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Biomedical subjects

T Sekine

Publications and source records attributed to T Sekine.

At least 235 records · Page 13Linked to original sources
PubMed1984

Spatial relationship between SH1 and the actin binding site on myosin subfragment-1 surface.

To examine the spatial relationship between SH1 thiol and actin binding site on subfragment-1 surface, we studied the interaction with actin of subfragment-1 whose SH1 was labeled with an iodoacetate derivative of biotin and covered with avidin. Subfragment-1--avidin complex bound F-actin and its Mg2+ ATPase activity was activated by actin. Considering the size and the location of biotin binding site on avidin, our results suggest that SH1 is separated from the actin binding site on subfragment-1 surface by at least 17-20 A.

Actins↗
PubMed1984

New fluorogenic, photoactivable, heterobifunctional crosslinking thiol reagents.

Properties of newly synthesized crosslinking reagents (ACM) and their applications to proteins are studied (ACM is the abbreviation for a series of photoactivable and heterobifunctional crosslinking thiol reagents, each of which has two reactive groups, maleimide and azide). These reagents bind specifically to the sulfhydryl residues of proteins in the first reaction step. Upon photoactivation, the azide group of the coumarin ring reacts with side or main chains of the proteins, and thus intra- or intermolecular crosslinking can be elicited. In addition, the coumarin moiety of the reagents becomes highly fluorescent after photolysis. Therefore, the crosslinking products can be detected by fluorometry with high sensitivity in the pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reaction of ACM with rabbit muscle aldolase led to extensive crosslinking between subunits of the enzyme and maximally 25% of the total subunits were found to be crosslinked to the dimer.

Acetylcysteine↗
PubMed1984

Selective modification of myosin SH1 with 1,2,4-trinitrobenzene. VIII. Thiols of myosin.

Myosin has 2 mol of the most reactive thiol, named SH1. 1,2,4-Trinitrobenzene (TNB), a novel dinitrophenyl(DNP)ating reagent [Takahashi et al. (1983) Chem. Lett. 1445-1448], was found to react only with SH1 without any other amino acid residues in myosin under the conditions used. Its reaction with myosin SH1 was about 30 times faster than that with N-acetylcysteine (NAC). The reaction rate of TNB with SH1 was about twice compared with that of NEM, the most reactive selective reagent for SH1 so far found, although its rate with NAC was only one sixtieth that of NEM. As to the lambda max of the absorption spectrum of SH1-DNP-myosin, a large red shift of as much as 20 nm was observed compared with low molecular S-DNP derivatives. This red shift disappeared in 8 M urea. This outstanding feature of SH1 modification with TNB was discussed in terms of affinity labeling by interaction with an aromatic amino acid near SH1.

Adenosine Triphosphatases↗
PubMed1984

Photocross-linking from SH1 of the myosin heavy chain to light chains through the dinitrophenyl group attached to SH1.

Trinitrobenzene selectively dinitrophenylates SH1, a specific thiol in the myosin heavy chain which contains 1 mol of this cysteinyl residue. When the SH1-DNP-myosin thus obtained was irradiated with a mercury lamp, a cross-linked product was formed with a molecular weight of about 220K daltons. It was shown that this product was composed of both heavy and light chains by fluorescence labeling of the heavy chain at SH2, another specific thiol, and immune reaction using an anti-light chain antibody, respectively.

Animals↗
PubMed1984

[A monoclonal antibody reactive with scirrhous carcinoma of the stomach].

A monoclonal antibody reactive with human gastric carcinoma was established. When formalin-fixed, paraffin-embedded histological sections were stained by avidin-biotin-peroxidase complex method using this monoclonal antibody, it was found to react with 89% (17/19) of the scirrhous carcinomas (diffuse type of carcinoma) and 72% (13/18) of the differentiated adenocarcinomas of the stomach tested. However, this antibody did not react with normal gastric mucosa or various other normal tissues except the mucosa of the large intestine.

Adenocarcinoma, Scirrhous↗
PubMed1984

Monoclonal antibody reactive with the sialyl-sugar residue of a high molecular weight glycoprotein in sera of cancer patients.

A monoclonal antibody, ST-4-39, was obtained by means of a unique immunization procedure using a gastric cancer xenograft as an immunogen. ST-4-39 reacted immunohistochemically with various cancers and a limited number of normal tissues. The antigen reactive with ST-4-39 was detected in sera of cancer patients, and possessed a molecular weight of greater than or equal to 1 X 10(6). The antigenic determinant was a sialyl-sugar residue different from sialyl-Lewis a recognized by NS 19-9.

Antibodies, Monoclonal↗
PubMed1984

A monoclonal antibody reactive with gastric carcinoma.

A monoclonal antibody reactive with gastric carcinomas of the stomach was established. This antibody reacted with 88% (15/17) of the scirrhous carcinomas, and 75% (6/8) of the well differentiated adenocarcinomas of the stomach tested, but did not react with normal gastric mucosa or various other normal tissues, with the exception of the mucosa of the large intestine.

Adenocarcinoma↗
PubMed1983

Post-gamma globulin. II. Radioimmunoassay determination of levels of post-gamma globulin and beta 2-microglobulin.

Post-gamma globulin previously isolated and partially sequenced in this laboratory was used for production of polyclonal and monoclonal (hybridoma) antibodies. A radioimmunoassay method was developed for quantitation of post-gamma globulin with either antibody. The titration curves obtained were treated statistically and found practically indistinguishable. The sensitivity of the method adopted for the quantitation of post-gamma globulin in a variety of biological fluids was 0.13 ng/ml and the upper limit of precision was 2.5 ng/ml. The following results were obtained (mean +/- 1 SD): normal sera, 0.96 +/- 0.20 microgram/ml; pregnancy sera, 1.08 +/- 0.28 micrograms/ml; cord blood 2.08 +/- 0.33 micrograms/ml; hospitalized patient's sera, 1.3 +/- 0.54 micrograms/ml; geriatric subjects' sera 2.26 +/- 1.10 micrograms/ml; cerebrospinal fluid, 5.37 +/- 3.36 micrograms/ml; saliva, 1.22 +/- 0.67 micrograms/ml; synovial fluid, 1.27 +/- 0.41 micrograms/ml and urine, 0.11 +/- 0.125 microgram/ml. To shed light on the catabolism of post-gamma globulin the levels of beta 2-microglobulin were also measured radiometrically. Correlative statistical analysis of all the data have shown that renal handling of post-gamma globulin and beta 2-microglobulin may be very similar but not necessarily identical.

Antibodies↗
PubMed1983

N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM): an alternative label for fluorescence tracing.

N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), a fluorescent thiol reagent, has a high molar extinction coefficient and a high quantum yield when it reacts with protein sulphhydryl bonds. DACM proved to be effective as a fluorescent probe when used for labelling rabbit immunoglobulin G anti-human gamma-chain (IgG). DACM-labelled rabbit immunoglobulin G anti-human gamma-chain conjugates (DACM-IgG) can be purified efficiently by Sephadex G-50 gel chromatography. The immunological properties of the IgG are not altered appreciably by conjugation. As a label DACM has advantages with regard to ease of preparation, specific immunofluorescence and strong photoresistance.

Animals↗
PubMed1983

Actin and actin-associated proteins of rabbit liver cell.

Rabbit liver actin and its associated proteins were prepared and their properties were studied. Liver cells were isolated from excised rabbit liver after perfusion in situ with calcium-free Lock's solution. Dried powder of acetone-treated liver cells was extracted with a buffer previously used to extract actin from skeletal muscle. The liver actin was recovered by adding skeletal myosin to trap actin as actomyosin and the resulting complex was purified by centrifugation. The actin was then dissociated from myosin by adding MgATP and was purified by centrifugation. This fraction showed the characteristic properties of F-actin and was composed of 42K, 53K, and 61K proteins. Further fractionation of these proteins into three components was carried out by centrifugation, DNase-1 affinity chromatography, and preparative gel electrophoresis. The 42K protein proved to be actin since it activated the myosin Mg2+-ATPase activity, interacted with DNase-1, and had a very similar amino acid composition to skeletal muscle actin. In these experiments, binding affinity among these proteins was apparent. Analysis of subcellular fractions combined with the above results indicated that the liver cell 53K and 61K proteins were not soluble fraction components in the cytosol. The physicochemical properties of 53K and 61K proteins were compared with those of gizzard desmin, a typical intermediate filament protein.

Actins↗
PubMed1983

Interaction of alkali light chain 1 with actin: effect of ionic strength on the cross-linking of alkali light chain 1 with actin.

To determine the spatial relationship between alkali light chain and actin in the actosubfragment-1 complex, we studied the cross-linking of actin and subfragment-1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. We found that (a) alkali light chain 1 was cross-linked to actin at two sites in the extrapeptide region, and (b) cross-linking of these two sites, especially the one which was very close to the NH2 terminal of the alkali light chain, to actin was inhibited drastically when the KCl concentration was increased from 0 to 100 mM. Since the inhibition of cross-linking with carbodiimide reagent means separation of amino and carboxyl groups in alkali light chain and actin, we suggest that this decrease in electrostatic attraction is the reason why subfragment-1 with alkali light chain 1 has higher affinity to actin than subfragment-1 with alkali light chain 2 at low ionic strength but has almost the same affinity at moderate ionic strength.

Actins↗
PubMed1983

Loss of Ca2+-dependent regulation in glycerinated skeletal muscle contraction.

Glycerinated muscle fiber from rabbit psoas muscle often lost Ca2+-dependent regulation of its contraction with long-term extraction in a 50% glycerol solution containing 5 mM EGTA at -20 degrees C, designated as Ca2+-insensitive muscle fiber (CaIS-fiber). About 30 or 40% of glycerinated muscle fibers were CaIS-fibers after 1 to 3 months in the glycerol solution. We investigated the cause of the loss of Ca2+-sensitivity of the glycerinated muscle fiber by tension mechanogram and SDS polyacrylamide gel electrophoresis. This natural CaIS-fiber showed a new band of 30K daltons peptide on SDS gels. On the other hand, Ca2+-sensitive fiber (CaS-fiber) changed to CaIS-fiber by trypsin digestion for 40 sec. The tryptic CaIS-fiber had no troponin C and 30K daltons peptide bands in the electrophoretograms. Incubating with 2 mM CaCl2 for 40 hr at 25 degrees C, CaS-fiber changed easily to CaIS-fiber which had 30K daltons peptide and faint troponin T and I bands, as in natural CaIS-fiber. All CaIS-fibers could recover their Ca2+-dependent regulation by incubating with native tropomyosin from rabbit skeletal muscle for 2 days at 4 degrees C. These results indicate that the loss of Ca2+-dependent regulation of glycerinated muscle fiber is due to degradation of regulatory protein system by endogenous Ca2+-activated proteolytic enzymes.

Animals↗
PubMed1983

[Ultrastructure of induced yolk sac tumor in the pregnant rats].

By the administration of carcinogens to the fetal membrane of pregnant rats, yolk sac tumors producing AFP was induced. The process of production of such tumors and AFP was studied at the ultrastructural level. Electron microscopy revealed that the structure of the tumor induced extended radiately around the tubal cavity, and the cubical cells were arranged in rows. Microvilli were observed on the cellular surface toward the tubal cavity. The adjacent cells were relatively diffused and an interdigitation was partly observed, but formation of incomplete desmosome was also apparence. The nuclei were generally large, some being irregular in shape. Nucleoli with a thread-like nucleolonema were also observed. There was rich organella in the cytoplasm and mitochondria and r-ER was particularly noted. In the r-ER the intracavity was enlarged containing some substances with moderate density. Some granules with the same density as said substance were present in the cytoplasm. This substance was recognized to be AFP. As the source of the material, rich free-ribosome and glycogen granules were observed around the r-ER with an amorphous substance. Due to the effect of functional collaboration, AFP was produced and accumulated in the r-ER according to the same process as protein synthesis.

Animals↗
PubMed1983

Studies on the histogenesis of experimentally induced yolk sac tumor in rats.

As a method to elucidate the histogenesis of this tumor, it is necessary to make a comparative study of the morphological relation between the histopathology of the tumor and the mother tissue which is considered to be the origin of the tumor. Therefore, as already reported, since we induced the yolk sac tumor by treating the fetal membrane of pregnant rats with carcinogenic agents, we have comparatively studied the tumor tissue and the fetal membrane, which is considered the mother tissue, to elucidate the histogenesis by seeking the similarity between them and the common features and obtained the following conclusions. First, the presence of PAS positive non-glycogenic granules was noted in both cytoplasms, and then the localization of AFP was confirmed in both cytoplasms also in the study with a fluorescence antibody method using anti-rat AFP antibody. Further, in the observation at the level of electron microscopy, as findings common to both, we noted the presence of round homogeneous non-structural substance and characteristically developed r-ER, and, on the other hand, immature nuclei containing thread-like nucleolonema, considered characteristic to germ cell tumor. From the above results, this induced tumor was considered to have developed from the rat fetal membrane as mother tissue.

Animals↗