Summary The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin associated proteins and histone modifications. PRC1-class Polycomb Group protein complexes are essential for development, and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. We find that when PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division.

Antimitochondrial antibodies (AMA) directed against the lipoyl domain of the E2 subunit of pyruvate dehydrogenase (PDC-E2) are detected in 95% of patients with PBC and are present before onset of clinical disease. The recent demonstration that AMA recognize xenobiotic modified PDC-E2 with higher titers than native PDC-E2, raises the possibility that the earliest events involved in loss of tolerance are related to xenobiotic modification. We hypothesized that reactivity to such xenobiotics would be predominantly IgM and using sera from a large cohort of PBC patients and controls (n=516), we examined in detail sera reactivity against either SAc-conjugated bovine serum albumin (BSA), recombinant PDC-E2 (rPDC-E2) or BSA alone. Further, we also defined the relative specificity to the SAc moiety using inhibition ELISA; SAc conjugate and rPDC-E2 specific affinity purified antibodies were also examined for antigen specificity, isotype and cross-reactivity. Reactivity to SAc conjugates is predominantly IgM; such reactivity reflects a footprint of previous xenobiotic exposure. Indeed, this observation is supported by both direct binding, cross reactivity, and inhibition studies. In both early and late stage PBC, the predominant Ig isotype to SAc is IgM, with titers higher with advanced stage disease. We also note that there was a higher level of IgM reactivity to SAc in early stage versus late stage PBC. Interestingly, this finding is particularly significant in light of the structural similarity between SAc and the reduced form of lipoic acid, a step which is similar to the normal physiological oxidation of lipoic acid. We submit that specific modifications of the disulfide bond within the lipoic-acid-conjugated PDC-E2 moiety, i.e. by an electrophilic agent renders PDC-E2 immunogenic in a genetically susceptible host.

Our laboratory has hypothesized that xenobiotic modification of the native lipoyl moiety of the major mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), may lead to loss of self-tolerance in primary biliary cirrhosis (PBC). This thesis is based on the finding of readily detectable levels of immunoreactivity of PBC sera against extensive panels of protein microarrays containing mimics of the inner lipoyl domain of PDC-E2 and subsequent quantitative structure-activity relationships (QSARs). Importantly, we have demonstrated that murine immunization with one such mimic, 2-octynoic acid coupled to bovine serum albumin (BSA), induces antimitochondrial antibodies (AMAs) and cholangitis. Based upon these data, we have focused on covalent modifications of the lipoic acid disulfide ring and subsequent analysis of such xenobiotics coupled to a 15mer of PDC-E2 for immunoreactivity against a broad panel of sera from patients with PBC and controls. Our results demonstrate that AMA-positive PBC sera demonstrate marked reactivity against 6,8-bis(acetylthio)octanoic acid, implying that chemical modification of the lipoyl ring, i.e. disruption of the S-S disulfide, renders lipoic acid to its reduced form that will promote xenobiotic modification. This observation is particularly significant in light of the function of the lipoyl1oiety in electron transport of which the catalytic disulfide constantly opens and closes and, thus, raises the intriguing thesis that common electrophilic agents, i.e. acetaminophen or non-steroidal anti-inflammatory drugs (NSAIDs), may lead to xenobiotic modification in genetically susceptible individuals that results in the generation of AMAs and ultimately clinical PBC.

Actinophyllic acid is a biologically active indole alkaloid with a unique structural framework that incorporates five contiguous stereocenters. A total synthesis of (±)-actinophyllic acid has been completed that proceeds in only 10 steps from readily available, known compounds and with the isolation of nine intermediates. The synthesis features a novel cascade of reactions of N-stabilized carbocations with π-nucleophiles to create the tetracyclic core of actinophyllic acid in a single chemical operation. This pivotal cascade sequence generates substructures of the actinophyllic acid core that are not otherwise accessible, and one key intermediate was modified to furnish several novel compounds having potentially promising anticancer activity, one of which induces cell death in a wide range of cancer cell lines.

The parent 5H-indazolo[3,2-b]benzo[d]-1,3-oxazine heterocycle as well as a series of novel analogues have been synthesized utilizing two subsequent intramolecular heterocyclizations in one pot. A variety of diversity groups were added to explore the scope of this reaction and to provide a number of new compounds for biological screening.