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Biomedical subjects

E Watanabe

Publications and source records attributed to E Watanabe.

At least 91 records · Page 5Linked to original sources
PubMed1994

Low density, but not high density, C6 glioma cells support dorsal root ganglion and sympathetic ganglion neurite growth.

Accumulating evidence suggests that an inhibitory influence of the environment on growth cones plays a crucial role in development and regeneration of neuronal projections. Oligodendrocyte-associated neurite growth inhibiting substance is one of the most extensively studied molecules. Molecular biological studies, however, remain slow in progress. Although finding clonal cells that express such factors would facilitate the analysis of inhibitory influences on neurite growth, few cell lines have been reported to express neurite growth inhibitor. We therefore investigated the possibility of a clonal glial cell line to differentiate and express inhibitory or non-permissive features for neurite outgrowth in culture. We chose the C6 glioblastoma cell line and examined neurite extension from chick dorsal root ganglion (DRG) explants. Neurites from embryonic day 9 DRG extensively grew on C6 cells that were cultured at low cell density, while they failed to grow on C6 cells cultured at high density, even in the presence of nerve growth factor in high concentrations. Membrane extract from high density C6 cells, when used as culture substratum, was less permissive for neurite outgrowth compared to extract from low density cells. Treatment of the membrane extract derived from high density C6 cells with trypsin made it less non-permissive for neurite growth. These results suggest that C6 cells are induced to express a non-permissive property for neurite outgrowth by culturing them at high density.

Animals↗
PubMed1994

Expression cloning of a novel alpha 1,3-fucosyltransferase that is involved in biosynthesis of the sialyl Lewis x carbohydrate determinants in leukocytes.

The sialyl Lewis x (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc-NAc) determinants serve as ligands in the selectin-mediated adhesion of leukocytes to activated endothelium or platelets. In our efforts to identify glycosyltransferases involved in the biosynthesis of those ligands, we achieved expression cloning of a novel human alpha 1,3-fucosyltransferase termed Fuc-TVII from a THP-1 cDNA library by enrichment of the Namalwa cells highly expressing that determinant with a fluorescence-activated cell sorter. Expression of the COOH-terminal catalytic domain of Fuc-TVII showed an alpha 1,3-fucosyltransferase activity for a type II oligosaccharide with a terminal alpha 2,3-linked sialic acid among various acceptors, consistent with that in vivo acceptor specificity. Alignment of the primary sequences of five alpha 1,3-fucosyltransferases and assignment of the chromosomal location of Fuc-TVII gene, together with that acceptor specificity, indicate that Fuc-TVII consists of a unique class of the alpha 1,3-fucosyltransferase family. Determination of the expression levels of these alpha 1,3-fucosyltransferases in various cells revealed that both Fuc-TVII and a myeloid fucosyltransferase Fuc-TIV were significantly expressed in myeloid lineage cells. Fuc-TVII-transfected Namalwa cells exhibited significant binding to E-selectin in contrast to little binding of the Fuc-TIV-transfected cells. These results suggest that Fuc-TVII may participate in the biosynthesis of the selectin ligands.

Antibodies, Monoclonal↗
PubMed1994

Selection of chromatographic protein purification operations based on physicochemical properties.

The charge characteristics of four proteins (conalbumin, ovalbumin, apotransferrin, and soybean trypsin inhibitor) were determined over a range of pH using an electrophoretic technique (titration curve). HPLC anion-exchange chromatography for each of the proteins was performed to investigate whether electrophoretic and chromatographic results could be correlated. It was found that charge density, estimated from the electrophoretic titration curves (as net charge divided by the protein molecular weight) in most cases correlates extremely well the retention of the protein in terms of time needed for the protein to be eluted from the column. This correlation was far better than that of net charge or that of surface charge (net charge/surface area). The performance of anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration using preparative columns on an FPLC were compared. The ability of each method to separate the components of the mixture of the four proteins was assessed by calculating the resolution of the chromatographic results obtained for each pair of proteins. Using the concept of the deviation factor (DF) between individual properties of pairs of proteins (charge density, molecular weight, hydrophobicity), the separation efficiency factor (eta s) could be calculated. Resolution results showed that anion exchange was the best technique for separating the proteins followed by hydrophobic interaction chromatography and gel filtration. Calculations for efficiency, eta s, confirmed the superiority of anion-exchange chromatography as a separation method over gel filtration. For hydrophobic interaction chromatography, it was not possible to find a suitable parameter to express DF and, consequently, eta s. An appropriate way to express the hydrophobicity of a protein, as a physicochemical property, that can be used in the expression of DF should be investigated.

Animals↗
PubMed1994

Brain development and multiple molecular species of proteoglycan.

The occurrence of multiple proteoglycan species is a characteristic of the brain. The structural features of individually characterized proteoglycans in the brain are first introduced in brief, then some examples are shown that suggest a relationship between multiple proteoglycans and the many distinct cell types and neural circuits in the brain. Typical experiments demonstrated the neuronal-activity-dependent expression of neural proteoglycans during the critical developmental period of some functional systems such as the visual and vibrissal barrel systems. In addition, the binding properties of neural proteoglycans to other cell surface molecules are discussed in conjunction with their involvement in cell-cell and cell-substratum interactions. This review also covers other potential functions of proteoglycans not only in the development and maintenance of the brain but also in the pathogenesis of Alzheimer's disease. Proteoglycans are really coming of age in neuroscience.

Alzheimer Disease↗
PubMed1994

Immunological identification of two proteoglycan fragments derived from neurocan, a brain-specific chondroitin sulfate proteoglycan.

Neurocan is a brain-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. One of the proteolytic products (C-terminal half) is known to be a CSPG with a 150 kDa core glycoprotein (CSPG-150). To identify the N-terminal half of neurocan, we raised an anti-neurocan polyclonal antibody (PAb 291) using a synthetic peptide whose amino acid sequence matched a part of the N-terminal half of neurocan. Western blots showed that PAb 291 recognized two CSPGs, one with a 220 kDa core glycoprotein (CSPG-220, namely neurocan) and one with a 130 kDa core glycoprotein (CSPG-130) isolated from young rat brains. CSPG-130 was co-purified along with CSPG-220 by PAb 291-immunoaffinity column chromatography. The amino acid sequence of the N-terminus of the immunopurified CSPG-130 was exactly the same as the N-terminal sequence of CSPG-220. These results suggest that not only the C-terminal half (CSPG-150) but also the N-terminal half (CSPG-130) of CSPG-220 exists in a CSPG form in rat brain. Using PAb 291 and monoclonal antibody 1G2 (MAb 1G2) which recognizes CSPG-150 in addition to CSPG-220, we found that the contents of CSPG-130 and CSPG-150 in the rat brain reached maximum levels around the time of birth. Both CSPG-130 and 150 were observed, while CSPG-220 was hardly detectable in extracts from the adult rat brain. Immunohistochemical investigation showed that the PAb 291 antigen had a similar distribution pattern to the MAb 1G2 antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Developmentally regulated expression of a brain specific species of chondroitin sulfate proteoglycan, neurocan, identified with a monoclonal antibody IG2 in the rat cerebrum.

The mammalian brain contains many species of proteoglycan. To identify each proteoglycan species, we have raised monoclonal antibodies against soluble chondroitin sulfate proteoglycans purified from 10-day-old rat brains. One monoclonal antibody, named monoclonal antibody 1G2, recognized two proteoglycan species with 220,000 and 150,000 mol. wt core glycoproteins (chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150). Partial amino acid sequences of N-termini of their core proteins coincided with those of neurocan, a brain-unique chondroitin sulfate proteoglycan species, whose complete coding sequence was recently reported [Rauch et al. (1992) J. biol. Chem. 269, 19,536-19,547]. Western blots revealed that chondroitin sulfate proteoglycan-220 became detectable in the rat cerebrum on embryonic day 14, and that it disappeared from the brain around postnatal day 30. In contrast, a fairly large amount of chondroitin sulfate proteoglycan-150 remained in the mature brain. Immunohistochemical studies revealed that 1G2 antigen was first localized in the preplate zone, then both in the marginal zone and in the subplate of the rat cerebrum on embryonic day 16, prior to arrival of the first thalamic afferents at the cortex. On embryonic day 20, immunolabeling with monoclonal antibody 1G2 began to spread from the subplate into the developing cortical plate. On postnatal day 10, the neuropil of the cerebrum, except for the barrel field, was diffusely stained with the antibody, intensely in the hippocampus and superficial layers (I-III) of the cerebral cortex and weakly elsewhere. The barrel hollows were stained very weakly compared with the barrel walls at this stage. The immunoreactivity in the hippocampus and superficial cortical layers was weakened in the mature brain, so that no particular staining pattern, but weak and diffuse staining was observed in the adult rat cerebrum. The 1G2 antigen was immunohistochemically associated largely with glial fibrillary acidic protein-positive cells in primary cultures of the neonatal rat cerebrum. Both chondroitin sulfate proteoglycan-220 and chondroitin sulfate proteoglycan-150 were detected in the conditioned media not only of highly enriched cultures of fetal rat cortical neurons but also of pure cultures of mature astrocytes; more (12- to 20-fold) in the astrocyte conditioned media. Astrocytes, in addition to neurons, may be a cellular source of neurocan in brain at least under certain physiological conditions. The spaciotemporal expression pattern of 1G2 epitope-bearing proteoglycan, or neurocan, suggests that this proteoglycan species plays some roles at least in forming the elongation pathway for early cortical afferent fibers as well as the functional barrel structure in the somatosensory cortex.

Amino Acid Sequence↗
PubMed1994

Spinal cord pencil-shaped softening: comparison between the clinical findings and the autopsy findings. Case report.

The neurological symptoms and the magnetic resonance imaging (MRI) findings in a patient with a spinal pencil-shaped form of softening were compared with the autopsy findings. The distribution and shape of the lesion on MRI, especially on T2-weighted images, correlated well with the autopsy findings at the level between the medulla oblongata and the T2 segment. But these findings did not necessarily correlate with the neurological abnormalities. A pencil-shaped lesion that had extended cephaladly, and which was seen on MRI and at autopsy, did not manifest neurological deficit. MRI is useful to aid the clinical diagnosis of spinal cord pencil-shaped softening.

Aged↗
PubMed1994

[A clinical study of sixteen polycythemia vera cases--acute myeloblastic leukemia in patients with polycythemia vera].

The clinical course of 16 patients with polycythemia vera (PV), treated in the period 1982 to 1993, was shown. Splenomegaly occurred in three fourths of these patients (75%), and hypertension was a major symptom. Thrombosis such as myocardial infarction and cerebral infarction was noted. Eight patients was treated with myelosuppressive agents and the 8 other patients were treated with phlebotomy. A 70-year-old male who was treated with mitobronitol (DBM) developed acute myeloblastic leukemia (AML) 11 years later. He was treated with multi-combination chemotherapy (BHAC-DMP), and entered complete remission, followed by early relapse. He became refractory to chemotherapy and died of acute pneumonia 6 months later. Median survival of 16 cases of PV was more than 10 years, and long-term treatment and care are necessary.

Adult↗
PubMed1994

[A case of essential thrombocythemia with clonal evolution in the terminal phase].

This 50-year-old male was admitted to the hospital on April 1983 with complaints of severe chest pain attacks 2 weeks previously. Laboratory data: On admission the blood findings were Hb 14.3 Gm/dl of blood, RBC 4.70 million/mm3, WBC 11,600/mm3 and a platelet count of 1.1 million/mm3. ECG showed elevation of ST-T in V1 to V4. Serum LDH and CPK levels were high. He was diagnosed as acute myocardial infarction with thrombocythemia. Three days after admission he abruptly fell into a semicomatose state and left hemiplesia. Head computed tomography showed a large, low-density lesion in the right mid-cerebral artery area, and we also diagnosed cerebral infarction. He was given nimustine (ACNU) 100 mg/week three times as remission induction therapy. For maintenance chemotherapy, at first we administered mitobronitol (DBM) 150 mg/day then changed to intermittent administration of ACNU 100 mg. On September 1991, the patient was admitted to the hospital with progressive anemia and uncontrollable thrombocythemia. Bone marrow chromosome analysis revealed aneuploidy. The patient received interferon alpha 3 million unit/day. The thrombocythemia could be controlled but his general condition deteriorated. On April 1992, he died of interstitial pneumonia.

Aneuploidy↗
PubMed1993

Expression cloning of a novel Gal beta (1-3/1-4) GlcNAc alpha 2,3-sialyltransferase using lectin resistance selection.

This report describes the isolation of a cDNA encoding a novel human Gal beta (1-3/1-4)GlcNac alpha 2,3-sialyl-transferase involved in the biosynthesis of the sialyl Lewis x determinant (NeuAc alpha 2-3 Gal beta 1-4(Fuc alpha 1-3)GlcNAc). A cDNA library of the human melanoma cell line WM266-4 was constructed in an Epstein-Barr virus-based cloning vector. Selection of the B-cell line Namalwa expressing transfected cDNAs in the presence of the cytotoxic lectin Ricinus communis agglutinin 120 gave a cDNA encoding a protein with type II transmembrane topology, as found for mammalian glycosyltransferases. The use of this lectin, which is specific to galactose residues (especially the Gal beta 1-4GlcNAc structure), originates from our prediction that the modification of the Gal beta 1-4GlcNAc structure (a backbone of the sialyl Lewis x structure) by glycosyltransferases may increase the levels of resistance to this lectin. Comparison of this cDNA sequence with those of three other cloned sialyltransferases revealed two conserved regions shared by all four enzymes. Expression of the COOH-terminal catalytic domain of this protein showed alpha 2,3-sialyltransferase activity with substrate specificity different from that of CMP-N-acetylneuraminate:N-acetyllactosaminide alpha-2,3-sialyltransferase (Gal-beta 1-3(4)GlcNAc alpha 2,3-sialyltransferase, EC 2.4.99.6). Furthermore, expression of this cDNA in Namalwa cells increased the level of sialyl Lewis x antigens. The cloning approach based on lectin resistance may be useful for the isolation of cDNAs encoding other mammalian glycosyltransferases.

Amino Acid Sequence↗
PubMed1993

Ability of peroxisome proliferators to induce cell transformation, chromosome aberrations and peroxisome proliferation in cultured Syrian hamster embryo cells.

Di(2-ethylhexyl)phthalate (DEHP), a commonly used plasticizer, induces proliferation of peroxisomes in liver cells and causes hepatocellular carcinomas when chronically administered in the diet to rodents. To examine possible mechanisms for DEHP-associated cancer, we have measured induction of morphological transformation, chromosome aberrations and peroxisome proliferations of cultured Syrian hamster embryo (SHE) cells by DEHP and other peroxisome proliferators. Morphological transformation of SHE cells was weakly induced by treatment for 48 h with DEHP and its metabolite mono(2-ethylhexyl)phthalate (MEHP). The transformation frequency by DEHP was enhanced by exogenous metabolic activation using rat liver postmitochondrial supernatants. Treatment for 24 h with DEHP resulted in chromosome aberrations of the cells only in the presence of exogenous metabolic activation. 2-(p-chlorophenoxy)-2-methylpropionic acid ethyl ester (clofibrate), a widely used hypolipidemic drug, failed to induce morphological transformation or chromosome aberrations of SHE cells. Treatment with [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (WY-14,643), which is a more potent carcinogen than DEHP or clofibrate, elicited a lower frequency of morphological transformation than DEHP in the presence of exogenous metabolic activation but was more active than DEHP at inducing chromosome aberrations. Similar levels of peroxisome proliferation, as determined by an intensity of diaminobenzidine staining, were observed in cultures treated for 2 h with DEHP, MEHP, clofibrate or WY-14,643. These results suggest a possible involvement of genetic damage by DEHP metabolites in the induction of cell transformation of SHE cells by DEHP; however, no clear relationship among induction of peroxisome proliferation, carcinogenicity in vivo and cell transformation was observed. Although the ability to induce cell transformation and chromosomal mutations is not adequate to explain the carcinogenicity of this class of compounds, these biological effects may be contributory to the carcinogenic activities of peroxisome proliferators.

Animals↗
PubMed1993

Nucleotide sequence of tobamovirus Ob which can spread systemically in N gene tobacco.

The genomic RNA sequence of tobamovirus Ob (Ob), which can spread systemically in tobacco carrying the N gene, was determined. It consists of 6507 nucleotides and contains four open reading frames, exactly corresponding to the genomic organization of tobamoviruses known so far, i.e. encoding the 130K, 180K, 30K and coat proteins. There were no nucleotide overlaps between any open reading frames. The Ob nucleic acid sequence, predicted protein sequences and gene organization were compared with those of other tobamoviruses reported previously. This virus was originally reported as a tomato mosaic virus; however, the nucleotide sequence data given here refute this classification. The determinants that allow tobamovirus Ob to overcome the N gene, a feature peculiar to this virus, were not identified apart from sequence data. This virus should be regarded as a new tobamovirus. The determinants interacting with the tentative N gene product have not yet been analysed.

Amino Acid Sequence↗
PubMed1993

[Identification of the central sulcus using magnetoencephalography and neuronavigator].

The brain-generated currents that produce potentials measured by the electroencephalogram also produce magnetic fields which can be measured by the magnetoencephalogram (MEG), N 20 compatible evoked field after median nerve stimulation is known to be generated in primary sensory cortex. Using MEG with 37 channel SQUIDs, a current dipole is back traced which corresponds to the sensory cortex. When the dipole is projected onto the MRI of the same patient, the primary sensory cortex is precisely identified in the MRI images. These data were used as the key images for navigator enabling a surgeon identify the central cortex in the surgical field. Seven patients with peri-central mass lesion (3 meningiomas, 1 metastatic tumors, 1 angiomas, 2 gliomas) underwent surgery under MEG-navigator method. In every case, the central sulcus and motor cortex were easily identified on the cortex and the tumor was removed as far as possible preserving the motor strip. There were no postoperative worsening of the motor paresis and no other complications were noticed. The method which combines the MEG functional mapping and navigator was considered to be a powerful tool in surgery of the pericentral mass lesions.

Adult↗