Quantitative analysis of Werner helicase activity using the single-molecule fluorescence detection system MF10S

We developed a system that uses the single-molecule fluorescence detection system MF10S to assess quantitatively the activity of WRN helicase, the product of the causative gene of Werner syndrome that includes premature ageing. Double-strand DNA substrates labeled with the fluorescence dye TAMRA at the 5' end and with a quencher at the 3' end of the counter strand were incubated with a single trapper oligonucleotide and Werner helicase, and the resultant single DNA fragments labeled with TAMRA produced by the unwinding of WRN helicase were detected using the MF10S. The results using this system and those using polyacrylamide gel electrophoresis were well correlated. The MF10S system provides a quantitative analysis that is much faster, simpler, and more economical than systems using polyacrylamide gel electrophoresis and radioisotopes, and could be used as a quantitative analysis system for Werner helicase and other DNA helicase activities.     

A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects

Natto, steamed soybeans fermented by Bacillus subtilis natto, is a traditional Japanese food. We derived a purified protein layer, called NKCP as a trade mark, from B. subtilis natto fermentation. In the present study, we examined the fibrinolytic and antithrombotic effects of NKCP and identified its active component to clarify the fibrinolytic effect of NKCP observed in preliminary clinical trials previously. The active component of NKCP was identified as a 34-kilodalton protein designated bacillopeptidase F. NKCP showed direct degradation of artificial blood clot in saline. The protease activity, accounting for the fibrinolytic effect of NKCP, was examined with a chromogenic substrate for plasmin. Dose-dependent prolongations of both prothrombin time and active partial thromboplastin time were observed in rats with intra-duodenum administration of NKCP. Our in vitro and in vivo studies suggest that NKCP has both a fibrinolytic effect and an antithrombotic effect similar to heparin. Because NKCP is derived from food and has safety data demonstrated by previous animal experiments and preliminary clinical trials, NKCP is considered as safe for clinical use.     

High iron storage levels are associated with increased DNA oxidative injury in patients on regular hemodialysis

Background Accumulating evidence suggests that oxidative stress is enhanced in patients on regular hemodialysis (HD). Iron supplementation is essential for the treatment of renal anemia, but there is a possibility that it could enhance oxidative stress by inducing the Fenton reaction. Here, we report our investigation of the relation between iron storage and DNA oxidative injury in HD patients.Methods The study subjects were 48 patients on regular HD (age, 62.7 ± 12.1 years; HD duration, 67.2 ± 62.5 months; non-diabetic/diabetic; 22:26). Patients who were positive for hepatitis C virus antibody (HCV Ab), or hepatitis B surface antigen (HBsAg), and those with inflammatory or malignant diseases were excluded. The serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) level, a marker of DNA oxidative injury, was measured before the first HD session of the week in all patients, and factors associated with high serum 8-OHdG were investigated. In 9 patients with a serum ferritin level of more than 1000 ng/ml at study entry, serum 8-OHdG levels were followed up for 6 months in the absence of iron supplementation.Results Multivariate analysis showed that the serum ferritin level was a significant and independent determinant of serum 8-OHdG, and serum ferritin correlated significantly with the total dose of iron supplementation during the 6-month period of the study. In the nine patients, without iron supplementation, serum 8-OHdG levels, as well as serum ferritin, decreased significantly during follow-up.Conclusions Our results suggest that increased iron storage may induce DNA oxidative injury in patients on regular HD, and that the serum ferritin level is a surrogate marker for this pathological condition.     

A Case of Vogt-Koyanagi-Harada Disease Associated with Malignant Lymphoma

Background Vogt-Koyanagi-Harada disease (VKH), an inflammatory ocular disorder characterized by bilateral granulomatous panuveitis and a variety of extraocular manifestations, has been reported to be associated with various immune disorders but has not been linked to malignant lymphoma (ML).Case We present here a case of VKH associated with a recurrence of ML.Observations A 69-year-old man who initially had ML presented with a history of sudden bilateral visual acuity loss. Funduscopy showed papilloedema and serous retinal detachment in both eyes, and a diagnosis of VKH was reached soon thereafter. Chest X-ray and an abdominal computed tomography scan indicated the metastatic focus of the ML. A recurrence was suspected because the ML-associated soluble interleukin-2 receptor (sIL-2R) in the serum was highly elevated. Treatment successfully resolved both the ML and the VKH. The inflammatory activities of VKH and ML were found to correlate with the serum levels of sIL-2R.Conclusions This case suggests an association between sIL-2R levels and disease activity in VKH and ML, and provides additional evidence that VKH can be induced by immune disorders caused by high sIL-2R levels in ML. Jpn J Ophthalmol 2005;49:253–256 © Japanese Ophthalmological Society 2005     

Biodistribution characteristics of all-trans retinoic acid incorporated in liposomes and polymeric micelles following intravenous administration

The aim of this study was to investigate the biodistribution characteristics of all-trans retinoic acid (ATRA) incorporated in liposomes and polymeric micelles following intravenous administration. [3H] ATRA were incorporated in distearoylphosphatidylcholine (DSPC)/cholesterol (6:4) liposomes. Two types of block copolymers, poly (ethylene glycol)-b-poly-(aspartic acid) derivatives with benzyl (Bz-75) groups, were synthesized to prepare the polymeric micelles for [(3)H]ATRA incorporation. ATRA were dissolved in mouse serum to analyze their inherent distribution. After intravenous administration, the blood concentration of [3H] ATRA in liposomes and polymeric micelles (Bz-75) was higher than that of inherent [3H]ATRA, suggesting that liposomes and polymeric micelles (Bz-75) control the distribution of ATRA. Pharmacokinetic analysis demonstrated that [3H]ATRA incorporated in polymeric micelles (Bz-75) exhibit the largest AUC(blood) and lowest hepatic clearance of ATRA, suggesting that polymeric micelles (Bz-75) are an effective ATRA carrier system for acute promyelocytic leukemia (APL) therapy. These results have potential implications for the design of ATRA carriers for APL patients.