Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Proteomic analyses of a Listeria monocytogenes mutant lacking σB identify new components of the σB regulon and highlight a role for σB in the utilization of glycerol

In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.

Atmospheric warming and the amplification of precipitation extremes

Climate models suggest that extreme precipitation events will become more common in an anthropogenically warmed climate. However, observational limitations have hindered a direct evaluation of model-projected changes in extreme precipitation. We used satellite observations and model simulations to examine the response of tropical precipitation events to naturally driven changes in surface temperature and atmospheric moisture content. These observations reveal a distinct link between rainfall extremes and temperature, with heavy rain events increasing during warm periods and decreasing during cold periods. Furthermore, the observed amplification of rainfall extremes is found to be larger than that predicted by models, implying that projections of future changes in rainfall extremes in response to anthropogenic global warming may be underestimated.

Linear amplification of marginally neutral baroclinic waves

Baroclinic wave development is investigated for unstable parallel shear flows in the limit of vanishing normal-mode growth rate. This development is described in terms of the propagation and interaction mechanisms of two coherent structures, called counter-propagating Rossby waves (CRWs). It is shown that, in this limit of vanishing normal-mode growth rate, arbitrary initial conditions produce sustained linear amplification of the marginally neutral normal mode (mNM). This linear excitation of the mNM is subsequently interpreted in terms of a resonance phenomenon. Moreover, while the mathematical character of the normal-mode problem changes abruptly as the bifurcation point in the dispersion diagram is encountered and crossed, it is shown that from an initial-value viewpoint, this transition is smooth. Consequently, the resonance interpretation remains relevant (albeit for a finite time) for wavenumbers slightly different from the ones defining cut-off points. The results are further applied to a two-layer version of the classic Eady model in which the upper rigid lid has been replaced by a simple stratosphere.

Rapid characterisation of archaeological midden components using FT-IR spectroscopy, SEM-EDX and micro-XRD

Samples taken from middens at the Neolithic site of Catalhoyuk in Turkey have been analysed using IR spectroscopy backed up by powder XRD and SEM-EDX. Microcomponents studied include fossil hack-berries (providing evidence of ancient diet and seasonality), mineral nodules (providing evidence of post-depositional change) and phytoliths (mineralised plant cells, providing evidence of usage of plant species). Finely laminated ashy deposits have also been investigated allowing chemical and mineralogical variations to be explored. It is found that many layers which appear visually to be quite distinctive have, in fact, very similar mineralogy. (C) 2009 Elsevier B.V. All rights reserved.

Rapid synthesis of carbohydrate derivatives, including mimetics of c-linked disaccharides and c-Linked aza disaccharides, using the Hetero-Diels−Alder Reaction

In this work we demonstrate the value of performing a Hetero Diels-Alder reaction (HDAR) between Danishefsky’s diene and a range of aldehydes or imines, under microwave irradiation. By using a range of aldehydes and imines, including those derived from carbohydrates, access to functionalised 2,3-dihydro-4H-pyran-4-ones or 2,3-dihydro-4-pyridinones in good to excellent synthetic yields is possible. A particular strength of the methodology is its ability to access mimetics of C-linked disaccharides and C-linked aza disaccharides, targets of current therapeutic interest, in a rapid, convenient and diastereoselective manner. The effect of high pressure on the HDARs involving carbohydrate derived aldehydes and imines is also explored, with enhancement in yields occurring for the aldehyde substrates. Finally, HDARs using carbohydrate derived ketones, enones and enals are described under a range of conditions. Optimum results were obtained under high pressure conditions, with highly functionalized carbohydrate derivatives being afforded, in good yields, in this way.

The rapid rise of supermarkets?

A series of articles, many of them published in this journal, have charted the rapid spread of supermarkets in developing and middle-income countries and forecast its continuation. In this article, the level of supermarket penetration (share of the retail food market) is modelled quantitatively on a cross-section of 42 countries for which data could be obtained, representing all stages of development. GDP per capita, income distribution, urbanisation, female labour force participation and openness to inward foreign investment are all significant explanators. Projections to 2015 suggest significant but not explosive further penetration; increased openness and GDP growth are the most significant factors.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Mapping antimalarial pharmacophores as a useful tool for the rapid discovery of drugs effective in vivo: design, construction, characterization, and pharmacology of metaquine

Resistant strains of Plasmodium falciparum and the unavailability of useful antimalarial vaccines reinforce the need to develop new efficacious antimalarials. This study details a pharmacophore model that has been used to identify a potent, soluble, orally bioavailable antimalarial bisquinoline, metaquine (N,N'-bis(7-chloroquinolin-4-yl)benzene-1,3-diamine) (dihydrochloride), which is active against Plasmodium berghei in vivo (oral ID50 of 25 mu mol/kg) and multidrug-resistant Plasmodium falciparum K1 in vitro (0.17 mu M). Metaquine shows strong affinity for the putative antimalarial receptor, heme at pH 7.4 in aqueous DMSO. Both crystallographic analyses and quantum mechanical calculations (HF/6-31+G*) reveal important regions of protonation and bonding thought to persist at parasitic vacuolar pH concordant with our receptor model. Formation of drug-heme adduct in solution was confirmed using high-resolution positive ion electrospray mass spectrometry. Metaquine showed strong binding with the receptor in a 1: 1 ratio (log K = 5.7 +/- 0.1) that was predicted by molecular mechanics calculations. This study illustrates a rational multidisciplinary approach for the development of new 4-aminoquinoline antimalarials, with efficacy superior to chloroquine, based on the use of a pharmacophore model.

Rapid characterisation of archaeological midden components using FT-IR spectroscopy, SEM-EDX and micro-XRD

Samples taken from middens at the Neolithic site of Catalhoyuk in Turkey have been analysed using IR spectroscopy backed up by powder XRD and SEM-EDX. Microcomponents studied include fossil hack-berries (providing evidence of ancient diet and seasonality), mineral nodules (providing evidence of post-depositional change) and phytoliths (mineralised plant cells, providing evidence of usage of plant species). Finely laminated ashy deposits have also been investigated allowing chemical and mineralogical variations to be explored. It is found that many layers which appear visually to be quite distinctive have, in fact, very similar mineralogy. (C) 2009 Elsevier B.V. All rights reserved.