Crystal Structure of Enzyme I of the Phosphoenolpyruvate Sugar Phosphotransferase System in the Dephosphorylated State

The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 Å crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable βα phospho-histidine (P-His) domain. EIC folds into an (βα)₈ barrel. The dimer interface of EIC buries 1833 Ų of accessible surface per monomer and contains two Ca²⁺ binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218–16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse.     

Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities

Biological soil characteristics such as microbial biomass, community structures, activities, and functions may provide important information on environmental and anthropogenic influences on agricultural soils. Diagnostic tools and detailed statistical approaches need to be developed for a reliable evaluation of these parameters, in order to allow classification and quantification of the magnitude of such effects. The DOK long-term agricultural field experiment was initiated in 1978 in Switzerland for the evaluation of organic and conventional farming practices. It includes three representative Swiss farming systems with biodynamic, bio-organic and conventional fertilization and plant protection schemes along with minerally fertilized and unfertilized controls. Effects on microbial soil characteristics induced by the long-term management at two different stages in the crop rotation, i.e. winter wheat after potato or corn, were investigated by analyzing soil bacterial community structures using analysis of PCR-amplified rRNA genes by terminal restriction fragment length polymorphism and ribosomal intergenic spacer analysis. Application of farmyard manure consistently revealed the strongest influence on bacterial community structures and biomass contents. Effects of management and plant protection regimes occurred on an intermediate level, while the two stages in the crop rotation had a marginal influence that was not significant.     

Interactions of Divalent Cations in Their Action on ATPases from Cucumber Roots

A microsomal fraction (10,000–30.000 g) was prepared from roots of Cucumis satirus L. (cv. Bestseller Fl) grown in solution culture. The ATPase activity was stimulated by Mg and Mn with optima between pH 6 and 7. Stimulation by Ca was obtained only above pH 7. Activations by Mg and Mn were inhibited by Ca, Mn and Mg interacted, so that Mn appeared strongly inhibitory for activation by Mg and Mg weakly inhibitory for activation by Mn: the simplest interpretation for this would be two separate enzymes. Cucumber accumulates and deposits Ca contrary to wheat and oat, which contain Ca-activated ATPase in the pH region 6 to 7. The Ca data for cucumber are compared with earlier findings from wheat and oat and are tentatively related to known physiological differences.     

Maturation of human lactase-phlorizin hydrolase. Proteolytic cleavage of precursor occurs after passage through the Golgi complex.

Maturation of human intestinal lactase-phlorizin hydrolase (LPH) requires that a precursor (pro-LPH) be proteolytically processed to the mature microvillus membrane enzyme (m-LPH). The subcellular site of this processing is unknown. Using low-temperature experiments and brefeldin A (BFA), intracellular transport was blocked in intestinal epithelial cells. In Caco-2 cells incubated at 18 degrees C, pro-LPH was complex-glycosylated but not cleaved, while at 20 degrees C small amounts of proteolytically processed LPH were observed. These data exclude a pre-Golgi proteolytic event. BFA completely blocked proteolytic maturation of LPH and lead to an aberrant form of pro-LPH in both Caco-2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a post-Golgi event, occurring either in the trans-Golgi network, transport vesicles, or after insertion of pro-LPH into the microvillus membrane.     

Apoptosis in sepsis: a new target for therapeutic exploration.

The treatment of sepsis and septic shock remains a clinical conundrum, and recent prospective trials with biological response modifiers aimed at the inflammatory response have shown only modest clinical benefit. Recently, interest has shifted toward therapies aimed at reversing the accompanying periods of immune suppression. Studies in experimental animals and critically ill patients have demonstrated that increased apoptosis of lymphoid organs and some parenchymal tissues contributes to this immune suppression, anergy, and organ system dysfunction. During sepsis syndromes, lymphocyte apoptosis can be triggered by the absence of IL-2 or by the release of glucocorticoids, granzymes, or the so-called 'death' cytokines: tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via auto-activation of cytosolic and/or mitochondrial caspases, which can be influenced by the pro- and anti-apoptotic members of the Bcl-2 family. In experimental animals, not only can treatment with inhibitors of apoptosis prevent lymphoid cell apoptosis; it may also improve outcome. Although clinical trials with anti-apoptotic agents remain distant due in large part to technical difficulties associated with their administration and tissue targeting, inhibition of lymphocyte apoptosis represents an attractive therapeutic target for the septic patient.     

Adenoviral delivery of human and viral IL-10 in murine sepsis.

Adenovirus (Ad) gene therapy has been proposed as a drug-delivery system for the targeted administration of protein-based therapies, including growth factors and biological response modifiers. However, inflammation associated with Ad transduction has raised concern about its safety and efficacy in acute inflammatory diseases. In the present report, intratracheal and i.v. administration of a first-generation adenoviral recombinant (E1,E3 deleted) either containing an empty cassette or expressing the anti-inflammatory cytokines viral or human IL-10 (IL-10) was administered to mice subjected to zymosan-induced multisystem organ failure or to acute necrotizing pancreatitis. Pretreatment of mice with the intratracheal instillation of Ad expressing human IL-10 or viral IL-10 reduced weight loss, attenuated the proinflammatory cytokine response, and reduced mortality in the zymosan-induced model, whereas pretreatment with a control adenoviral recombinant did not significantly exacerbate the response. Pretreatment of mice with pancreatitis using adenoviral vectors expressing IL-10 significantly reduced the degree of pancreatic and liver injury and liver inflammation when administered systemically, but not intratracheally. We conclude that adenoviral vectors can be administered prophylactically in acute inflammatory syndromes, and expression of the anti-inflammatory protein IL-10 can be used to suppress the underlying inflammatory process.     

Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation

Interferons (IFNs) are cytokines that possess potent anti-viral and immunoregulatory activities. In contrast, their potential role(s) in anti-bacterial defense and neutrophil activation mechanisms is less well explored. By comparing gene expression patterns between immature and mature human neutrophils, we obtained evidence that intracellular proteases and other anti-bacterial proteins are produced at earlier stages of maturation, whereas the genes for receptors and signaling molecules required for the release of these effector molecules are preferentially induced during terminal differentiation. For instance, mature neutrophils strongly expressed genes that increase their responses to type I and type II IFNs. Interestingly, granulocyte/macrophage colony-stimulating factor was identified as a repressor of IFN signaling components and consequently of IFN-responsive genes. Both IFN-alpha and IFN-gamma induced strong tyrosine phosphorylation of STAT1 in mature but not in immature neutrophils. Functional in vitro studies suggested that IFNs act as priming factors on mature neutrophils, allowing the formation of extracellular traps upon subsequent stimulation with complement factor 5a (C5a). In contrast, both IFN-alpha and IFN-gamma had only little capacity to prime immature cells in this system. Moreover, both IFNs did not have significant anti-proliferative effects on immature neutrophils. These data contribute to our understanding regarding changes of gene expression during neutrophil differentiation and IFN-mediated anti-bacterial defense mechanisms.