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N Usuda

Publications and source records attributed to N Usuda.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular cloning and characterization of a mitochondrial peroxisome proliferator-induced acyl-CoA thioesterase from rat liver.

We have previously reported the purification and characterization of the peroxisome proliferator-induced very-long-chain acyl-CoA thioesterase (MTE-I) from rat liver mitochondria [L.T. Svensson, S.E. H. Alexson and J.K. Hiltunen (1995) J. Biol. Chem. 270, 12177-12183]. Here we describe the cloning of the corresponding cDNA. One full-length clone was isolated that contained an open reading frame of 1359 bp encoding a polypeptide with a calculated molecular mass of 49707 Da. The deduced amino acid sequence contains a putative mitochondrial leader peptide of 42 residues. Expression of the cDNA in Chinese hamster ovary cells, followed by immunofluorescence, immunoelectron microscopy and Western blot analyses, showed that the product was targeted to mitochondria and processed to a mature protein of 45 kDa, which is similar to the molecular mass of the protein isolated from rat liver mitochondria. The recombinant enzyme showed the same acyl-CoA chain-length specificity as the isolated rat liver enzyme. Sequence analysis showed no similarity to known esterases, but a high degree (approx. 40%) of identity with bile acid-CoA:amino acid N-acyltransferase cloned from human and rat liver. A putative active-site serine motif (Gly-Xaa-Ser-Xaa-Gly) of several carboxylesterases and lipases was identified. Western and Northern blot analyses showed that MTE-I is constitutively expressed in heart and is strongly induced in liver by feeding rats with di(2-ethylhexyl)phthalate, a peroxisome proliferator, suggesting a role for the enzyme in lipid metabolism.

Amino Acid Sequence↗
PubMed1998

Immunohistochemical localization of taurine in various tissues of the mouse.

The localization of taurine was investigated in several tissues of the mouse. Immunohistochemical methods using a polyclonal antibody for taurine derived from rabbits was used in these studies. This method was used since it is a simple procedure and the results are clear and reliable. Tissues were fixed with paraformaldehyde, embedded in paraffin and treated in a microwave oven before using an avidin-biotin-complex method (ABC method). Control staining was accomplished by employing absorption staining using various amino acids: taurine, arginine, cysteine, hypotaurine and others. For purposes of comparison, radioautography (RAG) with 3H-taurine was performed to confirm the reliability of the immunohistochemical staining compared with the localization of the 3H-taurine incorporation in endothelial cells of the blood vessels of several tissues. In this investigation, immunoreactivity was broadly observed in many tissues: Purkinje cells of the cerebellum, glia cells of brain tissue, cardiac muscle cells, matrices of the bone, mucus granules of goblet cells of the intestines, and brown adipose cells of the fetus. Although the meaning of this widespread localization of taurine can not be explained completely, we surmise that taurine may have a different function in each of the tissues. In addition, taurine reactivity was observed in cell nuclei which was evidence of the presence of taurine in the nuclei.

Age Factors↗
PubMed1998

Ultrastructural and immunohistochemical analysis of biopsy-proven chronic active mycocarditis with numerous clusters of lymphocytes.

A 60-year-old women was admitted to our hospital with deteriorating congestive heart failure. Although the diagnosis of active myocarditis was confirmed by right ventricular endomyocardial biopsy, this patient died of refractory heart failure during corticosteroid treatment. Numerous lymphocytic clusters were observed microscopically in the heart at autopsy. Most of the infiltrating cells in the clusters were positive for CD 8, HAM 56 or MHC class 2 antigen; few cells were positive for CD 56. Expression of perforin was found in some of the infiltrating cells. Electron microscopic examination revealed small lymphocytes adhering to the surface of injured cardiac myocytes. Close contact of these lymphocytes to macrophages was shown in the clusters. ICAM-1 and MHC class 1 antigens were strongly expressed in the cardiac tissue. These results indicate that cytotoxic T lymphocyte-mediated cytotoxicity had continued to operate during immunosuppressive therapy. Corticosteroids may not be suitable for the treatment of chronic active myocarditis when persistent expression of ICAM-1 is observed.

Antibodies↗
PubMed1998

Immunohistochemical localization of peroxisomal enzymes in developing rat kidney tissues.

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, l-alpha-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1997

Electron microscopic immunohistochemistry of ocular and extraocular pseudoexfoliative material.

To investigate the nature of extraocular pseudoexfoliative (PSX) material, and to validate the hypothesis that the PSX syndrome is a systemic disorder, we studied the immunoreactivity of intraocular and extraocular PSX material from patients with the PSX syndrome using antibodies against proteins of extracellular matrices. Surgical specimens of four different tissues were obtained from human eyes with the PSX syndrome: nine trabecular tissues, three cataractous lenses, six bulbar conjunctivas, and seven lid skins. These tissues were processed for electron microscopic immunohistochemistry, and stained with antibodies against vitronectin, fibronectin, laminin, and elastin, by an indirect immunogold procedure. Density of the gold particles located on the PSX material was estimated by video image analysis. Statistical analysis of the data revealed that the PSX material from trabecular tissues, lenses, bulbar conjunctivas, and lid skins had almost identical immunoreactivity to the antibodies studied. PSX materials of trabecular tissues, lenses, bulbar conjunctivas, and lid skins were found to have not only a similar ultrastructure, but also almost identical immunohistochemical characteristics. These results support the hypothesis that the PSX syndrome is a systemic disorder.

Aged↗
PubMed1997

Immunohistochemical expression of bcl-2 protein in squamous cell carcinoma and basaloid carcinoma of the esophagus.

In the present study, the expression of bcl-2 protein in esophageal squamous cell carcinoma (SCC) and basaloid carcinoma (BC) was immunohistochemically examined, and its relation to tumor progression and postoperative survival was determined in SCC. A total of 42 SCC and 4 BC tumor samples were fixed with formalin, embedded in paraffin, and stained using monoclonal bcl-2 protein antibody, clone 124. Immunoreactivity was semiquantitatively scored, and the staining results were compared with the pathologic features and survival rates. The cytoplasm of basal cells from the normal esophageal epithelium was stained. In some well- and moderately differentiated SCCs, bcl-2 protein-positive reaction was observed in the peripheral part of the tumor cord, but in poorly differentiated SCC, the cells were weakly or hardly stained. However, in BC, the cells were strongly stained. The immunoreactivity was positive in 45.2% of the SCCs and all of the BCs. There were no significant differences in pathological features or patient survival between the bcl-2 protein-positive and protein-negative SCCs. In conclusion, the expression was not related to tumor progression and had no prognostic significance in SCC. Conversely, BC had strong immunohistochemical expression, probably associated with the differentiation of carcinoma cells simulating the basal cells of the esophagus.

Carcinoma, Squamous Cell↗
PubMed1997

Immunohistochemical studies of vascular volume and proliferative activity in squamous cell carcinoma of the esophagus.

For immunohistochemical investigation and clarification of the relationship between the vascular volume in esophageal carcinoma and the proliferative activity of its tumor cells, we examined surgical specimens of squamous cell carcinoma (SCC) of the esophagus from 15 patients. The vascular volume was evaluated by immunostaining for platelet-endothelial cell adhesion molecule-1 with monoclonal antibody JC70, and the proliferative activity of the carcinoma cells was evaluated by immunostaining for proliferating cell nuclear antigen (PCNA) with antibody 19A2. The ratio of the vascular area to the tumor area and the labeling index (LI) for PCNA in the carcinoma cells was then calculated. The antibody JC70 was useful for immunohistochemically detecting blood microvessels in esophageal carcinoma. The vascular volume, expressed as the ratio mentioned above, was higher in well- and moderately differentiated SCCs than in poorly differentiated SCC (P < 0.01), and the PCNA LI did not depend on the degree of differentiation. However, there was a significantly inverse relationship between the ratio of the vascular area to that of carcinoma and the PCNA LI of the carcinoma cells (P < 0.01). These findings show that angiogenesis is greater in esophageal carcinomas with little proliferative activity.

Aged↗
PubMed1997

Therapeutic efficacy of the angiogenesis inhibitor O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) for human anaplastic thyroid carcinoma in nude mice.

TNP-470 (AGM-1470), a synthetic analog of fumagillin (6-chloroacetyl-carbamoyloxy-4-(1,2-epoxy-1,5-dimethyl- 4-hexenyl)-5-methoxy-1-oxaspiro [2,5] octane 1, has been reported to reduce the supply of nutrients to experimental tumors by inhibiting angiogenesis. In this study, we investigated anti-tumor activity of TNP-470 against human thyroid anaplastic carcinoma with a view to developing a new treatment for this thyroid tumor. A transplantable tumor was established from thyroid anaplastic carcinoma of a 78-year-old woman, as a xenograft in nude mice (BALB/c, nu/nu, male). This transplantable tumor, with chromosomal abnormality was shown to be non-functional in excretory hormones and to preserve morphological characteristics of the original anaplastic tumor tissue. TNP-470 was given at a dose of 50 mg/kg b.w. to nude mice transplanted with human thyroid anaplastic carcinoma by different routes of administration: intratumoral, peritumoral, subcutaneous and intraperitoneal. Intratumoral and peritumoral administration were effective, and especially the TNP-470 administered by the former route completely inhibited tumor growth. Immunohistochemical analysis using anti-factor VIII antibody revealed the density of microvessels to be significantly decreased by local administration of TNP-470 (intratumoral administration, 7.8 +/- 2.9/mm2, control, 27.0 +/- 6.3/mm2; peritumoral administration, 9.7 +/- 2.6/mm2, control, 21.1 +/- 5.1/mm2). Our findings suggested the possibility of clinical application of TNP-470 to control the growth of human anaplastic thyroid carcinoma.

Aged↗
PubMed1996

Identification and purification of Ca2+/calmodulin-dependent protein kinase V from human gastric carcinoma.

We previously purified a novel Ca2+/calmodulin-dependent protein kinase (CaM kinase) V, which has proven to be a member of the CaM kinase I family. Immunohistochemical staining of surgically-resected specimens from human subjects using specific antibody which reacts with CaM kinases I and V demonstrated heterogeneous distribution of CaM kinase I/V in normal gastric mucosa. The kinase was located mainly at the bottom of foveoral epithelium and in the gastric gland (< 25% immunopositive). In contrast, this kinase was abundant in various types of gastric carcinomas (> 75%), but not in gastric adenomas. Preferential and consistent presence of this kinase was confirmed by immunoblot analysis of gastric carcinoma and human gastric cancer cell lines, Kato-III and MKN-45. CaM kinase I/V was co-purified with CaM kinase II from resected gastric carcinoma using anion-exchange chromatography followed by calmodulin-affinity chromatography. The two kinases were finally separated by HPLC-based gel filtration. Purified CaM kinase I/V from gastric carcinoma did not possess detectable autophosphorylating activity, in contrast to CaM kinase II. The findings suggest CaM kinase I/V may possess abnormal biochemical properties in human gastric carcinoma, and the kinase could participate in cell growth of the carcinoma.

Adenocarcinoma↗
PubMed1996

Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal beta-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal beta-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX -/-) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX -/- mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-month-old ACOX -/- mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX -/- mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6-7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the beta-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal beta-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

Acyl-CoA Oxidase↗
PubMed1996

Increase in lectin binding sites on epithelial cells by chronic bladder infection in rats.

Using lectin histochemistry we assessed whether chronic bladder infection modifies carbohydrate residues of glycoconjugates on uroepithelial cells in rats. The bladder infection was produced by implanting a knotted silk thread with Escherichia coli into the bladder. One or 4 weeks after the implantation the bladder was excised, incubated with sixteen biotinylated lectins and stained. The bladder epithelia as a whole stained more strongly positive for eight lectins in the infected rats than in the control rats having a sterile silk thread in the bladder. In the infected rats, the superficial epithelial layer that stained negative for Arachis hypogaea (PNA) in the controls became strongly positive for PNA, whereas the middle and deep epithelial layers increased in staining for Canavalia ensiformis and six other lectins. These results indicate that chronic bladder infection increases carbohydrate residues on uroepithelial cells and may facilitate bacterial adherence to uroepithelial cells.

Animals↗
PubMed1996

Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation.

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.

Amino Acid Sequence↗
PubMed1995

Immunogold studies on peroxisomes: review of the localization of specific proteins in vertebrate peroxisomes.

Peroxisomes, since their discovery as microbodies, have been studied mostly independently by electron microscopists and biochemists. The fine structure has been studied by electron microscopy, and the compositional enzymes and proteins by protein biochemistry. Electron microscopic histochemistry has been used to try to clarify the relationship between the fine structure and its constituents. The immunogold technique, a combination of electron microscopy and protein biochemistry, for the first time resolved this problem due to the high sensitivity and resolution power of the staining and the high reliability of the technique. The present paper reviews the way in which the immunogold techniques, especially the protein A-gold technique, revealed the localization of various enzymes or proteins in peroxisomes or peroxisomal subcompartments, and discusses why this technique should be employed in peroxisome research.

Animals↗