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Biomedical subjects

T Miyazawa

Publications and source records attributed to T Miyazawa.

At least 145 records · Page 8Linked to original sources
PubMed1998

Age-associated oxidative damage in microsomal and plasma membrane lipids of rat hepatocytes.

Phosphatidylcholine hydroperoxide (PC-OOH) and phosphatidylethanolamine hydroperoxide (PE-OOH) concentrations were determined in microsomes and plasma membranes prepared from 2- and 17-month-old male Sprague-Dawley rat hepatocytes, to verify the dissimilarity of age dependency of lipid peroxidation in organelle membranes. The hydroperoxides were directly measured by chemiluminescence detection-high-performance liquid chromatography (CL-HPLC), and 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH) and 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylethanolamine (PLPE-OOH) were enzymatically synthesized and utilized as standards for the calibration. Baseline concentrations of hydroperoxides (PC-OOH + PE-OOH) of the 17-month-old rats were 46 pmol per mg protein in microsomes (2.7 times higher than the 2-month-old rats) and 306 pmol per mg protein in plasma membranes (9.9 times higher than the 2-month-old rats). Both microsomal and plasma membrane lipids were severely peroxidized and converted to phospholipid hydroperoxides by NADPH-dependent lipid peroxidation in vitro, but the age-dependency was only observed in the plasma membranes. These results demonstrate that substantial oxidative damage to membrane phospholipids occurs with ageing both in microsomes and plasma membranes, but is more prevalent in plasma membranes in rat hepatocytes.

Aging↗
PubMed1998

Characterization of anti-feline CD8 monoclonal antibodies.

We generated three monoclonal antibodies (mAbs) (2D7, 10C7 and 12A3) reactive to the alpha-chain of feline CD8 (fCD8) molecule. Further we showed that reference anti-fCD8 mAbs, FT2, 3.357 and vpg9 recognize the beta-chain, alpha-chain and alphabeta-complex epitope, respectively. Flow cytometric analysis using these mAbs suggested that fCD8alpha(+)beta(-) cells were present in lymphocytes of spleen, but not significantly in those of thymus, lymph nodes and peripheral blood of normal kittens.

Animals↗
PubMed1998

Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics.

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha subunit (Gi1alpha) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgammaS binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 microg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in alpha helix content of the Gi1alpha by 7%. Likewise, mastoparan also enhanced the rate of GTPgammaS binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1alpha similar to compound 48/80. In corresponding experiments using a K349P-Gi1alpha, a Gi1alpha counterpart of the unc mutant in Gsalpha in which Pro was substituted for Lys349, enhancement of the GTPgammaS binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1alpha. Taken together, our results strongly suggest that the activator-induced unwinding of the alpha helix of the G protein alpha subunit is mechanically coupled to the enhanced release of bound GDP from the alpha subunit.

Circular Dichroism↗
PubMed1998

In vitro recombination of feline herpesvirus type 1.

We investigated the possibility of in vitro recombination of three different strains of feline herpesvirus type 1 (FHV-1), a virulent strain (C7805), a vaccine strain (F2), and a candidate vaccine strain with a deletion in the thymidine kinase gene (C7301dlTK). The results showed that recombination within different genotypes of FHV-1 strains could occur at a relatively high frequency ranging from 10.1% to 21.5% in vitro. These results indicate the generation of multiple mutant viruses by in vitro recombination experiments. The possibility for generating a virulent virus or novel type of FHV-1 by recombination in vivo is discussed.

Animals↗
PubMed1998

Cross virus neutralizing antibodies against feline immunodeficiency virus genotypes A, B, C, D and E.

Feline immunodeficiency virus (FIV) is classified into five genotypes, A, B, C, D and E, based on the phylogenetic analysis of the env V3-V5 region. However, whether there is correlation between phylogenetic and antigenic diversities remains unknown. In this study, we examined the cross virus neutralization of FIV genotypes A through E by sera from cats infected with a single genotype. The results indicated some relationships between phylogenetic genotype and neutralization serotype, and that cross-clade virus neutralization is possible. For example, serum from a cat infected with genotype E virus neutralized all five FIV genotypes. Our results suggest that the FIV subtyping according to the sequence diversity is partially reflected by antigenic diversity and serum neutralization.

Animals↗
PubMed1998

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus.

We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301 ddlTK-gag was confirmed to express the FIV Gag precursor protein in the cells.

Alphaherpesvirinae↗
PubMed1998

The roles of vif and ORF-A genes and AP-1 binding site in in vivo replication of feline immunodeficiency virus.

To examine the in vivo roles of auxiliary genes and regulatory elements of feline immunodeficiency virus (FIV), the provirus load in various tissues of cats infected with each of the mutant viruses (delta vif, delta ORF-A and delta AP-1) was studied. Although all mutant viruses could infect various tissues, provirus loads in various tissues especially those in cats infected with delta vif virus were lower than those with the wild-type virus. Our results indicate the significance of vif and ORF-A genes and AP-1 binding site of FIV for efficient viral replication and full pathogenicity in cats.

Animals↗
PubMed1998

Cats are protected against feline immunodeficiency virus infection following vaccination with a homologous AP-1 binding site-deleted mutant.

Following establishment, via the vaginal route, of infection with an AP-1 binding-site deleted mutant (delta AP-1) of feline immunodeficiency virus (FIV), cats were challenged with a homologous intact strain (TM2) of FIV. The cats were observed for 23 weeks to evaluate the efficacy of the delta AP-1 against the homologous TM2 strain challenge. These two viruses were differentiated by Southern blotting after amplification of proviral DNA by semi-nested polymerase chain reaction in DNAs of peripheral blood mononuclear cells and tissues. A TM2-specific band was detected in one cat exposed to but not infected with delta AP-1, but not in two delta AP-1-infected. These results indicate that delta AP-1 could protect against subsequent challenge with homologous FIV TM2 strain.

Animals↗
PubMed1998

Less invasive reconstruction of the temporalis muscle for pterional craniotomy: modified procedures.

BACKGROUND: I developed a modified pterional craniotomy technique which is less invasive for both the facial nerve and the temporalis muscle. METHODS: The keys to this procedure are subfascial dissection and reflection of the temporalis fascia without interfascial dissection, and posterior displacement of the temporalis muscle without muscular incision. The limited exposure from the bulky temporalis muscle can be avoided with this procedure. A small cuff of the fascia-periostium complex is left on a free bone flap along the superior temporal line to facilitate the subsequent muscle closure. RESULT: Since the facial nerve and temporalis muscle are not injured with this procedure, the functional and cosmetic results are satisfactory. CONCLUSION: To reduce functional and cosmetic complications, and the cost of pterional craniotomy, I believe that my procedure is recommended.

Craniotomy↗
PubMed1998

Molecular genotyping by pulsed-field gel electrophoresis of restricted genomic DNA of strains of Taylorella equigenitalis isolated in Ireland and in the United States.

The profiles, after digestion with ApaI or NotI and pulsed-field gel electrophoresis (PFGE), of genomic DNA from 18 strains of Taylorella equigenitalis isolated in Ireland were of three different types. The 13 strains in one of these types gave PFGE profiles identical to that of an American prototype strain, Kentucky 188, but different from strain NCTC11184T and from a strain isolated in Japan. Eight additional strains isolated in the United States gave four distinct types of genomic PFGE profiles. All four types were different from that of T. equigenitalis NCTC11184T or that of the Japanese strain. The profile of three strains of one type was identical to that of Kentucky 188.

Animals↗
PubMed1998

Molecular cloning and sequencing of feline stromal cell-derived factor-1 alpha and beta.

The stromal cell-derived factor-1 alpha and beta (SDF-1 alpha/beta) are the ligands of fusin/CXCR4, the co-receptors of human immunodeficiency virus type 1 and the feline immunodeficiency virus. We cloned the cDNA of feline SDF-1 alpha/beta. The open reading frames of feline SDF-1 alpha/beta were 267/279 base pairs and encoded 89/93 amino acid residues.

Amino Acid Sequence↗
PubMed1998

Effects of unilateral intrathecal administrations of low dose tissue-type plasminogen activator on clot lysis, vasospasm and brain phospholipid hydroperoxidation in a primate model of bilateral subarachnoid hemorrhage.

In order to clarify the effect of clot lysis by recombinant tissue-type plasminogen activator (tPA) on the brain lipid peroxidation, we measured phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) levels in a primate model of subarachnoid hemorrhage (SAH). Monkeys were assigned into two groups; a tPA-treated group receiving intrathecal injections of 0.02 mg tPA, and a placebo-treated group receiving saline. The tPA or placebo was injected into the right side of the basal cistern every 8 h for 6 days following bilateral SAH induction. The tPA cleared the right side clots (p < 0.0001), but not the left side clots. The degree of vasospasm in the right middle cerebral artery and the rCBF decrease in the right parietal cortex were significantly attenuated in the tPA group (p < 0.05). In the placebo group, more severe vasospasm and marked rCBF reduction were noted in comparison with those in the tPA group. PCOOH levels in the parietal cortex were significantly higher in the placebo group than in the tPA group (p < 0.05). There were no significant changes in brain PEOOH levels. These results may explain the limitations for clinical application of unilateral intrathecal administration of tPA.

Animals↗
PubMed1998

Protection of hippocampal neurons from ischemia-induced delayed neuronal death by hepatocyte growth factor: a novel neurotrophic factor.

Hepatocyte growth factor (HGF), a natural ligand for the c-met protooncogene product, exhibits mitogenic, motogenic, and morphogenic activities for regeneration of the liver, kidney, and lung. Recently, HGF was clearly shown to enhance neurite outgrowth in vitro. To determine whether HGF has a neuroprotective action against the death of neurons in vivo, we studied the effect of HGF on delayed neuronal death in the hippocampus after 5-minute transient forebrain ischemia in Mongolian gerbils. Continuous postischemic intrastriatal administration of human recombinant HGF (10 or 30 micrograms) for 7 days potently prevented the delayed death of hippocampal neurons under both anesthetized and awake conditions. Even when HGF infusion started 6 hours after ischemia (i.e., in a delayed manner), HGF exhibited a neuroprotective action. We conclude that HGF, a novel neurotrophic factor, has a profound neuroprotective effect against postischemic delayed neuronal death in the hippocampus, which may have implications for the development of new therapeutic strategies for ischemic neuronal damage in humans.

Animals↗
PubMed1998

Expansion of CD8alpha+beta- cells in cats infected with feline immunodeficiency virus.

CD8+ lymphocytes have been subdivided into CD8alphabeta and CD8alpha alpha populations in the peripheral blood lymphocytes (PBL) of humans and in several animal species but have not yet been investigated in cats. Feline immunodeficiency virus (FIV) causes progressive immunological disorders similar to human AIDS. In this study, we analysed CD8+ cells in PBL of FIV-infected or uninfected cats by two-colour flow cytometric analysis. In specific pathogen-free adult cats, feline CD8alpha+beta(high) cells were observed but CD8alpha+beta- cells were not found in significant numbers. On the other hand, not only CD8alpha+beta(high) but also CD8alpha+beta- and CD8alpha+beta(low) cell populations were observed in cats chronically infected with FIV. The expansion of the CD8beta(low) or CD8beta- subpopulations resulted in the apparent differences in CD4/CD8 ratios depending on the anti-CD8 MAb used. These findings suggest a need to reconsider the CD4/CD8 ratio in studies of FIV infection. Furthermore, we found that the CD8alpha+beta- cell population expressed CD5 at a low level.

Animals↗
PubMed1998

Protein-binding properties of the putative AP-1 and ATF sequences in the feline immunodeficiency virus long terminal repeat.

Electrophoresis-mobility-shift assays with nuclear extracts from a feline renal cell line and a T-lymphoblastoid cell line revealed that the AP-1 and ATF sites of feline immunodeficiency virus (FIV) TM2 strain had similar protein-binding properties to those of FIV Petaluma strain and consensus sequences of AP-1 and ATF sites, and that nuclear factors binding to these sites differed between the two cell lines. Cross-competition and gel-supershift assays demonstrated that the AP-1 and ATF sites had similar protein-binding properties. The effects of internal deletions of AP-1 and/or ATF sites on the basal promoter activity were also examined. Although deletion of either site moderately reduced activity, a mutant deleted in both sites had dramatically reduced activity. Therefore, we suggest that these two sites co-operatively regulate transcriptional activity of the promoter.

Activating Transcription Factors↗
PubMed1998

Identification and transcriptional analysis of the homologues of the herpes simplex virus type 1 UL41 to UL51 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 nonpathogenic phenotypes of MDV. We determined the 16770 bp nucleotide sequence of the MDV2 genome located in the right part the of unique long region. The analysis revealed 12 complete open reading frames (ORFs) with high amino acid sequence identities to the gene products of other alphaherpesviruses. The MDV2 ORFs were arranged collinearly with the prototype sequence of herpes simplex virus type 1 ranging from the UL41 to UL51 genes. Except for the MDV2 UL41 gene, all of the identified genes were confirmed to be transcribed with 3'-coterminal mRNAs and/or a unique transcript in the virus-infected cells. Transcriptional patterns for the regions of the MDV2 UL48 to UL49.5 genes were notably different from the similar area of MDV serotype 1.

Animals↗