Synthesis, stereochemical determination and biochemical characterization of the enantiomeric phosphate esters of the novel immunosuppressive agent FTY720

The novel immunosuppressive agent FTY720 (1) is phosphorylated in vivo in a variety of species yielding an active metabolite that is an agonist of four of the five known G-protein-coupled sphingosine-1-phosphate (S1P) receptors. A synthesis amenable to producing gram quantities of the stereoisomeric phosphate esters, a determination of their absolute stereochemistry via an enantioselective synthesis and their characterization as S1P receptor agonists and antagonists is reported.     

Nonlinear relationships and phylogenetically independent contrasts

The method of phylogenetically independent contrasts is commonly used for exploring cross-taxon relationships between traits. Here we show that this phylogenetic comparative method (PCM) can fail to detect correlated evolution when the underlying relationship between traits is nonlinear. Simulations indicate that statistical power can be dramatically reduced when independent contrasts analysis is used on nonlinear relationships. We also reanalyze a published data set and demonstrate that ignoring nonlinearity can affect biological inferences. We suggest that researchers consider the shape of the relationship between traits when using independent contrasts analysis. Alternative PCMs may be more appropriate if data cannot be transformed to meet assumptions of linearity.     

The independent cue and cus systems confer copper tolerance during aerobic and anaerobic growth in Escherichia coli

Copper is essential but can be toxic even at low concentrations. Coping with this duality requires multiple pathways to control intracellular copper availability. Three copper-inducible promoters, controlling expression of six copper tolerance genes, were recently identified in Escherichia coli. The cue system employs an inner membrane copper transporter, whereas the cus system includes a tripartite transporter spanning the entire cell envelope. Although cus is not essential for aerobic copper tolerance, we show here that a copper-sensitive phenotype can be observed when cus is inactivated in a cueR background. Furthermore, a clear copper-sensitive phenotype for the cus system is revealed in the absence of O(2). These results indicate that the cue pathway, which includes a copper exporter, CopA, and a periplasmic oxidase, CueO, is the primary aerobic system for copper tolerance. During anaerobic growth, however, copper toxicity increases, and the independent cus copper exporter is also necessary for full copper tolerance. We conclude that the cytosolic (CueR) and periplasmic (CusRS) sensor systems differentially regulate copper export systems in response to changes in copper and oxygen availability. These results underscore the increased toxicity of copper under anaerobic conditions and the complex adaptation of copper export in E. coli.     

Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR

Because copper ions are both essential cofactors and cytotoxic agents, the net accumulation of this element in a cell must be carefully balanced. Depending upon the cellular copper status, copper ions must either be imported or ejected. CopA, the principal copper efflux ATPase in Escherichia coli, is induced by elevated copper in the medium, but the copper-sensing regulatory factor is unknown. Inspection of the copA promoter reveals signature elements of promoters controlled by metalloregulatory proteins in the MerR family. These same elements are also present upstream of yacK, which encodes a putative multi-copper oxidase. Homologues of YacK are found in copper resistance determinants that facilitate copper efflux. Here we show by targeted gene deletion and promoter fusion assays that both copA and yacK are regulated in a copper-responsive manner by the MerR homologue, ybbI. We have designated ybbI as cueR for the Cu efflux regulator. This represents the first example of a copper-responsive regulon on the E. coli chromosome and further extends the roles of MerR family members in prokaryotic stress response.     

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.     

A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration

A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration. It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations. With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles. The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro. No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found. The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk.     

Strain-specific contribution of NS1-activated phosphoinositide 3-kinase signaling to influenza A virus replication and virulence

We generated influenza A viruses expressing mutant NS1 proteins unable to activate phosphoinositide 3-kinase (PI3K) in two mouse-lethal strains. The recombinant A/Puerto Rico/8/34 (rPR8) mutant virus strain was attenuated and caused reduced morbidity/mortality. For the recombinant A/WSN/33 (rWSN) virus strain, the inability to stimulate PI3K had minimal impact on replication or morbidity/mortality. Cell-based assays revealed subtly distinct intracellular sites of NS1 localization and PI3K activation between the strains. We hypothesize that specific spatially regulated NS1-activated PI3K signaling, rather than simply the total level of active PI3K, is important for virus replication and virulence.     

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen interacts with multifunctional angiogenin to utilize its antiapoptotic functions

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with the angioproliferative Kaposi's sarcoma (KS). KSHV infection and the expression of latency-associated nuclear antigen (LANA-1) upregulates the angiogenic multifunctional 123-amino-acid, 14-kDa protein angiogenin (ANG), which is detected in KS lesions and in KSHV-associated primary effusion lymphoma (PEL) cells. ANG knockdown or the inhibition of ANG's nuclear translocation resulted in decreased LANA-1 gene expression and reduced KSHV-infected endothelial and PEL cell survival (Sadagopan et al., J. Virol. 83:3342-3364, 2009). Further studies here demonstrate that LANA-1 and ANG colocalize and coimmunoprecipitate in de novo infected endothelial cells and in latently infected PEL (BCBL-1 and BC-3) cells. LANA-1 and ANG interaction occurred in the absence of the KSHV genome and other viral proteins. In gel filtration chromatography analyses of BC-3 cell lysates, ANG coeluted with LANA-1, p53, and Mdm2 in high-molecular-weight fractions, and LANA-1, p53, and Mdm2 also coimmunoprecipitated with ANG. LANA-1, ANG, and p53 colocalized in KSHV-infected cells, and colocalization between ANG and p53 was also observed in LANA-1-negative cells. The deletion constructs of ANG suggested that the C-terminal region of amino acids 104 to 123 is involved in LANA-1 and p53 interactions. Silencing ANG or inhibiting its nuclear translocation resulted in decreased nuclear LANA-1 and ANG levels, decreased interactions between ANG-LANA-1, ANG-p53, and LANA-1-p53, the induction of p53, p21, and Bax proteins, the increased cytoplasmic localization of p53, the downregulation of Bcl-2, the increased cleavage of caspase-3, and the apoptosis of cells. No such effects were observed in KSHV-negative BJAB cells. The phosphorylation of p53 at serine 15, which is essential for p53 stabilization and for p53's apoptotic and cell cycle regulation functions, was increased in BCBL-1 cells transduced with short hairpin RNA targeting ANG. Together, these studies suggest that the antiapoptosis observed in KSHV-infected cells and the suppression of p53 functions are mediated in part by ANG, and KSHV has probably evolved to utilize angiogenin's multiple functions for the maintenance of its latency and cell survival. Thus, targeting ANG to induce the apoptosis of cells latently infected with KSHV is an attractive therapeutic strategy against KSHV infection and associated malignancies.