A homogeneous caspase-3 activity assay using HTRF technology.

Caspases are cysteine proteases presenting a conserved active site that cleaves protein substrates at a highly specific position. They are involved in different aspects of the active cell death pathway. Most of them act through proteolytic degradations of cellular components. This paper describes the assay development, assay validation, and screening for inhibitors of this enzyme, which could be potential drug candidates. The assay uses homogeneous time-resolved fluorescence based on energy transfer from europium cryptate as donor to cross-linked allophycocyanin as acceptor (XL665). A double-tagged substrate, biotinyl-epsilon-aminocaproyl-L-aspartyl-L-glutamyl-L-valyl-Laspartyl-L-alanyl-L-propyl-N(epsilon)-(2,4-dinitrophenyl)-L-lysine-amide (biotin-X-DEVDAPK(dnp)-NH(2)), is conjugated with streptavidin cryptate and anti-dnp-XL665 monoclonal antibody. The close proximity between donor and acceptor induces a specific time-resolved fluorescence signal. In the presence of enzyme activity, the substrate cleavage induces an unlinking of the two fluorescent probes and, subsequently, the disappearance of the specific signal as a result of loss of proximity. Experiments to optimize the reagent concentration, incubation times, precision, reproducibility, and robustness are discussed in comparison with a fluorometric method.

High throughput screening for human interferon-gamma production inhibitor using homogenous time-resolved fluorescence.

An immunoassay for interferon-gamma (IFN-gamma) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IFN-gamma can be detected by simply adding a mixture of three reagents-biotinylated polyclonal antibody, europium cryptate (fluorescence donor, EuK)-labeled monoclonal antibody, and crosslinked allophycocyanin (fluorescence acceptor, XL665) conjugated with streptavidin-and then measuring the time-resolved fluorescence. The detection limit of IFN-gamma by the proposed method is about 625 pg/ml. We applied the method to the detection of IFN-gamma secreted from NK3.3 cells and employed it in high throughput screening for IFN-gamma production inhibitors. With this screening format, IFN-gamma can be measured by directly adding the above reagents to microplate wells where NK3.3 cells are being cultured and stimulated with interleukin-12. This "in situ" immunoassay requires only pipetting reagents, with no need to transfer the culture supernatant to another microplate or wash the plate. Therefore, this screening format makes possible full automation of cell-based immunoassay, thus reducing cost and experimental time while increasing accuracy and throughput.

Aldosterone antagonists. 1. Synthesis and biological activities of 11 beta,18-epoxypregnane derivatives.

Several steroid derivatives having the 11 beta,18-epoxypregnane skeleton, 7, 8, 19, 20, 21, and 31, were synthesized to evaluate their antialdosterone activity. Among them, 3-(9 alpha-fluoro-17 beta-hydroxy-3-oxoandrost-4-en-17 alpha-yl)propionic acid gamma-lactone (31) possessed fairly strong binding affinity for the cytoplasmic mineralocorticoid receptor of rat kidney and exhibited good aldosterone antagonist activity in an in vivo assay. However, its agonistic nature cannot be ignored. The properties of 31 as an aldosterone antagonist were enhanced by its very low to negligible binding affinity for the androgen, progestin, estrogen, and glucocorticoid receptors.

[Digital subtraction angiography of the cerebral vessels by intraarterial injection].

Three hundred and fifty-seven digital subtraction angiography (DSA) were performed in 184 neurosurgical patients by intraarterial injection. Examinations consisted of 192 carotid angiography, 110 vertebral angiography, 23 aortography, 11 spinal angiography and 21 other angiography. In all examinations, visualization of the vessels was excellent and the complications were never experienced. High contrast sensitivity of DSA resulted in better visualization of tumor stains, phlebogram, and arteries in cerebral arteriovenous malformations with large shunt blood flow than conventional angiography. Selective catheterization into each cerebral arteries was not necessarily demanded for good opacification of the vessels because of high sensitivity. High contrast sensitivity also permitted low concentration of contrast material, small dose of contrast material, and slow injection rate. Low concentration of contrast material reduced pain and heat during injection especially in the external carotid and vertebral angiography. Using slow injection, recoiling of catheter into the aorta was reduced, so that injection from the innominate and subclavian arteries for visualization of origin of the cerebral arteries were always successful. Full study of cerebral arteries by Seldinger's method, if necessary, was easily achieved using DSA even in patient with high age or with severe atherosclerosis. Bolus injection of small dose of contrast material as well as serial imaging was helpful in evaluating hemodynamics in the lesion. Real time display of DSA reduced the time required for angiography and was very convenient for artificial embolization. Besides these advantages, DSA became comparable to conventional angiography in special resolution by use of intraarterial injection and could be a preoperative genuine examination as well as a screening method.

[Monitoring system of cerebral blood flow and cerebral metabolism. Part II. Relationship between internal jugular O2 tention and cerebral blood flow (author's transl)].

Changes of PjO2 values of internal jugular vein were monitored in patients with various types of cerebrovascular disease. The significance of PjO2 monitoring was evaluated. 1) Control value of PjO2 in thirteen normal control cases was 36.7 +/- 1.9 mmHg (mean +/- SD). 2) Limit of brain hypoxia showing no neurological sign and symptom in patients with occlusive cerebrovascular disease was studied by the stepwise reduction of arterial blood pressure, using drip infusion of trymetaphan camsylate, under the careful monitoring of signs and symptoms and monitoring of PjO2 and EEG. Appearance of signs and symptoms of brain hypoxia were checked by yawning and EEG slowing. At the time of the appearance of brain hypoxia PjO2 was 28.6 +/- 3.2 mmHg. 3) CO2 reactivity of CBF was studied in patients with occlusive cerebrovascular disease. Relation between PaCO2 and PjO2 was as follows; PjO2 = 0.68 . PaCO2 + 7.55 4) Within 24 hours after the onset of stroke, ipsilateral PjO2 of the cases with disturbance of consciousness was lower than that of the cases without disturbance of consciousness, which might indicate the significant decrease of CBF in the former cases. During 3rd to 7th day after the onset the cases with disturbance of consciousness showed the significant elevation of PjO2, which might indicate the reduction of cerebral metabolism. 5) The elevation of ipsilateral PjO2 were well correlated to the degree of hemispheric brain swelling. 6) During general convulsion, high level of PjO2 values were observed. 7) PjO2 values were inversely correlated to the hemoglobin values after blood transfusion, which was mainly due to the decrease of CBF by high content of hemoglobin.