Biomedical subjects

Naoki Kobayashi

Publications and source records attributed to Naoki Kobayashi.

At least 19 recordsLinked to original sources

PubMed2006

Enhanced antigen-specific antibody production following polyplex-based DNA vaccination via the intradermal route in mice.

DNA vaccination is an attractive approach with various advantages over conventional vaccination. The present study was undertaken to examine whether polyplex-based DNA vaccination could be used to modulate immune responses by plasmid DNA (pDNA). Methylated bovine serum albumin (mBSA) was used as a model of a cationic macromolecular carrier of pDNA encoding obalbumin (OVA) and the effects of polyplex formation of pDNA with mBSA on the antigen-specific immune responses were examined. Anti-OVA IgG antibody production was significantly increased following intradermal immunization with the polyplex compared with naked pDNA, although the induction of cytotoxic T lymphocyte activity was lowered by polyplex formation. We also demonstrated that the disposition and gene expression of pDNA following intradermal injection could be manipulated by polyplex formation. Intriguingly, we also found that the migration of dendritic cells to the injected site could be induced by polyplex formation probably due to a high level of tumor necrosis factor alpha production from the keratinocytes treated with mBSA/pDNA complexeses. Thus, the present study has demonstrated that the immune responses could be biased towards a Th2-type response by polyplex-based DNA vaccination through manipulation of not only pDNA disposition but also dendritic cell migration.

Animals↗

PubMed2006

Confocal nonlinear optical microscopy for high-resolution measurement of absorptive objects.

We demonstrate a new scheme to enhance the spatial resolution of a confocal optical microscope. Our scheme improves the spatial resolution in the direction of the optical axis. The confocal optical microscope, by use of a four-wave mixing geometry, can obtain optical information about weakly absorptive objects with organic dyes. Owing to the third-order nonlinear dependence of absorptive objects, the optical signal is confined to the focal region of the focusing incident beam.

Journal Article↗

PubMed2006

magp4 gene may contribute to the diversification of cichlid morphs and their speciation.

Lake Victoria harbors more than 300 species of cichlid fish, which are adapted to a variety of ecological niches with various morphological species-specific features. However, it is believed that these species arose explosively within the last 14,000 years and transcripts among Lake Victoria cichlid species are almost identical in sequence. These data prompted us to develop a DNA chip assay to compare patterns of gene expression among cichlid species. We prepared a DNA chip spotted with 6240 elements derived from cichlid expressed sequence tag (EST) clones and successfully characterized gene expression differences between the cichlid species Haplochromis chilotes and Haplochromis sp. "rockkribensis". We identified 14 transcripts that were differentially expressed between these species at an early developmental stage, 15 days post-fertilization (dpf), and several were further analyzed using quantitative real-time PCR (qPCR). One of these differentially expressed transcripts was a homolog of microfibril-associated glycoprotein 4 (magp4), a putative causative gene for the human inherited disease, Smith-Magenis syndrome (SMS), for which facial defects are among the phenotypic features. Further analysis of magp4 expression showed that magp4 was expressed in the jaw portion of cichlid fry and that expression profiles between Haplochromis chilotes and Haplochromis sp. "rockkribensis" differed during development. These data suggest that the differential expression of a gene associated with human cranial morphogenesis may be involved in the diversification of cichlid jaw morphs.

Animals↗

PubMed2006

Mapping of panda plumage color locus on the microsatellite linkage map of the Japanese quail.

BACKGROUND: Panda (s) is an autosomal recessive mutation, which displays overall white plumage color with spots of wild-type plumage in the Japanese quail (Coturnix japonica). In a previous study, the s locus was included in the same linkage group as serum albumin (Alb) and vitamin-D binding protein (GC) which are mapped on chicken (Gallus gallus) chromosome 4 (GGA4). In this study, we mapped the s locus on the microsatellite linkage map of the Japanese quail by linkage analysis. RESULTS: Segregation data on the s locus were obtained from three-generation families (n = 106). Two microsatellite markers derived from the Japanese quail chromosome 4 (CJA04) and three microsatellite markers derived from GGA4 were genotyped in the three-generation families. We mapped the s locus between GUJ0026 and ABR0544 on CJA04. By comparative mapping with chicken, this locus was mapped between 10.0 Mb and 14.5 Mb region on GGA4. In this region, the endothelin receptor B subtype 2 gene (EDNRB2), an avian-specific paralog of the mammalian endothelin receptor B gene (EDNRB), is located. Because EDNRB is responsible for aganglionic megacolon and spot coat color in mouse, rat and equine, EDNRB2 is suggested to be a candidate gene for the s locus. CONCLUSION: The s locus and the five microsatellite markers were mapped on CJA04 of the Japanese quail. EDNRB2 was suggested to be a candidate gene for the s locus.

Animals↗

PubMed2006

Cell adhesion molecules for targeted drug delivery.

Rapid advancement of the understanding of the structure and function of cell adhesion molecules (i.e., integrins, cadherins) has impacted the design and development of drugs (i.e., peptide, proteins) with the potential to treat cancer and heart and autoimmune diseases. For example, RGD peptides/peptidomimetics have been marketed as anti-thrombic agents and are being investigated for inhibiting tumor angiogenesis. Other cell adhesion peptides derived from ICAM-1 and LFA-1 sequences were found to block T-cell adhesion to vascular endothelial cells and epithelial cells; these peptides are being investigated for treating autoimmune diseases. Recent findings suggest that cell adhesion receptors such as integrins can internalize their peptide ligands into the intracellular space. Thus, many cell adhesion peptides (i.e., RGD peptide) were used to target drugs, particles, and diagnostic agents to a specific cell that has increased expression of cell adhesion receptors. This review is focused on the utilization of cell adhesion peptides and receptors in specific targeted drug delivery, diagnostics, and tissue engineering. In the future, more information on the mechanism of internalization and intracellular trafficking of cell adhesion molecules will be exploited for delivering drug molecules to a specific type of cell or for diagnosis of cancer and heart and autoimmune diseases.

Animals↗

PubMed2006

Functional and phenotypic analysis of human memory CD8+ T cells expressing CXCR3.

Several chemokine receptors play an important role in the migration of naïve, memory, and effector T cells. Flow cytometric analyses showed that human CD8+ T cells with naïve (CD27+ CD28+ CD45RA+) or memory (CD27+ CD28+/- CD45RA+) phenotypes included a population expressing a high level of CXC chemokine receptor 3 (CXCR3high) and one expressing a low level of it (CXCR3low), but those with the effector phenotype (CD27- CD28- CD45RA+/-) included a population that did not express CXCR3 (CXCR3-) and a CXCR3low population. This relation between the expression level of CXCR3 and memory/effector phenotypes also applied to Epstein-Barr virus- or human cytomegalovirus-specific CD8+ T cells. CXCR3high cells were found predominantly in CC chemokine receptor 7 (CCR7)+ CCR5- and CCR7- CCR5- subsets of CD8+ T cells with the CD27+ CD28+ CD45RA- memory phenotype, suggesting that they are memory cells with intermediate differentiation. Indeed, CXCR3high CD27+ CD28+ CD45RA- CD8+ T cells had the ability to produce interleukin-2 and interferon-gamma. These results together indicate that the expression of CXCR3 is up-regulated on intermediately differentiated memory CD8+ T cells. CXCR3high CD8+ T cells had a greater ability to migrate in response to CXCR3 ligands than CXCR3low ones. As CXCR3high memory CD8+ T cells do not express CCR5, high expression of CXCR3 on these memory CD8+ T cells might play an important role in the migration of these cells to inflammatory sites and in their differentiation.

CD28 Antigens↗

PubMed2005

Inhibition of e-cadherin-mediated homotypic adhesion of Caco-2 cells: a novel evaluation assay for peptide activities in modulating cell-cell adhesion.

Transient modulation of E-cadherin-mediated cell-cell adhesion may improve paracellular drug delivery through biological barriers. Therefore, there is a need to develop an efficient method to evaluate cadherin peptides that can modulate the intercellular junctions. The objective of this study was to establish a novel assay to evaluate peptide activity in modulating E-cadherin-mediated homophilic interactions, based on the homotypic adhesion of Caco-2 cells. Fluorescence-labeled Caco-2 single cells were incubated with Caco-2 monolayers that were treated beforehand with Ca(2+)-free medium. The homotypic adhesion in the presence or absence of peptide and antibody was determined fluorometrically. The Ca(2+)-deficient pretreatment dramatically increased the number of single cells bound to the monolayers. Immunofluorescence staining showed that some of E-cadherins became accessible without surfactant-induced permeabilization of Caco-2 cell monolayers after the Ca(2+)-deficient pretreatment. The homotypic adhesion was largely dependent on extracellular Ca(2+) concentrations and significantly inhibited by the presence of anti-E-cadherin monoclonal antibody DECMA-1. In contrast, DECMA-1 did not inhibit E-cadherin-independent adhesion, such as the homotypic adhesion of Caco-2 cells in the absence of Ca(2+) or the heterotypic adhesion of Molt-3 T cells to Caco-2 monolayers. These results indicate the predominant involvement of E-cadherin-mediated cell-cell adhesion in this assay. E-cadherin-derived peptides, which had been shown in our previous studies to inhibit E-cadherin-mediated cell-cell adhesion, significantly inhibited homotypic adhesion in a dose-dependent manner. These results, taken together, suggest that the present assay can be used for evaluation of peptide, protein, or antibody activity in modulating the E-cadherin-mediated homophilic interactions in the context of whole live cells.

Antibodies, Monoclonal↗

PubMed2005

Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference.

PURPOSE: RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs. METHODS: The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively. RESULTS: Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b. CONCLUSIONS: Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

Animals↗

PubMed2005

A CpG-containing oligodeoxynucleotide as an efficient adjuvant counterbalancing the Th1/Th2 immune response in diphtheria-tetanus-pertussis vaccine.

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component(s). Diphtheria-tetanus-pertussis (DPT) vaccine contains not only aluminum hydrate (alum) to enhance the immune response to the vaccine ingredients, but also, both for that purpose and as a principal ingredient, pertussis toxin (PT). However, both adjuvants strongly promote T helper (Th) 2 type immune responses. Th1 and Th2 type immune responses are counterbalanced in vivo, and a Th2-prone immune response is not effective against intracellular infections but promotes IgE production, which is related to allergic disease. In this study, we used the CpG motif contained in oligodeoxynucleotide (CpG-ODN), which has an adjuvant effect and also induces the Th1 response, as an adjuvant to this vaccine, and we investigated its adjuvanticity and its potential to modulate immune responses to DPT vaccine. Administration of DPT vaccine with CpG-ODN (DPT-alum/ODN) to mice significantly reduced the total IgE levels and increased the anti-PT specific IgG2a titer in serum, in comparison with ordinary DPT vaccine (DPT-alum). Moreover, we investigated the antibody response to orally administrated ovalbumin (OVA) after vaccine administration. In the DPT-alum/ODN-administered group, the OVA specific IgE production in serum greatly decreased in comparison with that in the DPT-alum-administered group. These data indicate that CpG-ODN was not useful only as an efficient vaccine adjuvant but also shifted the immune responses substantially toward Th1 and modulated the Th1/Th2 immune response in DPT vaccine. These data suggested new applications of CpG-ODN as adjuvants in DPT vaccine.

Adjuvants, Immunologic↗

PubMed2005

Gravity-assisted pivot-shift test for anterior cruciate ligament injury: a new procedure to detect anterolateral rotatory instability of the knee joint.

The denominated gravity-assisted pivot-shift test was introduced as a new procedure to detect anterolateral rotatory instability of the knee joint. The patient lies in the supine position or slightly rotated onto the affected side. The affected knee flexed approximately 60 degrees and the ipsilateral hip flexed, abducted and externally rotated so that the plane of the knee motion runs parallel to the floor. The examiner instructs the patient to raise the affected leg off the examining table and to extend the affected knee gradually. If the lower leg is internally rotated suddenly, with the knee subluxated at an angle of approximately 20 degrees , followed by the reduction in flexion, this test is regarded as positive. This test was investigated on 51 anterior cruciate ligament (ACL) deficient knees, being positive in 30 knees (Group P) and negative in 21 (Group N) with the positive rate of 59%. There was no significant correlation between the result of this test and the clinical features, but Group N included relatively small number of females and recurrent injuries tended to occur more frequently in Group P. Thirty-six knees received ACL reconstruction subsequently. There was no statistically significant difference between the groups in the side-to-side difference in anterior knee laxity at one year postoperatively. However, three patients with the side-to side difference of more than 3 mm belonged to Group P. Relatively low positive rate in ACL deficient knees suggests that it may not be used as a diagnostic procedure for ACL injury. It is possibly used for the prediction of high risk patients for symptomatic giving-way and/or patients with poor prognosis after ACL reconstruction.

Adolescent↗

PubMed2005

Manipulation of local disposition and gene expression characteristics of plasmid DNA following intramuscular administration by complexation with cationic macromolecule.

To modulate the immune responses of DNA vaccine, it is very important to control the disposition and gene expression of plasmid DNA (pDNA) after local administration. We chose methylated bovine serum albumin (mBSA), a cationic macromolecule, as a carrier of pDNA. We examined the effects of complexation of pDNA with mBSA on the disposition and gene expression in mice after intramuscular administration. The elimination from injection site was retarded and the accumulation to lymph nodes was increased at the positively charged mBSA/pDNA complexes. As the charge ratios of mBSA/pDNA complexes were higher, the levels of gene expression were reduced. Antigen specific immune responses were evaluated using pDNA encoding ovalbumin (OVA), pCMV-OVA, as a model antigen-expressing pDNA. However, significant levels of production of anti-ovalbumin IgG antibody were obtained in mice immunized with a positively charged complex, mBSA/pCMV-OVA (8:1) (weight ratio). In vitro experiments using DC2.4 cells, a murine dendritic cell line, demonstrated that the levels of gene expression and cytokine release were increased by complexation. These results suggest that the immune responses might be manipulated by complexation presumably due to the altered disposition and gene expression of pDNA.

Animals↗

PubMed2005

The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels.

Hydrodynamics-based gene delivery, involving a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), gives a significantly high level of transgene expression in vivo. This has attracted a lot of attention and has been used very frequently as an efficient, simple and convenient transfection method for laboratory animals. Until recently, however, little information has been published on the pharmacokinetics of the injected DNA molecules and of the detailed mechanisms underlying the efficient gene transfer. We and other groups have very recently demonstrated that the mechanism for the hydrodynamics-based gene transfer would involve, in part, the direct cytosolic delivery of pDNA through the cell membrane due to transiently enhanced permeability. Along with the findings in our series of studies, this article reviews the cumulative reports and other intriguing information on the controlled pharmacokinetics of naked pDNA in the hydrodynamics-based gene delivery. In addition, we describe various applications reported so far, as well as the current attempts and proposals to develop novel gene medicines for future gene therapy using the concept of the hydrodynamics-based procedure. Furthermore, the issues associated with the clinical feasibility of its seemingly invasive nature, which is probably the most common concern about this hydrodynamics-based procedure, are discussed along with its future prospects and challenges.

Animals↗

PubMed2005

High-resolution optical storage by use of minute spheres.

We have developed a new optical storage system that uses minute spheres arranged upon a surface-relief grating. By using dye-doped minute spheres as recording bits we can limit a sensitive region within a sphere's diameter. Using a reflection-type confocal optical system, we read out shape signals from minute spheres at high resolution. The shape signals from minute spheres are utilized as clock signals in recording and readout.

Journal Article↗

PubMed2005

Determination of the standard value of circulating blood volume during anesthesia using pulse dye-densitometry: a multicenter study in Japan.

PURPOSE: The standard value for circulating blood volume (BV) during anesthesia was determined by a multicenter study in Japan. The significance of BV on the reduction of blood pressure after the induction of anesthesia was also examined. METHODS: The study included 184 patients from eight university hospitals. After the induction of anesthesia, pulse dye-densitometry was performed according to a uniform protocol. Factors contributing to reduced blood pressure after induction of anesthesia were examined by multiple logistic regression analysis. RESULTS: The mean and standard deviation of BV was 80.0 +/- 13.9 ml x kg(-1); for females and 84.2 +/- 15.3 ml x kg(-1) for males (P > 0.05). There was no age difference in terms of BV. After adjusting for the effects of height, weight, and age, the factors predisposing to a reduction in blood pressure of >20 mmHg after induction of anesthesia were found to be age (P < 0.01) and BV (ml x kg(-1)) (P < 0.001). CONCLUSION: We determined the BV of anesthetized patients before surgery in Japan using pulse dye-densitometry. It is suggested that age is not a factor regarding BV, and that blood pressure tends to be reduced in hypovolemic patients after induction of anesthesia.

Adult↗

PubMed2005

Pulse total-hemoglobinometer provides accurate noninvasive monitoring.

OBJECTIVE: Rapid noninvasive measurement of total hemoglobin would be extremely useful for various clinical situations. This study determined the clinical accuracy and utility for a pulse total-hemoglobinometer using four wavelengths: 660 nm (reduced hemoglobin), 805 nm (isosbestic point), 940 nm (oxygenated hemoglobin), and 1300 nm (water density). DESIGN: Clinical trial. SETTING: University school of medicine. PATIENTS: Patients were 122 individuals (age, 18-82 yrs; 49.4 +/- 16.0 yrs [mean +/- SD]), including 71 healthy volunteers, 24 patients undergoing surgery, and 27 patients undergoing hemodialysis. INTERVENTIONS: The hemoglobinometer probe, which simultaneously indicated peripheral oxygen saturation, pulse rate, and hemoglobin, was placed on the fingertip similarly to a regular pulse oximeter. The hemoglobin values were compared with those obtained by the co-oximeter or the sodium lauryl sulfate-methemoglobin method. Those hemoglobin values were assigned to either the training set or the validation set for statistical evaluation. MEASUREMENTS AND MAIN RESULTS: Multiple regression analysis including the ratio of the pulsatile optical density (phi(ij)) derived from the four wavelengths and other factors demonstrated that the mean value of the normalized pulse wave obtained from the photodiode at 805 nm (DC805) and the ratios of DC940 and DC1300 (DC940/DC1300) were the pivotal factors in the hemoglobinometer's increased accuracy in the clinically useful range. The coefficient of determination between both methods was r2 = .81 (p < .0001) in the training set and r2 = .75 (p < .0001) in the validation set. When the cutoff value of anemia was set at 10 g/dL, and anemia was defined as <10 g/dL, the respective sensitivity and specificity of hemoglobinometer values to detect anemia in intraoperative patients were 84.3% and 84.6% (n = 20). CONCLUSIONS: The data demonstrated the necessity for consideration of light scattering in red blood cells for pulse-spectrophotometric hemoglobin measurement. This was accomplished with additional factors, such as DC805 and DC940/DC1300. With these improvements, the pulse hemoglobinometer provided noninvasive, clinically acceptable measurement of hemoglobin. The pulse hemoglobinometer is a versatile tool that might be useful for routine health checkups of neonates and young children, intraoperative monitoring of bleeding, and emergency care.

Adult↗

PubMed2004

Extensive analysis of ORF sequences from two different cichlid species in Lake Victoria provides molecular evidence for a recent radiation event of the Victoria species flock: identity of EST sequences between Haplochromis chilotes and Haplochromis sp. "Redtailsheller".

The Lake Victoria Cichlid fishes have diverged very rapidly. The estimated 500 species inhabiting the lake are believed to have arisen within the last 14,000 years. The fishes' jaws and teeth have diverged markedly to adapt to different feeding behaviors and environments. To examine how the genomes of these fishes differentiated during speciation, we performed comparative analysis of expressed sequenced tag (EST) sequences. We constructed cDNA libraries derived only from the jaw portions of two cichlid species endemic to Lake Victoria. We sequenced 17,280 cDNA clones from Haplochromis chilotes and 9600 cDNA clones from Haplochromis sp. "Redtailsheller" and obtained 543 different genes common to both species. Of these genes, 441 were essentially identical between species and 102 contained base replacements in their open reading frame (ORF) or untranslated (UTR) regions. Comparative analysis of 71 selected sequences has revealed that while the degree of polymorphism is 0.0054/site for H. chilotes and 0.0047/site for H. sp. "Redtailsheller", genetic distance between the two species is 0.0031/site. The genetic distance particularly indicates that the two species diverged about 890,000 years ago.

Africa, Eastern↗

PubMed2004

Organotin compounds promote adipocyte differentiation as agonists of the peroxisome proliferator-activated receptor gamma/retinoid X receptor pathway.

Nuclear receptors play important roles in the maintenance of the endocrine system, regulation of organ differentiation, and fetal development. Endocrine disruptors exert their adverse effects by disrupting the endocrine system via various mechanisms. To assess the effects of endocrine disruptors on nuclear receptors, we developed a high-throughput method for identifying activators of nuclear receptors. Using this system, we found that triphenyltin and tributyltin were activators of peroxisome proliferator-activated receptor (PPAR) gamma and retinoid X receptor. Because PPARgamma is a master regulator of adipocyte differentiation, we assessed the effect of organotin compounds on preadipocyte 3T3-L1 cells. We found that organotin compounds stimulated differentiation of 3T3-L1 cells as well as expression of adipocyte marker genes.

3T3 Cells↗