Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.

SUMMARYIn rat liver epithelial (WB) cells, the protein kinase C inhibitor H7 blocked gap junctional intercellular communication (GJIC) and reduced virus infectivity. Octanol, 18-beta-glycyrrhetinic acid, and staurosporine, agents that reduce GJIC, had no effect upon virus infectivity. Previous studies demonstrated that herpes simplex virus-type 2 (HSV-2) infection was accompanied by attenuated GJIC. Of agents tested, only H7 reduced plaque forming unit (pfu) ability in a dose-dependent manner with 100% plaque reduction at 40 μM without evidence of cytotoxicity. Dye transfer indicated that H7 decreased GJIC, although Western blotting revealed that it did not alter phosphorylation of the gap junction protein, connexin 43 (Cx43). Using indirect immunofluorescence, Cx43 was found to localize in membrane plaques in uninfected cells and H-7 did not alter this distribution. However, Cx43 was lost from the membrane at 24 hrs in both H-7 treated and untreated cells infected with HSV-2. Viral infection increased serine phosphorylation, particularly in the nuclear region, and this effect was reduced following H-7 treatment. Thus, H-7 attenuated both GJIC and infectivity of HSV-2 in WB cells but the anti-viral effects were due to reduced nuclear protein phosphorylation rather than alterations in phosphorylation or localization of Cx43. KeywordsGap junctions; Connexin 43; protein kinase C; H7; HSV-2 1) INTRODUCTIONIt has long been known that certain RNA and tumor viruses decrease gap junctional communication among infected cells (Atkinson et al., 1981;Crow et al., 1990;Danave et al., 1994;Ennaji et al., 1995;Faccini et al., 1996, Jou et al., 1995. Recent studies have shown electrophysiologically that down-regulation of gap junctions by herpes simplex virus-type 2 (HSV-2) begins before viral DNA replication, and long before morphological signs of infection (Fischer et al., 2001). In addition, Musee et al. (2002) demonstrated that antiviral agents affect electrical coupling in HSV-2 infected cells in different ways, depending on the mechanism of action of the agent. However, it was not clear if GJIC down-regulation was initiated by the infected cells or by their surrounding uninfected neighbors. If the latter, drugs that reduce GJIC might protect neighboring cells from infection.* To whom all correspondence should be addressed: Phone -(610)-436-2985, Fax -(610)-436-2183, e-mail-mknabb@wcupa.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. How HSV-2 induces gap junctional down-regulation is unknown although both viral-or cellmediated mechanisms are possible. Gap junction channels are...

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