Yutaka Tajima

Phosphotungstate shows a heparin-like anticoagulant effect but inhibits heparin.

We examined the anticoagulant effects of phosphotungstate (PTA). PTA inhibited factors IIa and Xa activity only in the presence of antithrombin III (ATIII). The inhibitory effect of PTA on IIa was much smaller than that on Xa. When PTA was added to heparin (Hep), the inhibitory effect of Hep on IIa was reduced. As a result of charge interaction, PTA showed an inhibitory effect on anion transport activity of the erythrocyte, a potent salting-in effect and a metachromatic reaction on toluidine blue (TolB). Silicotungstate (STA) showed almost the same results as PTA. These effects of PTA and STA were not replaced by their constituents, tungstate, silicate and phosphate, but may arise from their high anionic valency.

Development of a sensitive enzyme-linked immunosorbent assay for measurement of DNase I in human serum.

BACKGROUND Serum deoxyribonuclease I (DNase I) activity was reported to increase in the early phase after onset of acute myocardial infarction (AMI). Up to now, DNase I activity has been quantified by the single radial enzyme diffusion (SRED) method, which unfortunately requires a long incubation time. Therefore it is necessary to develop another assay suitable for measurement of serum DNase I concentrations in a clinical setting. METHODS A sandwich ELISA was established for measurement of DNase I protein using a polyclonal antibody directed against DNase I protein and a biotinylated monoclonal for subsequent detection. Concentrations of serum DNase I protein were measured in healthy individuals and patients with AMI. RESULTS This method was as precise as SRED, and took less time than SRED. A significant correlation was observed between DNase I concentration and enzyme activity (r=0.839; P<0.001). The average of serum DNase I in AMI patients within 0-12 h of chest pain was significantly higher than that in healthy individuals (P<0.001), and decreased with time. CONCLUSIONS We have developed a sensitive ELISA capable of measuring DNase I protein concentrations. This method may be a useful alternative to SRED as an aid to diagnosis of AMI based on the serum DNase I level.

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I‐producing human pancreatic cancer cell line QGP‐1, and revealed a novel site ∼ 12 kb upstream of exon 1, which was previously believed to be the single transcription‐starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription‐starting exons in pancreas and QGP‐1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP‐1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT‐PCR analysis indicated alternative splicing of human DNASE1 pre‐mRNA in pancreas and QGP‐1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

An infant with asymptomatic hepatic granuloma probably caused by bacillus Calmette-Guérin (BCG) vaccination found incidentally at autopsy: a case report

IntroductionBacillus Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis. Usually, systemic complications due to BCG vaccination are quite rare. However, since BCG is a live vaccine, there is still a possibility that it may cause an infection.Case presentationHepatic granuloma was found incidentally in an asymptomatic 5-month-old infant who was found dead in his bed. The probable cause of death was asphyxia due to milk aspiration into the lungs. The granuloma was composed of epithelioid histiocytes with frequent multinucleated Langhans-type giant cells and a small number of lymphocytes.ConclusionThe cause of the asymptomatic granuloma was not identified, but was considered likely due to BCG vaccination.

Phosphotungstate as a useful eluent for antithrombin III purification by heparin-agarose affinity chromatography

We have recently found that phosphotungstate (PTA) has a heparin-like anticoagulant effect. In the present paper, we studied whether PTA is useful as an eluent of antithrombin III (ATIII) on heparin-Sepharose affinity chromatography. Human ATIII adsorbed on the heparin-Sepharose gel was eluted with NaCl buffer at the NaCl level of 1M. Whereas, PTA could elute ATIII at the level of less than 1mM and thus obtained ATIII fraction contained less impurities than such a fraction eluted with NaCl. Residual PTA in the eluate was easily decomposed by alkalization, being convenient for subsequent studies.

A case of acute myocardial infarction after intracoronary stent implantation: Demonstration of the stent location by postmortem X-ray examination

A 70-year-old man died 1 h after his car was involved in a minor collision with a stationary bus. One month before the accident, he had been diagnosed as having ischemic heart disease due to severe stenosis of the left anterior descending coronary artery (LADCA) by coronary angiography, followed by intracoronary stent implantation. Postmortem examination failed to show any potentially fatal injury, but macroscopic examination demonstrated myocardial necrosis accompanied by massive bleeding in the anterior left ventricle. Since it was difficult to delineate the precise site of the implanted stent in the heart by naked-eye examination, X-ray examination was performed. Guided by X-ray imaging, the stent, measuring 10 mm in length and 2 mm in diameter, was confirmed in the LADCA. Microscopic examination demonstrated myocardial necrosis accompanied by hemorrhage and granulation tissue in the anterior wall of the left ventricle, in the territory of the LADCA downstream from the implanted stent. However, there was no evidence of stent thrombosis. Therefore, it was likely that occlusion had occurred in a branch or branches of the LADCA downstream from the location of the stent. In conclusion, X-ray examination seems to be an effective adjunct in forensic pathology for localization of an implanted coronary stent and careful investigation of the coronary artery surrounding the stent.