Detection of total microcystin in fish tissues based on lemieux oxidation, and recovery of 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB) by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS).

Microcystins (MCs) are widespread cyanobacterial toxins in freshwater systems, and have been linked to both acute and chronic health effects. A growing number of studies suggest that MC can bioaccumulate in food webs. Although, several methods (i.e. ELISA, LC-MS) have been developed for analysis of MC in water, extraction (for subsequent analysis) of the toxin from biological matrices (i.e. animal tissues) is impeded owing to covalent binding of toxins and active sites of their cellular targets, i.e. protein phosphatases. As an alternative approach, chromatographic methods for analysis of a unique marker, 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), the product of the Lemieux oxidation of MCs, have been previously developed, and shown to measure total (bound and unbound) MC. Application, however, has been limited by poor recovery of the analyte. An improved recovery method is proposed - specifically the use of solidphase microextraction (SPME). The MMPB analogue, 4-phenylbutanoic acid (4PB), and oxidized MC, were used to develop methods, and we specifically investigated several SPME fibres, and post-oxidation steps. Specifically, a method employing post-oxidation methyl esterification, followed by headspace SPME recovery of MMPB, was developed, and subsequently applied to analysis of environmental samples (i.e. fish tissues) previously shown to contain MCs. The method shows high linearity for both water and tissues spiked with MC, and an improved limit of quantitation of approximately 140 ng g(-1). Evaluation of field samples by SPME-GC/MS detected considerably higher levels of MC, than detected by conventional methods (i.e. ELISA), and it is proposed that this technique reveals MC (particularly in the bound form) that is not detected by these methods. These results indicate that the developed method provides improved detection capability for MC in biological matrices, and will enhance our ability to understand bioaccumulation in freshwater food webs, as well as monitor exposure.

The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44.

The protein kinase pUL97 of human cytomegalovirus plays important but incompletely defined roles in viral replication. Concerning the early phase of infection, it is postulated that pUL97 possesses regulatory functions in gene expression and/or DNA synthesis. Here we report that pUL97 interacts with an essential component of the replication complex, the DNA polymerase processivity factor pUL44. Interaction was determined by yeast two-hybrid and coimmunoprecipitation analyses and was mapped to the pUL97 region 366-459. In vitro kinase assays demonstrated that pUL44, coimmunoprecipitated either from transfected or from infected cells, is phosphorylated by pUL97 (but not by a catalytically inactive pUL97-mutant). In infected fibroblasts, immunofluorescence analysis revealed that pUL97 and pUL44 accumulate in the nucleus and are both incorporated into viral replication centers. The treatment with inhibitors of DNA synthesis or pUL97 kinase activity largely prevented colocalization. Thus, pUL97 may be indirectly involved in viral genome replication by modifying the replication cofactor pUL44.