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↗
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↗
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↗
Establishment of a highly sensitive and specific exon-trapping system.
We have established a highly sensitive and specific exon-trapping system (SETS) with a specific plasmid vector in which an exon in a given DNA segment is identified by its ability to remain as a mature mRNA after splicing. The SETS provides us with the isolation of possible exons rapidly and easily from DNA fragments in chromosomal regions of more than 300 kilobase pairs. Genomic DNA fragments were partially digested and subsequently cloned into plasmid pMHC2, an exon-trapping vector we have constructed. These constructs were transfected into COS-7 cells, and consequent RNA transcripts were spliced in the cells. The resulting mature mRNA was harvested and amplified by using reverse transcription-PCR. Possible exons can be recognized by the sizes of PCR products and cloned into a plasmid vector. The SETS provides a direct means of cloning exons from genomic DNA of more than 300 kilobase pairs within a short period of time. Using this system, we have screened 300-kilobase-pair genomic DNA segments derived from human chromosome 11q13. Human chromosome 11q13 may contain genes responsible for human cancers, because DNA amplification is observed in several malignant tumors. We have successfully identified exon 2 of the HST1 gene and additional transcribed sequences.
Base Sequence↗
[Analysis of urinary tract infections in hospitalized elderly patients, with particular reference to the use of diapers].
We investigated the clinical and laboratory data of 215 hospitalized patients (mean age were 76.9 +/- 12.1) to analyze both the characteristics of senile UTI and the influence of the way of urination. UTI was present in 121 of 1897 patients (6.4%), 95 of whom (78.5%) were female. Comparison of the parameters between non-infected and infected patients were as follows: body temperature was 36.57 +/- 0.64 degrees C vs. 37.49 +/- 0.77 degree C; WBC, 5410 +/- 2040/microliters vs. 7260 +/- 3230/microliters; CRP, 1.2 +/- 2.4 mg/dl vs. 3.5 +/- 3.4 mg/dl; mean class of urinary RBC, 0-1/hpf vs. 3-5/hpf; and mean class of urinary WBC, 5-10/hpf vs. 30-50/hpf. All parameters were significantly elevated (p < 0.001) in the patients with UTI. The rate of detection of causative bacteria was 88.7%; with 14.8% Escherichia coli, 12.8% Providencia species, 9.6% Enterococci, and 8.7% Pseudomonas aeruginosa. Patients with UTI were divided into three groups according to their method of urination: normal urination, use of diapers and catheterization. Body temperature (> or = 37.5 degrees C) was 2.8%, 10.1% and 34.9%; WBC (> or = 9.000), 2.7%, 6.1%, and 14.3%; CRP, 16.9%, 36.1% and 51.1%; urine RBC (> or = 6-10/hpf), 8.4%, 7.1% and 36.1%; urine WBC (> or = 15-30/hpf), 20.4%, 44.4% and 76.9%, respectively. There was a significant difference (p < 0.05-0.001) between all parameters except for urine RBC between the normal-urination patients and diaper using patients. This investigation suggested that the use of diapers was a risk factor for UTI in elderly patients.
Adult↗
Biological significance of gene amplification in carcinogenesis.
K-SAM gene was originally isolated as an amplified gene in a stomach cancer cell line by in-gel DNA renaturation method. K-SAM encodes a membrane receptor with tyrosine kinase and is often amplified in poorly differentiated type of stomach cancer, while c-ERBB-2 is often amplified in well differentiated type of stomach cancer. There are several forms of K-SAM mRNAs which are generated by alternative splicing, and two types of K-SAM protein without transmembrane region. The ligand of K-SAM is considered to be growth factor(s) belonging to fibroblast growth factor (FGF) or heparin binding growth factor (HBFG) family. We have also frequently found amplification of HST-1 or HSTF1 gene in esophageal cancer. HST-1 gene, originally found as a transforming gene, is located on human chromosome 11q13, and it locates 35 kbp apart from its related gene, INT-2. Neither of the genes was expressed even in cancer cells with the co-amplification. By cosmid walking, we have identified at least two genes, designated tentatively as EXP1 and EXP2, on the same amplicon as HST-1 and INT-2, and the mRNAs for EXP1 and EXP2 genes were increased in amounts proportional to the degree of amplification.
Esophageal Neoplasms↗
[Placental transfer and pharmacokinetic parameters of flomoxef during the perinatal period].
Flomoxef (FMOX), a new oxacephem with low MIC values against not only Gram-negative bacilli (GNB) but also against Gram-positive cocci (GPC), was evaluated for its transfer into fetus, amniotic fluid, maternal milk, spinal fluid and urine during the perinatal period following a single intravenous drip infusion at a dose of 1 g for 30 minutes. The results obtained are summarized below. 1. High concentrations of FMOX were demonstrated in maternal serum, umbilical arterial serum and amniotic fluid with Cmax values of 48.0, 10.99 and 10.20 micrograms/ml, respectively. 2. Maternal urinary excretion rate was 65.4% in the first 6 hours after administration. 3. In contrast, maternal milk and spinal fluid levels were lower than 3 and 0.20 micrograms/ml, respectively. These results showed a good placental transfer of FMOX, which is very useful for various perinatal infections. No adverse effects were observed in mothers and neonates during the course of this study.
Adult↗
Structural kinetics of the allosteric transition of aspartate transcarbamylase produced by physiological substrates.
We have studied the kinetics of the quaternary structure change associated with the allosteric transition of aspartate transcarbamylase (ATCase) (E. coli), inducing this change by exposure to the natural substrates (carbamyl phosphate and L-aspartate). The presence of 30% ethylene glycol slowed the quaternary structure change sufficiently for it to be followed by stopped-flow X-ray scattering at -5 degrees C. After adding substrates to the enzyme, the change occurred, with a half-life of a few seconds, yielding a mixture of the two standard quaternary structures (or, conceivably, a state intermediate between them). This mixture persisted until the enzyme reduced the substrate concentration below a threshold value.
Allosteric Regulation↗
A fluorescence temperature-jump study on Ca2(+)-induced conformational changes in calmodulin.
Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.
Calcium↗
[Retrograde continuous cold blood cardioplegia for aortic valve surgery].
From September 1983 to February 1987, 45 consecutive aortic valve operations were performed using the retrograde continuous cold blood cardioplegia (RCCBC) via the coronary sinus. Except one patient died of stuck valve after aortic and mitral valve replacements, 44 patients were subjected for this study and all survived patients were symptomatically improved. They were divided into two groups according to the aortic cross clamps time (ACCT). Group 1; 23 patients, ACCT within 120 minutes, and Group 2; 21 patients, ACCT exceeding 120 minutes. After the operation blood was taken periodically to check creatine phosphokinase (CPK) and its iso-enzyme creatine-kinase (CK-MB) and GOT etc. Then their levels were compared between the two groups. Although there seemed to be a tendency that the highest levels of these enzymes increase according to the ACCT, there was no significant difference in CK-MB level between the two groups. Judging from the operative results and the postoperative study of enzymatic level, RCCBC is safe and useful for aortic valve surgery, especially when long ACCT is necessary for correcting complicated lesions or when the coronary orifice is not suitable for coronary perfusion in aortic dissection.
Aortic Valve↗
Kinetic study on the dimer-tetramer interconversion of phosphorylase b by a stopped-flow X-ray scattering method.
The dimer-tetramer interconversion of phosphorylase b induced by the binding of AMP and Mg2+ was monitored using a stopped-flow X-ray scattering method. The rate constants of this second-order reaction have been determined by a nonlinear least-squares method. Burst phases in both radii of gyration and zero-angle intensities were detected at the initial step of the reaction. This suggests that rapid association might take place, followed by a slow association process of which the kinetics were measured in the present study. The radius of gyration of tetrameric phosphorylase b was determined and found to be in excellent agreement with that of phosphorylase a, but different from that of phosphorylase b reported elsewhere (G. Puchwein, O. Kratky, C. F. Golker and E. Helmreich, Biochemistry 9 (1970) 4691). The reason for this inconsistency is discussed.
Animals↗
Skin absorption of organic solvent vapors in nude mice in vivo.
Nude mice each attached to a respirator to avoid pulmonary uptake were exposed in a glass exposure chamber to 200, 1000 or 3000 ppm of benzene, toluene or tetrachloroethylene (perclene) for 2, 4 or 6 h. The animals were killed at the end of the study and the amount of each solvent retained in the whole body was determined by gas chromatography. Skin absorption rates were calculated from the amount retained in the whole body using the single compartment model (elimination rate constant) obtained in a previous experiment. There was a linear relationship between the amount of skin absorption and exposure time, and also a linear relationship between the skin adsorption rate and concentration of exposed vapors. Skin absorption of solvent vapors occurs by passive diffusion as defined by Fick's law. The skin absorption coefficient (cm/h) of each solvent vapor was calculated by dividing the skin absorption rate by exposure concentration; the values were 1.24 for toluene, 1.00 for perclene and 0.619 for benzene. The coefficient may be useful for evaluating the amount of skin absorption (ng) was calculated by multiplying the skin absorption coefficient (cm/h), concentration of solvent vapor (ng/cm3), exposure time (h) and exposed skin area (cm2).
Animals↗