The radius of gyration of an apomyoglobin folding intermediate.
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Apoproteins↗
Biomedical subjects
H Tsuruta
Publications and source records attributed to H Tsuruta.
Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments.
Solution X-ray scattering experiments have been carried out on recombinant bovine Hsc70 (with 650 amino acid residues), a 60 kDa subfragment (residues 1-554) which has ATPase- and peptide-binding activities, a 44kDa subfragment (residues 1-386) which has only ATPase activity, and a peptide-binding fragment (residues 388-554). Modeling based on steady-state values of radii of gyration (Rg's) and P(r) functions shows that the 44 kDa and peptide-binding domains are oblate fragments while Hsc70 and the 60 kDa fragment are prolate and relatively elongated. Rg values decrease significantly in the presence of MgATP relative to their values in the presence of MgADP (delta Rg approximately 4-5 A) for Hsc70 and the 60 kDa fragment; in contrast, they are essentially equal in the presence of either nucleotide for the 44 kDa ATPase fragment. The kinetics of the change of Rg for Hsc70 and the 60 kDa fragment under single-ATPase cycle conditions show that the transition to the ATP-induced Rg occurs significantly more rapidly than ATP hydrolysis while the reverse transition to the larger Rg value does not occur before product release. Altogether, the solution scattering data support a model in which a conformational change in Hsc70 (presumably to the low-peptide-affinity state) is predicated on ATP binding while the reverse transition is predicated on product release.
Adenosine Triphosphatases↗
An automated ELISA system using a pipette tip as a solid phase and a pH-sensitive field effect transistor as a detector.
A fully automated ELISA system was constructed using a pipette tip as a solid phase, urease as a detecting enzyme, and a pH-FET as a detector of urease activity. The inner wall of the end part of a pipette tip was used as a solid phase, and the urease activity of the conjugate, captured after a two-step immunoreaction, was measured by coupling the pipette tip with the pH-FET in a pH-measuring cell. Full automation of the ELISA system was achieved by using a disposable reagent cartridge and three pipetters for all mechanical operations, including sample dilution and B/F separation. This system can treat 60 samples per hour with an assay time of 21 min for all assay configurations. The system was applied to two-step sandwich assays for AFP, CEA, HBsAg, and HBsAb, a two-step competition assay for HBcAb, and a second antibody assay for HTLV-I Ab.
Carcinoembryonic Antigen↗
Quantitation of IL-1 beta mRNA by a combined method of RT-PCR and an ELISA based on ion-sensitive field effect transistor.
A method for quantitative RT-PCR using ELISA detection was developed and applied to the quantitation of IL-1 beta mRNA in clinical samples. To compensate for the 'tube effect' of RT-PCR, a synthetic RNA, pRSET RNA, was added to sample solutions as an internal standard and co-amplified with IL-1 beta mRNA. Sense primers for IL-1 beta and pRSET were labeled with digoxigenin and FITC, respectively, while anti-sense primers for both were labeled with biotin. Double-stranded PCR products were captured by two solid-phase pipettetips. One was coated with an anti-digoxigenin antibody and another with an anti-FITC antibody, and sandwiched by avidin-urease, and their activities were measured by coupling them with a pH-FET in a pH-measuring cell containing urea solution. The ratio of the signal intensity for IL-1 beta to that for pRSET was used to quantify the concentration of IL-1 beta mRNA. A calibration curve was obtained by using a known amount of AW 109 RNA as an external standard of IL-1 beta RNA. It was found that 10(2)-10(6) copies of IL-1 beta mRNA were measurable by the present method. Expression levels of IL-1 beta mRNA in clinical samples, such as monocytes of peripheral blood or synovial cells from patients with RA or OA, were determined.
Base Sequence↗
The effect of intravenous and intra-tumoural chemotherapy using a monoclonal antibody-drug conjugate in a xenograft model of pancreatic cancer.
In order to investigate the efficacy of the intra-tumoural administration of an anticancer drug-monoclonal antibody conjugate in athymic nude mice bearing xenografts of a human pancreatic carcinoma, we examined the clearance of the murine monoclonal antibody A7 from the xenografts after intravenous or intra-tumoural administration and measured the antitumour effect of neocarzinostatin conjugated to MAb A7 following intravenous or intra-tumoural injection. Compared with 125I-labelled normal mouse IgG, a larger amount of 125I-labelled A7 remained in the tumour after both intravenous and intra-tumoural injection, and a significantly larger amount of 125I-labelled A7 remained in the tumour after intra-tumoural injection than that after intravenous injection. Moreover, a larger amount of 125I-labelled A7-NCS localized in the tumour after intra-tumoural injection than that after intravenous injection. Neocarzinostatin conjugated to MAb A7 showed greater activity against human pancreatic cancer than neocarzinostatin alone after both intravenous and intra-tumoural administration. Tumour growth was suppressed completely by the intra-tumoural administration of A7-NCS at a dose that did not suppress tumour growth via the intravenous route. These observations suggest that the intra-tumoural injection of neocarzinostatin conjugated to MAb A7 offers promise in treating pancreatic carcinoma.
Adenocarcinoma↗
ATP-hydrolyzing excitation state of the reconstituted alpha 3 beta 3 complex of ATP synthase from the thermophilic bacterium PS3: structural characteristics shown by time-resolved small-angle X-ray scattering with synchrotron radiation.
The ATP-hydrolyzing excitation state of the alpha 3 beta 3 complex of the ATP synthase from the thermophilic bacterium PS3 was investigated using time-resolved small-angle X-ray scattering with synchrotron radiation. The results showed the presence of the alpha 3 beta 3 complex at a steady state during ATP hydrolysis when the alpha 3 beta 3 hexamer reacted with Mg-ATP. The radius of gyration of the complex in the steady state was significantly larger than that of the Mg-AMP-PNP-hexamer complex, indicating a conformational change to an expanded structure during catalysis. This alpha 3 beta 3 complex dissociated into alpha 1 beta 1 heterodimers with apparent first-order reaction kinetics after all the ATPs were converted to ADPs. In contrast, when the alpha 3 beta 3 complex reacted with Mg-ADP, the complex dissociated into dimers with apparent first-order reaction kinetics without showing the steady state of the complex. The dimers, however, re-associated into the hexamer when Mg-ATP was added. The results were well-explained by a computer simulation based on non-linear chemical dynamics, in which a reaction mechanism that incorporates the dynamic structure of the hexamer in the steady state was considered.
Adenosine Triphosphate↗
Cytochrome P4502E1 (CYP2E1) genetic polymorphism in a case-control study of gastric cancer and liver disease.
Cytochrome P4502E1 (CYP2E1) activates carcinogenic N-nitrosamines, benzene, urethane and other low molecular weight compounds. This enzyme is also inducible by ethanol, and metabolizes alcohol. A restriction fragment length polymorphism (RFLP) using the Rsa I restriction enzyme has been identified in the CYP2E1 transcription regulatory region; recent studies suggest that this polymorphism may affect gene expression. We investigated the frequency of the Rsa I RFLP in a Japanese population in relation to gastric cancer and liver disease susceptibility. The frequency of this polymorphism was determined in 150 gastric cancer, 16 hepatocellular cancer, 48 liver cirrhosis and 203 benign gastric disease (controls) patients. This preliminary study shows no association of the specific genotype with gastric cancer in all subjects (odds ratio = 1.04, 95% CI = 0.74-3.08 for the heterozygote and 0.57, 95% CI = 0.22-1.50 for the homozygous rare allele, respectively). To further confirm this lack of association, an age and gender matched case-control study should be performed. Separately, there was no association of the Rsa I RFLP with hepatocellular carcinoma (p = 0.911), but there was a suggested difference between the non-viral associated liver cirrhosis patients and control patients. Thus, this polymorphism may be related to ethanol metabolism and consequential liver diseases in a Japanese population.
Base Sequence↗
Detection of the products of polymerase chain reaction by an ELISA system based on an ion sensitive field effect transistor.
An ELISA system was developed using a pH sensitive ISFET (pH-FET) as a detector, a pipette tip as a solid phase, and urease as a detecting enzyme. Double stranded PCR products with digoxigenin and biotin at both terminals were obtained by using digoxigenin- and biotin-labeled primers. 1 microliters of the PCR solution was directly introduced into the end part of a pipette tip coated with anti-digoxigenin antibody. Biotin-labeled PCR products captured at the solid phase were detected with avidin-urease, of which the activity was measured by a pH-FET in a pH-measuring cell containing urea solution. The assay was used to detect HTLV-I provirus gene integrated in the genome of a human MT-1 cell, and it was found that 100 pg of the genomic DNA of MT-1 cell was specifically detectable after 35 cycles of PCR. Also the detection limit of the present ELISA system itself was determined by using known amounts of purified PCR product labeled with digoxigenin and biotin, and it was found that 10 amol of the labeled DNA in 1 microliter of sample was detectable.
Base Sequence↗
Kinetics of the quaternary structure change of aspartate transcarbamylase triggered by succinate, a competitive inhibitor.
The quaternary structural change of Escherichia coli aspartate transcarbamylase (ATCase) was studied by time-resolved X-ray solution scattering following the binding of carbamoyl phosphate and of succinate, a competitive inhibitor of the natural substrate L-aspartate. Stopped-flow experiments at sub-zero temperatures in the presence of 30% ethylene glycol allowed us to monitor the evolution of the scattering pattern, including the characteristic scattering peak in an s (=2 sin theta/lambda) range of 0.01-0.06 A-1. The inhibitor binding promotes a quaternary structure change from the T state toward the R state, and as expected for a simple ligand binding process, ATCase remains in the R state, unlike the physiological enzyme reaction [Tsuruta, H., et al. (1990) FEBS Lett. 263, 66-68]. After equilibrium had been established, the final scattering pattern was recorded. When the succinate concentration was sufficiently high, this pattern was the same as that given by ATCase saturated with the bisubstrate analogue N-phosphonoacetyl-L-aspartate (PALA). This implies that, under cryogenic conditions, succinate and carbamoyl phosphate promote the same quaternary structure change as PALA, which is in good agreement with the crystallographic studies of Gouaux and Lipscomb [Gouaux, J.E., & Lipscomb, W.N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4205-4208]. Scattering patterns recorded during the course of the structural transition were satisfactorily reproduced by a linear combination of the initial and final patterns, suggesting that there is no significant concentration of quaternary structure intermediates between the T and R states. This is consistent with a concerted structural transition of ATCase.(ABSTRACT TRUNCATED AT 250 WORDS)
Aspartate Carbamoyltransferase↗
MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering.
Small angle x-ray scattering experiments have been carried out on the purified iron proteins of nitrogenase from wild-type Azotobacter vinelandii and from a Nif- mutant strain, A. vinelandii UW91 (which has an A157S mutation). This study was designed to investigate the influence of MgATP and MgADP binding on the protein structure in solution. For the wild-type protein, the binding of MgATP induces a significant conformational change that is observed as a decrease of about 2.0 A in the radius of gyration. In contrast, the binding of MgADP to the wild-type iron protein does not detectably affect the radius of gyration. In the absence of nucleotides, the radius of gyration for the UW91 mutant is indistinguishable from that of the wild-type. However, unlike for the wild-type protein, the radius of gyration of the UW91 iron protein is unaffected by the addition of MgATP. We have previously shown that the UW91 iron protein has a normal [4Fe-4S] cluster and MgATP binding ability but that it is completely blocked for electron transfer and MgATP hydrolysis (Gavini, N., and Burgess, B. K. (1992) J. Biol. Chem. 267, 21179-21186). These x-ray scattering measurements suggest that a conformation different from that of the native state is therefore required for the iron protein to perform electron transfer to the MoFe protein. These results also support the hypothesis that Ala-157 is crucial for the iron protein to establish the electron-transfer-favored conformation induced by MgATP binding.
Adenosine Diphosphate↗
Messenger RNA levels of five genes located at chromosome 11q13 in B-cell tumors with chromosome translocation t(11;14)(q13;q32).
To identify genes activated by chromosome translocation t(11;14)(q13;q32), mRNA levels of five genes (cyclin D1, EXP1, MB38, HST1, and INT2) at chromosome 11q13 were investigated. The cyclin D1 mRNA increased in BCL-1-rearranged B-cell tumor cell lines SP-49, NOP-2, FLAM-76, KMS-12-PE, and KMS-12-BM cells, while it was not detected in cell lines without the translocation, Raji, U266, and HEL cells. A significant amount of the MB38 mRNA was detected irrelevantly to the translocation in all of these cell lines. The mRNAs of EXP1, HST1, and INT2 were undetectable in these cells. The results suggested that the translocation activates cyclin D1 alone, while the mRNA levels of the other four genes are regulated independently of the translocation.
Blotting, Northern↗
The kinetics of conformational changes of alpha 2-macroglobulin determined by time resolved X-ray solution scattering.
The rate of gross conformational change of alpha 2-macroglobulin (alpha 2M) during its proteinase trapping was directly determined for the first time using time-resolved X-ray solution scattering. Decrease of radius of gyration was observed under pseudo-first-order conditions with excess proteinases, which exhibited a monophasic time-course. The rate constants were 0.5 +/- 0.1 s-1 and 0.8 +/- 0.2 s-1 for the reaction with chymotrypsin and trypsin, respectively. There was no concentration dependence of the observed rate constants. Therefore, the rate-limiting step of the gross conformational change was not the bimolecular encounter reaction between alpha 2M and proteinases, which requires a new proposal of pre-trapping of proteinases before the gross conformational change.
Chymotrypsin↗
Mathematical model of cardiovascular mechanics for diagnostic analysis and treatment of heart failure: Part 1. Model description and theoretical analysis.
The planning of drug therapy for heart failure should involve both the diagnostic analysis of the patient's defective state and a prediction of the drug effects on the identified state. We have devised a mathematical model of cardiovascular system mechanics, on which both quantitative diagnosis and evaluation of drug effects can be made. The model was composed of systemic and pulmonary circulatory networks including the dynamics of the left and right ventricles. The model of the ventricles can represent both systolic and diastolic problems in heart failure through the parameters of ventricular contractility and diastolic stiffness. Each vascular network was composed of arterial and venous resistances and total vascular capacitance. Patient's ventricular and vascular parameters were estimated simultaneously from the clinically measurable haemodynamic variables based on the model. Despite the simplicity of the model, the results showed good agreement with clinical and experimental data. The clinically significant haemodynamic classification of heart failure by Forrester et al. (Forrester et al., 1977) was simulated well by the model. This model provides a useful basis for analysing pathophysiological states in heart failure and evaluating drug effects on the disease.
Heart↗
Mathematical model of cardiovascular mechanics for diagnostic analysis and treatment of heart failure: Part 2. Analysis of vasodilator therapy and planning of optimal drug therapy.
Using a mathematical model of cardiovascular mechanics, various complicated responses to vasodilator therapy for heart failure have been well accounted for through common logic: (i) the differential effects of various vasodilators on cardiac output; (ii) the opposite response of cardiac output to sodium nitroprusside in a normal state and heart failure state; (iii) the different responses of cardiac index, arterial pressure and left ventricular end-diastolic pressure to hydralazine in different types of heart failure. The response to combined vasodilator-inotropic agent therapy was simulated well by the model. The optimal therapeutic regimen was then formulated to simultaneously control the cardiac output, systemic and pulmonary arterial and venous pressures, and the degree of coronary ischaemia by multiple drug delivery, and the problem was solved using the model. We conclude that the model provides a useful basis for obtaining a guidance for more appropriate therapeutic regimen in heart failure.
Cardiotonic Agents↗
Age-related changes in electrocardiographic responses to trichloroethylene inhalation in conscious rats.
Age-related effects of trichloroethylene (TRI) inhalation on heart rate (HR), its circadian rhythm, the incidence of spontaneous bradyarrhythmias (BA) and ventricular premature contractions (VPC) were examined in conscious rats, as was the dependence of arrhythmias on sleep-wakefulness. Indwelling electrodes were used for simultaneous electrocardiographic (ECG), electroencephalographic (EEG) and electromyographic (EMG) measurements in 2, 13, 20 and 26-month old rats. The rats were exposed for 8 hours to 300 ppm TRI followed by exposure to clean air for 7 days, after which they were exposed to 1,000 ppm for 8 hours. The polygraphic recordings were made during 8-hr exposures and for 28 hours thereafter. Control values for all physiological parameters were measured during 36-hr exposure to clean air. The exposure to TRI exacerbated an age-dependent decrease in HR and its circadian amplitude. Although the spontaneous BA incidence decreased with advancing age, the ratio of the number of BA episodes during the post-exposure period after TRI exposure to those during the corresponding period of clean-air exposure increased more pronouncedly for 20 and 26-month old rats than for 2 and 13-month old rats. The number of spontaneous VPC episodes increased for 20 and 26-month old rats but was not affected by the exposure to TRI. Gas chromatographic analysis of TRI and free trichloroethanol (TRI-OH) in the brain and blood of the TRI-exposed rats revealed the prolonged half-life of TRI and the delayed clearance of free TRI-OH from the tissues with advancing age. The age-related exacerbation of those ECG responses to TRI inhalation appears to be brought about in part by the age-related change in the pharmacokinetics of TRI and TRI-OH.
Age Factors↗
Amplification and overexpression of EXP1 and EXP2/Cyclin D1 genes in human esophageal carcinomas.
We previously identified two genes, EXP1 and EXP2/Cyclin D1, about 120 kb apart from HST1 and INT2 on human chromosome 11q13. Here we report the frequent amplification and overexpression of these genes in surgically resected esophageal carcinomas. Amplification was observed in 8 out of 22 cases. In the tumors with the amplification, these genes were overexpressed, while in the tumors without the amplification, expression levels of these genes were almost the same as those with normal mucosa. These results suggest that EXP1 and/or EXP2/Cyclin D1 may play an important role in the progression of human esophageal carcinomas.
Adult↗
An accelerative enumeration algorithm for the bottle combination problem and the menu planning problem.
In the clinical practice, there are a number of instances in which the request tailored to each patient must be satisfied with a combination of ready-made medical supplies. For example, in intravenous infusion and feeding, an appropriate combination of commercially available bottles must be chosen such that fluid, electrolyte, and nutritional requirements can be satisfied for the individual patient. Menu planning also has the same feature. However, the problem of the optimal combination of medical supplies is difficult to solve exactly and quickly even with the aid of the computer, because of the combinatorial nature of this problem. We formulate the combinatorial problem of medical supplies as a nonlinear all-integer programming problem and present a fast exact algorithm to solve the problem. The proposed algorithm is proved to find an optimal combination in an acceptable calculation time for medical problems of the usual size.
Algorithms↗
In vivo differences between Asian, black and white in the stratum corneum barrier function.
The stratum corneum barrier function of Blacks, Caucasians and Asians were compared in vivo. A noninvasive technic, laser doppler velocimetry (LDV), was used to evaluate the cutaneous penetration of nicotinates by the determination of the lag time before vasodilatation induced by the application of those local vasodilator drugs. The study was performed on untreated skin and after removal of the stratum corneum by 12 strips. The influence of molecular weight and solubility of different nicotinates (methyl, ethyl, hexyl and vitamin E) were also studied on Japanese skin. Vasodilatation lag times assessed by LDV, with methyl nicotinate (MN), showed that skin permeability was more important in Asians (P < 0.01) and in Caucasians (P < 0.05) than in Blacks. Moreover Asian skin was significantly more sensitive to stripping (P < 0.05) than Black skin. A significant shorter lag time was obtained with small and hydrophilic nicotinates (methyl and ethyl) (P < 0.01) compared to a lipophilic one (hexyl). The alteration of the stratum corneum barrier function by stripping showed a more important modification with MN (P < 0.05) than with hexyl nicotinate. Consequently, this noninvasive method can evaluate the modifications of the stratum corneum barrier function and racial origin has to be taken into account in the determination of skin absorption.
Adult↗