Biomedical subjects

Yasuhiro Ikeda

Publications and source records attributed to Yasuhiro Ikeda.

49 records · Page 3Linked to original sources

PubMed2003

Continuous high-titer HIV-1 vector production.

Human immunodeficiency virus type 1 (HIV-1)-based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced. Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20-80 ng/ml), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 Gag-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10(7) 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.

Gene Expression Regulation, Viral↗

PubMed2003

Experimental mucosal infection with molecularly cloned feline immunodeficiency viruses.

Four of six specific pathogen-free cats were infected after intravaginal exposure to molecularly cloned lymphotropic but non-Crandell feline kidney (CRFK)-tropic feline immunodeficiency virus strain TM2 and its AP-1 deletion mutant. The sequences of the env V3-to-V5 region which defines the CRFK tropism were unchanged in the infected cats through the infection. These data suggest that the strain was transmitted across the mucosal epithelium without a broadening of cell tropism.

Amino Acid Sequence↗

PubMed2003

Local changes of IGF-I mRNA, GH receptor mRNA, and fiber size in rat plantaris muscle following compensatory overload.

We investigated whether local insulin-like growth factor-I (IGF-I) mRNA and growth hormone (GH) receptor mRNA expressions in plantaris muscle were related to the region-specific hypertrophy following compensatory overload. Adult male normal or hypophysectomized rats were subjected to unilateral distal-half removals of the gastrocnemius and soleus muscles. The contralateral hindlimb was used as the control. Two weeks later, fiber areas in the distal and proximal parts of the plantaris muscle were measured. All the fiber areas measured in the distal part of the plantaris muscle in normal and hypophysectomized rats were significantly increased following the compensatory overload. In the proximal part, fiber areas of type I, IIA, and IIC were significantly increased, but fiber area of type IIB did not change. IGF-I mRNA expressions in the distal and proximal parts were increased 3 d after the compensatory overload in normal and hypophysectomized rats. The increase of IGF-I mRNA expression in the distal part 3 d after compensatory overload was greater than those in the proximal part. IGF-I mRNA expressions in the distal and proximal parts were increased 14 d after the compensatory overload in hypophysectomized rats, but not in normal rats. GH receptor mRNA expressions were decreased following compensatory overload, and almost disappeared 14 d after the compensatory overload in hypophysectomized rats. Thus muscle fiber hypertrophy following compensatory overload was different among the parts in a muscle and IGF-I mRNA was expressed in concert with the region-specific hypertrophy, but not GH receptor mRNA.

Adaptation, Physiological↗

PubMed2002

Chronic suppression of heart-failure progression by a pseudophosphorylated mutant of phospholamban via in vivo cardiac rAAV gene delivery.

The feasibility of gene therapy for cardiomyopathy, heart failure and other chronic cardiac muscle diseases is so far unproven. Here, we developed an in vivo recombinant adeno-associated virus (rAAV) transcoronary delivery system that allows stable, high efficiency and relatively cardiac-selective gene expression. We used rAAV to express a pseudophosphorylated mutant of human phospholamban (PLN), a key regulator of cardiac sarcoplasmic reticulum (SR) Ca(2+) cycling in BIO14.6 cardiomyopathic hamsters. The rAAV/S16EPLN treatment enhanced myocardial SR Ca(2+) uptake and suppressed progressive impairment of left ventricular (LV) systolic function and contractility for 28-30 weeks, thereby protecting cardiac myocytes from cytopathic plasma-membrane disruption. Low LV systolic pressure and deterioration in LV relaxation were also largely prevented by rAAV/S16EPLN treatment. Thus, transcoronary gene transfer of S16EPLN via rAAV vector is a potential therapy for progressive dilated cardiomyopathy and associated heart failure.

Adenosine Triphosphatases↗

PubMed2002

Restoration of deficient membrane proteins in the cardiomyopathic hamster by in vivo cardiac gene transfer.

BACKGROUND: One of the most important problems in developing in vivo cardiac gene transfer has been low transfection efficiency. A novel in vivo technique was developed, tested in normal hamsters, and the feasibility of restoring a deficient structural protein (delta-sarcoglycan) in the cardiomyopathic (CM) hamster evaluated. METHODS AND RESULTS: Adenoviral (AdV) vectors encoding either the lacZ gene or delta-sarcoglycan gene were constructed. Hypothermia was achieved in hamsters by external body cooling to a rectal temperature of 18 to 25 degrees C. Through a small thoracotomy, the ascending aorta and the main pulmonary artery were occluded with snares, and cardioplegic solution containing histamine was injected into the aortic root; viral constructs were delivered 3 to 5 minutes later followed by release of the occluders and rewarming. Four days later, homogeneous beta-galactosidase expression was detected throughout the ventricles of the normal hearts (average 77.3+/-9.0% [SEM] of left ventricular myocytes). At 1 and 3 weeks after transfection, immunostaining showed extensive restoration of delta-sarcoglycan as well as alpha- and beta-sarcoglycan proteins to the myocyte membranes, despite loss of beta-galactosidase expression at 3 weeks. Also, at 3 weeks after gene transfer, there was significantly less progression of left ventricular dysfunction assessed as percent change in fractional shortening compared with controls. CONCLUSIONS: This study demonstrates the feasibility of high efficiency in vivo myocardial gene transfer and shows application in improving the level of a deficient cardiac structural protein and cardiac function in CM hamsters. The approach should be useful for assessing effects of expressing other genes that influence the structure or function of the normal and failing heart.

Adenoviridae↗

PubMed2002

Fibulin-5/DANCE is essential for elastogenesis in vivo.

The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-beta-binding proteins) and polymerized elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins alphavbeta3, alphavbeta5 and alpha9beta1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.

Animals↗

PubMed2002

Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure.

To determine the usefulness of recombinant Sendai virus (SeV) for ocular gene transfer, the authors characterized SeV-mediated gene transfer to the retinal tissue of adult rats via subretinal injection. Recombinant SeV encoding the lacZ gene achieved frequent transgene expression in the retinal pigment epithelium (RPE) (mean=38.76%), while gene transfer to other retinal cells was rare. These findings are similar to those of previous reports using adenoviruses. Peak reporter gene expression of SeV in cultured RPE cells was similar to that of adenovirus at the same titer; however, SeV achieved high levels of expression after a brief vector-cell contact time, while adenovirus required over 3hr for efficient gene transfer. This finding was also observed in vivo following a brief SeV filling in the subretinal space, and may therefore provide a clinical advantage in avoiding retinal damage due to prolonged detachment. The observed SeV-mediated gene expression in the rat retina was transient. The initial phase of the decrease in luciferase activity could be prevented by daily eye drops of dexamethasone, suggesting that the corticosteroid-sensitive host reaction may affect early clearance of the virus. The late decline of transgene expression (2 weeks) was inhibited by the immunosuppressant, cyclosporin A, in a dose-dependent manner, suggesting that the cytotoxic T-lymphocyte response may be important in this phase. This work represents the first report of SeV-mediated gene transfer to ocular tissue, and identifies recombinant SeV as a new tool for studies of retinal gene transfer and gene therapy.

Adenoviridae↗

PubMed2002

Apoptosis and oncosis in the early progression of left ventricular dysfunction in the cardiomyopathic hamster.

The genetic defect in the cardiomyopathic (CM) hamster is a mutation in the glycoprotein-sarcoglycan (a component of the dystrophin-glycoprotein complex). Apoptosis has been identified in skeletal muscle of dystrophin-deficient mice, and therefore the role of myocardial apoptosis in relation to oncosis in causing myocardial necrosis was assessed at the onset of left ventricular (LV) dysfunction in CM hamsters. LV size and function were evaluated in normal and CM hamsters (CHF147 line) by echocardiography at 1, 2, 3, and 5 months (mo) of age. The decrease of LV fractional shortening was found to be most marked (45%) between 1 and 2 mo of age. Apop totic nuclei were identified at each time point using in situ end-labeling of DNA strand breaks (TUNEL), together with immunolabeling of myocytes; DNA fragmentation (laddering) and nuclear morphology were also assessed. Myocyte oncotic necrosis was assessed at 2 mo by Evans blue dye (EBD), wheat germ agglutinin, hematoxylin/eosin staining, and electron microscopy. Apoptotic nuclei were not detected in age-matched normal hamsters. In the CM hamsters apoptotic myocyte nuclei comprised an average of 0.041% of myocyte nuclei between 1 and 5 mo, an increase at 2 mo (to 0.076%) was not significant, and DNA laddering was not detected. The number of myocyte nuclei per unit area decreased by 32% between 1 and 2 mo, and in 2 mo old CM hamsters myocardial staining with EBD was positive in 9.82% of the myocardial cross sectional areas examined, most of which was consistent with sarcolemmal rupture and oncosis with inflammatory cell infiltration. It is concluded that myocyte oncosis provides the major mechanism for the decreased number of myocyte nuclei and the early decrease of cardiac function between 1 and 2 mo of age in the CM hamster, with only a small contribution of myocyte apoptosis.

Animals↗

PubMed2002

Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats.

Monocyte/macrophage chemoattractant protein-1 (MCP-1), a potent chemoattractant chemokine and an activator for mononuclear cells, may play a role in the initiation and/or progression of pulmonary hypertension (PH). To determine whether blockade of a systemic MCP-1 signal pathway in vivo may prevent PH, we intramuscularly transduced a naked plasmid encoding a 7-NH(2) terminus-deleted dominant negative inhibitor of the MCP-1 (7ND MCP-1) gene in monocrotaline-induced PH. We also simultaneously gave a duplicate transfection at 2-wk intervals or skeletal muscle-directed in vivo electroporation (EP) to evaluate whether a longer or higher expression might be more effective. The intramuscular reporter gene expression was enhanced 10 times over that by EP than by simple injection, and a significant 7ND MCP-1 protein in plasma was detected only in the EP group. 7ND MCP-1 gene transfer significantly inhibited the progression of MCT-induced PH as evaluated by right ventricular systolic pressure, right ventricular hypertrophy, medial hypertrophy of pulmonary arterioles, and mononuclear cell infiltration into the lung. Differential effects of longer or higher transgene expression were not apparent. Although the in vivo kinetics of 7ND MCP-1 gene therapy should be studied further, these encouraging results suggest that an anti-inflammatory strategy via blockade of the MCP-1 signal pathway may be an alternative approach to treat subjects with PH.

Animals↗

PubMed2002

Effect of ischemic preconditioning and mitochondrial KATP channel openers on chronic left ventricular remodeling in the ischemic-reperfused rat heart.

The influence of ischemic preconditioning (IP) and mitochondrial ATP-sensitive potassium (mito-KATP) channel openers on chronic left ventricular (LV) remodeling remains unknown, so the effect of IP and mito-KATP channel openers on the LV pressure-volume curve was assessed in rats subjected to 30 min ischemia followed by a 3-week reperfusion. Infarct size was histologically determined at 3 weeks after reperfusion. The LV pressure-volume curve was significantly shifted left by IP, diazoxide and nicorandil compared with the controls. These effects were blocked by the selective mito-KATP channel blocker 5-hydroxydecanoate. The LV remodeling and the infarct size at 3 weeks after reperfusion correlated well, indicating that the reduction of LV remodeling in the ischemic-reperfused model was strongly influenced by attenuation of the ischemic injury. LV remodeling in the chronic phase is attenuated by IP and mito-KATP channel openers with concomitant reduction of infarct size.

Animals↗

PubMed2002

Feline host range of canine parvovirus: recent emergence of new antigenic types in cats.

Since the emergence of Canine parvovirus (CPV-2) in the late 1970s, CPV-2 has evolved consecutively new antigenic types, CPV-2a and 2b. Although CPV-2 did not have a feline host range, CPV-2a and 2b appear to have gained the ability to replicate in cats. Recent investigations demonstrate the prevalence of CPV-2a and 2b infection in a wide range of cat populations. We illustrate the pathogenic potential of CPV in cats and assess the risk caused by CPV variants.

Amino Acid Sequence↗

PubMed2002

[Multicenter clinical trial of angiotensin receptor blocker in patients with chronic heart failure: review of recent clinical trial].

Angiotensin receptor blocker(ARB) is expected to be a substitute of ACE inhibitor for ACE inhibitor(ACEi) incompetent patient or an additive drug for the treatment of severe heart failure. Two large mega-trials(ELITE II & Val-HEFT) have been reported so far and one large trial(CHARM) is ongoing. In ELITE II study, there was no additional beneficial effects of ARB compared to ACEi(captopril). On the other hand, additive ARB may be beneficial for most of patients with heart failure, according to the Val-Heft trial. However, some adverse effects of ARB by the combinatory use with ACEi and beta-blocker should be taken into careful consideration. Ongoing CHARM trial may answer if ARB is ultimately useful for the treatment of heart failure.

Aged↗

PubMed1999

Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL30 to UL40 genes in the genome of nononcogenic Marek's disease virus serotype 2.

Studies on Marek's disease virus serotype 2 (MDV2) are important for understanding the natural nononcogenic phenotype of MDV. This study reports a 27,535 bp nucleotide sequence of part of the MDV2 genome located in the central unique long (U(L)) region. The analysis revealed 11 complete ORFs with high amino acid sequence identities to the products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1, ranging from the UL30 to UL40 genes. Sequences that were particularly well conserved among alphaherpesviruses were the putative functional domain of the DNA polymerase (UL30) and the ribonucleotide reductase large and small subunits (UL39 and UL40). On the other hand, in contrast to oncogenic MDV1, MDV2 did not contain the conserved proline-repeat region in the UL36 homologue. All the genes identified were confirmed to be transcribed as 3'-coterminal mRNAs and/or unique transcripts in virus-infected cells.

Base Sequence↗