Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd²⁺ and Cu²⁺ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu²⁺ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu²⁺ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu²⁺ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.     

Genetic imbalances in preleukemic thymuses

To understand the molecular mechanisms involved in preleukemia, the suppression subtractive hybridization method was used in a murine radiation-induced thymic lymphoma model. Seventeen mRNAs overexpressed in preleukemic thymuses were identified: mouse laminin binding protein (p40/37LBP), E25 protein, Rattus norvegicus clone BB.1.4.1, profilin, poly(A) binding protein (PABP), mouse high mobility group protein 1, topoisomerase I, clusterin, proteasome RC1 subunit, rat prostatein C3 and C1 subunits; two ESTs and four unknown genes. The overexpression of PABP, clusterin, profilin, and the p40/37LBP mRNAs was confirmed in preleukemic thymuses and can be related to some cellular events observed during the preleukemic period, i.e., alterations of cell cycle and apoptosis properties. The p40/37LBP and 67-kDa laminin receptor proteins were upregulated during the preleukemic period. The data suggest that additional studies on p40/37LBP and 67-kDa laminin receptor regulation are required to evaluate their potential role in the lymphoma prevention by TNF-alpha and IFN-gamma.     

Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.     

Neutrophil priming mechanisms of sulfolipid-I and n-formyl-methionyl-leucyl-phenylalanine

The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O ₂ ^– ) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca²⁺), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O ₂ ^– generation by FMLP and SL-I required increases in intracellular Ca²⁺, an increase in intracellular calcium concentration ([Ca²⁺]_i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca²⁺, Ca²⁺ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells.     

Modulation of PAI-1 and proMMP-9 syntheses by soluble TNFalpha and its receptors during differentiation of the human monocytic HL-60 cell line

During phorbol ester-induced differentiation of HL-60 monocytic cells, tumor necrosis factoralpha (TNFalpha) synthesis and secretion are increased, which contributes to the autocrine regulation of TNFalpha-responsive genes. We investigated how, during phorbol ester-induced differentiation of HL-60 cells, the secreted TNFalpha modulated plasminogen activator inhibitor type I (PAI-1) and gelatinase B (MMP-9) syntheses, two proteins involved in pericellular proteolysis. The differentiation-induced release of TNFalpha, was abolished by the hydroxamate-based matrix metalloproteinase (MMP) inhibitor, RU36156. RU36156 or a neutralizing anti-TNFalpha significantly down-regulated PAI-1 synthesis exclusively during the early phases of differentiation (from promyelocyte to monocytic-like cells), which underlined the activating role of autocrine TNFalpha during this time range. As cells progressed to monocyte/macrophage phenotype, they still released TNFalpha, but RU36156 or anti-TNFalpha no longer had an effect on PAI-1 synthesis. This lack of effect was not due to a default of TNFalpha signaling since PAI-1 synthesis was still stimulated in response to exogenous TNFalpha. TNFalpha receptor RI was also actively released and was shown to reduce TNFalpha activity which may account for the inability of soluble TNFalpha to up-regulate PAI-1 synthesis. In later mature stage, cells became susceptible to exogenous TNFalpha-induced apoptosis and rapidly lost their ability to respond to TNFalpha. The MMP-9 synthesis followed similar regulation as PAI-1. Isolated human blood monocytes-derived macrophages behave like HL-60-derived macrophages. In conclusion, these results show that during leukocyte differentiation, time windows exist during which the autocrine TNFalpha is active and then down-regulated by RI, which may temper a continuous up-regulation of the synthesis of proteins involved in pericellular proteolysis.     

The fine structure of polychaete septate junctions

Summary Epidermal septate junctions of Nereis sp. and Cirriformia sp. fixed with OsO₄ or glutaraldehyde/OsO₄ display variable structure in electron micrographs. In transverse section the septa are often indistinct and obscured by opaque material that fills the junctional cleft. Septa (spaced at 180–280 Å) are more clearly defined in slightly oblique transverse section; they exhibit an electron lucent center and appear to be linked by arms. En face views of the junction show a honeycomb pattern. Cytoplasmic faces of junctional membranes are backed with plaques opposite the septa. Lanthanum used as a tracer delineates junctional structure in negative contrast. In transverse section a chain-like lattice is present in the junctional cleft. En face views show parallel rows of pleated elements often linked by arms into honeycomb arrays. Oblique sections demonstrate that these pleated elements are continuous with the chain-like lattice seen in transverse sections. Lanthanum does not pass entirely through the junction. Lanthanum reveals that the septa have a very intricate substructure, but it is difficult to visualize the architecture that could generate the various images presented by these junctions when seen in different orientations. However, it is clear that these junctions possess some features that are diagnostic of several supposedly different types of septate junctions in invertebrates.Supported by USPHS grants NIH 5 P01 NS-07512, NIH 2701 GM-00102, and NB-00840, and by a grant from the Pomona College Research CommitteeI thank Sarah Wurzelmann, Stanley Brown, Nancy Kelly, and Gerhard Ott for excellent technical assistance. Portions of this study were carried out while I was a Postdoctoral Fellow in the Department of Anatomy, Albert Einstein College of Medicine. I dedicate this article to Berta Scharrer as a token of appreciation and affection for her guidance, encouragement, inspiration, and example of excellence     

Litter decay controlled by temperature,not soil properties,affecting future soil carbon

Widespread global changes, including rising atmospheric CO₂ concentrations, climate warming and loss of biodiversity, are predicted for this century; all of these will affect terrestrial ecosystem processes like plant litter decomposition. Conversely, increased plant litter decomposition can have potential carbon‐cycle feedbacks on atmospheric CO₂ levels, climate warming and biodiversity. But predicting litter decomposition is difficult because of many interacting factors related to the chemical, physical and biological properties of soil, as well as to climate and agricultural management practices. We applied ¹³C‐labelled plant litter to soil at ten sites spanning a 3500‐km transect across the agricultural regions of Canada and measured its decomposition over five years. Despite large differences in soil type and climatic conditions, we found that the kinetics of litter decomposition were similar once the effect of temperature had been removed, indicating no measurable effect of soil properties. A two‐pool exponential decay model expressing undecomposed carbon simply as a function of thermal time accurately described kinetics of decomposition. (R² = 0.94; RMSE = 0.0508). Soil properties such as texture, cation exchange capacity, pH and moisture, although very different among sites, had minimal discernible influence on decomposition kinetics. Using this kinetic model under different climate change scenarios, we projected that the time required to decompose 50% of the litter (i.e. the labile fractions) would be reduced by 1–4 months, whereas time required to decompose 90% of the litter (including recalcitrant fractions) would be reduced by 1 year in cooler sites to as much as 2 years in warmer sites. These findings confirm quantitatively the sensitivity of litter decomposition to temperature increases and demonstrate how climate change may constrain future soil carbon storage, an effect apparently not influenced by soil properties.     

Metabolomics of the human aqueous humor

Introduction

The optical elements of the eye—cornea, lens, and vitreous humor—are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of AH and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues.

Objectives

The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids.

Methods

The quantitative metabolomic profiling was carried out with the use of two independent methods—high-frequency ¹H NMR spectroscopy and HPLC with high-resolution ESI-MS detection.

Results

The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2–5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes.

Conclusion

The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.