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Biomedical subjects

K Fujiwara

Publications and source records attributed to K Fujiwara.

At least 379 records · Page 21Linked to original sources
PubMed1995

Functional chicken gizzard heavy meromyosin expression in and purification from baculovirus-infected insect cells.

The heavy chain and the essential and the regulatory light chains of chicken gizzard heavy meromyosin (HMM) were coexpressed in Spodoptera frugiperda (fall armyworm) cells infected with a mixture of two recombinant Autographa californica baculoviruses. Soluble HMM consisting of the heavy chain and the two types of light chains was obtained. The recombinant HMM was purified from the virus-infected cells and characterized. The regulatory light chain of the isolated recombinant HMM was phosphorylated by myosin light chain kinase in the presence of calmodulin in a Ca(2+)-dependent manner. The ATPase of the recombinant HMM was activated by rabbit skeletal muscle actin when myosin light chain kinase, calmodulin, and Ca2+ were present in the reaction medium. Chicken gizzard tropomyosin enhanced the actin-activated ATPase activity. The recombinant HMM decorated actin filaments, displaying the characteristic arrowhead pattern along the filaments. This report on a functional recombinant double-headed smooth muscle myosin fragment opens the way to detailed studies on the molecule.

Actins↗
PubMed1995

Focal adhesion proteins associated with apical stress fibers of human fibroblasts.

Human fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti-alpha-actinin, and antimyosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types of stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron-dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber-membrane association site by using immunocytochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including beta 1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti-integrin alpha v subunit or anti-focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber-membrane contact has some important functions different from those of the focal adhesion.

Antibody Specificity↗
PubMed1995

Mononuclear cell thyroiditis in rats with graft-versus-host disease.

Rats with graft-versus-host disease (GVHD), induced by injecting spleen cells of parental strain rats (Brown Norway) into non-irradiated (Brown Norway x Lewis) F1 hybrid rats, develop thyroiditis. This is characterized by mononuclear cell infiltration with destruction of the epithelium and a significant reduction in serum thyroid hormone levels. Immunohistochemically, the mononuclear cells consisted mainly of CD8-positive cells and macrophages. These findings provide evidence that the thyroid gland can now be enumerated as one of the target organs during GVHD. The CD8-positive cells may serve as important effector cells in lesion development, either by direct cytotoxicity or by supporting the cytotoxic potential of macrophages.

Animals↗
PubMed1995

Mature cystic teratomas of the ovary with intestinal wall structures harboring intestinal- type epithelial neoplasms.

Mature cystic teratomas of the ovary frequently contain intestinal-type tissues, but these rarely organize into complete intestinal walls, including all layers, and even more rarely develop secondary neoplasms. The present report documents two mature teratomas with formation of complete segments of intestinal wall, one also containing a benign mucinous cystadenoma of the appendiceal type and the other an intestinal-type adenocarcinoma infiltrating into the neoplastic bowel wall but not the surrounding ovarian parenchyma. The latter patient is alive and well 5 years after surgery. These cases expand the repertoire of secondary neoplasms, benign and malignant, developing in mature cystic teratomas.

Adenocarcinoma↗
PubMed1995

Clinical implications of metastases to the ovary.

Predictive factors for ovarian metastases were analyzed in 313 autopsy cases of women dying of malignancy. Genital, hematopoietic, brain tumors, and double primaries were excluded. Ovarian metastases were found in 60 of 313 (19.2%) cases. The primary sites of origin were stomach (18 cases), breast (13), pancreas (7), bile duct (7), large intestine (4), kidney (3), liver (2), lung (1), and other (5). The percentages of ovarian metastasis in each primary site were 50% (3/6) in the kidney, 38.3% (18/47) in the stomach, 30.8% (4/13) in the large intestine, 22.9% (13/57) in the breast, 22.6% (7/31) in the bile duct, 19.4% (7/36) in the pancreas, 5.4% (2/37) in the liver, and 3.4% (1/29) in the lung. Seventy-five percent (75%) of ovaries with metastasis were 5 cm or less in the greatest dimension. Seventy-eight percent (78%) of ovarian metastases were bilateral. Fifty-one percent (51%) of the involved ovaries were cystic. Twenty-four percent (24%) of the metastasis were occult in which microscopic disease was present in normal appearing ovaries. The mean age of death in the cases with ovarian metastasis was 53.8 +/- 16.8 years. This was significantly younger than the mean age without ovarian metastasis that mainly was attributable to the younger ages for breast and bile duct cancer. The number of other extraovarian metastatic sites for positive ovarian cases was significantly greater than for the negative ovarian cases (7.6 +/- 2.4 and 5.8 +/- 2.5, respectively). This difference was dominant in gastric and bile duct cancers. The other metastatic sites that accompanied ovarian metastases were the adrenal gland in gastric cancer, the spleen and small intestine in breast cancer, and the adrenal gland and colon in bile duct cancer. The frequency of peritoneal dissemination was essentially the same between cases with positive and negative ovarian metastasis. Scirrhous adenocarcinoma of the stomach more commonly metastasized to the ovary than any other stomach histological type. These findings suggest that although the impact upon outcome of bilateral salpingo-ophorectomy at the initial operation is unknown, the discovery of ovarian metastases in a large proportion of patients may dramatically alter initial management in patients otherwise thought to have only local disease.

Adolescent↗
PubMed1995

Calcitonin gene-related peptide- and substance P-immunoreactive axons in the nucleus gracilis of the rat with special reference to axonal dystrophy: light and electron microscopic observations.

Calcitonin gene-related peptide (CGRP) and substance P (SP)-immunoreactive (IR) axons in the nucleus gracilis of normal rats (1-15 months of age) were studied by light and electron microscopy. Besides many CGRP-IR and SP-IR varicosities with normal appearance, we found a few swollen (nearly round or oval) varicosities with either CGRP or SP immunoreactivity. Swollen CGRP-IR varicosities were more frequently seen than SP-IR ones, appearing from 3 months of age and increasing in number and size (up to approximately 25 microns in diameter) with advancing age. At the electron microscopic (EM) level, CGRP-IR and SP-IR swollen varicosities showed dystrophic changes, i.e., many membranous dense bodies, and proliferation of microtubules and neurofilaments. CGRP-IR or SP-IR dystrophic axons also contained many mitochondria and sometimes made synaptic contacts with nonreactive dendrites (occasionally with non-IR axons). These findings suggest that the dystrophic CGRP and SP axonal profiles represent a functionally distinct subpopulation of axonal dystrophy in the nucleus gracilis and use CGRP or SP as a neuroactive substance. Using a double-immunostaining method, many of normal CGRP-IR axons were identified to be SP-IR. However, no single dystrophic varicosity was found to contain both CGRP and SP immunoreactivities. These findings suggest that CGRP and SP afferents are independently affected and progress to dystrophic changes.

Animals↗
PubMed1995

Monoclonal antibody against the glutaraldehyde-conjugated polyamine, spermine.

We developed a mouse monoclonal antibody (ASPM-29, mAb) against spermine (Spm) conjugated to human serum albumin (HSA) using glutaraldehyde-sodium borohydride, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections. ASPM-29 showed an almost equal immunoreactivity to Spm and spermidine (Spd) but no reactivity to any of the other polyamine (PA)-related compounds tested. By use of this antibody, indirect immunoperoxidase staining was observed in different tissues fixed with glutaraldehyde in combination with borohydride reduction. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the immunostaining could be completely inhibited by 50 micrograms/ml of Spm or Spd and partially inhibited by N-acetylspermine (Ac-Spm), N1-acetylspermidine (N1-Ac-Spd), or N8-acetylspermidine (N8-Ac-Spd), but was hardly inhibited at all by other PA-related compounds or amino acids. The reactivity of the antibody with Spm conjugated on wells in an ELISA plate was inhibited by micromolar concentrations of Spm, Spd, Ac-Spm, N1-Ac-Spd, or N8-Ac-Spd, in decreasing order, but not by other small molecules. Dense ICC staining was observed in the paranuclear and basal cytoplasm of acinar cells of rat pancreas, submandibular gland and paratid gland, these results being in complete agreement with our recent ICC methods using other mAbs produced against N-(gamma-male-imidobutyryloxy) succinimide-conjugated Spm.

Animals↗
PubMed1995

Use of prostaglandin I2 analog in treatment of massive hepatic necrosis associated with endothelial cell injury and diffuse sinusoidal fibrin deposition.

Endothelial cell damage causes massive hepatic necrosis as a result of fibrin deposition in the hepatic sinusoids. When a stable analog of prostaglandin I2, beraprost sodium, was administered to rats given either dimethylnitrosamine, carbon tetrachloride, or endotoxin following Corynebacterium parvum administration, the hepatic necrosis produced in each was attenuated, but to a greater extent in the dimethylnitrosamine and endotoxin/Corynebacterium parvum models, where fibrin deposition in the hepatic sinusoids occurs, as compared to the carbon tetrachloride model, where such fibrin deposition does not occur. Beraprost sodium reduced the expected increase of portal venous pressure in the endotoxin/Corynebacterium parvum model without affecting plasma thrombin-antithrombin III complex levels. Beraprost sodium also significantly reduced cell killing of both isolated rat hepatocytes and hepatic sinusoidal endothelial cells exposed to tert-butyl hydroperoxide when compared to controls. Beraprost sodium could prove to be a therapeutic candidate for the treatment of hepatic necrosis, particularly in cases associated with fibrin deposition in the hepatic sinusoids because of its fibrin clot-clearing action.

Animals↗
PubMed1995

Lipidosis of the dorsal root ganglia in rats treated with an almitrine metabolite.

Toxic effects of a detriazinyl metabolite of almitrine (DTMA) were evaluated in rats and on cultured rat macrophages. In rats daily treated with DTMA for 16 weeks, spastic gaits with heel-lifting appeared, and lamellated and/or crystalloid bodies formed in sensory neurons, satellite cells, Schwann cells, and vascular endothelial cells of the dorsal root ganglia. The lysosomal lamellated bodies, which were not induced by almitrine, were produced also in cultured rat macrophages exposed to over 1 x 10(-5) M DTMA.

Administration, Oral↗
PubMed1995

Regulation of hepatic macrophage function by oral administration of xiao-chai-hu-tang (sho-saiko-to, TJ-9) in rats.

The effect of Xiao-Chai-Hu-Tang (Sho-saiko-to, TJ-9), the extract of a mixture of 7 herbs, on hepatic macrophage function was studied using rats. Hepatic macrophages were activated by injection of Corynebacterium parvum or 70% partial hepatectomy. Oral administration of TJ-9 for 3 weeks did not affect the ability of these macrophages to produce superoxide anions evaluated in situ by liver perfusion with nitro blue tetrazolium (NBT) and phorbol myristate acetate (PMA). However, the similar administration of TJ-9 attenuated the blocking of the activation after partial hepatectomy produced by pretreatment with gum arabic, a polysaccharide of high molecular weight. When gum arabic was added to the medium of rat hepatic macrophages cultured with normal rat sera, their ability to produce superoxide anions was reduced in a dose-related manner. This reduction was attenuated by changing the sera to the sera obtained from rats given oral doses of TJ-9 for 3 weeks. These results suggest that TJ-9 may improve the blocked function of hepatic macrophages in activation.

Administration, Oral↗
PubMed1995

Immunohistochemical demonstration of TGF-beta-receptor type II in human pituitary adenomas.

To investigate whether the signalling pathway of transforming growth factor-beta (TGF-beta) is active in human pituitary tumors, an antibody against TGF-beta-receptor type II (TGF beta-R-II) was prepared to demonstrate immunohistochemically TGF beta-R-II expression in 48 cases of human pituitary adenoma. Positive immunoreactivity for TGF beta-R-II was found in 26 adenomas: 1 of 2 somatotroph cell adenomas, 1 of 9 mammotroph cell adenomas, 0 of 3 mixed somatotroph and mammotroph cell adenomas, 5 of 9 corticotroph cell adenomas, 3 of 3 gonadotroph cell adenomas, 12 of 15 plurihormonal adenomas, and 4 of 7 null cell adenomas. Expression of TGF beta-R-II varied between histological types of adenomas and increased with decreasing grades of differentiation. It is concluded that TGF-beta-mediated signals may be passed via TGF beta-R-II in pituitary tumors.

Adenoma↗
PubMed1995

Immunohistochemical demonstration of tenascin in human pituitary glands and adenomas.

The expression of tenascin in 8 cases of normal fetal and adult human pituitary glands and in 51 cases of human pituitary adenomas was investigated immunohistochemically with the use of polyclonal antibodies against human tenascin. The immunohistochemical study demonstrated that tenascin is expressed in both fetal and adult normal epithelial cells of the anterior lobe of pituitary glands and in tumor cells of pituitary adenomas, which suggests that tenascin is synthesized by normal pituitary cells as well as by pituitary adenoma cells. Plurihormonal pituitary adenomas, especially those producing 4 or 5 different hormones, tended to show the highest incidence of positive immunoreactivity for tenascin, suggesting that tenascin may be involved in the aggressive behaviour of plurihormonal adenomas.

Adenoma↗
PubMed1995

Determination of urinary acetylpolyamines by a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA).

A monoclonal antibody (mAb), ASPM-2, produced against N-(gamma-maleimidobutyryloxy)-succinimide (GMBS)-conjugated polyamine spermine [Spm; Fujiwara et al. (1994) Histochemistry 102, 397-404] was used for the development of an enzyme-linked immunosorbent assay (ELISA) for acetylpolyamines (Ac-PAs) in human urine. The ELISA is based on the principle of competition between an analyte and Spm-glutaraldehyde-bovine serum albumin conjugate-coated polystyrene microtiter wells for the mAb, followed by immunoreaction with biotinylated anti-mouse immunoglobulin and horseradish peroxidase-streptavidin. The ASPM-2 mAb showed strong immunoreaction with N1,N12-diacetylspermine (2Ac-Spm), N-monoacetylspermine (Ac-Spm), and N1-acetylspermidine (N1-Ac-Spd), the EC50 values being 29, 50, and 51 microM, respectively, but no cross-reaction with other PA-related compounds or amino acids. The method was used to measure urinary Ac-PA levels in healthy subjects and cancer patients, without pretreatment of the specimens, mean concentrations of 3.25 and 2.80 mumol per 24-h urine, respectively (as N1-Ac-Spd), being found. The ASPM-2 ELISA for N1-Ac-Spd, which is the PA most relevant to the analysis of human urine among the three Ac-PAs mentioned above, is specific and accurate, and can easily be used to analyze large numbers of specimens in parallel. It should thus have potential for studying the relationship between urinary N1-Ac-Spd levels and cancer.

Animals↗