Biomedical subjects

Noriyuki Yamamoto

Publications and source records attributed to Noriyuki Yamamoto.

7 recordsLinked to original sources

PubMed2003

Decrease in heart rates by artificial CO2 hot spring bathing is inhibited by beta1-adrenoceptor blockade in anesthetized rats.

To investigate the effects of carbon dioxide (CO2) hot spring baths on physiological functions, head-out immersion of urethane-anesthetized, fursheared male Wistar rats was performed. Animals were immersed in water (30 or 35 degrees C) with high-CO2 content ( approximately 1,000 parts/million; CO2-water). CO2-water for bathing was made by using an artificial spa maker with normal tap water and high-pressure CO2 from a gas cylinder. When a human foot was immersed for 10 min in the CO2-water at 35 degrees C, the immersed skin reddened, whereas skin color did not change in normal tap water at the same temperature. Arterial blood pressure, heart rate (HR), underwater skin tissue blood flow, and temperatures of the colon and immersed skin were continuously measured while animals were immersed in a bathtub of water for approximately 30 min at room temperature (26 degrees C). Immersed skin vascular resistance, computed from blood pressure and tissue blood flow, was significantly lower in the CO2-water bath than in tap water at 30 degrees C, but no differences were apparent at 35 degrees C. HR of rats in CO2-water was significantly slower than in tap water at 35 degrees C. Decreased HR in CO2-water was inhibited by infusion of atenolol (beta1-adrenoceptor blocker), but it was unaffected by atropine (muscarinic cholinoceptor blocker). Theses results suggest that bradycardia in CO2 hot spring bathing is caused by inhibition of the cardiac sympathetic innervation. This CO2-water maker should prove a useful device for acquiring physiological evidence of balneotherapy.

Adrenergic beta-Antagonists↗

PubMed2003

The orally available spleen tyrosine kinase inhibitor 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]nicotinamide dihydrochloride (BAY 61-3606) blocks antigen-induced airway inflammation in rodents.

Spleen tyrosine kinase (Syk) tyrosine kinase plays essential roles in receptors for Fc portion of immunoglobulins and B cell receptor complex signaling in various inflammatory cells; therefore, inhibitors of Syk kinase may show potential as antiasthmatic/allergic therapeutics. We identified 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY 61-3606), a potent (Ki = 7.5 nM) and selective inhibitor of Syk kinase. BAY 61-3606 inhibited not only degranulation (IC50 values between 5 and 46 nM) but also lipid mediator and cytokine synthesis in mast cells. BAY 61-3606 was highly efficacious in basophils obtained from healthy human subjects (IC50 = 10 nM) and seems to be at least as potent in basophils obtained from atopic (high serum IgE) subjects (IC50 = 8.1 nM). B cell receptor activation and receptors for Fc portion of IgG signaling in eosinophils and monocytes were also potently suppressed by BAY 61-3606. Oral administration of BAY 61-3606 to rats significantly suppressed antigen-induced passive cutaneous anaphylactic reaction, bronchoconstriction, and bronchial edema at 3 mg/kg. Furthermore, BAY 61-3606 attenuated antigen-induced airway inflammation in rats. Based on these anti-inflammatory effects of BAY 61-3606 both in vitro and in vivo, it was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions.

Administration, Oral↗

PubMed2003

Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors.

Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS.

Buffers↗

PubMed2003

Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines.

Adenovirus (Ad) vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by immune responses that reduce transgene expression and decrease the efficiency of repeated vector administration. In this study, the efficacy of gene transduction and the tumor-cell killing effect on four human oral (SAS, HSC-2, HSC-3, HSC-4) and one murine squamous cell carcinoma cell (SCC-7, a kind gift of Dr. M. Hiraoka, Kyoto University) lines in vitro with Ad vector conjugated with catioic liposome (Ad/SUV) was evaluated. Ad/SUV resulted in two to five-fold over higher transduction efficiency in four human and one murine cell lines in vitro than Ad vector alone. The optimal Ad-SUV ratio was determined as 10(6) pfu of Ad vector with 1 micromol SUV. Ad/SUV showed more tumor-cell killing effect than Ad vector alone. Furthermore, the shielding effects of Ad vector with Ad/SUV from neutralizing antibody were evaluated. We also found that Ad/SUV is less susceptible to inactivation by neutralizing antibodies in vitro. The efficacy of gene transduction with Ad vector was blocked more than 70% with neutralizing serum, while Ad/SUV retained approximately 50% of the control activity in vitro. On the basis of these results, the anti-tumor effect with suicide gene therapy using Ad/SUV in vivo was evaluated. Three injections of Ad/SUV showed the inhibition of tumor growth compared with control in vivo. Our results suggested that an enhanced anti-tumor effect on human oral squamous cell carcinoma would be obtained with repeated administrations of Ad/SUV.

Adenoviridae↗

PubMed2003

Effects of autologous serum on osteoblastic differentiation in human bone marrow cells.

BACKGROUND: Expanding cells ex vivo is an important step in tissue engineering and the culture medium is usually supplemented with bovine serum. When a patient receives bone marrow stromal cells (BMSCs) grown in a medium containing bovine serum, infection of bovine diseases and/or the patient's unfavorable immune reaction to bovine proteins are of concern. To overcome these problems, we examined whether a patient's autologous serum could support the growth and differentiation of his/her BMSCs. METHODS: Bone marrow was collected from the iliac by aspiration and cultured in a medium supplemented with 10% autologous serum or 10% fetal bovine serum (FBS). Number and area of the colonies of fibroblast-like cells (colony-forming unit fibroblast, CFU-f) were measured 8 days after the cell inoculation (day 8). Number and area of the alkaline phosphatase (ALP) positive colonies were measured on day 10. On day 40, the cultures were stained with alizarin red S. RNA was prepared from the culture on day 20, and the mRNA expression of osteoblastic genes was examined by reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: BMSCs, which were cultured in the medium supplemented with autologous serum, produced CFU-f, ALP-positive area and mineralized nodules, which is comparable to the BMSCs in the culture supplemented with FBS. The mRNA expressions of osteopontin, parathyroid hormone receptor, osteocalcin, and bone sialoprotein were detected in the culture supplemented with autologous serum. CONCLUSIONS: A patient's autologous serum could expand BMSCs without losing their potentiality for osteoblastic differentiation. Patients' autologous serum could be efficient to expand BMSCs and to be utilized safely for bone regeneration therapy.

Adolescent↗

PubMed2002

Establishment of the superoxide production assay with human monocytic cell line, U937, for the evaluation of Syk kinase inhibitors.

Protein tyrosine kinase Syk is known to play critical roles in the signal transduction from receptors for Fc portion of immunoglobulins (FcRs) and B cell receptor complex (BCR). Its importance was well studied in Fc epsilon RI-induced activation of mast cells; therefore, Syk inhibitors are expected to have anti-allergic effects and to be novel therapy for allergic diseases, such as asthma, allergic rhinitis and atopic dermatitis. We previously developed an enzyme assay of recombinant human Syk kinase for the high throughput screening. In order to evaluate the Syk kinase inhibitors in a human cell system, we have developed an assay with human monocytic cell line, U937, to monitor FcgammaRI-mediated superoxide production. We treated cells with IFN-gamma to enhance the expression of FcgammaRI and to obtain enough production of superoxide. Engagement of FcgammaRI stimulated superoxide production, which was accompanied with Syk phosphorylation. PMA, an activator of protein kinase C, also evoked superoxide production, but Syk was not phosphorylated. Moreover, the treatment of cells with antisense oligonucleotide against syk attenuated Syk protein expression and suppressed superoxide production induced by FcgammaRI-engagement, but not by PMA. These results confirm that Syk is involved in the signal transduction from FcgammaRI upstream of PKC in U937 cells and we can evaluate the efficacy and the selectivity of Syk inhibitors with this assay system.

Enzyme Precursors↗

PubMed2002

Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure.

It has been shown that norepinephrine (NE) can mediate vasodilatation by stimulating the production of nitric oxide (NO) in brown adipose tissue (BAT), resulting in an increase in BAT blood flow. We speculated that constitutive NO synthase (NOS) is involved in this NO production. However, it is not known whether constitutive NOS is expressed in BAT. To answer this question, we assessed the expression of two types of constitutive NOS, endothelial (eNOS) and neuronal NOS (nNOS), in BAT of rats. eNOS was abundantly expressed in both BAT and isolated brown adipocytes, whereas nNOS was not. Cold exposure, which is known to stimulate NE release from sympathetic nerve terminals in BAT, led to a significant increase in eNOS mRNA in this tissue. In contrast, very low levels of inducible NOS (iNOS) mRNA were expressed, and cold stimulation failed to increase iNOS mRNA levels in BAT. These results suggest that eNOS is the primary isoform that is responsible for NO production in BAT and that its expression may be under sympathetic control.

Adaptation, Physiological↗