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Biomedical subjects

H Ueda

Publications and source records attributed to H Ueda.

At least 361 records · Page 20Linked to original sources
PubMed1997

Immunohistochemical analysis of hepatocyte growth factor in human coronary atherectomy specimens: comparison with transforming growth factor beta isoforms.

The expression and localization of hepatocyte growth factor/scatter factor (HGF/SF) were examined immunohistochemically in 59 human coronary artery lesions retrieved by directional coronary atherectomy and compared with the localization of transforming growth factor beta isoforms (TGF-beta 1, -beta 2, and -beta 3). In 21 of the 59 specimens (35.6%) HGF-like immunoreactivity (HGF-IR) was revealed. The HGF immunopositivity rate of 45% (14/31) in thrombotic tissue was significantly (P < 0.05) higher than the rates of 7.3% (4/55), 7.1% (3/42), and 0% (0/14) in fibrous tissue, neointimal hyperplasia and atheromatous gruel, respectively. Immunoreactivity for HGF was much weaker than that for TGF-beta isoforms in these components except in thrombotic tissue. These cells exhibiting strong HGF-IR were inflammatory cells such as monocytes/macrophages in thrombotic tissue, in tissue lesions adjacent to a thrombus, and outside the capillary walls in a portion of the neovascularized lesions. Smooth muscle cells (SMCs) hardly demonstrated HGF-IR. In contrast, in control coronary arteries obtained at autopsy, the HGF-IR was strongly expressed in SMCs. These findings suggest that HGF produced by macrophages play a part in the process of coronary plaque formation attributable to thrombus in man.

Adult↗
PubMed1997

Plasma total homocysteine concentrations in epileptic patients taking anticonvulsants.

Plasma total homocysteine (tHcy) and serum folate (FA) concentrations were measured in 130 epileptic patients taking anticonvulsant drugs. A significant inverse correlation was found between FA and tHcy. This was greater in the older group (> or = 15 years) than in the younger group (1 to 14 years). There were four FA-deficient patients (FA concentration < 3 ng/mL regardless of symptoms), including three patients in the older group and one in the younger group. All FA-deficient patients had received long-term treatment (> 7 years) with multiple anticonvulsants. Their tHcy levels were higher than the 90th percentile of those in control subjects. Two patients showed extremely high levels of tHcy (57.9 and 29.1 mumol/L) and subnormal plasma methionine levels. After FA therapy, their tHcy decreased to levels the same as or less than those of control subjects and FA increased to above the normal range. Based on these findings, we conclude that measuring FA and tHcy concentrations may be useful for preventing thrombosis due to hyperhomocysteinemia in epileptic patients taking anticonvulsants, particularly those who receive long-term treatment with multiple agents.

Adolescent↗
PubMed1997

Multiple forms of AMPA-type glutamate receptor mRNA phenotypes in goldfish retina and tectum.

1. A goldfish AMPA-type glutamate receptor cDNA (GFGR49) was cloned from a goldfish retinal cDNA library. 2. The GFGR49 clone is an immature product of pre-mRNA, possessing the C-flip exon and flanking introns. 3. RNase protection assays revealed multiple mRNAs that contain the C-flip exon. In addition, these assays indicated differential processing of these RNAs between retina and brain. 4. Finally, RNase protection assays employing probes spanning the 3'-coding region of GFGR49 revealed at least two different mRNA phenotypes. 5. Comparison with the sequence of genomic DNA, which was obtained by polymerase chain reaction amplification, suggests that such multiple mRNA phenotypes are attributed to the use of alternative intron-exon splicing junctions and to RNA editing.

Amino Acid Sequence↗
PubMed1997

Changes in cochlear function after double-membrane rupture in the guinea pig.

We measured the transiently evoked otoacoustic emissions (TEOAEs), compound action potentials (CAPs) and cochlear microphonics (CMs) in guinea pigs after rupture of the round window membrane alone (n = 5) or of the round window membrane with localized cochlear damage (n = 10). The localized cochlear damage entailed rupture of Reissner's membrane with damage to the stria vascularis. We determined the time course of changes in the total echo power (TEP) in TEOAEs and the minimal detectable levels of CAPs and CMs. The endocochlear potential (EP) was measured in the cochlea with localized damage. There were no changes in TEOAEs, CAPs or CMs in the guinea pigs subjected to round window membrane rupture alone, but the minimal detectable levels of CAPs and CMs were increased in all the guinea pigs in which TEOAEs were absent after rupture of the round window membrane with localized cochlear damage. Our results suggest that double-membrane rupture (rupture of the round window membrane with localized cochlear damage) produces acute sensorineural hearing loss. The hearing loss appeared to be related to damage to the cochlea, which may be induced by influx of potassium-rich endolymph into the perilymph, and by morphological damage to the scala media.

Acoustic Stimulation↗
PubMed1997

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels.

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not.

Animals↗
PubMed1997

Transcriptional activation through interaction of MBF2 with TFIIA.

BACKGROUND: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ-F1 or its silkworm counterpart BmFTZ-F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ-F1 (or BmFTZ- F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription. RESULTS: To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ-F1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acid sequence of the factor showed no homology with proteins in the databases. Farwestern analyses and glutathione S-transferase interaction assays demonstrated that MBF2 makes a direct contact with the beta-subunit of TFIIA. In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires the MBF2-TFIIA interaction. When recombinant MBF2 was added to the HeLa cell nuclear extract in the presence of MBF1 and FTZ622 bearing the DNA-binding region of FTZ-F1, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the MBF2-TFIIA interaction. CONCLUSION: MBF2 activates transcription through its interaction with TFIIA. Selective transcriptional activation occurs when MBF2 is recruited to a promoter carrying the FTZ-F1 binding site by FTZ-F1 and MBF1.

Amino Acid Sequence↗
PubMed1997

Temporal regulation of the mid-prepupal gene FTZ-F1: DHR3 early late gene product is one of the plural positive regulators.

BACKGROUND: Various ecdysteroid responsive genes play important roles in insect moulting and metamorphosis. Late FTZ-F1, a member of the nuclear receptor superfamily, is a unique transcription factor which is induced by a pulse exposure of 20-hydroxyecdysone. Elucidation of the regulation mechanism of this gene during prepupal period will help our understanding of metamorphosis at a molecular level. RESULTS: Using transgenic fly lines carrying various transcription regulatory regions of the FTZ-F1 gene fused to the LacZ gene, we investigated cis-regulatory elements in the late FTZ-F1 transcription unit. The region which governs the stage-specific expression during prepupal period was narrowed down to 1.2kb, from -0.7 to +0.5kb relative to the transcription start site. Electrophoresis mobility shift assays using staged extracts and various probes within the stage-specific region allowed us to identify binding sites for DHR3, an early late gene product, around 170 and 450bp downstream of the transcription initiation site. Mutations disrupting these binding sites reduced the reporter gene expression without affecting the stage specificity. CONCLUSIONS: Our deletion and mutation studies of the cis-regulatory element of the FTZ-F1 gene suggest that the DHR3 binding sites located in the 5' non-coding region are involved in the prepupal expression of the gene. These DHR3 binding sites confer high level expression while other elements are also involved in stage-specific expression.

Animals↗
PubMed1997

An immunocytochemical study of changes in the human erythrocyte membrane skeleton produced by stretching examined by the quick-freezing and deep-etching method.

A quick-freezing and deep-etching method in combination with erythrocyte splitting was used to examine the cytoplasmic aspect of whole-mount human erythrocyte membranes. Various external forces induced alterations in membrane skeletal organisation during the splitting procedure. The initial change was elongation in the peripheral part of the membrane skeleton, examined by immunostaining with a monoclonal antispectrin antibody. Under severe stretching conditions, a linear rearrangement of filamentous components was evident; these were disposed parallel to the rim of the erythrocyte, while the central part of the concavity exhibited a more compacted structure. These changes resulted in a different distribution of membrane skeletal components between central rigid and peripheral flexible areas in biconcave erythrocytes. It is suggested that the reversible membrane skeletal changes in the flexible areas which resist the external forces are important for maintaining the normal framework of biconcave human erythrocytes.

Antibodies, Monoclonal↗
PubMed1997

Tissue-specific and glucose-dependent expression of receptor genes for glucagon and glucagon-like peptide-1 (GLP-1).

Both glucagon and glucagon-like peptide-1 (GLP-1) play an important role in the regulation of nutrient homeostasis. In this study, the tissue distributions of the expression of receptor genes for glucagon and GLP-1 were examined. Expression of glucagon receptor gene was detected in liver, kidney, ileum and pancreatic islets but not in brain. In contrast, expression of GLP-1 receptor gene was detected in brain, pancreas and pancreatic islets but not in liver, kidney, or ileum. To investigate the existence and characteristics of glucagon and GLP-1 receptors on pancreatic beta cells, expression of the receptor genes and translational regulation of the expression of the receptor genes by glucose were analyzed in a mouse pancreatic beta cell line, MIN6 cells. In the cDNA pool of MIN6 cells, both glucagon and GLP-1 receptor genes were identified and showed higher expression level in MIN6 cells cultured under high glucose condition than in those cultured under low glucose condition. These results suggest that glucagon and GLP-1 receptor genes are expressed in pancreatic beta cells and their expression is upregulated by glucose.

Animals↗
PubMed1997

Construction, bacterial expression, and characterization of hapten-specific single-chain Fv and alkaline phosphatase fusion protein.

We have designed and constructed a bacterial expression vector to produce a fusion protein of hapten-specific single-chain Fv (ScFv) and alkaline phosphatase (PhoA) in Escherichia coli. The ScFv gene was assembled using genes encoding the heavy and light chain variable domains of anti-NP (4-hydroxy-3-nitrophenyl acetyl) mouse monoclonal antibody. The ScFv gene was then fused to the 5' terminus of the E. coli PhoA coding region. The expressed fusion protein ScFv(NP)-PhoA was purified using an NP affinity column, and gel-filtration. Characterization of the fusion protein was then performed. The estimated molecular weight by gel filtration was approximately 151 kDa, suggesting the dimerization of the protein. Kinetic constants of ScFv(NP)-PhoA were calculated and compared with those of wild-type PhoA. The k(cat) values of ScFv(NP)-PhoA and wild-type PhoA were 103 (s(-1)) and 96.1 (s(-1)), respectively, showing that PhoA activity was somewhat increased by tethering the molecules. The equilibrium binding constant of ScFv(NP)-PhoA was determined using two different haptens, NP-capronate and NIP(3-iodo-4-hydroxy-5-nitrophenyl acetyl) by means of fluorescence quenching measurements. The obtained binding constants were 2.2 x 10(5) (M-1) for NP-capronate and 1.O x 10(6) (M(-1)) for NIP, respectively. No apparent difference in binding constants was seen between ScFv(NP) and ScFv(NP)-PhoA, showing that sufficient specificity and binding affinity were retained when ScFv(NP) was tethered to alkaline phosphatase. ScFv(NP)-PhoA can be used to detect nanogram concentrations of NP-BSA in ELISA without the use of chemically conjugated secondary antibodies.

Alkaline Phosphatase↗
PubMed1997

Electron microscopic study of erythroblastic islands obtained by 'tissue-stamp culture' method.

A new 'tissue-stamp culture' method was developed for stamping proliferating erythroblasts of mouse spleens on collagen-coated coverslips after inducing haemolytic anaemia by administration of 1-acetyl-2-phenylhydrazine, and then adherent splenic cells were cultured for a few days. We could obtain many erythroblastic islands, where cultured erythroblasts were located over macrophages and were proliferated synchronously for 10-30 h, and then the erythroblasts were differentiated and enucleated after 30-50 h in the presence of erythropoietin. To observe three-dimensional structures of the erythroblastic islands, a scanning electron microscope was used for the cultured cells treated with critical point-drying method. Immature wrinkled erythroblasts with many micropinocytic pits were attached to the central area of the flattened macrophages with many cytoplasmic projections, though matured erythroblasts were localized on their peripheral areas. Moreover, cytoplasmic projections of underlying macrophages, which were attached to the matured erythroblasts, were decreased in number. At a late stage, deep cytoplasmic invaginations of erythroblasts observed at a middle stage became shallow after their enucleation and flattened to form their concave shapes. This 'tissue-stamp culture' system would be useful for studying specific interaction between stromal macrophages and haematopoietic cells.

Anemia, Hemolytic↗
PubMed1997

Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen.

Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen. This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones with specificity toward target antigens. The results can be summarized as follows. (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized. (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library. (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry. These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.

Amino Acid Sequence↗
PubMed1997

Insulin-dependent diabetes mellitus associated with autoimmune thyroiditis and rheumatoid arthritis.

A case associated with insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), and autoimmune thyroid disease (AITD) was reported. A high titer of anti-glutamic acid decarboxylase antibody (GAD) and a positive islet cell antigen were observed. The patient's human leukocyte antibody (HLA) haplotype was a homozygote of DQA1*0301, DQB1*0401, and DRB1*0405. Because this haplotype was in linkage disequilibrium with DPB1*0501, an allele associated with AITD in Japanese patients, the patient was homozygous for alleles susceptible to IDDM, RA, and AITD. A specific HLA haplotype susceptible to several autoimmune disease may result in the development of IDDM, RA, and AITD.

Alleles↗
PubMed1997

Induction of tumor necrosis factor in a murine tumor by systemic administration of a novel synthetic lipid A analogue, ONO-4007.

The effect of a novel synthetic lipid A analogue, ONO-4007, on tumor necrosis factor (TNF) production was investigated in normal and tumor-bearing mice. When vehicle was administered to normal mice, slight TNF activity was detected in some organs, but more TNF activity was detected in spleen, liver, lung, kidney, and serum when ONO-4007 (300 micrograms/mouse) was administered. When vehicle was given to tumor bearers, on the other hand, little TNF was detected in most organs, but when ONO-4007 was given, more TNF was produced in many organs, particularly spleen, liver, and tumor tissue. TNF production of spleen and liver reached a maximum 1-2 h after ONO-4007 injection and then decreased rapidly, but that of tumor remained high for at least 6 h after administration. When mice were pretreated with dexamethasone, TNF activity of normal organs were completely inhibited, but that of tumor was only partially decreased. We have shown that ONO-4007 causes rapid, definite growth inhibition of solid tumor, and speculate that long-sustained intratumoral TNF is the main cause of this beneficial anti-tumor effect. We report here that ONO-4007 can induce TNF in tumor locus, and its utilization may offer a new therapeutic method.

Animals↗
PubMed1997

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes.

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.

Apocrine Glands↗
PubMed1997

Changes in evoked otoacoustic emissions in the guinea pig after pure-tone acoustic overstimulation.

To test if click-evoked otoacoustic emissions (CEOAEs) have frequency specificity, continuous changes in CEOAEs (especially frequency components of the CEOAE power spectrum) after pure-tone exposure in guinea pigs were examined. Pure-tone stimuli (0.5 kHz, 120 dB SPL; 2 kHz, 115 dB SPL; 4 kHz, 110 dB SPL) were given in a closed system for 3 min. After exposure, the frequency components in the CEOAE power spectrum decreased maximally at one-half octave or more above the overstimulation frequency. They partially recovered 2 h after exposure. The time course of compound action potential (CAP) thresholds after exposure was similar to that of the frequency components of the CEOAE power spectrum. It was concluded that some local damage caused by outer hair-cell dysfunction in the guinea pig cochlea can be detected by measuring shifts in frequency components in the CEOAE power spectrum.

Animals↗
PubMed1997

Generation and molecular characterisation of monoclonal IgG4 rheumatoid factor from a patient with rheumatoid arthritis.

OBJECTIVE: To characterise IgG4 rheumatoid factor (RF) at the molecular level from a patient with rheumatoid arthritis. METHODS: B cells were cloned from the peripheral blood of a patient with rheumatoid arthritis, using EB virus transformation. The supernatants of the clones were screened for IgG RF activity by ELISA. Nucleotide sequences of the expressed immunoglobulin heavy and light chain genes of one IgG RF producing clone were determined by direct sequencing of the products of a polymerase chain reaction. RESULTS: One clone producing monospecific IgG4 RF was obtained. Sequence analysis of the heavy and light chain genes suggested the accumulation of somatic mutations resulting in amino acid replacement in complementarity determining regions. CONCLUSIONS: The results may suggest an antigen driven response in the generation of IgG4 RF in rheumatoid arthritis disease processes.

Amino Acid Sequence↗