PubMed · 3174028
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1988. Traumatic hyphema therapy.. https://doi.org/10.1016/s0161-6420(88)33127-1
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An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening.
Using a high throughput gene microarray technology that detects approximately 22 000 genes, we found that arginase I was the most significantly up-regulated gene in the murine spinal cord during experimental autoimmune encephalomyelitis (EAE). By Northern blot and arginase enzyme assay, we detected high levels of arginase I mRNA and protein, respectively, in the spinal cord of EAE mice, but not in the spinal cord of normal mice or mice that had recovered from EAE. In vitro, both microglia and astrocytes produced arginase and nitric oxide synthase, two enzymes that are involved in arginine metabolism. To explore the roles of arginase in EAE, we injected the arginase inhibitor amino-6-boronohexanoic acid (ABH) into mice during the inductive and effector phases of the disease. Compared with mice that received vehicle control, mice treated with ABH developed milder EAE with delayed onset, reduced disease score and expedited recovery. Spleen mononuclear cells from ABH-treated mice produced more nitric oxide and secreted less interferon-gamma and tumour necrosis factor-alpha as compared to control mice. These results indicate that arginase plays important roles in autoimmune inflammation in the central nervous system.
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