An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04 pmol (25 pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40 s.

A compact electron cyclotron resonance negative hydrogen ion source for evaluation of plasma electrode materials.

A compact ion source that produces hydrogen plasma with an electron cyclotron resonance (ECR) configuration combined with a 2-stage extraction system with a single aperture of 6 mm diameter has been designed and built to study the performance of different materials as the plasma electrode (PE) of a negative hydrogen ion source. The source has the capability to electrically bias the PE with respect to the ECR plasma. The first experiment with low ECR power input (less than 40 W) was carried out. The PE of the C12A7 electride showed the largest H- current among aluminum, molybdenum, and the C12A7 electride.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.