Yuichiro Cho

Development of a new measurement method for serum calcium with chlorophosphonazo-III

Background Although serum calcium has been measured using the o-cresolphthalein complexone (oCPC) method in the clinical laboratory, this method still has some problems regarding linearity and reagent stability. We developed a new measurement procedure using chlorophosphonazo-III (CPZ-III: 2,7-bis (4-chloro-2-phosphonophenylazo) -1,8- dihydroxy-3, 6-naphthalenedisulphonic acid, disodium salt) as a chelator with an acid medium for serum calcium measurement. The present method showed better linearity and reagent stability compared with the oCPC method. Methods Characteristics were studied in optimized conditions measuring wavelength by absorption spectra analysis, and interference of protein and metals with Mg2+, Fe2+, Cu2+ and Zn2+. The method was applied to an automated analyser (7170; Hitachi High Technologies Corp). The measurement performance was evaluated for accuracy, precision, recovery rate, linearity and reagent stability with a comparison study against atomic absorption spectrophotometry (AAS). Results The within-run and between-run variations (coefficient of variation [CV]) were 0.92–1.01% and 0.75–1.43%, respectively. The linearity was 0–7.0 mmol/L. The comparison study obtained y = 1.002x (AAS) – 0.10, Sy/x = 0.18 mmol/L, n = 50. Reagent stability was at least 20 d at 4°C without daily calibration. Conclusion The new calcium measurement method in serum was demonstrated to have reliable and acceptable performances as a routine test in clinical laboratory.

The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

Morphological analysis of the external anal sphincter motor nerve and its motoneurons in the cat

To investigate the spinal neural circuitry that controls the tonus of the external anal sphincter (EAS) in the cat, the size distribution of EAS motor fibers and their motoneurons (MN) was examined, and the presence of muscle spindles in EAS musculature was also tested for. The size distribution of EAS motor fibers was examined after degeneration of afferent fibers and that of their MN was measured, after labeling the cells with horseradish peroxidase. Both distributions were unimodal, thereby demonstrating the difficulty of distinguishing between alpha and potential gamma MN; but muscle spindles were not found in the musculature. Mechanisms underlying the spinally controlled tonus of the EAS remain unclear, including the nature and role of spinal reflexes. It is argued that non-spindle sensory receptors in the anal canal may provide the sensory component of a reflex circuit that contributes to this tonus.