomes (progressive brain injury), such as "talk and deteriorate" types, could be readily identified on admission.
Adolescent↗
Effects of lactitol-oligosaccharides on calcium and magnesium absorption in rats.
Lactitol-oligosaccharide (LO) was prepared from lactitol by transglycosylation reaction with Aspergillus oryzae beta-galactosidase. LO is resistant to metabolism in the small intestine but not in the large intestine. The effects of LO, lactose (Lac), lactitol (Lacol) and galactooligosaccharide (GL) on calcium and magnesium absorption were determined by feeding 8-week-old Sprague-Dawley male rats diets containing 5% of the above carbohydrates for two weeks. The results obtained were as follows. 1) A significant increase of calcium absorption was observed in the LO diet. 2) A significant increase of magnesium absorption was observed in the LO, GL and Lacol diets. 3) The concentration of total volatile fatty acids (VFA) in the cecal contents increased significantly in the LO, GL and Lacol diets. The main constituent of VFA in the cecal contents was found to be acetic acid. 4) The correlations between calcium and magnesium absorption ratios and total VFA concentration in the cecum were found to be significantly related. These findings suggest that LO is metabolized to VFA, among which acetic acid concentration seems to have the most stimulatory effect on the absorption of calcium.
Animals↗