Biomedical subjects

T Hayakawa

Publications and source records attributed to T Hayakawa.

At least 19 recordsLinked to original sources

PubMed2006

Characterization of capsid-modified adenovirus vectors containing heterologous peptides in the fiber knob, protein IX, or hexon.

Adenovirus (Ad) vectors are widely used in gene therapy and in vitro/in vivo gene transfer because of their high transduction efficiency. However, Ad vector application in the gene therapy field is limited by poor transduction into cells not expressing the primary receptor, coxsackievirus and adenovirus receptor. To overcome this problem, several types of capsid-modified Ad vectors have been developed. The HI loop or C-terminus of the fiber knob, the C-terminus of the protein IX (pIX) and the hypervariable region 5 of the hexon are promising candidate locations for displaying foreign peptide sequences. In the present study, we constructed Ad vectors in which each of the above region was modified by a simple in vitro ligation-based method, and examined the characterization of each Ad vector containing the FLAG tag (DYKDDDDK) or RGD (CDCRGDCFC) peptide. Enzyme-linked immunosorbent assay examining the surface expression of foreign peptides on the virus suggested that foreign peptides are exposed on virion surfaces in all types vectors and that the hexon was the most efficiently reacted, reflecting the copy number of the modification. However, in the case of the transduction efficiency of Ad vectors containing the RGD peptides, the modification of pIX and the hexon showed no effect. The modification of the HI loop of the fiber knob was the most efficient, followed by the modification of the C-terminus region of the fiber knob. These comparative analyses, together with a simple construction method for each modified Ad vector, could provide basic information for the generation of capsid-modified Ad vectors.

Adenoviridae↗

PubMed2006

Functionalization of multilayered DNA-coatings with bone morphogenetic protein 2.

The focus of the present study was to functionalize multilayered DNA-coatings with the osteoinductive factor bone morphogenetic protein 2 (BMP-2) using different loading modalities. The multilayered DNA-coatings were built up from either poly-d-lysine (PDL) or poly(allylamine hydrochloride) (PAH) and DNA using electrostatic self-assembly (ESA). The amounts of BMP-2 loaded into the multilayered DNA-coatings and its subsequent release characteristics were determined using radiolabeled BMP-2. Additionally, the effect of BMP-2 functionalized multilayered DNA-coatings on the in vitro behavior of bone marrow-derived osteoblast-like cells was evaluated in terms of proliferation, differentiation, mineralization, and cell morphology. The results demonstrate the feasibility of multilayered DNA-coatings to be functionalized by embedding BMP-2 according to three different loading modalities: superficial (s), deep (d), and double-layer (dl). BMP-2 was incorporated proportionally into the multilayered DNA-coatings as: s+(4*d)=dl. All differently loaded multilayered DNA-coatings showed an initial burst release followed by an incremental sustained release of the remaining BMP-2. In vitro experiments demonstrated that the loaded factor remained biologically active, as an accelerated calcium deposition was observed on s- and dl-loaded multilayered DNA-coatings, without affecting cell proliferation. In contrast, d-loaded multilayered DNA-coatings influenced osteoblast-like cell behavior by decreasing the deposition of calcium.

Alkaline Phosphatase↗

PubMed2006

Adenovirus serotype 35 vector-mediated transduction into human CD46-transgenic mice.

We previously demonstrated that systemic administration of adenovirus serotype 35 (Ad35) vectors to mice does not mediate efficient transduction in organs, probably because expression of the mouse analog of the subgroup B Ad receptor, human CD46 (membrane cofactor protein), is limited to the testis. Here, we describe the in vitro and in vivo transduction characteristics of Ad35 vectors by using homozygous and hemizygous human CD46-transgenic (CD46TG) mice, which ubiquitously express human CD46. An Ad35 vector more efficiently transduced the primary dendritic cells and macrophages prepared from CD46TG mice than those from wild-type mice. In vivo transduction experiments demonstrated that CD46TG mice are more susceptible to Ad35 vector-mediated in vivo transduction than are wild-type mice. In particular, homozygous CD46TG mice, which express higher levels of CD46 in the organs than hemizygous CD46TG mice, tend to exhibit higher transduction efficiencies after intraperitoneal administration than hemizygous CD46TG mice. Intraperitoneal administration of Ad35 vectors resulted in efficient transduction into the mesothelial cells of the peritoneal organs in homozygous CD46TG mice. These results indicate that an Ad35 vector recognizes human CD46 as a cellular receptor in CD46TG mice. However, the in vivo transduction efficiencies of Ad35 vectors in CD46TG mice are much lower than those of conventional Ad5 vectors in wild-type mice.

Adenoviridae↗

PubMed2006

Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species.

In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity.

Animals↗

PubMed2006

Are comprehensive environmental changes as effective as health education for smoking cessation?

OBJECTIVES: To compare the effectiveness of health education on smoking cessation for all smokers regardless of their willingness to quit smoking and cumulative environmental changes including designation of smoking places, legislation, and price rise. DESIGN: Comparison of smoking cessation rates over two time periods: the period of health education on smoking cessation (1997-1999), and the period of cumulative environmental changes (2002-2004). SETTING: An occupational setting in a radiator manufacturing factory in Japan. SUBJECTS: All habitual male smokers who remained in the worksite through the pertinent time period (n = 202 in the period of health education and n = 170 in the period of environmental changes). MAIN OUTCOME MEASUREMENTS: Smoking cessation rates at the end of each time period. RESULTS: The smoking cessation rates over the periods of health education and environmental changes were 8.9% and 7.1%, respectively. There was no difference between these two proportions in a chi2 test (p = 0.513). The age adjustment did not significantly alter the cessation rate. CONCLUSIONS: Cumulative environmental changes are fairly effective in promoting smoking cessation, and may yield similar smoking cessation rates as a health education intervention reaching all smokers regardless of their willingness to quit smoking.

Adolescent↗

PubMed2005

Screening of novel nuclear receptor agonists by a convenient reporter gene assay system using green fluorescent protein derivatives.

Nuclear receptors represent a very good family of protein targets for the prevention and treatment of diverse diseases. In this study, we screened natural compounds and their derivatives, and discovered ligands for the retinoic acid receptors (RARs) and the farnesoid X receptor (FXR). In the reporter assay systems of nuclear receptors presented here, two fluorescent proteins, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), were used for detection of a ligand-based induction and as an internal control, respectively. By optimizing the conditions (e.g., of hormone response elements and promoter genes for reporter plasmids), we established a battery of assay systems for ligands of RARs, retinoid X receptor (RXR) and FXR. The screening using the reporter assay system can be carried out without the addition of co-factors or substrates. As a result of screening of more than 140 compounds, several compounds were detected which activate RARs and/or FXR. Caffeic acid phenylethyl ester (CAPE), known as a component of propolis from honeybee hives, and other derivatives of caffeic acid up-regulated the expression of reporter gene for RARs. Grifolin and ginkgolic acids, which are non-steroidal skeleton compounds purified from mushroom or ginkgo leaves, up-regulated the expression of the reporter gene for FXR.

Animals↗

PubMed2005

Roles of cyclooxygenase 2 and microsomal prostaglandin E synthase 1 in rat acid reflux oesophagitis.

BACKGROUND: Although prostaglandin E2 (PGE2), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES-1) are known to play a role in various inflammatory events, their roles in the pathogenesis of gastro-oesophageal reflux disease are not known. AIMS: We examined the dynamics of COX-1, COX-2, mPGES-1, mPGES-2, cytosolic PGES (cPGES), and PGE2 synthetic activity in rat acid reflux oesophagitis and the effects of COX-2 inhibitors on the severity of oesophagitis. METHODS: Acid reflux oesophagitis was induced by ligating the transitional region between the forestomach and the glandular portion and wrapping the duodenum near the pylorus. Rats were killed on day 3 (acute phase) or day 21 (chronic phase) after induction of oesophagitis. RESULTS: Expression of COX-2 and mPGES-1 was markedly increased in oesophagitis while modest changes in COX-1, cPGES, and mPGES-2 expression were observed. COX-2 and mPGES-1 were colocalised in epithelial cells of the basal layer, as well as inflammatory and mesenchymal cells in the lamina propria and submucosa. COX-2 inhibitors significantly reduced the severity of chronic oesophagitis but did not affect acute oesophageal lesions. COX-2 inhibitors significantly inhibited the increase in PGE2 synthesis in oesophageal lesions on both days 3 and 21. Epithelial proliferation was significantly increased in the basal layer on day 21. Inflammatory cells and epithelial cells of the basal layer exhibited reactions for EP4 in oesophagitis. CONCLUSION: PGE2 derived from COX-2 and mPGES-1 plays a significant role in the pathogenesis of chronic acid reflux oesophagitis, and possibly in basal hyperplasia and persistent inflammatory cell infiltration.

Animals↗

PubMed2005

Quartz-crystal microbalance-dissipation technique for the study of initial adsorption of fibronectin onto tresyl chloride-activated titanium.

The immobilization of cell-adhesive proteins onto titanium implants improves biological response at the implant-tissue interface. Previous studies demonstrated the easy and direct attachment of fibronectin onto titanium with the use of a 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride) activation technique. The present study investigated the initial adsorption behavior of fibronectin on tresyl chloride-activated titanium by the quartz-crystal microbalance-dissipation (QCM-D) technique. The crystal resonant frequency and the dissipation shift of the oscillator were simultaneously measured by the injection of fibronectin/phosphate-buffered saline solution (pH = 7.4). The tresyl chloride-activated titanium surface showed a faster and greater decrease in frequency than that of untreated titanium, indicating that a greater amount of fibronectin was adsorbed in the former case during a 120-min adsorption. The dissipation-frequency plots revealed that, during the initial stage of adsorption, the bond between fibronectin and tresyl chloride-activated titanium is stronger than that between fibronectin and untreated titanium. The QCM-D technique can provide new insights into the adsorption mechanism of fibronectin.

Adsorption↗

PubMed2005

Augmentation of the migratory ability of DC-based vaccine into regional lymph nodes by efficient CCR7 gene transduction.

Although dendritic cell (DC)-based immunotherapy is considered a promising approach for cancer treatment, a large quantity of DC vaccine is required for effective sensitization/activation of immune cells because of the poor migratory ability of administered DCs into regional lymphoid tissue. In this study, we created a DC vaccine sufficiently transduced with CC chemokine receptor-7 gene (CCR7/DCs) by applying RGD fiber-mutant adenovirus vector (AdRGD), and investigated its immunological characteristics and therapeutic efficacy. CCR7/DCs acquired strong chemotactic activity for CC chemokine ligand-21 (CCL21) and exhibited an immunophenotype similar to mature DCs but not immature DCs with regard to major histocompatibility complex/costimulatory molecule-expression levels and allogenic T cell proliferation-stimulating ability, while maintaining inherent endocytotic activity. Importantly, CCR7/DCs injected intradermally into mice could accumulate in draining lymph nodes about 5.5-fold more efficiently than control AdRGD-applied DCs. Reflecting these properties of CCR7/DCs, DC vaccine genetically engineered to simultaneously express endogenous antigen and CCR7 could elicit more effective antigen-specific immune response in vivo using a lower dosage than DC vaccine transduced with antigen alone. Therefore, the application of CCR7/DCs having positive migratory ability to lymphoid tissues may contribute to reduction of efforts and costs associated with DC vaccine preparation by considerably reducing the DC vaccine dosage needed to achieve effective treatment by DC-based immunotherapy.

Adenoviridae↗

PubMed2005

Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference.

Increase of Skp-2, which is involved in the degradation of cell cycle regulators including p27Kip1, p21 and c-myc, is one of the important mechanisms for dysregulation of cell cycles in various cancers. We applied RNA interference (RNAi) for Skp-2 by using HIV-lentiviral or adenoviral vectors for a human small-cell lung carcinoma cell line with increased Skp-2 to evaluate RNAi strategy for cancer gene therapy. HIV-lentivirus-mediated RNAi for Skp-2 resulted in efficient inhibition of the in vitro cell growth of cancer cells with increased Skp-2 through the increase of p27Kip1 and p21, but no significant effect on the growth of cells without high Skp-2 expression. Furthermore, intratumoral administration of adenovirus siRNA vector for Skp-2 efficiently inhibited growth of established subcutaneous tumor on NOD/SCID mice. These results indicate that the Skp-2 RNAi may be a useful strategy for gene therapy of cancers with high Skp-2 expression.

Adenoviridae↗

PubMed2005

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities.

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

Actins↗

PubMed2005

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

AIDS Vaccines↗

PubMed2005

Different effects of blood pressure on mortality from stroke subtypes depending on BMI levels: a 19-year cohort study in the Japanese general population--NIPPON DATA80.

To evaluate the relationship between blood pressure and cerebrovascular death depending on body mass index (BMI) levels, we analysed a database of 9338 subjects from the National Integrated Project for Prospective Observation of Non-communicable Disease and its Trends in the Aged, which was originally conducted a baseline survey in 1980 and followed up in 1999. Relative risk (RR) and a 95% confidence interval (CI) of death from total stroke, cerebral infarction, and intracerebral haemorrhage after adjusting for age, sex, serum cholesterol, albumin, glucose, the use of antihypertensive agents, a past history of diabetes, BMI, smoking, and drinking were estimated with the Cox-proportional hazard model in the BMI tertile groups of a representative Japanese population. Cutoff points of BMI tertiles are 21.2 and 23.8 kg/m2. The results indicated that a 10 mmHg systolic blood pressure (SBP) increase was associated with mortality from intracerebral haemorrhage at low and middle BMI groups (RR= 1.38 and 1.23; 95% CI=1.17-1.62 and 1.03-1.47, respectively). SBP was positively associated with mortality from cerebral infarction in middle and high BMI groups (RR=1.19 and 1.21; 95% CI=1.06-1.33 and 1.06-1.38, respectively). The effects of diastolic blood pressure on intracerebral haemorrhage and infarction had the same tendency as those of SBP. These results suggested that the causal effect of blood pressure on stroke subtypes might be modified by BMI.

Adult↗

PubMed2004

Comparison of transgene expression mediated by several Fiber-modified adenovirus vectors in trophoblast cells.

The transfer of genes of interest is a useful method for studying placental biology. Recombinant adenovirus (Ad) vector is an efficient vector for transgene expression. An interaction between the fiber of Ad and the coxsackievirus and adenovirus receptor on the cell membrane is the first step in infection. We previously developed fiber-modified Ad vectors and showed that they improved transgene activity in several cell lines when compared to wild-type vector. In the present study, we examined the ability of three fiber-modified Ad vectors to transduce human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and rat trophoblast cell lines (Rcho-1, TR-TBT 18d-1 and TR-TBT 18d-2). We compared the transgene efficacy of wild-type Ad-L2 vector, Ad-RGD(HI)-L2 vector containing an Arg-Gly-Asp motif, Ad-K7(C)-L2 vector containing a 7-tandem lysine motif, and Ad-RGD(HI)K7(C)-L2 vector containing both motifs in the fiber. We used the luciferase gene as a reporter gene. In the human and rodent trophoblast cell lines, Ad-RGD(HI)-L2 had the greatest infectious potential, followed by Ad-RGD(HI)K7(C)-L2, Ad-K7(C)-L2 and Ad-L2. Compared to the amount of luciferase produced by wild-type vector, Ad-RGD(HI)-L2 mediated 8.1-fold the amount of luciferase in JEG-3 cells, 13.5-fold in JAR cells, 76.8-fold in BeWo cells, 5.0-fold in Rcho-1, 19.4-fold in TR-TBT 18d-1 and 15.0-fold in TR-TBT 18d-2. These results indicate that Ad-RGD(HI) is a potential recombinant Ad vector for transgene expression in some trophoblast cell lines.

Adenoviridae↗

PubMed2004

MEG latency difference measurement for priming experiments.

Latency analysis of magnetoencephalography (MEG) for priming experiments, not in motor readiness level but in cognition level, may be useful for identification of the brain system. We performed a masked priming experiment for repetitive presentation of visual words. The subjects of the experiment were asked to perform a categorization task and button-pressing. The stimuli of the experiment consisted of mask (duration: 700 ms) - prime (70 ms) - target (1000 ms). There were four types of experiment which depended on the combinations of primes and targets: #1) in-category/in-category; #2) pseudo-characters/in-category; #3) out-category/out-category; and #4) pseudo-characters/out-category. As a result, the order of reaction times (RTs) were #1 < #2 < #3 approximately #4. We performed MEG recording with the above experiment simultaneously. Due to such a short stimulus onset asynchrony (SOA) of 70 ms and higher-order brain activity for language processing, the MEG activity continued for several hundred milliseconds, did not have conspicuous peaks, and could not be separated in the prime and target responses. This kind of MEG data is difficult to investigate with conventional signal processing methods, such as subtraction or signal source estimation. We applied a pattern analyzing method that measured the similarity time course between two sets of MEG data. The similarity time courses between experiments #3 and #4 and the other experiments were calculated. The order of the peak latencies of the time course was the same as that of RTs.

Humans↗

PubMed2004

Hypernuclear fine structure in (16)(Lambda)O and the LambdaN tensor interaction.

We have observed two gamma-ray transitions in (16)(Lambda)O from the 6.6 MeV excited 1(-)(2) state to both ground-state spin-doublet members (1(-)(1),0(-)) by the (K-,pi(-)gamma) reaction. We have obtained the ground-state doublet spacing to be 26.4+/-1.6(stat)+/-0.5(syst) keV and the excitation energy of the 1(-)(2) state to be 6561.7+/-1.1(stat)+/-1.7(syst) keV. The ground-state doublet spacing provides a small but nonzero strength of the tensor interaction between a Lambda and a nucleon. This is the first experimental result on the LambdaN tensor interaction.

Journal Article↗

PubMed2004

Compact 10-GHz Nd:GdVO4 laser with 0.5-W average output power and low timing jitter.

We demonstrate a compact, diode-pumped Nd:GdVO4 laser with a repetition rate of 9.66 GHz and 0.5-W average output power. The laser is passively mode locked with a semiconductor saturable absorber mirror (SESAM), yielding 12-ps-long sech2-shaped pulses. For synchronization of the pulse train to an external reference clock, the SESAM is mounted on a piezoelectric transducer. With an electronic feedback loop of only a few kilohertz loop bandwidth we achieved a rms timing jitter of 146 fs (integrated from 10 Hz to 10 MHz). This is an upper limit because it is mostly limited by the measurement system. The laser setup with a simple linear cavity has a footprint of only 130 mm x 30 mm.

Journal Article↗

PubMed2004

Evidence for nucleosynthesis in the supernova gamma process: universal scaling for p nuclei.

Analyzing the solar system abundance, we find two universal scaling laws concerning the p and s nuclei. They indicate that the gamma process in supernova (SN) explosions is the most probable origin of the p nuclei that has been discussed with many possible nuclear reactions and sites in about 50 years. In addition, the scalings lead to new concepts: a universality of the gamma process and a new nuclear cosmochronometer. We carry out gamma-process nucleosynthesis calculations for typical core-collapse SN explosion models, and the results satisfy the observed scalings.

Journal Article↗