Minoru Kino

Detection of adenovirus DNA in clinical samples by SYBR Green real-time polymerase chain reaction assay

Abstract Background : Adenoviruses are associated with a variety of diseases including upper respiratory tract infections, acute conjunctivitis, cystitis and gastroenteritis. Adenoviruses can also cause fatal disseminated infections in patients undergoing stem cell transplantation. Measurement of adenovirus load in clinical samples from localized adenovirus infections or disseminated adenovirus infections may provide important information for analyzing the pathogenesis of various adenovirus infections. The purpose of the present study was to develop and optimize a highly sensitive real‐time polymerase chain reaction (PCR) assay to detect a wide range of adenoviruses and to detect adenovirus DNA in clinical samples from immunocompetent children.

Endothelin-1 has a priming effect on production of superoxide anion by alveolar macrophages : its possible correlation with bronchopulmonary dysplasia

The purpose of this study was to clarify the role of endothelin (ET)-1 in the development of bronchopulmonary dysplasia (BPD). Tracheal aspirates were obtained from 27 newborn babies with respiratory distress (13 with BPD and 14 without BPD) who were mechanically ventilated. Production of superoxide anion(O-2) by rabbit alveolar macrophages was determined by preincubation with the tracheal aspirate supernatant (TAS) and stimulation with phorbol myristate acetate (PMA). O-2 production was demonstrated only when PMA was added to the experimental system and was enhanced with TAS of infants who later developed BPD compared with TAS from infants without BPD. The effects of ET-1 and ET-3 on O-2 production and the blockade by anti-ET-1 antibody and BQ123 (ET A receptor antagonist) were also examined. The enhancing effect was blocked by either anti-ET-1 antibody or BQ123. PMA-stimulated production of O-2 increased when cells were preincubated with several doses of ET-1 (5 × 10-13 to 2 × 10-12 M), whereas ET-3 was without effect. TAS contained significant amounts of immunoreactive ET-1, and there was a close positive correlation (r = 0.764) between the activity of O-2 production and immunoreactive ET-1 levels in TAS samples. These results may be interpreted to indicate that ET-1 synthesized by and secreted from tracheal epithelial cells and/or alveolar macrophages has a priming effect on alveolar macrophages to produce O-2, thus possibly contributing to the development of BPD.

Vanadate effect on the Na,K-ATPase and the Na-K pump in in vitro-grown rat vascular smooth muscle cells.

The impact of vanadate on the Na,K-ATPase system in the vascular smooth muscle cell is poorly understood. The present study describes the kinetics of the effect of vanadate on Na,K-ATPase and the Na-K pump in in vitro grown rat VSMCs. Vanadate interaction with the Na,K-ATPase system in vascular smooth muscle cells was examined by observing its influence on ouabain-sensitive adenosine triphosphate hydrolysis in disrupted cells rendered permeable by osmotic shock, and the uptake of rubidium by intact cells. The I50 for vanadate inhibition of ouabain-sensitive hydrolysis of adenosine triphosphate occurred at vanadate concentrations of 10−* to 10″7 M. This inhibition was potassium dependent. The maximal inhibitory effect of vanadate occurred at potassium concentrations of 10–20 mEq/liter. Sodium exerted a moderate antagonistic influence on vanadate inhibition of ouabain-sensitive adenosine triphosphate hydrolysis. Rubidium uptake by vascular smooth muscle cells was not altered within 120 minutes when 10−1 M vanadate was added to the medium containing intact vascular smooth muscle cells. Yet, vanadium concentrations in the vascular smooth muscle cells within this incubation period reached levels 1.48-fold higher than the extracellular vanadate concentrations of 10″5 M. These observations indicate that vanadate is a potent inhibitor of the VSMC Na,K-ATPase in disrupted vascular smooth muscle cells. However, in intact vascular smooth muscle cells vanadium gaining access into the vascular smooth muscle cells interior does not inhibit the Na-K pump, probably because of its binding to intracellular proteins and/or conversion from the vanadate to the vanadyl ion.

Angiotensin II effect on 22Na+ transport in vascular smooth muscle cells.

It is well established that angiotensin II (AII) rapidly increases free cytosolic Ca2+ in vascular smooth muscle cells (VSMCs). Several studies have indicated that the hormone also plays a role in Na+-K+ regulation of these cells. In this study, we explored the mechanism of AII effect on 22Na+ transport in cultured rat VSMCs. The 22Na+ washout from these cells was described by three exponents with exponential factors k1 greater than k2 greater than k3. In 1.8 mM Ca2+ medium, AII (10(-9)-10(-6) M) increased (in a dose response manner) the k1 value, and consequently the initial washout rate constant (kei) for the isotope. AII had no effect on kei in Ca2+-deficient medium or in the presence of ouabain. Amiloride (10(-3) M) and verapamil (10(-5) M) abolished the AII induced increase in kei. These findings are consistent with angiotensin II stimulation of an amiloride-sensitive Na+ transport, which is likely to represent the Na+/H+ antiport. In cultured VSMCs, the sustained stimulation by AII of this transport system requires the presence of extracellular Ca2+ and its influx into these cells.

Possible splenic tuberculosis presenting as unexplained fever

problem to pediatricians. The patient described in the present report visited us with a 3 week history of remittent fever. Among various diagnostic approaches investigated, high serum adenosine deaminase (ADA) activity initially raised the possibility of tuberculosis and repeated ultrasonography (US) of the abdomen eventually helped us to make a diagnosis of possible splenic tuberculosis. The purpose of the present article is to report on such a patient and to direct attention towards abdominal tuberculosis as a possible cause of unknown fever in children.

Changes in Plasma Nitrate Levels in the Acute Phase of Kawasaki Disease

Aim: Plasma nitrate, the stable end product of nitric oxide(NO), has been reported as an indirect measure of the whole body NO production. The purpose of this study is to measure plasma nitrate in the acute phase of Kawasaki disease, and to evaluate NO pr oduction in patients with and without coronary artery lesions. Methods: Thirty patients aged 3 months to 6 years were enrolled in this study. Blood samples were obtained serially on the 1, 2, 3, 4, and 8th week of illness. Plasma nitrate was measured by h igh-performance liquid chromatography. Twenty-six patients were treated with aspirin (10-30mg/kg/day) and intravenous immunoglobulin (2g/kg single dose). Four patients received only aspirin. Results: We classified the subjects into 3 groups: normal coro nary artery (group N, n=15), coronary dilatation and aneurysm(group D, n=9), transient coronary dilatation (group T, n=6). In all groups, plasma nitrate increased significantly from the 1st week to the 2nd week (p<0.05). Peak levels of nitrate (mean+ mn;SEM, μmol/L) in each group were as follows: group N=73.0±15.8, group D=52.3±12.9, group T=58.5±4.4, respectively. Plasma nitrate fell from the 3rd week to the 4th and 8th weeks, but still elevated in each group in compariso n with age-matched healthy controls (22.1±8.8): group N=50.9±5.3, group D=46.8±9.3, group T=29.0±1.8. There were no correlations between plasma nitrates and C-reactive protein, neutrophil counts and the Harada score, respective l y. Conclusion: Increased production of NO in the first 2nd to 3rd weeks of the acute phase was observed. It was consistent with the pathological stage of generalized microvasculitis and myocarditis. Plasma nitrates in group D were lower than those in gr oup N through the course of 8 weeks, indicating decreased NO production due to impairment of the endothelial function.

The Effect of Melittin on Na+ and Rb+ Transport in Cultured Skin Fibroblasts of the Spontaneously Hypertensive Rat

Melittin effect on transport of Na+ and Rb+(K+ analog) was examined in cultured skin fibroblasts originating from the Spontaneously Hypertensive, Wistar Kyoto and Wistar rats. Melittin increased both Na+ (22Na+) uptake and 86Rb+ efflux as well as the activity of the Na+-pump (ouabain sensitive 86Rb+ uptake) in all three preparations. The effect of the toxin was maximal at a dose of 160-240ng/10(5) cells/ml. At this dose, fibroblasts of the Spontaneously Hypertensive rat demonstrated the greatest response to melittin with respect to the increase in Na+ and Rb+ fluxes and increase in the intracellular Na+ concentrations. It is concluded that melittin can be utilized as a probe to delineate subtle differences in the cellular regulation of Na+ and K+ in the Spontaneously Hypertensive rat as compared with its normotensive controls.