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T Kohno

Publications and source records attributed to T Kohno.

At least 325 records · Page 18Linked to original sources
PubMed1991

Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity.

Development of ultrasensitive enzyme immunoassays for antigens, haptens and antibodies is reviewed with emphasis on factors which limit the sensitivity. One of the most important conditions for ultrasensitive immunoassays is the use of non-competitive solid phase assay systems rather than competitive ones. Although non-competitive immunoassays are available for antigens and antibodies, there are only competitive immunoassays for hapten molecules which can not be bound simultaneously by two different antibodies. In order to overcome this difficulty, methods have been developed to derivatize haptens with amino groups so that the derivatized haptens may be measured by two-site assays. The other condition for ultrasensitive immunoassays is to minimize the non-specific binding of labelled reactants. This has been achieved by developing methods to transfer the complex of analytes and labelled reactants from solid phase to solid phase without dissociation. Thus, the sensitivity for antigens, haptens and antibodies has been markedly improved. However, the sensitivity for the detection of label enzymes remains to be improved.

Antibodies↗
PubMed1991

Role of actin in the myosin-linked Ca(2+)-regulation of ATP-dependent interaction between actin and myosin of a lower eukaryote, Physarum polycephalum.

Actin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend, Pharmacol. Sci. 11, 433-435]. The activity of dephosphorylated myosin remained at a low level both in the presence and absence of Ca2+, although Ca(2+)-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum, myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo. Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin. Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955-957].(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1991

Mechanisms of molecular recognition of tRNAs by aminoacyl-tRNA synthetases.

Interactions of Escherichia coli isoleucyl- and glutamyl-tRNA synthetases and their cognate tRNAs were analyzed by phosphate-alkylation mapping with N-nitroso-N-ethylurea and/or by 1H-NMR analysis. When E. coli tRNA(Ile) was bound with isoleucyl-tRNA synthetase, many of the phosphate groups in the anticodon loop and stem and in the D-stem were protected from alkylation. This result is consistent with that of analysis of imino proton resonances due to the secondary and tertiary base pairs. These analyses also suggested that the L-shaped tertiary structure of tRNA(Ile) is distorted upon complex formation with IleRS because of disruption of some tertiary base pairs. In the case of E. coli tRNA(Glu), several phosphate groups in the D-stem and the variable loop were significantly protected by the cognate synthetase. These results indicate that the two tRNAs, unlike other tRNAs studied so far, have some of the "identity determinants" in the D-stem and/or in the anticodon stem.

Alkylation↗
PubMed1991

[Analysis of factors relating to early repairment of the surgical margin following liver resection--factors relating to duration of hospital stay].

To analyze relating factors to early repairment of the surgical margin of the remnant liver we measured plasma fibronectin (FN), coagulation factor XIII (XIII), polymorphonuclear leukocyte elastase (PMNE), platelet counts (Plt), prothrombin time (PT%) before and at the first, third, 7th and 14th days after liver resection in 25 patients. Changes in these factors (1) were compared with their clinical status, such as liver cirrhosis, high fever, abscess formation, duration of drainage and use of microwave tissue coagulator (MTC) (2). The multivariate analysis about the factors influencing the duration of hospital stay (3) were carried out. (1) In the all cases FN, XIII (14th), PT%, Plt decreased and PMNE increased significantly versus pre-operative data. There were significant correlations between FN and XIII, FN and PMNE. (2) In the cirrhotic group FN, XIII, PT% and Plt were significantly lower than those of non cirrhotic group. In the abscess formation group PT% was significantly lower than the no abscess formation group. In the MTC group XIII, PT% and Plt were significantly lower than those of the no-MTC group. In conclusion, FN, XIII, Plt and PT% in the cirrhotic, high fever, abscess formation or longer drainage group were in lower levels compared with the each control group. (3) By the multivariate analyses, abscess formation, high fever and liver cirrhosis were the most influencing factors for the duration of hospital stay.

Adult↗
PubMed1991

[Ocular manifestations of Yusho 22 years after the onset].

Ocular manifestations were studied in 91 Yusho patients who took the contaminated rice oil 22 years ago. Tear film dysfunction was examined using the rose bengal test. Eighty-seven percent of the patients complained eye discharge, although edema of the eyelid and pigmentation of the conjunctiva were rarely seen. Cystic swelling of the tarsal glands was seen in 16 patients (18%) and white cheese-like contents in the tarsal glands were detected in 7 patients (8%). Twenty-five patients showed positive staining with the rose bengal test, although the intensity of the staining was slight. From this study it appears that there may not be tear film dysfunction in Yusho patients.

Adult↗
PubMed1991

[Dural arteriovenous malformation in the falx with subarachnoid hemorrhage].

A case of falx dural arteriovenous malformation was reported. A 62 year old man was admitted to Nakamura City Hospital on August 15, 1989, with severe headache as his chief complaint. On admission, his consciousness was lethargic. CT scan showed subarachnoid hemorrhage with ventricular perforation and hematoma of the corpus callosum. Angiograms demonstrated a dural arteriovenous malformation (DAVM) in the frontal falx, which was fed by bilateral middle meningeal arteries and the left anterior falx artery and drained into the superior sagittal sinus via the dural vein. Bifrontal craniotomy was performed. At first, bilateral middle meningeal arteries were coagulated, and the frontoparietal dura was excised widely. Then, the falx was cut at the crista galli. The DAVM was found in the falx, including a vascular sac embedded in the brain tissue. The DAVM was coagulated as much as possible. Carotid angiograms revealed complete disappearance of the DAVM, 4 months after the operation. Although angiograms performed after only one month still showed a small residual DAVM. On reviewing the literature we found only 5 patients with the DAVM in the falx. In 6 cases including our own, intracranial hemorrhage occurred in 4 cases (3 cases were subarachnoid hemorrhage). Vascular sacs were seen in 4 cases, and drainage to the pial vein was noted in 3 cases. It seemed to be rare that the DAVM drained into the dural vein. In our particular case, operative findings showed the DAVM drained into the dural vein without the pial vein, and intracranial hemorrhage was attributed to rupture of the vascular sac.

Dura Mater↗
PubMed1990

Ultrasensitive enzyme immunoassay.

Ultrasensitive enzyme immunoassay methods not only for antigens but also for antibodies and haptens are reviewed. These methods are based on a noncompetitive type of assay using solid phase rather than a competitive one. One of the greatest obstacles limiting the sensitivity of noncompetitive immunoassay methods using solid phase is the nonspecific binding of labeled reactants to solid phase. A novel method (immune complex transfer immunoassay method) to overcome this difficulty has been developed. In the initially developed method, antibodies in test serum were reacted with dinitrophenylated biotinylated antigen. The complex formed was trapped onto (anti-dinitrophenyl group) IgG-coated solid phase. The solid phase was washed to eliminate nonspecific immunoglobulins. The complex was eluted from the solid phase with dinitrophenyl-L-lysine, again trapped onto avidin(streptavidin)-coated solid phase and finally measured by reaction with (anti-immunoglobulin) Fab'-enzyme conjugate. Namely, the complex of antibodies and dinitrophenylated biotinylated antigen was transferred from one solid to another, effectively eliminating nonspecific immunoglobulins nonspecifically adsorbed onto the first solid phase. By this method with and without further modifications, the sensitivity for antibodies in serum has been considerably improved. The same principle of immune complex transfer has been applied to the detection of antigens, and one milliattomole (600 molecules) of human ferritin has been detected. Ultrasensitive, noncompetitive immunoassay methods to measure haptens with amino groups have also been developed to measure attomole amounts of haptens.

Animals↗
PubMed1990

Nonprotein amino acid furanomycin, unlike isoleucine in chemical structure, is charged to isoleucine tRNA by isoleucyl-tRNA synthetase and incorporated into protein.

Nonprotein amino acid furanomycin was found to bind with Escherichia coli isoleucyl-tRNA synthetase (IleRS) almost as tightly as the substrate L-isoleucine. The conformation of furanomycin bound to the enzyme was determined by NMR analyses including the transferred nuclear Overhauser effect method. The conformation of IleRS-bound furanomycin was similar to that of L-isoleucine, although the chemical structure of furanomycin is unlike that of L-isoleucine. By E. coli IleRS, E. coli tRNAIle was charged with furanomycin as efficiently as with L-isoleucine. Furthermore, furanomycyl-tRNAIle was bound to polypeptide chain elongation factor Tu as tightly as isoleucyl-tRNAIle. Furanomycin was found to be incorporated into beta-lactamase precursor by in vitro protein biosynthesis. A newly designed amino acid will probably be incorporated into proteins, provided that the new amino acid takes a similar conformation as a protein-constituting amino acid in the active site of an aminoacyl-tRNA synthetase.

Amino Acids↗
PubMed1990

Specific enzyme immunoassay for alpha-fetoprotein from hepatocellular carcinoma.

alpha-Fetoprotein in sera from healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak) was found in all the serum samples, but the other two peaks (the second and third peaks) were found only in patients with hepatocellular carcinoma. For alpha-fetoprotein in the second and third peaks, a specific enzyme immunoassay was developed. Lens culinaris agglutinin-coated polystyrene balls were incubated with alpha-fetoprotein and, after washing, with affinity-purified anti-alpha-fetoprotein Fab'-beta-D-galactosidase conjugate. beta-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry. The maximal volume of serum that could be used without interference was 0.2 microliters, and the minimal detectable serum concentrations of alpha-fetoprotein in the second and third peaks were 3.5 mg/liter and 0.1 mg/liter, respectively. The maximal serum concentration of alpha-fetoprotein in the first peak that did not affect the detection limits of alpha-fetoprotein in the second and third peaks was 10 mg/liter. It was possible to confirm the presence of alpha-fetoprotein in the second and third peaks by a significant difference between bound beta-D-galactosidase activities in the absence and presence of alpha-methyl-D-mannoside or D-glucose. This assay may be useful for diagnosis of hepatocellular carcinoma, although further improvements remain to be made.

Carcinoma, Hepatocellular↗
PubMed1990

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for alpha-fetoprotein from hepatocellular carcinoma.

Alpha-fetoprotein in sera from patients with hepatocellular carcinoma was fractionated into three peaks by affinity chromatography on a column of Lens culinaris agglutinin-Sepharose 4B. One peak (the first peak), which passed through the column without adsorption, was found in both healthy subjects and patients with liver cirrhosis and hepatocellular carcinoma. The second and third peaks were reactive with L. culinaris agglutinin and found only in patients with hepatocellular carcinoma. For alpha-fetoprotein in the second and third peaks, a novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) was developed. Alpha-fetoprotein in test serum was reacted with dinitrophenyl affinity-purified anti-alpha-fetoprotein IgG, and the complex formed was trapped onto affinity-purified (antidinitrophenyl bovine serum albumin) IgG-coated polystyrene balls. The polystyrene balls were washed to eliminate substance(s) other than alpha-fetoprotein in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine. The eluted complex containing alpha-fetoprotein in the second and third peaks was trapped onto L. culinaris agglutinin-coated polystyrene balls and reacted with affinity-purified anti-alpha-fetoprotein Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene balls was assayed by fluorimetry. The maximal volume of serum that could be used without interference was 20 microliters, which was 100-fold larger than that in the previous enzyme immunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Biomarkers, Tumor↗
PubMed1990

Novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups.

A novel and sensitive noncompetitive (two-site) enzyme immunoassay for haptens with amino groups is described. L-Thyroxine (T4) was used as a model hapten. T4 was indirectly biotinylated with glutathione as spacer between T4 and biotin molecules and trapped onto anti-(T4-bovine serum albumin) IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate unreacted biotin and other biotinylated substances, biotinylated T4 was eluted from the polystyrene balls with HCl and was reacted with anti-(T4-bovine serum albumin) Fab'-horseradish peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorimetry. The detection limit of T4 was 78 fg (0.1 fmol)/tube, which was 50-fold lower than the limit by competitive enzyme immunoassay using the same antibody and T4-peroxidase conjugate. This noncompetitive enzyme immunoassay was applied to the measurement of total T4 in serum. Serum levels of total T4 in 10 healthy subjects aged 25-40 yr were 94 +/- 13 (SD) micrograms/L (range, 78-114). The principle of this noncompetitive enzyme immunoassay may allow it to measure other haptens with amino groups more sensitively than can competitive immunoassays.

Adult↗
PubMed1990

Sensitive time-resolved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum.

A sensitive time-resolved fluorimetric immune-complex-transfer immunoassay for antithyroglobulin IgG in serum is described. Antithyroglobulin IgG in test serum was reacted with dinitrophenyl europium ion-labeled thyroglobulin. The complex formed of antithyroglobulin IgG and dinitrophenyl europium ion-labeled thyroglobulin was trapped onto two polystyrene balls coated with affinity-purified rabbit antidinitrophenyl bovine serum albumin IgG. The polystyrene balls were washed to eliminate nonspecific IgG in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to two polystyrene balls coated with affinity-purified rabbit antihuman IgG gamma-chain IgG. Europium ion bound to the polystyrene balls was measured by time-resolved fluorimetry. Antithyroglobulin IgG was demonstrated in all patients with Graves' disease and all patients with chronic thyroiditis. This immunoassay was more sensitive than the conventional enzyme immunoassay and less time-consuming than the previously described immune-complex-transfer enzyme immunoassays, although there were larger assay variations.

Adolescent↗
PubMed1990

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG in human serum using recombinant gag-env hybrid protein as antigen.

A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.

Blotting, Western↗
PubMed1990

A second tumor necrosis factor receptor gene product can shed a naturally occurring tumor necrosis factor inhibitor.

An inhibitor of tumor necrosis factor (TNF) has been isolated from the human histiocytic lymphoma cell line U-937 that is capable of inhibiting both TNF-alpha and TNF-beta. Protein sequencing has verified that it is distinct from a previously described TNF inhibitor that is a soluble fragment of a TNF receptor molecule (TNFrI). The cDNA sequence of this second TNF inhibitor clone suggests that it is also a soluble fragment of a TNF receptor. Expression of this cDNA sequence in COS-7 cells verified that it encodes a receptor for TNF-alpha (TNFrII) that can give rise to a soluble inhibitor of TNF-alpha, presumably through proteolytic cleavage. The extracellular domain of TNFrII has significant homology with that of TNFrI and these two receptors share a striking conservation of cysteine residue alignment with the extracellular domain of the nerve growth factor receptor. These three receptor molecules are therefore members of a family of polypeptide hormone receptors.

Amino Acid Sequence↗
PubMed1990

Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods.

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.

Amino Acid Sequence↗
PubMed1990

Recognition of tRNA identity determinants by aminoacyl-tRNA synthetases.

By analyzing aminoacylation activities of variants of isoleucine tRNAs and glutamic acid tRNA from Escherichia coli, it was found that the anticodons are the major determinants for "identities" of these tRNAs. It was also shown that the post-transcriptional modifications are essential to aminoacylation of these tRNAs. Interactions between the tRNAs and the aminoacyl-tRNA synthetases were analyzed by NMR spectroscopy.

Amino Acyl-tRNA Synthetases↗