Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.</p

Phosphorylation of Serine 248 of C/EBPα Is Dispensable for Myelopoiesis but Its Disruption Leads to a Low Penetrant Myeloid Disorder with Long Latency

BACKGROUND: Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. CONCLUSIONS/SIGNIFICANCE: Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context

IL-3 and oncogenic Abl regulate the myeloblast transcriptome by altering mRNA stability

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. The degree to which IL-3 acts at the posttranscriptional level is largely unknown. We have conducted global mRNA decay profiling and bioinformatic analyses in 32Dcl3 myeloblasts indicating that IL-3 caused immediate early stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Stabilized transcripts were enriched for AU-Response elements (AREs), and an ARE-containing domain from the interleukin-6 (IL-6) 3′-UTR rendered a heterologous gene responsive to IL-3-mediated transcript stabilization. Many IL-3-stabilized transcripts had been associated with leukemic transformation. Deregulated Abl kinase shared with IL-3 the ability to delay turnover of transcripts involved in proliferation or differentiation blockade, relying, in part, on signaling through the Mek/ Erk pathway. These findings support a model of IL-3 action through mRNA stability control and suggest that aberrant stabilization of an mRNA network linked to IL-3 contributes to leukemic cell growth. © 2009 Ernst et al

Ferromagnetism in Mn-implanted epitaxially grown Ge on Si(100)

We have studied ferromagnetism of Mn-implanted epitaxial Ge films on silicon. The Ge films were grown by ultrahigh vacuum chemical vapor deposition using a mixture of germane (GeH{sub 4}) and methylgermane (CH{sub 3}GeH{sub 3}) gases with a carbon concentration of less than 1 at. %, and observed surface rms roughness of 0.5 nm, as measured by atomic force microscopy. Manganese ions were implanted in epitaxial Ge films grown on Si (100) wafers to an effective concentration of 16, 12, 6, and 2 at. %. Superconducting quantum interference device measurements showed that only the three highest Mn concentration samples are ferromagnetic, while the fourth sample, with [Mn] = 2 at. %, is paramagnetic. X-ray absorption spectroscopy and x-ray magnetic circular dichroism measurements indicate that localized Mn moments are ferromagnetically coupled below the Curie temperature. Isothermal annealing of Mn-implanted Ge films with [Mn] = 16 at. % at 300 C for up to 1200 s decreases the magnetization but does not change the Curie temperature, suggesting that the amount of the magnetic phase slowly decreases with time at this anneal temperature. Furthermore, transmission electron microscopy and synchrotron grazing incidence x-ray diffraction experiments show that the Mn-implanted region is amorphous, and we believe that it is this phase that is responsible for the ferromagnetism. This is supported by our observation that high-temperature annealing leads to recrystallization and transformation of the material into a paramagnetic phase

Spectroscopic investigation of tetrahydroisoquinoline in supersonic jet

Fluorescence excitation and dispersed emission spectra of tetrahydroisoquinoline are presented here. Two bands at 36781 and 36884 cm<SUP>-1</SUP> are confirmed from the spectral hole burning studies as two origins for two conformers. These bands correspond to the inequivalent twist conformers with the hydrogen atom attached to the nitrogen atom at axial and equatorial positions, respectively. The former is found to be the most stable one. SVL spectra are explained on the basis of two theoretically calculated low-frequency vibrations. These correspond to the butterfly and puckering motions of the benzene chromophore, respectively