Biomedical subjects

Y Hagiwara

Publications and source records attributed to Y Hagiwara.

At least 55 records · Page 3Linked to original sources

PubMed1999

Effective adenovirus-mediated gene expression in adult murine skeletal muscle.

We established an efficient method for obtaining expression of a foreign marker gene transferred in vitro into myoblasts and in vivo into adult mouse skeletal muscles using adenovirus vector. After infection of the C2 myoblasts with the adenovirus vector containing the beta-actin promoter with cytomegalovirus (CMV) enhancer (CAG promoter) AxCALacZ, significantly greater number of cells express beta-galactosidase when compared with the adenovirus vector expressing the lacZ gene under the control of the SR alpha viral terminal repeat promoter (AxSRLacZL) or the myosin heavy chain (MHC) IIB promoter (AxMHCLacZ). We also injected AxCALacZ into the skeletal muscles of 5- to 6-week-old C57BL/10 mice and determined that more than 60% of their muscle fibers expressed the lacZ gene 7 days after injection. The CAG promoter may have application in the development of gene therapy for Duchenne muscular dystrophy (DMD) using adenovirus vector.

Adenoviridae↗

PubMed1999

Regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization.

A reporter open reading frame (ORF) coding for a fusion of bacterial beta-glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.

Cells, Cultured↗

PubMed1999

Evidence for the involvement of platelet-derived growth factor in the angiotensin II-induced growth of rat vascular smooth muscle cells.

This study examines the possible involvement of endogenous platelet-derived growth factor (PDGF) in the angiotensin II-induced growth of rat aortic smooth muscle cells. In quiescent confluent cells, anti-PDGF-AB neutralizing antibody inhibited angiotensin II-induced DNA synthesis and protein synthesis. PDGF-AA, -AB, and -BB produced concentration-dependent increases in DNA synthesis and protein synthesis. Genistein did not inhibit PDGF-AB-induced [3H]thymidine incorporation and [3H]leucine incorporation. PDGF-AB stimulated mitogen-activated protein (MAP) kinases, and PDGF-induced MAP kinase activation was inhibited by genistein. Angiotensin II induced PDGF-A chain messenger RNA expression, and genistein inhibited angiotensin-induced PDGF gene expression. These findings suggest that endogenous PDGF is, at least in part, involved in angiotensin II-induced cell growth in rat vascular smooth muscle cells. It appears that genistein inhibits angiotensin II-induced DNA synthesis partly by inhibiting PDGF-A gene expression.

Angiotensin II↗

PubMed1999

Application of CT imaging for dental implant simulation.

Accurate diagnosis and exact treatment planning are very important for successful implant treatment. Pretreatment examination and simulations using CT can be especially effective information sources, and diagnosis by CT imaging before treatment substantially enhances the security and safety of the treatment plan. New interactive CT software (SIM/Plant) which enables diagnosis of bone morphology and quality, and also implant simulation on a personal computer through multiplanar reformation of CT images, has been developed. Stereolithography models (SLMs) are reproduced anatomical morphological models of an individual patient's bone structure from the information obtained by the CT scan, and is fabricated out of light cure resin. In this article, SLMs and SIM/Plant for simulation of various examinations and diagnosis incorporating CT information are described, and their features are introduced.

Computer Simulation↗

PubMed1999

Creatine kinase, cell membrane and Duchenne muscular dystrophy.

In 1958 Professor Setsuro Ebashi found that serum creatine kinase activity is increased in patients suffering from various muscular dystrophies, especially Duchenne muscular dystrophy (DMD). He and others proposed that creatine kinase passes through the cell membrane as it is released from DMD muscle fibers. Since then, it has been found that dystrophin and dystrophin-associated proteins are connected to several other components, including the basal lamina and subsarcolemmal cytoskeletal networks on the cell membrane, while dystrophin anchors these dystrophin-associated proteins to the actin filaments inside the muscle cell. In DMD muscle, dystrophin has been found to be absent and dystroglycans and sarcoglycans decreased. However, how creatine kinase molecules can pass through the DMD muscle cell membrane still remains unanswered. On the basis of recent findings on the structure of the protein layers which sandwich the lipid bilayer of muscle cell membranes, this essay stresses the importance of these lipid bilayers in protecting creatine kinase release from protoplasma in normal muscle. It further indicates the possibility that the absence of dystrophin in DMD muscle during muscle contraction may result in temporal damage to the lipid bilayer.

Binding Sites↗

PubMed1998

Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency.

Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.

Adenoviridae↗

PubMed1998

Evidence that angiotensin II, endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat aorta.

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.

Angiotensin II↗

PubMed1998

Evidence for involvement of the lateral parabrachial nucleus in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla of the rat.

We examined whether sites in the lateral parabrachial nucleus (PBN) where L-glutamate produced increases in arterial pressure were involved in mediation of cholinergic inputs to neurons in the rostral ventrolateral medulla (RVLM). Male Wistar rats were anesthetized, paralyzed and artificially ventilated. Unilateral microinjection of L-glutamate into the lateral PBN produced a pressor response. Microinjection of the muscarinic receptor antagonist scopolamine into the unilateral RVLM inhibited the pressor response to L-glutamate injected ipsilaterally into the lateral PBN, whereas microinjection of the cholinesterase inhibitor physostigmine into the RVLM enhanced it. PBN microinjection of L-glutamate also enhanced the firing rate of RVLM sympathoexcitatory neurons and the enhancement of the firing rate was inhibited by scopolamine iontophoretically applied on neurons. PBN injection of L-glutamate produced a tetrodotoxin (TTX)-sensitive release of ACh in the RVLM. Unilateral microinjection of TTX into the lateral PBN inhibited the pressor response induced by RVLM microinjection of physostigmine. These results provide evidence that neurons in the pressor sites of the lateral PBN are involved in mediation of cholinergic inputs responsible for pressor responses in the RVLM.

Acetylcholine↗

PubMed1998

Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA.

Two forms of a proteinase, KN-BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN-BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N-terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low-molecular-mass kininogen. KN-BJ 1 and KN-BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin-releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN-BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin-like proteinases, also inhibited the amidolytic activity of KN-BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N-terminus and three tryptic peptides of KN-BJ 2. Therefore, the cDNA is believed to represent the gene of KN-BJ 2. The deduced amino acid sequence indicates that KN-BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.

Amino Acid Sequence↗

PubMed1998

From dystrophinopathy to sarcoglycanopathy: evolution of a concept of muscular dystrophy.

Duchenne and Becker muscular dystrophies are collectively termed dystrophinopathy. Dystrophinopathy and severe childhood autosomal recessive muscular dystrophy (SCARMD) are clinically very similar and had not been distinguished in the early 20th century. SCARMD was first classified separately from dystrophinopathy due to differences in the mode of inheritance. Studies performed several years ago clarified some immunohistochemical and genetic characteristics of SCARMD, but many remained to be clarified. In 1994, the sarcoglycan complex was discovered among dystrophin-associated proteins. Subsequently, on the basis of our immunohistochemical findings which indicated that all components of the sarcoglycan complex are absent in SCARMD muscles, and the previous genetic findings, we proposed that a mutation of any one of the sarcoglycan genes leads to SCARMD. This hypothesis explained and predicted various characteristics of SCARMD at the molecular level, most of which have been verified by subsequent discoveries in our own as well as various other laboratories. SCARMD is now called sarcoglycanopathy, which is caused by a defect of any one of four different sarcoglycan genes, and thus far mutations in sarcoglycan genes have been documented in the SCARMD patients. In this review, the evolution of the concept of sarcoglycanopathy separate from that of dystrophinopathy is explained by comparing studies on these diseases.

Animals↗

PubMed1998

Fiber-type-dependent expression of adenovirus-mediated transgene in mouse skeletal muscle fibers.

We show the efficient transduction and expression of the lacZ gene in the skeletal muscle of adult C57BL/10ScSn mice after adenovirus-mediated gene transfer. Of the myofibers in the tibialis anterior muscle 62% were beta-galactosidase positive after injection of the lacZ gene under the control of the chicken beta-actin promoter and the cytomegalovirus enhancer. The transduced gene was preferentially expressed in type IIA and IIX fibers, which were richer in oxidative enzymes than type IIB fibers.

Actins↗

PubMed1998

Elevated serum level of thioredoxin in patients with hepatocellular carcinoma.

Thioredoxin (TRX) is known to contain an active site with a redox-active disulfide and has various biological activities. The objective of the present study was to investigate whether circulating TRX levels are elevated in patients with chronic hepatitis (CH) or liver cirrhosis (LC) and hepatocellular carcinoma (HCC). An anti-TRX monoclonal antibody and polyclonal antibodies that specifically recognize TRX, were generated and used for the development of an ELISA system to measure TRX levels in human serum. The geometric mean and its 95% confidence interval of serum level of TRX in healthy volunteers was 81.75 ng/ml (74.60-89.59 ng/ml). The serum level of TRX in LC/CH patients without HCC was 80.87 ng/ml (69.66-93.88 ng/ml). The value was not statistically different from that in serum from normal volunteers (p=0.69). In contrast, the serum level of TRX in patients with HCC was 147.35 ng/ml (125.53-172.96 ng/ml), which was significantly higher when compared with the level in serum of normal volunteers (p<0.001) and in serum of LC/CH patients without HCC (p<0.001). In four patients with HCC, the initially high level of serum TRX (>150 ng/ml) decreased below 150 ng/ml after surgical removal of the tumor. The data reported herein revealed that patients with HCC had a significantly elevated serum level of TRX, suggesting that measurement of serum of TRX might be a useful clinical parameter when HCC is suspected.

Adult↗

PubMed1998

Effects of isoflurane on oxygenation during one-lung ventilation in pulmonary emphysema patients.

BACKGROUND: Hypoxic pulmonary vasoconstriction has an important role in human one-lung ventilation (OLV) in the lateral decubitus position under general anesthesia. During OLV, inhalational anesthesia may inhibit hypoxic pulmonary vasoconstriction and the decrease in arterial oxygenation. We studied the effect of isoflurane administration on arterial oxygen tension in chronic obstructive pulmonary disease patients. METHODS: Ten patients who had thoracoscopic laser ablation of bullous emphysema were studied. Patients received 2% isoflurane in oxygen from induction until the first 20 min of OLV in the lateral decubitus position, then were switched to 1% isoflurane lasting 20 min and next were switched to 0.5% isoflurane lasting 20 min. After each 20-min inhalation, pulmonary and hemodynamic parameters were measured. The given concentrations for isoflurane were merely vapor meter concentrations. RESULTS: PaO2/FIO2, Qs/Qt respiratory rate peak inspiratory pressure and PaCO2 showed no significant changes at each point of isoflurane. Expiratory tidal volume significantly decreased (P < 0.05) with 0.5% isoflurane compared to that with 2% isoflurane. Cardiac output, mean arterial pressure, mean pulmonary arterial pressure, systemic vascular resistance and pulmonary vascular resistance showed no significant changes at each point of isoflurane. CONCLUSIONS: In patients with pulmonary emphysema, arterial oxygenation is not affected by low isoflurane concentration during OLV in the lateral decubitus position.

Aged↗

PubMed1998

Genes required for replication of the 15.5-kilobase RNA genome of a plant closterovirus.

A full-length cDNA clone of beet yellows closterovirus (BYV) was engineered and used to map functions involved in the replication of the viral RNA genome and subgenomic RNA formation. Among 10 open reading frames (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication and transcription. The proteins encoded in these ORFs harbor putative methyltransferase, RNA helicase, and RNA polymerase domains common to Sindbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5'-proximal region of ORF 1a was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L-Pro and the remainder of the ORF 1a product was essential for replication of RNA. Second, an additional L-Pro function that was separable from proteolytic activity was required for efficient RNA accumulation. The deletion of a large, approximately 5.6-kb, 3'-terminal region coding for a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, minor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of mutants with replacements of start codons in each of these seven 3'-terminal ORFs revealed that p21 functions as an enhancer of genome amplification. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronavirus-like viruses are discussed.

Closterovirus↗

PubMed1998

Recombinant human acid alpha-glucosidase corrects acid alpha-glucosidase-deficient human fibroblasts, quail fibroblasts, and quail myoblasts.

Acid alpha-glucosidase (GAA) deficiency causes Pompe disease, a lethal lysosomal glycogen storage disease for which no effective treatment currently exists. We investigated the endocytic process in deficient cells of human recombinant GAA produced in Chinese hamster ovary cells, and the potential of GAA-deficient Japanese acid maltase-deficient quail as a model for evaluating the enzyme replacement therapy for Pompe disease. After 24-h incubation with a single dose of recombinant enzyme, intracellular GAA and glycogen levels in deficient human fibroblasts were normalized, and this correction lasted for 7 d. The 110-kD precursor recombinant enzyme was processed to the 76-kD mature form within 24 h after uptake. Intracellular GAA levels in deficient quail fibroblasts and myoblasts were similarly corrected to their average normal levels within 24 h. Differences existed in the efficiency of endocytosis among subfractions of the enzyme, and among different cell types. Fractions with a larger proportion of precursor GAA were endocytosed more efficiently. Quail fibroblasts required a higher dose, 4200 nmol.h-1.mL-1 to normalize intracellular GAA levels than human fibroblasts, 1290 nmol.h-1.mL-1, whereas primary quail myoblasts required 2800 nmol.h-1.mL-1. In all three cell lines, the endocytosed enzyme localized to the lysosomes on immunofluorescence staining, and the endocytosis was inhibited by mannose 6-phosphate (Man-6-P) added to the culture medium. Despite structural differences in Man-6-P receptors between birds and mammals, these studies illustrate that Man-6-P receptor mediated endocytosis is present in quail muscle cells, and demonstrate the potential of acid maltase-deficient quail to test receptor mediated enzyme replacement therapy for Pompe disease.

Animals↗

PubMed1998

Effects of genistein and staurosporine on angiotensin II-induced DNA synthesis, protein synthesis and mitogen-activated protein kinase activation in vascular smooth muscle cells.

This study examines the effects of protein kinase inhibitors and activator on angiotensin II-induced DNA synthesis and protein synthesis of rat aortic smooth muscle cells. In quiescent confluent cells, angiotensin II induced a concentration-dependent increase in thymidine incorporation and leucine incorporation. The tyrosine kinase inhibitor genistein caused an inhibition of the angiotensin II-induced DNA synthesis but not of the agent-induced protein synthesis. The protein kinase C inhibitors staurosporine and calphostin C caused an inhibition of the angiotensin II-induced protein synthesis but not of the agent-induced DNA synthesis. The protein kinase C activator phorbol 12-myristate 13-acetate stimulated protein synthesis. Angiotensin II stimulated mitogen-activated protein (MAP) kinases and the angiotensin II-induced MAP kinase activation was inhibited by genistein but not by staurosporine. These findings suggest that angiotensin II-induced DNA synthesis is at least partly mediated via protein-tyrosine phosphorylation and angiotensin II-induced protein synthesis is at least partly mediated by activation of protein kinase C. It seems likely that MAP kinase activation is involved in DNA synthesis but not in protein synthesis induced by angiotensin II.

Angiotensin II↗

PubMed1997

Molecular cloning of acid alpha-glucosidase cDNA of Japanese quail (Coturnix coturnix japonica) and the lack of its mRNA in acid maltase deficient quails.

Acid alpha-glucosidase (GAA) hydrolyzes alpha-1, 4 and alpha-1, 6 glucosidic linkages of oligosaccharides and degrades glycogen in the lysosomes. The full-length GAA I cDNA, pQAM8, was isolated from a cDNA library derived from Japanese quail liver. The cDNA is 3569 base pairs long and has an open reading frame capable of coding 932 amino acids. The deduced amino acid sequence shares 52% identity with human GAA. Transfection of expression vector pETAM8 into COS-7 cells or acid maltase deficient (AMD) quail embryonic fibroblasts increased the level of GAA 20-50-fold. Compared to normal quail, the levels of GAA I mRNA were significantly reduced in the muscle, liver, heart, and brain of AMD quails, suggesting the GAA deficiency in AMD quail is due to a lack of GAA I mRNA. A second GAA II cDNA was identified after probing the cDNA library from the ovarian large follicles of quails with a PCR product derived from cultured quail skin fibroblasts. This clone having 3.1 kb insert, has GAA activity as well (3 to 10 fold increase). This cDNA, designated GAA II, predicted an 873 amino acid polypeptide showing 63% identity to human GAA and 51% identity to the GAA I. The RT-PCR analysis demonstrated that GAA II mRNAs were barely detectable in normal tissues, while they were enhanced to higher levels in AMD tissues. These results suggest that GAA II expression is up-regulated at the transcription levels, and quail GAA gene redundancy performs the same function of satisfying GAA demand at the two different phases represented by normal and AMD.

Amino Acid Sequence↗

PubMed1997

Evidence for the involvement of endogenous aspartate in the mediation of carotid chemoreceptor reflexes in the rostral ventrolateral medulla of the rat.

2-Amino-5-phosphonovalerate (AP5; 153 pmol) injected into the rostral ventrolateral medulla (RVLM) inhibited pressor responses induced by carotid chemoreceptor stimulation. AP5 also inhibited pressor responses to aspartate (0.75 nmol) but not to glutamate (0.53 nmol) similarly injected. High K+ (50 mM) released endogenous aspartate and glutamate in a Ca2+-dependent manner from the RVLM. Chemoreceptor stimulation caused a release of aspartate but not of glutamate in the RVLM, and sinus nerve denervation abolished the release of aspartate. Increases in blood pressure induced by intravenous phenylephrine did not release aspartate. These results support the hypothesis that endogenous aspartate in the rat RVLM is involved in the mediation of chemoreceptor reflexes.

2-Amino-5-phosphonovalerate↗