Tadashi Okada

c-kit mutation in a population of patients with mastocytosis

The c-kit Asp816Val activating mutation is found in all patients with mastocytosis with an associated hematologic disorder, and at least in a subset of patients with indolent mastocytosis. The case of an 11-month-old child is presented who was categorized as having indolent mastocytosis, and where the Asp816Val mutation was identified in lesional skin, but not in bone marrow or in peripheral blood mononuclear cell populations. The significance of these findings is discussed.

Protein separation by nonsynchronous coil planet centrifuge with aqueous–aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.

Treadmill running and static stretching improve long-lasting hyperalgesia, joint limitation, and muscle atrophy induced by cast immobilization in rats

The effects of exercise on chronic pain induced by immobilization are incompletely understood. The purpose of this study was to investigate whether 30min of treadmill running (TR; active exercise) and 10min of static stretching (SS; passive exercise) of the immobilized hindlimb reduce widespread chronic pain, joint limitation, and hindlimb muscle atrophy induced by cast immobilization in rats. One hindlimb of Sprague Dawley (SD) rats was immobilized for 2 weeks with a cast, and remobilization was conducted for 7 weeks. MRI study showed that cast immobilization had induced inflammatory changes in the immobilized hindlimb, beginning as early as 2h after cast removal; these changes continued for 2-3 days. Mechanical hyperalgesia in the calf and hindpaw developed as early as 2h after cast removal and continued for 7 weeks. TR and SS were initiated 3 days after cast removal and were continued 3 times per week for 2 weeks. Both forms of exercise significantly inhibited mechanical hyperalgesia in the calf and hindpaw in immobilized rats. Range-of-motion limitations in the knee and ankle joints and calf muscle atrophy after cast removal were also decreased by both TR and SS. This study is the first to demonstrate the beneficial effect of TR and SS on widespread chronic pain, joint limitation, and muscle atrophy in a cast-immobilized rat model.

Lansiumamide B and SB-204900 isolated from Clausena lansium inhibit histamine and TNF-α release from RBL-2H3 cells

Aims and objectiveMast cells play a central role in allergic and chronic inflammation. Extracts from Clausena lansium (Lour.) Skeels (Rutaceae) possess many pharmacological effects including anti-inflammatory, anti-oxidant, anti-cancer, and anti-trichomonal activities. In addition, the leaves and fruit are used in Chinese folk medicine. We have isolated and identified four known cinnamamides from this plant: lansiumamide C, lansamide I, lansiumamide B, and SB-204900. However, the biological activities of these compounds are not yet understood. The purpose of this paper is to clarify the pharmacological effects of these compounds on mast cells.MethodsWe measured inflammatory molecules in A23187-stimulated rat basophilic leukemia cells (RBL-2H3) treated with these compounds using HPLC, ELISA, and immunoblotting methods. In addition, some signaling molecules were investigated by immunoblotting.ResultsLansamide I, lansiumamide B, and SB-204900 significantly decreased histamine release. Furthermore, lansiumamide B- and SB-204900-treated cells also reduced the protein and/or mRNA levels of TNF-α. SB-204900 markedly suppressed the phosphorylation of p38 MAPK.ConclusionOur findings suggest that lansiumamide B and SB-204900 attenuate mast-cell-induced inflammation.

Effect of natsudaidain isolated from Citrus plants on TNF‐α and cyclooxygenase‐2 expression in RBL‐2H3 cells

Objectives Flavonoids inhibit the activity of chemical mediators released from mast cells. Our aim was to investigate the effects of natsudaidain, a polymethoxyflavone isolated from Citrus plants, on mast cells.

Purification of Mast Cells with an Improved Nonsynchronous Flow-Through Coil Planet Centrifuge

A method for cell purification was designed without using high-density media which may impair membrane receptors. Rat and mouse mast cells were separated with an improved nonsynchronous flow-through coil planet centrifuge. Peritoneal cells were suspended at a concentration of 2-3x10(7) cells/ml in conditioned RPMI 1640, supplemented with 50% heat-inactivated FCS and 0.32% sodium citrate. In each separation 3 ml of cell suspension were loaded into the coiled column and elutriated at 4 degrees C. Several conditions, including the centrifugal force, revolution/rotation ratio, density of separation media, flow speed, and designs of both coiled column and flow tubes, were examined and optimized for mast cell purification. Rat mast cells were separated at the purity of 99.2%, with an average yield of 40% under sterile conditions. Nearly 90% pure mouse mast cells were harvested, despite a very low population of mast cells available in murine peritoneal cells. Purified cells were morphologically intact and discharged granules by exocytosis as indicated by electron-microscopic observations. The average histamine release with antigenic specificity was 34 and 61%, in passive sensitization in vitro and in vivo, respectively. Mast cells sensitized with mouse monoclonal IgE antibody released histamine, similar to cells sensitized with homologous antibody. This newly devised method of cell separation will be useful to purify biologically intact mast cells.

Application of a Novel Continuous‐Flow Cell Separation Method for Separation of Cultured Human Mast Cells

Abstract A novel, continuous cell separation method, based on density differences, is developed for application to transfusion medicine. Performance of a miniature separation column with ca. 8 mL capacity was examined on separation of human buffy coat (containing 107 nucleated cells and 1010 erythrocytes/mL). Differential leukocyte counts revealed that lymphocytes were concentrated in two fractions of density 1.065 to 1.070. Neutrophils were located in another fraction (density 1.080), while some cells present in early eluted low density fractions were difficult to identify by microscope examination. Flow cytometry analysis revealed that CD34‐positive cells, which are considered to be stem cells, were relatively concentrated in an early eluted fraction. Comparing the data obtained from hemocytometer and flow cytometry suggested the presence of immature CD45‐negative nucleated cells in the same fraction. These results indicate that the present density method might be capable of collecting CD34‐negative stem cells from peripheral blood. The present method was applied to the separation of cultured human mast cells. When the co‐cultured cell suspension containing 3.5×106 cells (approximately 10% of mast cells and 90% of fibroblasts) was separated with a set of media (density: 1.065–1.085); fibroblasts were concentrated in a fraction of density 1.065 with a few young mast cells. Densely granulated matured mast cells were collected in a fraction of density 1.085. The method could also be used to detect minute changes in cell density under various physiological and pathological conditions.

Protein and Cell Separations using Nonsynchronous Coil Planet Centrifuge

Abstract The nonsynchronous coil planet centrifuge has a unique mode of planetary motion in that it allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. This paper describes a series of studies using the apparatus fabricated in our laboratory on the countercurrent chromatographic separation of proteins with aqueous‐aqueous polymer phase systems, experimental and theoretical analysis of the effect of planetary motion on protein separation, and application to elutriation of cells such as blood cell components and mast cells using a single‐phase physiological buffer solution.

Lapachol suppresses cell proliferation and secretion of interleukin-6 and plasminogen activator inhibitor-1 of fibroblasts derived from hypertrophic scars

Objectives  The pathogenesis and therapy of hypertrophic scar have not yet been established. Our aim was to investigate the antiproliferative and antisecretory effects of lapachol, isolated from the stem bark of Avicennia rumphiana Hall. f., on hypertrophic scar fibroblasts.

Elutriation of Blood Cell Components and Mast Cells by Nonsynchronous Coil Planet Centrifuge

Abstract The nonsynchronous coil planet centrifuge was applied to the separation of blood cell components and mast cells using a physiological buffer solution. The optimal separation of sheep blood samples was performed at the counterclockwise revolution and the clockwise rotation of coiled separation column by the head to tail elution mode. Under the high speed of revolution of 600–1000 rpm, sheep and human blood samples were completely separated and eluted into two peaks, where all cell components, other than erythrocytes such as platelets and leukocytes, were eluted at 0 rpm and erythrocytes at 10 rpm of coil rotation. The normal elution pattern was achieved at 800 rpm of revolution for sheep blood and at 700 rpm for human blood samples. The separation of mast cells, large cells with a high density, was also performed using an eluent composed of RPMI 1640 medium+10% FCS. Mast cells were satisfactorily separated from other cell components under a high speed revolution combined with a low speed of coil rotation (2–5 rpm). The overall results indicate that the nonsynchronous CPC can be effectively used for separation of blood cell components and mast cells according to their sedimentation rates.