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T Miyagi

Publications and source records attributed to T Miyagi.

At least 55 records · Page 3Linked to original sources
PubMed1999

Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides.

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19-38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha2-->3 and alpha2-->8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.

Amino Acid Sequence↗
PubMed1999

Left vocal cord paralysis as a primary manifestation of invasive pulmonary aspergillosis in a nonimmunocompromised host.

We report the first case (to our knowledge) of vocal cord paralysis as a primary manifestation of invasive pulmonary aspergillosis, which occurred in a 69-year-old woman without immunodeficiency. Her chest radiograph showed left upper lobe infiltration with pleural thickening, and a computed tomogram of her chest showed a thick pleural reaction and fibrosis around the arch of the aorta. A transbronchial biopsy specimen revealed Aspergillus infection. The patient was treated with oral itraconazole. However, since vocal cord paralysis persisted, the patient underwent type I thyroplasty to improve vocal function. A review of the literature showed that the incidence of invasive pulmonary aspergillosis has increased, even in nonimmunocompromised subjects, and that the disease has a potential for recurrent laryngeal nerve palsy. Therefore, invasive pulmonary aspergillosis should be considered in patients with vocal cord paralysis.

Administration, Oral↗
PubMed1999

Synthesis of basement membrane by gastrointestinal cancer cell lines.

Gastrointestinal adenocarcinoma-derived cell lines were studied in order to determine their pattern of expression of basement membrane components and their ability to form a basement membrane. In contrast to the well-preserved expression of laminin beta 2, beta 3, gamma 1, and gamma 2 chain mRNAs, five of eight gastrointestinal cancer cells lacked alpha 3 mRNA. Immunohistochemical and electron microscopic examination of four cell lines transplanted subcutaneously to SCID mice demonstrated the presence of both alpha 3 and alpha 5 chains and the formation of a basal lamina in two cases. The other two cell lines lacked both alpha 3 and alpha 5 chains and could not form a basal lamina, suggesting that this deficiency may be a factor which affects their ability to form a basement membrane. This abnormality might play some role in stromal invasion by tumour cells in gastrointestinal cancer.

Animals↗
PubMed1999

Apolipoprotein A-I of hyperlipidemia atherosclerosis prone (LAP) quail: cDNA sequence and tissue expression.

Apolipoprotein A-I (apo A-I) has an important role in the transport of cholesterol. This study describes the complete nucleotide and deduced amino acid sequence for apo A-I of LAP quail. A full length apo A-I cDNA clone for hyperlipidemia atherosclerosis prone (LAP) quail was isolated from a lambda gt10 liver cDNA library. The DNA sequence of LAP apo A-I cDNA was similar to that of normal Japanese quail. The deduced amino acid sequence of LAP apo A-I was hence identical to that of normal Japanese quail. LAP apo A-I mRNA is about 1.4 kilobases in length and expressed in a variety of tissues including small intestine, liver, lung, breast muscle, testis, and heart. Although the tissue distribution of apo A-I was similar between strains, LAP quail expressed more apo A-I mRNA than normal Japanese quail in all tissues examined. This tendency was pronounced with the small intestine. Although the concentration of serum apo A-I did not correlate with the tissue expression of mRNA, the observation may suggest that the increased apo A-I expression in LAP strain had some relevance to the susceptibility of this strain to the experimental atherosclerosis.

Amino Acid Sequence↗
PubMed1998

Partial purification and characterization of RalF40I, a class II restriction endonuclease from Ruminococcus albus F-40, which recognizes and cleaves 5'-/GATC-3'.

Restriction endonuclease RalF40I was purified from cell-free extracts of the rumen cellulolytic bacterium Ruminococcus albus F-40 heparin-Sepharose chromatography. The preparation was active only on DNA substrates that were not Dammethylated. RalF401 recognizes the 4-bp palindrome, 5'-/GATC-3', and cleaves DNA at the 5' side of G in the sequence, producing 5' tetranucleotide protruding ends. RalF40I is a class II restriction endonuclease and an isoschizomer of MboI and DpnII.

Animals↗
PubMed1998

Structural analysis of a new cryptic plasmid pAR67 isolated from Ruminococcus albus AR67.

The complete nucleotide sequence of a new cryptic plasmid pAR67 isolated from a rumen bacterium Ruminococcus albus AR67 has been determined. The plasmid pAR67 was 3419 bp in size with a 45% GC content. Two open reading frames, ORF1 and ORF2, encoding potential polypeptides of 285 and 165 amino acids, with limited sequence similarity to replication and mobilization proteins, respectively, were identified within the sequence. The region upstream of ORF1 had an AT-rich (80%) segment followed by four 19-bp direct repeats, which is similar to the structural organization characteristic of replication origins of some bacterial plasmids.

Amino Acid Sequence↗
PubMed1998

Transcript analysis of the tobacco plastid operon rps2/atpI/H/F/A reveals the existence of a non-consensus type II (NCII) promoter upstream of the atpI coding sequence.

The plastid ATP synthase complex is composed of nine subunits, of which six are encoded in the plastome. The plastid-encoded genes are arranged in two transcriptional units: atpB/E and atpI/H/F/A. We have recently reported that besides containing four -10 and -35 consensus-type (CT) promoters, the atpB/E operon also contains a non-consensus type (NCII) promoter that alone is responsible for its expression in non-photosynthetic plastids. As the functionality of ATP synthase requires expression of all nine subunits, NCII promoter-driven transcription of the atpI/H/F/A operon is to be expected in non-photosynthetic plastids. Therefore, a detailed transcriptional analysis of this operon was carried out using RNA samples from tobacco leaf, cultured cells (BY-2) and seedlings grown on streptomycin and spectinomycin; which contain chloroplasts, translationally active non-photosynthetic plastids and translationally inactive plastids, respectively. We identified a total of three transcription initiation sites (TIS) and four transcript processing sites in the non-coding regions of this operon. Our results also demonstrate that rps2 is co-transcribed with the atpI/H/F/A genes. One of the TIS (-208 atpI) is characterized by an NCII type promoter, while other two primary transcripts (-131 atpI and -384 atpH) initiate from CT promoters. In non-photosynthetic plastids the atpI/H/F/A-specific transcript pool seems to be solely contributed by initiation at the -208 atpI (NCII type) promoter, because transcripts from CT promoters do not accumulate in these plastid types.

Base Sequence↗
PubMed1998

A novel single cell PCR assay: detection of human T lymphotropic virus type I DNA in lymphocytes of patients with adult T cell leukemia.

The abnormal lymphocytes in adult T cell leukemia (ATL) reveal a peculiar morphology that is characterized by indented or lobulated nuclei. While human T lymphotropic virus type I (HTLV-I) is thought to be integrated in ATL cells, the correlation between the nuclear irregularities and HTLV-I infection is obscure. We have devised a novel single cell polymerase chain reaction (PCR) technique to examine the integration of HTLV-I provirus genome in cells from two patients with ATL. To isolate single cells, peripheral blood smears were prepared on thin polyester slides and stained with May-Grünwald-Giemsa. Morphologically defined single cells were cut out after light microscopy. The HTLV-I DNA sequences were detected not only in ATL cells but also in normal-looking lymphocytes. This novel PCR method may provide a valuable tool for understanding the molecular events associated with HTLV-I infection at the single cell level.

Adult↗
PubMed1998

Purification and characterization of a membrane-associated ganglioside sialidase from bovine brain.

A membrane-associated ganglioside-hydrolyzing sialidase was purified to apparent homogeneity from bovine brain. The enzyme was solubilized with Triton X-100 plus sodium cholate from the particulate fraction and purified over 100,000-fold by sequential chromatography on DEAE-cellulose, octyl-Sepharose, heparin-Sepharose, Sephacryl S-200, MonoQ, RCA-agarose, thiol-activated Sepharose, and ganglioside-affinity Sepharose. The final enzyme preparation exhibited a specific activity of 4,851.3 micromol/h/mg protein and an apparent molecular mass of 52 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme preferentially hydrolyzed gangliosides other than GM1 and GM2 but demonstrated hardly any activity against glycoproteins and oligosaccharides. Gangliosides GD3, GD1a, and GT1b were much better substrates than GM3 and GD1b in the presence of Triton X-100, but the latter became more sensitive to the sialidase with addition of sodium cholate. The enzyme was activated by dithiothreitol, strongly inhibited by 4-hydroxy-mercuribenzoate, and firmly adsorbed to thiol-activated Sepharose, indicating that free sulfhydryl groups are essential for its catalytic activity. Subcellular fractionation experiments revealed that the enzyme is mainly located in the synaptosomal fraction.

Animals↗
PubMed1998

Femoral condyle geometry in the normal and varus knee.

Femoral condyle geometry was evaluated in 30 normal and 30 varus knees using magnetic resonance imaging. In the sagittal view, the distal part of the medial condyle was deformed in the varus knees, but there was no significant difference in the posterior part of the medial condyle between the normal and varus knees. In the transverse view, the transepicondylar axis (a reliable rotational landmark) showed approximately 6 degrees external rotation relative to the posterior condylar axis in the varus knees and the normal knees. The results of this study suggest that no hypoplasia exists at the posterior part of the medial condyle in varus knees. In addition, the posterior condylar axis may be a reliable rotational landmark but is in relative internal rotation in varus knees when total knee arthroplasty is performed.

Aged↗
PubMed1998

Evaluation of highly sensitive thyrotropin assay for detecting thyroid diseases in neonatal screening: preliminary studies.

TSH concentrations in dried blood samples on filter paper were determined by a conventional enzyme-linked immunosorbent assay (ELISA), used for routine neonatal screening for primary hypothyroidism, and a highly sensitive bioluminescence ELISA (BL-ELISA) using firefly luciferase to examine whether central hypothyroidism and hyperthyroidism can be efficiently detected as cases of primary hypothyroidism. Samples were obtained from 3 patients with congenital central hypothyroidism, 5 patients with congenital primary hypothyroidism, 6 patients with hyperthyroidism, 31 neonatal babies with low birth weight (premature babies) and 242 newborn babies with normal birth weight from the general population (normal babies). The TSH values were low in central hypothyroidism and hyperthyroidism. Their deviations from the mean TSH value for normal babies by the BL-ELISA method (-3.12 SD and -4.79 SD in central hypothyroidism and hyperthyroidism respectively) were greater than those by the ELISA method (-2.00 SD and -2.97 SD respectively). The TSH values were high in primary hypothyroidism and normal in premature babies while deviations were the same when BL-ELISA and ELISA were used. These findings indicate that the highly sensitive TSH assay (BL-ELISA) can be used for detecting both primary and central hypothyroidism as well as hyperthyroidism in neonatal screening.

Adult↗
PubMed1998

[Pulmonary metastasis from renal cell carcinoma diagnosed by using thoracoscopic biopsy: a case report].

A 70-year-old woman underwent radical nephrectomy for right renal cell carcinoma and received prophylactic interferon-alpha (rHuIFN-alpha) administration for one year. Followup computerized tomography scan showed a small nodule in the right lung 39 months postoperatively. Pulmonary nodules had become multiple and increased in size (6 mm) at 53 months. To examine the pulmonary lesions, histopathologically thoracoscopic biopsy of the right pulmonary nodule was performed and the diagnosis of metastatic renal cell carcinoma was confirmed. Because of its minimal invasiveness, thoracoscopic biopsy may be indicated in selected cases.

Aged↗
PubMed1998

Comparative study of sialidase activity and G(M3) content in B16 melanoma variants with different metastatic potential.

We previously demonstrated that transfection of a sialidase cDNA into B16-BL6 cells, a highly metastatic and invasive cell line derived from the mouse B16 melanoma, resulted in a marked suppression of metastasis accompanied by decreased cellular content of the GM3 that is one of the target molecules of the sialidase expressed (Tokuyama et al., 1997 Int. J. Cancer, 73, 410-415). To obtain further insight into the involvement of sialidase in metastasis, we made a comparison of the levels of sialidase activity and GM3 content between B16 melanoma cell lines with low (B16-F1) and high (B16-F10 and -BL6) metastatic potential. The cells exhibited sialidase activity towards 4-methylumbelliferyl N-acetylneuraminic acid (4MU-Neu5Ac) and gangliosides at acidic pH in the particulate fractions, but not in the cytosol. The activity toward 4MU-NeuAc was significantly lower in highly metastatic cells. The activity toward gangliosides, on the other hand, varied independently of metastatic potential: B16-F10 cells with a high potential for experimental metastasis showed the lowest level and B16-BL6 cells having high invasiveness had rather a higher level of ganglioside sialidase along with a much greater GM3 synthase activity than the other two cell lines. Flow cytometric analysis with anti-GM3 antibody revealed that highly metastatic cell lines were higher in the binding affinity as compared to B16-F1 cells, B16-BL6 cells containing twice as much cellular GM3 as B16-F1 cells on thin-layer chromatography.

Animals↗
PubMed1998

[A case of crushing head injury with bilateral abducens and facial nerve palsies].

A 20-year-old male was admitted to our hospital suffering from a crushing head injury. At accident, his head had been compressed by the printing machine on both temporal regions. He remained at dull conscious. On admission one hour after the injury, he showed bilateral sixth-nerve and seventh nerve palsies and bleeding from the nose. CT scan showed marked pneumocephalus, traumatic subarachnoid hemorrhage, fluid collections in the bilateral sphenoid sinuses and right mastoid air cells. Bone CT disclosed bilateral temporal bone fractures. MRI did not show cerebral parenchymal damage. He recovered fully conscious at four hours after the injury, but cranial nerve palsies sustained over 30 days after the injury. Bilateral decompression of the facial canal were performed at day 31. At one year after the injury, bilateral abducens nerve palsies and facial nerve palsies recovered incompletely (grade III). The case report and the mechanism of such cranial nerve injuries by low-velocity crushing head injury is described.

Abducens Nerve↗
PubMed1997

Suppression of pulmonary metastasis in murine B16 melanoma cells by transfection of a sialidase cDNA.

A cytosolic sialidase cDNA was transfected into a highly metastatic and invasive cell line, B16-BL6, derived from the murine B16 melanoma. Stable transfection of a cytosolic sialidase expression vector yielded 4 transfectants with high content of the exogenous sialidase protein as well as enzyme activity. These transfectants exhibited markedly decreased experimental pulmonary metastasis, invasiveness in collagen gels and cell motility on colloidal gold-coated glass plates but no change in cell attachment to fibronectin, collagen type VI or laminin. To cast light on the underlying mechanisms, cellular constituents of the transfectants were analyzed. Sialidase over-expression did not lead to any significant changes in cell surface carbohydrates or intracellular glycoproteins, as revealed by lectin flow cytometry and lectin blotting, respectively. Thin layer chromatography of intracellular glycolipids, however, revealed decreased ganglioside GM3 and increased lactosylceramide as major changes.

Animals↗