Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
Development and application of an assay for 2',5'-oligoadenylate synthetase activity in human cells.
Diss Abstr Int [B]; 53(5):2261 1992. Unique Identifier : AIDSLINE ICDB/93688580 Miele ME; Pennsylvania State Univ.
Abstract:
2,5A synthetase catalyzes the polymerization of ATP into 2',5'-linked oligoadenylates of varying chain lengths (2,5A). To identify these multiple oligoadenylates, a method was devised utilizing electrophoretic separation of 32P-labeled 2,5A in a thin 20% polyacrylamide gel containing 7 M urea. A mixture of 2,5A synthesized from rat liver nuclear suspension was fractionated by this method. Each species eluted from the gel was characterized by specific nucleotidylic enzymes. All major species in the gel were identified, as well as dephosphorylated species. Thus, in a single step, this method produced a more complete assessment of newly synthesized 2,5A and their degradative products than DEAE-cellulose chromatography. It also allowed more rapid screening of multiple samples than HPLC. In addition, densitometry provided quantitation of 2,5A products. This method was used to assay 2,5A synthetase activity induced by interferon-alpha (IFN-alpha) in human CD4+ cell lines, H9, CEM-CM3, Molt-4 and U937. Total 2,5A synthetase activity was measured in human PBMC isolated by Ficoll-Hypaque density gradient. Basal 2,5A synthetase activities were studied in normal subjects, HIV-positive subjects, and renal cell carcinoma (RCC) patients (pts). In all populations studied, basal activity of 2,5A synthetase was lower in females than males. In vitro cytokine effects on 2,5A synthetase activity were studied in normal donors and RCC pts. In all populations, in vitro treatment with IFN-alpha or IL-2 + IFN-alpha induced 2,5A synthetase activity above basal and untreated levels. However, combination treatment did not induce 2,5A synthetase activity above that of IFN-alpha treatment alone. To examine effects of cytokines in vivo, pts receiving IFN-alpha or IL-2 and IFN-alpha therapy were studied. In response to IFN-alpha or combination IL-2 and IFN-alpha therapy, 2,5A synthetase activities increased in most pts. In RCC pts, clinical response was evaluated by CT scan. A correlation was observed between clinical response and 2,5A synthetase activity detected 24 hr after combination cytokine treatment. In summary, a method has been developed to measure 2,5A synthetase activity. Using PBMC, this assay revealed cellular responses to cytokines which may have several clinical implications. (Full text available from University Microfilms International, Ann Arbor, MI, as Order No. AAD92-26749.)
Keywords: Carcinoma, Renal Cell/ENZYMOLOGY Human HIV Infections/ENZYMOLOGY Interferon-alpha/PHARMACOLOGY Interleukin-2/PHARMACOLOGY Kidney Neoplasms/ENZYMOLOGY Phosphorylation Tumor Cells, Cultured 2',5'-Oligoadenylate Synthetase/*METABOLISM THESIS 930530
M9350990
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Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.
