Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

Structural and mechanistic studies of pesticin, a bacterial homolog of phage lysozymes

Yersinia pestis produces and secretes a toxin named pesticin that kills related bacteria of the same niche. Uptake of the bacteriocin is required for activity in the periplasm leading to hydrolysis of peptidoglycan. To understand the uptake mechanism and to investigate the function of pesticin, we combined crystal structures of the wild type enzyme, active site mutants, and a chimera protein with in vivo and in vitro activity assays. Wild type pesticin comprises an elongated N-terminal translocation domain, the intermediate receptor binding domain, and a C-terminal activity domain with structural analogy to lysozyme homologs. The full-length protein is toxic to bacteria when taken up to the target site via the outer or the inner membrane. Uptake studies of deletion mutants in the translocation domain demonstrate their critical size for import. To further test the plasticity of pesticin during uptake into bacterial cells, the activity domain was replaced by T4 lysozyme. Surprisingly, this replacement resulted in an active chimera protein that is not inhibited by the immunity protein Pim. Activity of pesticin and the chimera protein was blocked through introduction of disulfide bonds, which suggests unfolding as the prerequisite to gain access to the periplasm. Pesticin, a muramidase, was characterized by active site mutations demonstrating a similar but not identical residue pattern in comparison with T4 lysozyme.     

Do increasingly depleted δ<Superscript>15</Superscript>N values of atmospheric N<Subscript>2</Subscript>O indicate a decline in soil N<Subscript>2</Subscript>O reduction?

Growing concentrations of N₂O within the atmosphere have been accompanied by decreasing δ¹⁵N values, provoking the hypothesis of a global decline in the rate of N₂O reduction relative to its production in soil. We estimate that the ratio of N₂O produced to N₂O reduced within the soil profile has declined by about 10–25% relative to its pre-industrial value. To a smaller extent, a reduction in the uptake of atmospheric N₂O at the soil surface relative to its emission could also have contributed to the reported isotopic signal. This calls for a greater consideration of the process of N₂O reduction in soil and its role in the global turnover of N₂O.     

Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.     

Proteomic analysis of Bacillus cereus growing in liquid soil organic matter

Bacillus cereus is believed to be a soil bacterium, but studied solely in laboratory culture media. The aim of this study was to assess the physiology of B. cereus growing on soil organic matter by a proteomic approach. Cells were cultured to mid-exponential phase in soil extracted solubilized organic matter (SESOM), which mimics the nutrient composition of soil, and in Luria-Bertani broth as control. Silver staining of the two-dimensional gels revealed 234 proteins spots up-regulated when cells were growing in SESOM, with 201 protein spots down-regulated. Forty-three of these differentially expressed proteins were detected by Colloidal Coomassie staining and identified by matrix assisted laser desorption ionization-time of flight MS of tryptic digests. These differentially expressed proteins covered a range of functions, primarily amino acid, lipid, carbohydrate and nucleic acid metabolism. These results suggested growth on soil-associated carbohydrates, fatty acids and/or amino acids, concomitant with shifts in cellular structure.     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.