Discovery of a new class of glucosylceramide synthase inhibitors

A novel series of potent inhibitors of glucosylceramide synthase are described. The optimization of biochemical and cellular potency as well as ADME properties led to compound 23c. Broad tissue distribution was obtained following oral administration to mice. Thus 23c could be another useful tool compound for studying the effects of GCS inhibition in vitro and in vivo.     

The SUMO isopeptidase Ulp2p is required to prevent recombination-induced chromosome segregation lethality following DNA replication stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.     

Going nuclear: transcribers in transit

Recent experiments have identified novel RNA polymerase-associated proteins with roles in assembly and nuclear transport of multisubunit eukaryotic RNA polymerases. In this issue of Molecular Cell, Czeko et al. (2011) characterize a novel Pol II transport factor that is conserved from yeast to humans.     

Polysaccharides isolated from Açaí fruit induce innate immune responses

The Açaí (Acai) fruit is a popular nutritional supplement that purportedly enhances immune system function. These anecdotal claims are supported by limited studies describing immune responses to the Acai polyphenol fraction. Previously, we characterized γδ T cell responses to both polyphenol and polysaccharide fractions from several plant-derived nutritional supplements. Similar polyphenol and polysaccharide fractions are found in Acai fruit. Thus, we hypothesized that one or both of these fractions could activate γδ T cells. Contrary to previous reports, we did not identify agonist activity in the polyphenol fraction; however, the Acai polysaccharide fraction induced robust γδ T cell stimulatory activity in human, mouse, and bovine PBMC cultures. To characterize the immune response to Acai polysaccharides, we fractionated the crude polysaccharide preparation and tested these fractions for activity in human PBMC cultures. The largest Acai polysaccharides were the most active in vitro as indicated by activation of myeloid and γδ T cells. When delivered in vivo, Acai polysaccharide induced myeloid cell recruitment and IL-12 production. These results define innate immune responses induced by the polysaccharide component of Acai and have implications for the treatment of asthma and infectious disease.     

Cell cycle control and DNA damage response of conditionally immortalized urothelial cells

Background

Children with complex urogenital anomalies often require bladder reconstruction. Gastrointestinal tissues used in bladder augmentations exhibit a greatly increased risk of malignancy, and the bladder microenvironment may play a role in this carcinogenesis. Investigating the influences of the bladder microenvironment on gastrointestinal and urothelial cell cycle checkpoint activation and DNA damage response has been limited by the lack of an appropriate well-differentiated urothelial cell line system.

Methodology/Principal Findings

To meet this need, we have developed a well-differentiated conditionally immortalized urothelial cell line by isolating it from the H-2K^b-tsA58 transgenic mouse. These cells express a thermosensitive SV40 large T antigen that can be deactivated by adjustment of cell culture conditions, allowing the cell line to regain normal control of the cell cycle. The isolated urothelial cell line demonstrates a polygonal, dome-shaped morphology, expresses cytokeratin 18, and exhibits well-developed tight junctions. Adaptation of the urothelial cell line to hyperosmolal culture conditions induces expression of both cytokeratin 20 and uroplakin II, markers of a superficial urothelial cell or “umbrella cell.” This cell line can be maintained indefinitely in culture under permissive conditions but when cultured under non-permissive conditions, large T antigen expression is reduced substantially, leading to increased p53 activity and reduced cellular proliferation.

Conclusions/Significance

This new model of urothelial cells, along with gastrointestinal cell lines previously derived from the H-2K^b-tsA58 transgenic mouse, will be useful for studying the potential mechanisms of carcinogenesis of the augmented bladder.