Electroreflectance intensity for resonant coupling between Wannier-Stark localization states in a GaAs/AlAs superlattice.
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Journal Article↗
Biomedical subjects
H Nishimura
Publications and source records attributed to H Nishimura.
Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue.
We have recently discovered unusual sugar chains (xylose (Xyl)-glucose (Glc) and (Xyl)2-Glc) linked to a serine residue in the epidermal growth factor (EGF)-like domains of human and bovine clotting factors VII (Ser-52), IX (Ser-53), and protein Z (Ser-53), in addition to bovine platelet glycoprotein thrombospondin. We now have evidence of another modification in the first EGF-like domain of human factor IX, which proved to be a tetrasaccharide O-fucosidically linked to Ser-61. Two large peptides containing Ser-61 (positions 44-63), named hIX-GP1 and hIX-GP2, were first isolated from the lysyl endopeptidase-digest of human factor IX, by reversed-phase high performance liquid chromatography (HPLC). Data on the component sugar analysis after pyridylamination (PA) and sialic acid analysis of the isolated peptides indicated that they contained 1 mol each of galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (NeuAc), in addition to Glc and Xyl. hIX-GP1 was further digested with asparaginyl endopeptidase, and two glycopeptides containing Ser-61, named N-3 (positions 59-63) and N-9 (positions 55-63), were isolated, respectively. These glycopeptides were both composed of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc but did not contain Xyl and Glc. Moreover, the data on beta-elimination for N-9 and of the fast atom bombardment mass spectrometric analysis on peptide N-3 suggested the presence of a tetrasaccharide linked to Ser-61. An analysis of the PA-oligosaccharide released from hIX-GP1 by hydrazinolysis followed by pyridylamination revealed that the reducing end was PA-Fuc. All the results support the proposal that human factor IX has a novel tetrasaccharide consisting of 1 mol each of Gal, Fuc, GlcNAc, and NeuAc, which is O-glycosidically linked to Ser-61 through the Fuc residue.
Amino Acid Sequence↗
Tissue factor potentiates the factor VIIa-catalyzed hydrolysis of an ester substrate.
We designed a simple and sensitive method to assay the activity of the factor VIIa-tissue factor complex, using as a substrate N alpha-benzyloxycarbonyl-L-arginine p-nitrobenzyl ester (Z-Arg-ONb) (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The principle was to measure the amount of p-nitrobenzyl alcohol released during ester hydrolysis using reversed-phase high performance liquid chromatography. Z-Arg-ONb had a broad specificity for plasma serine proteases and factor IXa. Using this method, we examined the effect of tissue factor on the esterase activity of factor VIIa under various conditions. We found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb. Phospholipids were not required for the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, even in the presence of tissue factor. The Km value of factor VIIa alone toward the ester substrate was six times higher than that of a VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of the VIIa-tissue factor complex (14.3 versus 173 s-1). Thus, tissue factor apparently affects the catalytic site of factor VIIa and enhances hydrolysis of the ester substrate. This enhancing effect of tissue factor disappeared on removal of the gamma-carboxyglutamic acid domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification. The gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.
Amino Acid Sequence↗
[MR imaging of thymoma--comparison with CT, operative, and pathological findings].
Twenty-six patients with thymoma, who had magnetic resonance (MR) imaging and computed tomography (CT) before surgery, were studied. Twenty-six thymomas were classified into 11 non-invasive thymomas (Masaoka's clinical stage I) and 15 invasive thymomas (stage II, III, and IV). On MR imaging compared with histological findings, low signal intensity rim of the tumor was corresponded to fibrous capsule of the tumor, and linear and/or reticular low signal intensity lines in the tumor were corresponded to the fibrous septae dividing thymoma into lobules. The detectability of these findings by MR imaging was superior to that by CT. Margin of the tumor was smooth in non-invasive thymoma rather than invasive thymoma. The diagnostic accuracy of invasion to vessel, and pleura or lung on MR imaging and CT was compared with operative and histological findings. MR imaging was same as CT in its ability to detect tumoral invasion to vessel, and slightly superior to pleura or lung. In conclusion MR images clearly show the findings corresponding to pathologic specimens, and MR imaging combining with CT is useful to differentiate non-invasive thymoma to invasive thymoma.
Adult↗
Upstream activation element of the PH03 gene encoding for thiamine-repressible acid phosphatase in Saccharomyces cerevisiae.
The PH03 gene of Saccharomyces cerevisiae encodes thiamine-repressible acid phosphatase and requires the positively acting regulatory protein THI2 for its expression. Deletion analysis of the 5'-flanking region of PH03 gene revealed that an activating region located at nucleotide position -234 to -215 relative to the translation initiation codon is required for the expression and sensitivity to thiamine. A chemically synthesized DNA fragment covering -234 to -215 showed a significant level of expression when inserted in front of the PH03 promoter lacking the activating region. Electrophoretic mobility shift assay demonstrated the presence of proteins that bound to the above DNA fragment in the nuclear extract from cells grown in thiamine-free medium. These findings suggested that this region between -234 and -215 acts as an upstream activation element of the PH03 gene that can interact with regulatory proteins.
Acid Phosphatase↗
Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.
Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.
Angioplasty, Balloon↗
Identification of three types of PDGF-A chain gene transcripts in rabbit vascular smooth muscle and their regulated expression during development and by angiotensin II.
PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.
Aging↗
[Prediction for effectiveness of steroid pulse therapy by MRI in Graves' ophthalmopathy].
Fifteen patients with Graves' ophthalmopathy (GO) were treated with intravenous methylprednisolone (steroid pulse therapy, 1g daily for 3 days a week, 2-4 times) and followed up by ophthalmological assessment and magnetic resonance imaging (MRI). The signal intensity of enlarged eye muscle and retrobulbar fat was examined with MRI at 0.5T with short inversion time inversion recovery (STIR) sequences. The signal intensity of eye muscle and retrobulbar fat tissue in STIR was evaluated as the ratio to cerebral substantia alba (signal intensity ratio). The thickness of enlarged eye muscle was measured by T1-weighted coronal images. The signal intensity ratios of enlarged eye muscle of GO patients were significantly higher than those of eight normal subjects. Although the signal intensity ratios of muscle and retrobulbar fat before therapy were not related to the severity of clinical findings of GO assessed by ophthalmopathy index, the initial signal intensity ratios of eye muscle and retrobulbar fat of ten patients with improved clinical findings of GO after steroid pulse therapy tended to be higher than those of five patients without improvement by the therapy. After the therapy the signal intensity ratios of muscle and retrobulbar fat were significantly decreased in ten patients with favorable response. Our data suggested that high signal intensity in STIR may reflect edema caused by acute inflammation associated with GO. In conclusion, MRI may be a useful tool for determining the indication and prognosis of steroid pulse therapy. We strongly recommend measuring the signal intensity of eye muscle as well as muscle thickness in MRI to evaluate the activity of GO.
Adipose Tissue↗
Experimental investigation of radiation heat waves driven by laser-induced Planck radiation.
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Journal Article↗
Cloning and characteristics of a positive regulatory gene, THI2 (PHO6), of thiamin biosynthesis in Saccharomyces cerevisiae.
A thi2(pho6) mutant of Saccharomyces cerevisiae, defective in the expression of structural genes for thiamin-repressible acid phosphatase and enzymes involved in thiamin biosynthesis, was found to retain sufficient thiamin transport activity. The transport activity was repressed by thiamin in growth medium. We isolated from a S. cerevisiae genomic library two hybrid plasmids, pTSR1 and pTSR2, containing 10.2- and 12.0-kilobase (kb) DNA fragments, respectively, which complement the thi2(pho6) mutation of S. cerevisiae. This gene was localized on a 6.0-kb ClaI-ClaI fragment in the subclone pTSR3. Complementation of the enzyme activities for thiamin metabolism in the thi2(pho6) mutant transformed by some plasmids with the TH12(PHO6) gene was also examined.
Cloning, Molecular↗
Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter.
Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.
Amino Acid Sequence↗
Somatically mutated IgG anti-DNA antibody clonally related to germ-line encoded IgM anti-DNA antibody.
Among 15 anti-DNA antibody-producing hybridomas derived from a single NZB X NZW F1 mouse, an IgM and an IgG were shown to use the same VH gene of the Q52 family. Using a combination of two primers both consisting of a mixture of oligonucleotides (one complementary to the 5' end of VH segment and one to the 3' end of VH segment of Q52 family) we determined the sequences of several members of germ-line VH genes in the Q52 family derived from NZB and NZW strains. Comparison of the sequences with those of cloned VH cDNA obtained from the hybridomas revealed that the VH sequence of the IgM anti-DNA antibody was identical to that of a cloned NZW germ-line VH gene, except for the priming sites. In contrast, the VH sequence of the IgG counterpart contained somatically mutated nucleotides. Because the IgG anti-DNA antibody showed a higher DNA binding activity than did the IgM antibody, we conclude that these changes in nucleotide sequences were induced and selected through an antigen-driven mechanism as is the case in a normal immune response. It is tempting to speculate that the germ-line encoded, low-affinity IgM autoantibody undergoes somatic mutations and isotype switching, resulting in generation of pathogenic, high-affinity autoantibodies in autoimmune diseases.
Amino Acid Sequence↗
Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli alpha-hemolysin membrane translocation system.
Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli alpha-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a beta-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.
Amino Acid Sequence↗
Efficient selection of mu m-mutants from mu m-expressing myeloma cells by treatment with ricin A-conjugated anti-mu antibody.
We have developed an efficient system for obtaining myeloma mutants defective in trans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the mu gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold mu on the cell surface and whose CH1 sequence was removed to prevent mu from being retained in the endoplasmic reticulum. It efficiently and stably expressed mu chains of IgM on the cell surface (mu m+) without light chains. To obtain mutants lacking mu m (mu m-) from the mu m+ cell line by selectively killing mu m+ cells, a method with ricin A-conjugated anti-mu antibody was more reliable than complement lysis mediated by anti-mu antibody. Applying the system, we obtained a variety of mu m- mutants.
Animals↗
Cloning and sequencing of influenza C/Yamagata/1/88 virus NS gene.
It was previously shown that the shortest RNA of influenza C/California/78 virus contains 934 nucleotides and codes for two nonstructural proteins of 286 amino acids (NS 1) and 121 amino acids (NS 2). In this report, we determined the nucleotide sequence of the NS gene of the recently isolated influenza C/Yamagata/1/88 strain by using cloned cDNA derived from the viral RNA. Compared with the NS gene of C/California/78, one nucleotide insertion has occurred in the NS gene of C/Yamagata/1/88. This caused frame shifts of both the NS 1 and NS 2 reading frames, directing the synthesis of the NS 1 and NS 2 proteins consisting of 246 and 182 amino acids, respectively.
Amino Acid Sequence↗
Protective effect of serum antibody on respiratory infection of influenza C virus in rats.
The effects of serum antibody on the replication of influenza C virus in the nose and lung were evaluated in rats challenged with the virus by the intranasal and endotracheal routes, respectively. Convalescent rat serum administered intraperitoneally prior to infection suppressed virus replication significantly in both the nasal and pulmonary tissues. Resistance achieved was however much greater in the lung than in the nose. Rats with a serum neutralizing antibody titer of 1:800 showed almost complete resistance to pulmonary virus infection, and virus yield from the lung was reduced 10- to 100-fold in animals with the antibody titer of 1:80-160 or less. In contrast, significant decrease in virus shedding from the nose was observed only in animals with a serum antibody titer of 1:800 or greater. The effect of adoptive transfer of monoclonal antibodies (MAbs) to haemagglutinin-esterase (HE) glycoprotein and matrix (M) protein on pulmonary virus replication was also examined. Anti-HE MAbs with neutralization activity prevented virus shedding from the lung almost completely whereas non-neutralizing anti-HE MAb and anti-M Mab showed no inhibitory effect.
Animals↗
Adjuvant radiotherapy after complete resection of thymoma.
Seventy patients were studied after undergoing complete resection of thymoma to determine the effect of postoperative adjuvant mediastinal radiotherapy on prognosis, with regard to clinical stage, histological type, and pleural factor. Pleural factor was defined as follows: p0, no adhesion to the mediastinal pleura; p1, fibrous adhesion to the mediastinal pleura without microscopic invasion; and p2, microscopic invasion of the mediastinal pleura. Recurrence of thymoma after complete resection was observed in 13 patients, 12 (92%) with pleural dissemination, 6 (46%) with local recurrence, and 2 (15%) with distant metastasis (types of recurrence are overlapping). In stage I and stage II p0 patients, no recurrence was observed, regardless of mediastinal radiotherapy. Whereas mediastinal irradiation completely prevented recurrence in stage II p1 patients, 4 (36.4%) nonirradiated stage II p1 patients experienced recurrence. In stage II p2 patients, 75% had pleural dissemination even after radiotherapy. A high incidence of recurrence was also observed in stage III, nonirradiated (25%) and irradiated (30%) patients. The results suggest that mediastinal irradiation for stage I and II p0 patients is not always necessary, and that therapy for stage II p1 is essential and also expected to decrease the recurrence rate. On the other hand, in stage II p2 and stage III thymomas, mediastinal irradiation is not sufficient to prevent pleural recurrence even after complete resection. Our classification based on pleural factor is useful for better selection of appropriate postoperative treatment for thymoma patients.
Adolescent↗
Differential expression of a CD45R epitope(6B2) on murine CD5+ B cells: possible difference in the post-translational modification of CD45 molecules.
Although murine peritoneal B cells were homogenously positive for an epitope Lp-2, coded for by the alternative exon 4 of the CD45 gene, they were heterogenous with respect to the expression of another CD45R epitope, 6B2, of unknown exon dependency. While the majority of 6B2-high peritoneal B cells was composed of CD5- B cells, those with low or negative 6B2 were CD5+ B cells. Both 6B2+ and 6B2- peritoneal B cells expressed mainly the same largest CD45R transcripts, with all three alternative exon (4, 5, and 6) sequences. Further, a CD5+ B lymphoma cell line, BCL-1, which was found to be Lp-2+6B2- also had the largest isoform of CD45R molecules with all three alternate structures. Although enzyme digestion studies suggested that the 6B2 epitope resides in protein, not in sugar structures, it is likely that a post-translational modification of CD45R molecules is responsible for the presence or absence of 6B2 epitope expression on peritoneal CD5+ B cells. This event may be related to the differential role of CD45R molecules in regulating lymphocyte function.
Animals↗