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Biomedical subjects

Hiroyuki Koyama

Publications and source records attributed to Hiroyuki Koyama.

At least 19 recordsLinked to original sources
PubMed2006

Serological differentiation of FIV-infected cats from dual-subtype feline immunodeficiency virus vaccine (Fel-O-Vax FIV) inoculated cats.

Feline immunodeficiency virus (FIV) vaccine, Fel-O-Vax FIV, was released for sale in the US in 2002. The antibodies of vaccinated cats interfere with serological assays by currently available FIV diagnostic kits. In this study, we investigated whether it is possible to distinguish serologically cats vaccinated with Fel-O-Vax FIV from cats experimentally or naturally infected with FIV. A total of 153 sera taken from 97 cats were used as serum samples. Enzyme linked immunosorbent assay (ELISA) was performed using whole FIV antigen and formalin treated whole FIV antigen, recombinant-gag (r-gag) antigen, and transmembrane (TM) peptide. Statistical analysis was performed using ELISA optical density (O.D.) values obtained with each antigen as variables. Except for the ELISA O.D. values obtained with r-gag antigen, a significant difference in ELISA O.D. values was observed between the vaccinated and the infected groups. However, it was not possible to distinguish both groups unequivocally. Using discriminant analysis, it was possible to distinguish the two groups with an accuracy of 97.1% with two discriminating variables (ELISA O.D. values obtained with formalin treated whole FIV antigen, and TM peptide), 97.8% with three discriminating variables (ELISA O.D. values obtained with whole FIV antigen, formalin treated whole FIV antigen, and TM peptide). Therefore, it was considered possible to distinguish cats vaccinated with Fel-O-Vax FIV from FIV-infected cats by ELISA using two types of antigens including formalin treated whole FIV antigen and TM peptide, or three types of antigens including formalin treated whole FIV antigen, TM peptide and whole FIV antigen.

Animals↗
PubMed2006

A "possible" involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis.

Feline infectious peritonitis (FIP) cats show a decrease in peripheral blood lymphocyte counts, and a particularly marked decrease in T cells including CD4+ and CD8+ cells. In this study, we showed that lymphopenia observed in FIP cats was due to apoptosis, and that the ascitic fluid, plasma, and culture supernatant of peritoneal exudate cells (adherent cells with macrophage morphology, or PEC) from FIP cats readily induced apoptosis in specific pathogen-free cat peripheral blood mononuclear cells, particularly CD8+ cells. In addition, TNF-alpha released from macrophages and TNF-receptor (TNFR) 1 and TNFR2 mRNA expression in lymphocytes were closely involved in this apoptosis induction. In particular, in CD8+ cells cultured in the presence of the PEC culture supernatant, the expression levels of TNFR1 and TNFR2 mRNA were increased, indicating that CD8+ cells are more susceptible to apoptosis induction by TNF-alpha than other lymphocyte subsets, particularly B cells (CD21+ cells). The results of this study suggest that TNF-alpha, produced by virus-infected macrophages, is responsible for induction of apoptosis in uninfected T cells, primarily CD8+ T cells.

Animals↗
PubMed2006

AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis.

Aluminum (Al) tolerance in Arabidopsis is a genetically complex trait, yet it is mediated by a single physiological mechanism based on Al-activated root malate efflux. We investigated a possible molecular determinant for Al tolerance involving a homolog of the wheat Al-activated malate transporter, ALMT1. This gene, named AtALMT1 (At1g08430), was the best candidate from the 14-member AtALMT family to be involved with Al tolerance based on expression patterns and genomic location. Physiological analysis of a transferred DNA knockout mutant for AtALMT1 as well as electrophysiological examination of the protein expressed in Xenopus oocytes showed that AtALMT1 is critical for Arabidopsis Al tolerance and encodes the Al-activated root malate efflux transporter associated with tolerance. However, gene expression and sequence analysis of AtALMT1 alleles from tolerant Columbia (Col), sensitive Landsberg erecta (Ler), and other ecotypes that varied in Al tolerance suggested that variation observed at AtALMT1 is not correlated with the differences observed in Al tolerance among these ecotypes. Genetic complementation experiments indicated that the Ler allele of AtALMT1 is equally effective as the Col allele in conferring Al tolerance and Al-activated malate release. Finally, fine-scale mapping of a quantitative trait locus (QTL) for Al tolerance on chromosome 1 indicated that AtALMT1 is located proximal to this QTL. These results indicate that AtALMT1 is an essential factor for Al tolerance in Arabidopsis but does not represent the major Al tolerance QTL also found on chromosome 1.

Aluminum↗
PubMed2006

Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress.

The present study was conducted to investigate the cell wall properties in two wheat (Triticum aestivum L.) cultivars differing in their sensitivity to Al stress. Seedlings of Al-resistant, Inia66 and Al-sensitive, Kalyansona cultivars were grown in complete nutrient solutions for 4 days and then subjected to treatment solutions containing Al (0, 50 microM) in a 0.5 mM CaCl(2) solution at pH 4.5 for 24 h. Root elongation was inhibited greatly by the Al treatment in the Al-sensitive cultivar compared to the Al-resistant cultivar. The Al-resistant cultivar accumulated less amount of Al in the root apex than in the Al-sensitive cultivar. The contents of pectin and hemicellulose in roots were increased with Al stress, and this increase was more conspicuous in the Al-sensitive cultivar. The molecular mass of hemicellulosic polysaccharides was increased by the Al treatment in the Al-sensitive cultivar. The increase in the content of hemicellulose was attributed to increase in the contents of glucose, arabinose and xylose in neutral sugars. Aluminum treatment increased the contents of ferulic acid and p-coumaric acid especially in the Al-sensitive cultivar by increasing the activity of phenylalanine ammonia lyase (PAL, EC 4.3.1.5). Aluminum treatment markedly decreased the beta-glucanase activity in the Al-sensitive cultivar, but did not exert any effect in the Al-resistant cultivar. These results suggest that the modulation of the activity of beta-glucanase with Al stress may be involved in part in the alteration of the molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar. The increase in the molecular mass of hemicellulosic polysaccharides and ferulic acid synthesis in the Al-sensitive cultivar with Al stress may induce the mechanical rigidity of the cell wall and inhibit the elongation of wheat roots.

Adaptation, Physiological↗
PubMed2006

Inflammatory responses involving tumor necrosis factor receptor-associated factor 6 contribute to in-stent lesion formation in a stent implantation model of rabbit carotid artery.

OBJECTIVE: Inflammatory responses are considered to represent a unique property after stent implantation, and we previously demonstrated that inflammatory signaling involving tumor necrosis factor receptor-associated factor 6 (TRAF6) contributes to neointimal formation in a balloon injury model of rabbit carotid artery. The purpose of this study was to examine the role of TRAF6 in in-stent lesion formation after stent implantation in the rabbit carotid artery. METHODS: Rabbit carotid arteries were injured with a 2F Fogarty catheter, and 28 days later, the same arteries were implanted with a 3-mm-diameter Palmaz-Schatz stent. A dominant negative (DN) form of TRAF6 (pME-FLAG-T6deltaRZ5) was then transferred using a plasmid-based electroporation method. Its effects were evaluated compared with the findings in arteries treated with control plasmid (pME-FLAG). RESULTS: Immunostaining with anti-FLAG tag antibody showed that an expression plasmid vector containing the DN-TRAF6 sequence was successfully transferred to the arterial intima and media. Morphometric analyses revealed that the increase of intimal area in in-stent lesions was significantly inhibited by DN-TRAF6 14 days after stent implantation (DN-TRAF6 group, 3.01 +/- 0.25 x 10(5) microm2 vs control group, 4.25 +/- 0.23 x 10(5) microm2, P < .01), and the cell density was increased compared with that in the control group. In the DN-TRAF6 plasmid-treated vessels, cell replication was prevented in both the intima and media, and fewer leukocytes adhered to the luminal surface. Moreover, DN-TRAF6 suppressed macrophage infiltration, activation of proteases, and proteoglycan accumulation in the in-stent intima. CONCLUSIONS: These findings suggest that TRAF6 plays an important role in cell replication, inflammatory cell infiltration, protease activity, and extracellular matrix accumulation that contributes to in-stent lesion development.

Animals↗
PubMed2006

Immediate effects of the use of modified take-off boards on the take-off motion of the long jump during training.

We investigated the immediate effects of the combined use of inclined and raised flat boards on the take-off motion of the long jump. Eight male long jumpers were videotaped with two high-speed video cameras (250 Hz) set perpendicular to the runway. The athletes jumped with three modified take-off boards: upward-inclined boards of two different inclinations (2.5 and 5.0 degrees), and a raised flat board (50 mm high). The jumpers performed pre- and post-jumps using their own techniques before and after use of the boards to test their effects. The post- jump revealed significantly less reduction in the horizontal velocity during the take-off than the pre-jump, and the effectiveness of converting the velocity from horizontal to vertical increased significantly in the post-jump. The post-jump demonstrated significantly less knee flexion of the take-off leg during take-off. The reduced knee flexion and slower extension velocity of the take-off leg in the second phase of the post-jump contributed to increasing the knee extension torque in the second phase and resulted in the increases in vertical ground reaction force and vertical velocity. These results suggest that the combined use of the inclined and raised flat boards induced immediate effective changes in the kinematics and kinetics of the take-off motion and represent appropriate training tools for take-off techniques of the long jump.

Biomechanical Phenomena↗
PubMed2006

Simple identification of transgenic Arabidopsis plants carrying a single copy of the integrated gene.

Transgenic Arabidopsis thaliana plants carrying a single copy of integrated DNA can be identified by single-step genomic polymerase chain reaction. The reaction employs two sets of primer pairs with the same melting temperature that amplify the amplicons derived from the integrated T-DNA together with those from an endogenous single-copy gene as a reference. When the band intensity ratio is one, this means that the transgenic plants are carrying a single copy of the integrated gene per haploid.

Arabidopsis↗
PubMed2006

The prevalence of a group 2 coronavirus in dogs in Japan.

Canine coronavirus (CCoV) has been reported to cause acute diarrhea mainly in young pups. CCoV and feline coronavirus are classified as group 1 coronaviruses. However, it has recently been reported in the United Kingdom that the group 2 coronavirus gene, which is more closely related to the bovine coronavirus (BCoV) and human coronavirus strain OC43, has been detected in respiratory tract tissue samples from dogs with respiratory disease. In this study, we examined the prevalence of antibodies to group 2 coronaviruses in domestic dogs and cats in Japan by a neutralization test using BCoV. All 104 feline serum samples were negative (<1:5) for anti-BCoV antibodies. In contrast, of the 898 canine serum samples, 160 (17.8%) were positive for anti-BCoV antibodies, and the antibody titers ranged from 1:5 to more than 1:640, with 1:160 being the most frequent. No correlation was found between the titers of the anti-BCoV and anti-CCoV antibodies in the 198 serum samples of dogs with a known history of CCoV vaccination. We amplified, by RT-PCR, group 2 coronavirus-specific hemagglutination/esterase genes in the oral swabs of a total of 10 young pups presenting with or having recovered from respiratory signs, or having anti-BCoV antibodies, with the result that 2 pups were positive for the hemagglutination/esterase genes. These results strongly suggest that an unknown group 2 coronavirus as well as the known enteritis-causing CCoV (group 1 coronavirus) is prevalent among domestic dogs in Japan.

Animals↗
PubMed2005

Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer.

The control of gene transfection in the body is a core issue in gene therapy. Photochemical internalization is a technology that allows light-induced delivery of DNA, drugs or other biological factors directly inside cells. Usually it requires that a photosensitizer be added to the drug-delivery system to photochemically destabilize the endosomal membrane. Here we present a system for in vivo DNA delivery in which these two components are assembled into one structure. This is a ternary complex composed of a core containing DNA packaged with cationic peptides and enveloped in the anionic dendrimer phthalocyanine, which provides the photosensitizing action. The ternary complex showed more than 100-fold photochemical enhancement of transgene expression in vitro with reduced photocytotoxicity. In an animal experiment, subconjuctival injection of the ternary complex followed by laser irradiation resulted in transgene expression only in the laser-irradiated site. This work demonstrates a new biomedical application for dendrimers, and the first success in the photochemical-internalization-mediated gene delivery in vivo.

Animals↗
PubMed2005

Dual-subtype vaccine (Fel-O-Vax FIV) protects cats against contact challenge with heterologous subtype B FIV infected cats.

Fel-O-Vax FIV is a dual-subtype vaccine consisting of inactivated whole viruses of subtype A (Petaluma strain) and subtype D (Shizuoka strain). The efficacy of this vaccine against heterologous subtype A strain challenge was demonstrated, but it is unclear whether the result reflects efficacy in the field. In this study, we evaluated the efficacy of this vaccine against contact challenge by exposing both vaccinated and unvaccinated control animals with cats infected with Aomori-2 strain belonging to subtype B, a subtype prevalent in many regions of the world. Nineteen specific-pathogen-free (SPF) cats were divided into a vaccinated group (six cats), an unvaccinated control group (eight cats), and a challenge group (five cats), and maintained in the same room. Cats were monitored for FIV proviral DNA by nested PCR and for FIV-specific antibody levels by ELISA. After 1 year of commingling, each cat in the vaccinated group was given a booster dose. In addition, the original challenge group was removed and replaced with another challenge group of SPF cats, which were inoculated with the Aomori-2 strain. FIV infection was confirmed in four of the eight animals in the unvaccinated control group by the 29th week in the second year of commingling. In contrast, all of the animals were negative in the vaccinated group. These findings confirmed the efficacy of this vaccine against heterologous stains classified as subtype B, and suggested that the vaccine exhibits broad efficacy against genetically diverse FIV.

Animals↗
PubMed2005

Mutations in feline immunodeficiency (FIV) virus envelope gene V3-V5 regions in FIV-infected cats.

The envelope (Env) gene V3-V5 regions of the feline immunodeficiency virus (FIV) encode the neutralizing epitopes. Since mutations in these regions induce resistance to viral neutralizing antibodies, they may influence the effects of vaccines. To examine the in vivo mutation rate in these regions, we cloned cDNA for the Env gene V3-V5 regions from the PBMC of experimentally FIV-infected cats, and compared the deduced amino acid sequences. Blood or plasma from an FIV Shizuoka strain-infected cat was inoculated into a second group of SPF cats, and their blood or plasma was inoculated into the third group. The amino acid sequence encoded by the viral gene of the first cat was compared with those encoded by the viral genes of a total of eight cats in the second and third groups (two and six cats, respectively). The amino acid sequences in two cats in the second and third groups were 100% homologous and in one cat in the third group was 98.3% homologous to that in the first infected cat. Five cats had the same sequence, which was 97.8% homologous to that in the first infected cat. Three kittens, born 2 months after the inoculation of the FIV Aomori-2 strain into the mother cat, were anti-FIV negative at 4 weeks after birth, but became seropositive at 33 weeks after birth, confirming FIV infection. Comparison of the encoded amino acid sequences of the viral gene in two cats at 48 weeks after birth showed 100% homology to that of the virus inoculated into the mother cat, and the remaining one cat had a single residue substitution, resulting in 99.4% homology. These results suggest that the FIV Env gene V3-V5 regions are stably maintained for at least 1-2 years after infection.

Amino Acid Sequence↗
PubMed2005

Preparation and biological properties of dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt)-loaded polymeric micelles.

Block copolymer micelles, containing dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt), the oxaliplatin parent complex, were prepared through polymer-metal complex formation of DACHPt with poly(ethylene glycol)-poly(glutamic acid) block copolymer [PEG-P(Glu)] in distilled water. By dynamic light scattering (DLS) measurement, the micelle size was determined to be 40 nm with narrow distribution. The release of platinum complexes from the micelle core was measured in phosphate buffer saline (pH 7.4) at 37 degrees C. DACHPt-loaded micelle showed a sustained release rate of platinum after an induction period of 12 h. In the same conditions, the kinetic stability of DACHPt-loaded micelle was measured. The micelle was found to be very stable, keeping the initial size, for 240 h. Against murine colon adenocarcinoma 26 (C-26) cells, DACHPt-loaded micelle exhibited considerable in vitro cytotoxicity, lower than oxaliplatin but increasing with exposure time as a result of the release of platinum complexes from the micelle. In vivo biodistribution assay performed on tumor-bearing mice demonstrated that the micelle showed prolonged blood circulation due to its high stability and high tumor accumulation for a prolonged time.

Animals↗
PubMed2005

Mechanical properties of the take-off leg as a support mechanism in the long jump.

The aim of this study was to establish the functions of the support leg in the long jump take-off with a three-element mechanical model spring, damper, and actuator The take-off motions of eleven male long jumpers, with personal bests from 6.45 to 7.99 m, were videotaped at 250 Hz and ground reaction forces were simultaneously recorded at 1 kHz. A two-dimensional 14-segment linked model was used to collect basic kinematic parameters. The spring, damper and actuator forces were determined from the displacement and velocity of the centre of mass and from ground reaction forces. Large spring and damper forces were exerted, and absorbed the impact force immediately after the touch-down. The spring force was also exerted from 25 to 75% of the take-off phase. The actuator force was dominant in the latter two-thirds of the take-off phase. Statistically significant correlations were found between the spring force impulse and the knee flexion during the take-off phase (r = 0.699, p < 0.05), and between the knee flexion and the angular velocity of the thigh at the touch-down (r = 0.726, p < 0.05). These results indicated that the jumper should retain less flexion of the take-off leg knee to increase the spring force, after a fast extension of the hip, and use a more extended knee at the touch-down to prevent excessive knee flexion.

Biomechanical Phenomena↗
PubMed2005

Effects of an inclined board as a training tool on the take-off motion of the long jump.

In this study, we investigated the effects of inclined and raised flat boards on the take-off motion of the long jump to determine the effectiveness of these boards as training tools. Eight male long jumpers were videotaped with two high-speed video cameras (250 Hz) set perpendicular to the runway. Four different jumps were performed with in four conditions, all with their medium length run-up: a long jump with their normal technique, long jumps on upward inclined boards of two different inclinations (2.5 and 5.0 degrees), and a long jump on a raised flat board (0 degrees, 50 mm). The raised flat board enhanced the pivot of the body over the take-off foot, and reduced flexion of the take-off leg knee. However, the inclined boards did not produce the same effects as the raised flat board. The use of the inclined boards increased the vertical velocity of the centre of mass of the body at toe-off, resulting in increased airborne time. These results suggest that the raised flat board and inclined boards would be effective in improving long jumpers' techniques during the take-off and airborne phases.

Biomechanical Phenomena↗
PubMed2005

Ability of CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load in mononuclear cells in FIV-infected cats.

We investigated the relationship between CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load integrated in mononuclear cells. The anti-FIV activity and the proviral DNA load were correlated, and the number of proviral DNA copies was high in cats with decreased anti-FIV activity. Particularly, no anti-FIV activity was detected in the cats staged as having an acquired immunodeficiency syndrome (AIDS)-related complex or AIDS, and the number of proviral DNA copies was obviously increased compared to those in the cats in the asymptomatic stage. These results suggest that decreased anti-FIV activity destroys the control of in vivo FIV replication, which leads to an increased proviral DNA load with the progression of the clinical stage of disease.

Animals↗
PubMed2004

Gelatin hydrogel microspheres enable pinpoint delivery of basic fibroblast growth factor for the development of functional collateral vessels.

BACKGROUND: Various growth factors promote collateral vessel development and are regarded as promising for the treatment of vascular occlusive diseases. However, an efficacious delivery system for them has yet to be established. We devised a strategy to augment functional collateral vessels by using acidic gelatin hydrogel microspheres (AGHMs) incorporating basic fibroblast growth factor (bFGF). The aim of the present study was to investigate the hypothesis that by intra-arterial (IA) administration of bFGF-impregnated AGHMs, bFGF could be delivered from AGHMs trapped in distal small-diameter vessels and thereby induce functional collateral vessels with an assured blood supply through the process of arteriogenesis. METHODS AND RESULTS: Various sizes of AGHMs (3 mg) incorporating 125I-labeled bFGF were injected into the left internal iliac artery of a rabbit model of hindlimb ischemia. Less than 50% of radioactivity accumulated in the ischemic hindlimb after injection of AGHMs that were 10 mum in diameter, whereas approximately 80% of radioactivity was counted in the ischemic limb after administration of 29- or 59-microm-diameter AGHMs. Calf blood pressure ratio and the ratio of regional blood flow of the bilateral hindlimbs immediately before and after IA administration of 29-microm-diameter AGHMs showed no significant change. Then we evaluated the function of the developed collateral vessels 28 days after IA administration of bFGF-impregnated, 29-microm-diameter AGHMs. IA administration of bFGF-impregnated AGHMs induced marked collateral vessel improvement compared with IA administration of phosphate buffered saline-treated AGHMs and intramuscular administration of bFGF-impregnated AGHMs. CONCLUSIONS: IA administration of bFGF-impregnated, 29-microm-diameter AGHMs strongly induced functional collateral vessels without worsening ischemia, indicating the possible therapeutic usefulness of this approach.

Animals↗
PubMed2004

Inflammatory signaling pathway containing TRAF6 contributes to neointimal formation via diverse mechanisms.

OBJECTIVE: The purpose of this study was to investigate the contribution of inflammatory signaling containing tumor necrosis factor receptor-associated factor 6 (TRAF6) to neointimal formation in a balloon injury model of rabbit carotid artery. METHODS: Male Japanese white rabbits fed a normal diet were used. We transferred the dominant negative (DN) form of TRAF6 to a rabbit carotid artery that was subjected to balloon injury by in vivo electroporation method, and then evaluated its effect on intimal lesion formation after balloon injury. RESULTS: An expression plasmid vector containing the TRAF6 DN sequence was successfully transferred to arterial wall cells, and its inhibitory effect on inflammatory signaling was confirmed by the marked suppression of nuclear factor-kappaB (NFkappaB) activity after injury. Morphometric analyses revealed significant inhibition of intimal lesion formation at 7 days after injury. Cell replication and accumulation of macrophages in the media were significantly decreased, and apoptosis was enhanced on day 2. Cell migration to the intima was suppressed on day 4. Extracellular signal-regulated kinase1/2 (ERK1/2) activity at 2 h after injury was also down-regulated. Interestingly, intimal cell replication was significantly blocked when TRAF6 DN was transfected at 7 days after injury. CONCLUSION: TRAF6 plays important roles in cell replication and migration, besides promotion of inflammatory cell infiltration and suppression of apoptosis.

Animals↗