Biochemical engineering of the N-acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells

Sialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells.     

Mitochondrial data support an odd-nosed colobine clade

To obtain a more complete understanding of the evolutionary history of the leaf-eating monkeys we have examined the mitochondrial genome sequence of two African and six Asian colobines. Although taxonomists have proposed grouping the "odd-nosed" colobines (proboscis monkey, douc langur, and the snub-nosed monkey) together, phylogenetic support for such a clade has not been tested using molecular data. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian methods support a monophyletic clade of odd-nosed colobines consisting of Nasalis, Pygathrix, and Rhinopithecus, with tentative support for Nasalis occupying a basal position within this clade. The African and Asian colobine lineages are inferred to have diverged by 10.8 million years ago (mya or Ma). Within the Asian colobines the odd-nosed clade began to diversify by 6.7 Ma. These results augment our understanding of colobine evolution, particularly the nature and timing of the colobine expansion into Asia. This phylogenetic information will aid those developing conservation strategies for these highly endangered, diverse, and unique primates.     

Granule-mediated killing by granzyme B and perforin requires a mannose 6-phosphate receptor and is augmented by cell surface heparan sulfate

During granule-mediated killing by cytotoxic T lymphocytes or natural killer cells, the serine protease granzyme B enters the target cell by endocytosis and induces apoptosis. Previous studies suggested a role for the mannose 6-phosphate receptor, but further experiments with purified granzyme B indicated this was not essential. Additionally, it is now clear that grB is exocytosed from killer cells in a high-molecular-weight complex with the proteoglycan serglycin. Here granzyme B was delivered as a purified monomer, or in complex with either glycosaminoglycans or serglycin, and killing was evaluated. When granzyme B was a monomer, soluble mannose 6-phosphate had a limited impact, whereas apoptosis induced by the complexed grB was effectively inhibited by mannose 6-phosphate. Most importantly, when granzyme B and perforin were delivered together from granules, inhibition by mannose 6-phosphate was also observed. In pulldown assays mediated by the cation-independent mannose 6-phosphate receptor, granzyme B bound to the receptor more intensely in the presence of immobilized heparan sulfate. We therefore propose the model that under physiological conditions serglycin-bound granzyme B is critically endocytosed by a mannose 6-phosphate receptor, and receptor binding is enhanced by cell surface heparan sulfate.     

CD2AP regulates SUMOylation of CIN85 in podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.     

Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.     

Proteome analysis of the farnesol-induced stress response in Aspergillus nidulans--The role of a putative dehydrin

The isoprenoid alcohol farnesol represents a quorum-sensing molecule in pathogenic yeasts, but was also shown to inhibit the growth of many filamentous fungi. In order to gain a deeper insight into the antifungal activity of farnesol, we performed 2D-differential gel electrophoretic analysis (2D-DIGE) of Aspergillus nidulans exposed to farnesol. We observed an increased abundance of antioxidative enzymes and proteins involved in protein folding and the ubiquitin-mediated protein degradation. A striking finding was the strong up-regulation of a dehydrin-like protein (DlpA). Expression analyses suggested the involvement of DlpA in the cellular response to oxidative, osmotic and cold stress. In line with these data, we demonstrated that dlpA expression was regulated by the MAP kinase SakA/HogA. The generation of both a dlpA Tet(on) antisense RNA-producing A. nidulans strain (dlpA-inv) and a ΔdlpA deletion mutant indicated a role of DlpA in conidiation and stress resistance of dormant conidia against heat and ROS. Furthermore, the production of the secondary metabolite sterigmatocystin was absent in both strains dlpA-inv and ΔdlpA. Our results demonstrate the complexity of the farnesol-mediated stress response in A. nidulans and describe a farnesol-inducible dehydrin-like protein that contributes to the high tolerance of resting conidia against oxidative and heat stress.     

Acquired amegakaryocytic thrombocytopenia purpura in a Rhesus macaque (Macaca mulatta)

A 10-y-old multiparous rhesus macaque presented for an annual routine physical examination. Clinically, the animal had pale mucous membranes, petechial and ecchymotic hemorrhages in multiple sites, and a laceration at the tail base. Severe pancytopenia was noted on hematologic evaluation. The monkey was seronegative for SIV, simian T-lymphotropic virus, simian retrovirus type D, and Macacine herpesvirus 1. Bone marrow evaluation revealed a paucity of megakaryocytic precursors in a hypercellular marrow with marked erythroid hyperplasia. In light of these findings, the diagnosis was acquired amegakaryocytic thrombocytopenia purpura. Due to the poor prognosis of the syndrome and clinical deterioration of the monkey, euthanasia was elected. A definitive cause of the thrombocytopenia was not identified; however, the syndrome may have developed secondary to a recent spontaneous abortion. To our knowledge, this case represents the first reported observation of acquired amegakaryocytic thrombocytopenia purpura in a rhesus monkey.