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Biomedical subjects

T Sekine

Publications and source records attributed to T Sekine.

At least 199 records · Page 11Linked to original sources
PubMed1988

The sites on calmodulin cross-linked to myosin light chain kinase and troponin I with water-soluble carbodiimide.

To elucidate the interaction of calmodulin with calmodulin binding proteins, we studied the location of the interaction sites on calmodulin by using a chemical cross-linking reagent. Calmodulin prepared from wheat germ was cross-linked to myosin light chain kinase and troponin-I with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked products were cleaved partially with cyanogen bromide and cross-linked sites were determined by peptide mapping analysis using SDS-urea polyacrylamide gel electrophoresis. Peptides which contain the cross-linked site were displaced from their position because of the attached fragments of myosin light chain kinase or troponin I. The peptide of calmodulin from the N-terminal to Met-73 in the cross-linked product with myosin light chain kinase had the same mobility as that of uncross-linked calmodulin on the map though the amount of the peptide was decreased in the cross-linked product. The peptide from the N-terminal to Met-110 in the cross-linked product was displaced from its position. Similar change in the mobility of the calmodulin peptides was also observed in the cross-linked products with troponin I. It was concluded, therefore, that at least one cross-linked site for myosin light chain kinase and one for troponin I were located between Met-73 and Met-110 of the wheat germ calmodulin.

Binding Sites↗
PubMed1988

Photocross-linking from dinitrophenylated SH1 in myosin head. II. Cross-linked site on 50-kDa fragment.

We reported in the preceding paper [Muno, D., et al. (1987) J. Biochem. 101, 661-669] that the dinitrophenyl group exclusively introduced to SH1 on the 20-kDa fragment of myosin subfragment 1 was cross-linked to the 50-kDa fragment by irradiation, and that limited trypsinolysis of the cross-linked S1 generated an 83-kDa peptide, a cross-linking product between the 20- and 50-kDa fragments. This paper will deal with the location of the cross-linked residue on the 50-kDa fragment. When the 83-kDa fragment labeled at SH2 with a fluorogenic SH reagent was subjected to bromocyanolysis, a main fluorescent band, which implied a cross-linked peptide, appeared in the position with an apparent molecular mass of 18.5-kDa on SDS-PAGE. On the other hand, another cross-linked peptide was obtained from a complete tryptic digest of a 83-kDa fragment rich fraction. Amino acid sequence analysis of the two cross-linked peptides revealed that the DNP moiety attached at SH1 was cross-linked with a residue in the segment of the heavy chain spanning the 485-493 region from the N-terminus of the heavy chain.

Amino Acid Sequence↗
PubMed1988

Immunocytochemical localization of myosin in rabbit liver cell.

It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.

Actins↗
PubMed1988

[A case of rectal carcinoma that disappeared following preoperative chemotherapy with an FT-207 suppository--confirmed by a thorough histologic examination of the resected specimen].

Presented is the case of a patient, a 56-year-old female, who had complained of bloody stool and constipation. A barium enema and endoscopic examination revealed a tumor (Type 2) with a crater surrounded by a thick embankment, extending from the anterior wall to the left wall of the lower rectum. Biopsy specimens of the tumor disclosed a well-differentiated adenocarcinoma. A FT-207 suppository (1500 mg/day) was administered preoperatively for 50 days (total dose 75 g). On February 16, 1987, the patient underwent an abdominoperineal excision. The resected specimen took on the appearance of a chronic ulcer with an irregular depression, measuring 2.0 x 3.0 cm in size, in the lower rectum. The histology of the lesion also indicated a chronic ulcer, the base of which consisted of fibrosis covered with regenerative mucosa. No cancer cells or nests were demonstrated even serial tissue sections. As far as the rectal carcinoma is concerned, there has been no reported case of its disappearance by preoperative chemotherapy. The above results suggest that preoperative adjuvant chemotherapy can be quite effective against an advanced rectal carcinoma.

Adenocarcinoma↗
PubMed1988

Potassium permanganate used to eliminate interference with the Porter-Silber reaction in the butanol extraction method for determination of urinary 17-hydroxycorticosteroids.

We describe a simple, rapid, and less interference-susceptible method for using the Porter-Silber reaction for determination of urinary 17-hydroxycorticosteroids. The procedure is based on butanol extraction of the steroids after the urine sample is treated with potassium permanganate and sodium bisulfite. These treatments, together with an additional acid- and alkali-washing of the extract, could eliminate most of the substances in urine that interfere with the Porter-Silber reactions. Values so obtained correlated well (r = 0.95) with those by Furuya's method in which beta-glucuronidase is used. Our method may be useful and suitable for a screening test of adrenocortical and pituitary functions.

1-Butanol↗
PubMed1988

[An autopsy case of parathyroid carcinoma with primary hyperparathyroidism].

An autopsy case of a 72-year-old woman who was determined as having had a parathyroid carcinoma with primary hyperparathyroidism is reported. On autopsy, a tumor, measuring 6 X 3 X 2 cm, was found beneath the left lobe of the thyroid gland, adherent to the left recurrent nerve and the trachea, though there was no metastasis evident. The tumor was found to be lobulated on cross section inspection. Histologically, the tumor cells were arranged in trabecular and in solid patterns with fibrous bands. A few tumor cells showed mitosis. Invasions to the capsules and the blood vessels were found present. Immunohistochemically, the parathyroid hormone was found positive in the cytoplasm of the tumor cells. Otitis fibrosa generalisata, metastatic calcification, acute pancreatitis, and core pulmonale also were observed.

Aged↗
PubMed1987

A new, smaller actin-activatable myosin subfragment 1 which lacks the 20-kDa, SH1 and SH2 peptide.

The actin-dependent ATPase activity of myosin is retained in the separated heads (S1) which contain the NH2-terminal 95-kDa heavy chain fragment and one or two light chains. The S1 heavy chain can be degraded further by limited trypsin treatment into characteristic 25-, 50-, and 20-kDa peptides, in this order from the NH2-terminal end. The 20-kDa peptide contains an actin-binding site and SH1 and SH2, two thiols whose modification dramatically affects ATPase activity. By treating myosin filaments with trypsin at 4 degrees C in the presence of 2 mM MgCl2, we have now obtained preferential cleavage at the 50-20-kDa heavy chain site without any cleavage at the head-rod junction and hinge region in the rod. Incubation of these trypsinized filaments at 37 degrees C in the presence of MgATP released a new S1 fraction which lacked the COOH-terminal 20-kDa heavy chain peptide region. This fraction, termed S1'(75K), has more than 50% of the actin-activated Mg2+-ATPase activity of S1 and the characteristic Ca2+-ATPase and K+-EDTA ATPase activities of myosin. These results show that SH1 and SH2 are not essential for ATPase activity and that binding of actin to the 20-kDa region is not essential for the enhancement of the Mg2+-ATPase activity.

Actins↗
PubMed1987

Analyses of avidin-biotin complexes with lectins of membrane glycoproteins in the urinary bladder of rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.

The bladder epithelium was examined by staining with avidin-biotin complexes with lectins to determine the early membrane changes during bladder carcinogenesis in rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BHBN). Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), Ricinus communis (RCA) and Arachis hypogaea (PNA) were used as probes. The cellular distributions of lectin particles were distinguished into membranous and cytoplasmic patterns. Normal bladder cells stained very slightly and showed a spotty membranous pattern. After treatment of rats with BHBN for two or three weeks, staining became stronger and its pattern changed from a membranous to a cytoplasmic type. The staining of bladder cancer cells induced by BHBN varied from area to area and with different lectins. These data indicated that changes in carbohydrates occur in the bladder cell membrane during the early phase of carcinogenesis.

Animals↗
PubMed1987

Photocross-linking from DNPated SH1 in myosin head. I. Cross-linking to the 50-kDa fragment.

When DNP-SH1-myosin, selectively dinitrophenylated at SH1 by 1,2,4-trinitrobenzene, was irradiated with a high-pressure mercury lamp equipped with a UV cut filter, a new 220-kDa band called the X-band appeared right above the heavy chain band (200 kDa) on SDS-PAGE (Laemmli). The time course of the X-band formation was composed of two phases, the initial one being rapid, and the second slow. Immune reaction experiments using antibodies specific for heavy or light chains indicated that the X-band in the initial phase contained heavy chain alone, but no light chains. Such an extra band (106 kDa) was also observed in the initial phase of photolysis of DNP-SH1-Subfragment-1 (heavy chain: 96 kDa) obtained from DNP-SH1-myosin. Trypsinolysis of the 106-kDa product generated a 83-kDa band. N-Terminal sequence analysis and the amino acid composition of the band revealed that the X-band is an intraheavy chain cross-linking product between the 20- and the 50-kDa fragments. This presents a striking contrast to the other cross-linking from SH1 using benzophenone-4-iodoacetamide which reacted with the 25-kDa fragment alone (Lu, R.C. et al. (1986) Proc. Natl. Acad. Sci. U.S. 83, 6392-6396). Based upon the result obtained, the spatial arrangement of the three tryptic domains around SH1 is discussed.

Amino Acid Sequence↗
PubMed1987

Effect of avidin binding to SH1 on the interface between subfragment-1 and F-actin.

The subfragment-1-avidin complex, in which avidin is attached to a well defined thiol group called SH1, was purified by CM cellulose column chromatography or affinity chromatography using lipoic acid agarose. The interaction of the purified complex with F-actin was compared to that of normal subfragment-1 using chemical cross-linking and limited tryptic digestion techniques. It was found that the binding of avidin to SH1 lowered the extent of cross-linking between the subfragment-1 heavy chain and actin. The amount of the 175K product decreased to about 50% of the normal level and that of the 165K product decreased to about 35%. It was also found that the binding of avidin abolished the protective effect of F-actin on the 50K-22K junction of the S-1 heavy chain against tryptic attack. Since more than 95% of the S-1-avidin complex was attached to F-actin under our experimental conditions, these changes are due to an alteration of the S-1-actin interface. Considering the facts that SH1 is located on the side of S-1 facing the F-actin, in the tertiary structure, and is close to the cross-linked site and to the 50K-22K junction, in the primary structure, it is quite likely that avidin bound to SH1 causes these effects by sterically preventing the close contact of S-1 and actin.

Actins↗
PubMed1987

Derivation of a murine monoclonal antibody useful for immunohistochemical diagnosis of human adenoid cystic carcinoma and stomach cancer.

A monoclonal antibody, NCC-SG-007, was raised with a formalin-fixed adenoid cystic carcinoma of the salivary gland as the immunogen. The reactivity of the antibody was tested on paraffin sections using the avidin-biotin-peroxidase complex (ABC) method. NCC-SG-007 reacted with 36% of the adenoid cystic carcinomas, 42% of the pleomorphic adenomas and 14% of the mucoepidermoid tumors of the salivary gland which were tested. The antibody also reacted with some normal tissues and various other tumors. Surprisingly, NCC-SG-007 showed a reactivity as high as 94% with gastric carcinomas. The antigenic determinant was revealed to be a carbohydrate chain with no terminal sialic acid, the molecular weight of which was estimated to be 1.5 X 10(6) daltons. This antigen differed from other previously reported gastrointestinal cancer-associated antigens. The antibody should be useful for studying adenoid cystic carcinomas of the salivary gland and gastric cancers.

Animals↗
PubMed1987

Serological and immunohistochemical detection of a gastrointestinal cancer-associated asialoglycoprotein antigen of human with a murine monoclonal antibody.

A murine monoclonal antibody (mAb), NCC-AS 13 (IgM, kappa), was produced after immunization with ascites from a gastric cancer patient. In a sandwich enzyme immunoassay, NCC-AS 13 reacted with the sera from 32% of 58 gastric cancer patients, 44% of 9 hepatocellular cancer patients and 33% of 6 colorectal cancer patients. Immunohistological analysis showed NCC-AS 13 to react with approximately 90% of 51 gastric carcinomas, 12 out of 12 colon carcinomas and 3 out of 4 lung carcinomas. The NCC-AS 13 defined antigen was determined to be a carbohydrate without terminal sialic acid on macromolecular glycoproteins. This mAb NCC-AS 13, detecting a novel antigen associated with gastrointestinal cancers, may have potential uses for tumor immunoscintigraphy and for the therapy of gastric cancer patients.

Antibodies, Monoclonal↗