Search PubMed⌕ Search

Biomedical subjects

T Mikami

Publications and source records attributed to T Mikami.

At least 307 records · Page 17Linked to original sources
PubMed1996

High-performance liquid chromatographic separation of ultraviolet-absorbing bile alcohol derivatives.

This paper describes the high-performance liquid chromatographic separation of UV-absorbing bile alcohol derivatives. Bile alcohols were treated with 3 alpha-hydroxysteroid dehydrogenase to form the corresponding 3-keto bile alcohols. The 3-keto bile alcohols produced were converted to the 2,4-dinitrophenylhydrazone derivatives, separated using a Nova-Pak Phenyl column, and monitored at 364 nm. The separation of stereoisomers related to the configuration of hydroxyl groups on the side chain of the bile alcohols, which was not achieved by gas chromatography, could also be accomplished.

Animals↗
PubMed1996

Enhanced expression of beta-adrenergic receptor kinase 1 in the hearts of cardiomyopathic Syrian hamsters, BIO53.58.

We cloned an entire encoding sequence of beta-adrenergic receptor kinase 1 (beta ARK1) cDNA from the hearts of Syrian hamsters through reverse transcription and subsequent polymerase chain reaction. The cloned cDNA contained 2067 nucleotides coding 689 amino acids. The sequence had 95% homology to rat beta ARK1 and 90% homology to human homologue. Cardiomyopathic Syrian hamster, BIO53.58, has been used as a model animal of congestive heart failure. M-mode echocardiography confirmed that left ventricular contractility of 20-week-old BIO53.58 was markedly reduced. The expression of beta ARK1 mRNA in the hearts of BIO53.58 was significantly increased compared to control hamsters, F1b, suggesting that the enhanced beta ARK1 expression is acting as a negative feedback mechanism in order to maintain intracellular homeostasis against accelerated stimulation by catecholamines via phosphorylation of beta-adrenergic receptor.

Aging↗
PubMed1996

Identification and nucleotide sequence of the thymidine kinase gene of canine herpesvirus.

This paper presents the entire nucleotide sequence of the thymidine Kinase (TK) gene of canine herpesvirus (CHV). The gene was located within a 2.1 kbp EcoRV fragment by Southern-blot hybridization with a probe derived from the known feline herpesvirus type 1 (FHV-1) TK gene. An open reading frame (ORF) of 987 nucleotides, capable of encoding a TK translation product of 328 amino acids, was identified. Alignment of the predicted amino acid sequence of the CHV TK with other herpesvirus TKs revealed homologies of 25-47%. The proposed nucleotide-binding site and thymidine-binding site sequences of known herpesvirus TKs could be aligned with similar sequences in CHV TK. Northern-blot analysis revealed 1.3 kb and 5.0 kb mRNAs as the TK-specific transcripts. It is probable that the 1.3 kb transcript codes for the CHV TK and that the 5.0 kb transcript codes for the CHV TK and the downstream sequence.

Amino Acid Sequence↗
PubMed1996

Quantification of left ventricular size on exercise thallium-201 single-photon emission tomography.

The purposes of this study were to determine whether quantification of the left ventricular size on exercise thallium-201 single-photon emission tomography (SPET) correlates with echocardiographic measurements, whether the quantification reflects the severity of coronary artery disease, and whether it can provide supplementary information regarding the severity of coronary artery disease. In 42 control subjects and 110 patients who underwent coronary angiography, we performed exercise 201Tl SPET and quantified six non-regional markers: lung 201Tl uptake on an initial planar image (Lung/Heart), left ventricular width on a tomogram (Width), change in the Width from the initial to delayed tomograms (DeltaWidth), count ratio of the left ventricular cavity to the myocardium (C/M), count ratio of the lung to the myocardium (L/M), and count ratio of the lung to the left ventricular cavity (L/C). In 76 patients, furthermore, the Width was compared with echocardiographic measurements. The Width correlated with echocardiographic measurements (P<0.001). The Width and DeltaWidth were significantly different among zero-, one-, two- and three-vessel disease (P<0.001). However, the Width and DeltaWidth could not improve the power of discrimination for multi-vessel disease derived from the Lung/Heart. The six non-regional markers correlated with each other (P<0.001). Among the six markers, the Lung/Heart was only the independent discriminator for multi-vessel disease. In conclusion, quantification of the left ventricular size on exercise 201Tl SPET correlated with echocardiographic measurements and reflected the severity of coronary artery disease, but may be replaced with quantitation of the lung 201Tl uptake.

Cardiac Catheterization↗
PubMed1996

Identification of a potential Marek's disease virus serotype 2 glycoprotein D gene with homology to herpes simplex virus glycoprotein D.

The gene of Marek's disease virus (MDV) serotype 2 (MDV2) homologous to glycoprotein D (gD) of herpes simplex virus (HSV) was identified and characterized by its nucleotide and predicted amino acid sequences. The MDV2 gD homologous gene contains an open reading frame capable of specifying a polypeptide of 385 amino acids, which include N- and C-terminal hydrophobic domains consistent with signal and anchor regions, respectively, and two potential N-linked glycosylation sites, one of which was located in a highly conserved region when compared to MDV serotype 1 (MDV1) and herpesvirus of turkeys (HVT). By northern blot analysis using a MDV2 gD-specific DNA probe, two highly abundant polycistronic 6.0 and 4.2 kb transcripts were detected in MDV2-infected cells. The genes encoding MDV2 protein kinase (PK), gD, and glycoprotein I (gI) homologues are transcribed to form 3' coterminal mRNAs of 6.0 kb (encoding PK, gD and gI) and 4.2 kb (encoding gD and gI), respectively. By using rapid amplification cDNA end (RACE) method, several RNA start sites, to be thought those of the 4.2 kb mRNA, were detected in the upstream of MDV2 gD homologue.

Amino Acid Sequence↗
PubMed1996

Pathogenicity and vaccine efficacy of a thymidine kinase-deficient mutant of feline herpesvirus type 1 in cats.

We constructed a recombinant feline herpesvirus type 1 (FHV-1) which was deleted in a defined region (450 bp) within the thymidine kinase (TK) gene (C7301dlTK) [Yokoyama et al. (1995) J Vet Med Sci 57: 709-714]. In this report, we carried out two experiments to assess the pathogenicity and vaccine efficacy of the recombinant C7301dlTK in cats. The first experiment showed that, following multiple inoculation of the recombinant C7301dlTK by intraocular, intranasal and oral routes, the virus was sufficiently attenuated in cats, although a high titer of the virus was recovered from target organs (eye, nose, and mouth). In the second experiment, two intramuscular vaccinations with the recombinant C7301dlTK protected cats to a significant degree against subsequent challenge with the parent FHV-1 strain C7301 at 4 weeks after the last vaccination. These results demonstrate that the recombinant C7301dlTK is effective as a live attenuated vaccine with a clear genetic marker.

Alphaherpesvirinae↗
PubMed1996

Vaccine efficacy of recombinant feline herpesvirus type 1 expressing immunogenic proteins of feline calicivirus in cats.

We previously constructed a recombinant feline herpesvirus type 1 (FHV1), C7301dlTK-Cap, which contains an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain in the deleted locus of the thymidine kinase (TK) deficient mutant (C7301dlTK) of FHV1. In this report, we carried out in vivo experiments to assess the vaccine efficacy of the recombinant C7301dlTK-Cap against FCV and FHV1 infections in cats. As a result, two vaccinations with the C7301dlTK-Cap by intraocular, intranasal and oral routes protected cats to a significant degree against subsequent virulent challenges with both parent FCV F4 and FHV1 C7301 strains. The results are applicable for the further development of a new genetically engineered polyvalent vaccine for cats.

Administration, Intranasal↗
PubMed1996

Regulatory properties of the integrated long terminal repeat of the feline immunodeficiency virus.

To examine the regulatory properties of feline immunodeficiency virus (FIV) long terminal repeat (LTR) integrated into host chromatin, Crandell feline kidney cells were stably transfected with the FIV LTR that directs the bacterial chloramphenicol acetyltransferase (CAT) gene. Using these cells, we examined the effects of treatment with several chemical agents, infection with feline viruses, or transfection with effector plasmids expressing FIV gene products on FIV LTR-directed gene expression. Among them, treatment with the phorbol ester (a strong activator of protein kinase C), forskolin (an inducer of cyclic-AMP), 5-azacytidine (a DNA methylation antagonist), or infection with feline herpesvirus type 1 (FHV-1), resulted in induction of CAT activity in the cells. These results suggest that the integrated FIV LTR is stimulated by cellular transcriptional factors induced by phorbol ester, forskolin and FHV-1, and is also inactivated by DNA methylation. Furthermore, this permanent cell line can be used as a screening system of activators of the FIV LTR.

Animals↗
PubMed1996

Detection of feline immunodeficiency virus transcripts by quantitative reverse transcription-polymerase chain reaction.

We developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure to estimate replication status of feline immunodeficiency virus (FIV) in peripheral blood mononuclear cells (PBMCs) of cats. Primers used for the RT-PCR were designed to detect only multiple spliced transcripts of FIV and allowed us to detect the specific transcripts with high specificity. By using limiting-cell-dilution RT-PCR, we demonstrated that the specific transcripts were quantitatively detected in a single infected cell in a background of 1 x 10(6) uninfected cells without Southern blot hybridization. Furthermore, the transcripts were observed efficiently in all PBMCs of the chronically FIV-infected cats when examined by this RT-PCR technique. There results demonstrated that this RT-PCR method is applicable for specific detection of the FIV-specific transcripts in the PBMCs and for estimation of the viral replication status in vivo.

Animals↗
PubMed1996

A new approach for evaluation of left ventricular diastolic function: spatial and temporal analysis of left ventricular filling flow propagation by color M-mode Doppler echocardiography.

OBJECTIVES: To evaluate left ventricular diastolic function and differentiate the pseudonormalized transmitral flow pattern from the normal pattern, the propagation of left ventricular early filling flow was assessed quantitatively using color M-mode Doppler echocardiography. BACKGROUND: Because the propagation of left ventricular early filling flow is disturbed in the left ventricle with impaired relaxation, quantification of such alterations should provide useful indexes for the evaluation of left ventricular diastolic function. METHODS: Study subjects were classified into three groups according to the ratio of early to late transmitral flow velocity (E/A ratio) and left ventricular ejection fraction: 29 subjects with an ejection fraction > or = 60% (control group); 34 with an ejection fraction < 60% and E/A ratio < 1 (group I); and 25 with ejection fraction < 60% and E/A ratio > or = 1 (group II). The propagation of peak early filling flow was visualized by changing the first aliasing limit of the color Doppler signals. The rate of propagation of peak early filling flow velocity was defined as the distance/time ratio between two sampling points: the point of the maximal velocity around the mitral orifice and the point in the mid-left ventricle at which the velocity decreased to 70% of its initial value. High fidelity manometer-tipped measurement was performed in 40 randomly selected subjects. RESULTS: The rate of propagation decreased in groups I and II compared with that in the control group (33.8 +/- 13.8 [mean +/- SD] and 30.0 +/- 8.6 vs. 74.3 +/- 17.4 cm/s, p < 0.001, respectively) and correlated inversely with the time constant of left ventricular isovolumetric relaxation and the minimal first derivative of left ventricular pressure (peak negative dP/dt) (r = 0.82 and r = 0.72, respectively). CONCLUSIONS: Spatial and temporal analysis of filling flow propagation by color M-mode Doppler echocardiography was free of pseudonormalization and correlated well with the invasive variables of left ventricular relaxation.

Blood Flow Velocity↗
PubMed1996

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

Animals↗
PubMed1996

Expression and properties of feline herpesvirus type 1 gD (hemagglutinin) by a recombinant baculovirus.

We constructed a recombinant baculovirus expressing feline herpesvirus type I (FHV-1) gD in insect cells (Sf9 cells). The expressed product was identified as FHV-1 gD by a panel of monoclonal antibodies specific for the FHV-1 gD, and had an apparent molecular mass of approximately 49 kDa, which was less than that of the authentic FHV-1 gD. When the FHV-1 gD protein were expressed in Sf9 cells and CRFK cells in the presence of tunicamycin, the FHV-1 gD exhibited a molecular mass of 41 kDa. It was shown that the gD protein was transported to the surface of recombinant virus-infected Sf9 cells when examined by membrane-immunofluorescence analysis, and that the gD expressed on the surface of Sf9 cells adsorbed feline erythrocytes. Mice inoculated with a lysate of Sf9 cells expressing FHV-1 gD induced antibodies with virus-neutralizing and hemagglutination-inhibition activities. Therefore, the expressed gD appears to be biologically authentic. These data suggested that recombinant FHV-1 gD produced in Sf9 cells may be a useful immunogen as a feline vaccine.

Animals↗
PubMed1996

Recombinant feline herpesvirus type 1 expressing immunogenic proteins inducible virus neutralizing antibody against feline calicivirus in cats.

In this study, an entire open reading frame encoding the capsid protein of feline calicivirus (FCV) F4 strain was inserted into the deletion locus (SmaI site) of the thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) and the resulting recombinant virus was designated as C7301dlTK-Cap. Expression of the FCV antigens by C7301dlTK-Cap was confirmed by indirect immunofluorescence assay and immunoblot analysis. To assess whether the recombinant virus can induce virus neutralizing (VN) antibody against FCV in the natural host, three cats were inoculated intranasally and orally with C7301dlTK-Cap (two cats) or C7301dlTK (one cat). As a result, sera collected from cats inoculated with the C7301dlTK-Cap possessed VN antibody against FCV. This recombinant virus is expected as a new polyvalent recombinant vaccine against FHV-1 and FCV infections.

Animals↗
PubMed1996

Molecular cloning of the feline CD8 beta-chain.

Human mouse and rat CD8 have been described as being disulphide-linked heterodimers of alpha and beta chains. More recently the chicken alpha and beta chains were described. In the bovine and feline immune system only the z-chain was reported. In this study we have cloned and determined the nucleotide sequence of a cDNA encoding the beta-chain of the feline T-cell surface antigen CD8. Using a nested polymerase chain reaction- (PCR) and two primer pairs designed from the human CD8 beta cDNA nucleotide sequence, we amplified a 430 base pair fragment from a feline thymus cDNA library which was used as a probe for screening the feline library at high stringency. After three rounds of screening, five clones were isolated. A clone, named FTb-6, containing a 3.8 kilobase pair insert was mapped, sequenced and compared with the published sequences of the genes encoding the human, mouse, rat and avian CD8 beta. We have determined the primary structure of the feline CD8 beta. The feline CD8 beta has an open reading frame, 630 nucleotides in length encoding a protein with 210 amino acid residues and its composition showed that the feline molecule is a member of the immunoglobulin gene super family.

Amino Acid Sequence↗
PubMed1996

A critical role of IL-2 for the production and gene transcription of IL-5 in allergen-specific human T cell clones.

The role of IL-2 in IL-5 synthesis of human helper T cells was investigated. All of the Der f II (a major allergen of house dust mite)-specific T cell clones established from atopic asthmatic patients produced both IL-2 and IL-4 upon activation (Th0 phenotypes). Recombinant IL-2 induced gene expression and protein synthesis of IL-5 in T cell clones that produced IL-5 upon antigenic stimulation. Human IL-5 promoter/enhancer-luciferase gene construct transfected to T cell clones was clearly transcribed in response to IL-2, indicating that the approximately 500 bp gene segment 5' upstream of the coding region was functionally sufficient for the gene transcription induced by IL-2. IL-2-induced IL-5 synthesis as well as proliferation was dependent on tyrosine kinases. Moreover, IL-5 production by T cell clones stimulated with immobilized anti-CD3 antibody was completely abrogated by anti-IL-2 neutralizing antibody, suggesting that IL-5 (a Th2 cytokine) synthesis of human helper T cells is dependent on IL-2 (a Th1 cytokine). Our present findings clearly demonstrated that IL-2, known as a T cell growth factor, exerts a cytokine promoting activity on T cells. IL-2 produced at the site of allergic inflammation might facilitate eosinophilic inflammation by inducing IL-5 production in T cells.

Allergens↗
PubMed1996

Feline immunodeficiency virus can infect a human cell line (MOLT-4) but establishes a state of latency in the cells.

Infectivity of feline immunodeficiency virus (FIV) in feline and human lymphoblastoid cell lines was examined using homogeneous populations of FIV derived from infectious molecular clones of strains TMZ and Petaluma, and two recombinant chimeric clones carrying gag, pol, vif and ORF-A from the heterologous virus. FIV from the clones with the env region of the Petaluma strain was shown to infect and establish provirus in a human lymphoid cell line (MOLT-4), although the FIV-infected cells did not produce any infectious viruses. By treatment of the infected MOLT-4 cells with a phorbol ester, infectious virus was rescued. To examine which stage of the life-cycle of FIV is blocked in these cells, we analysed transcription of FIV-14 in the cells by RT-PCR. FIV-specific RNA expression could not be detected. These results strongly suggest that latency of the virus in MOLT-4 cells is due to a failure in transcription.

Antigens, Viral↗