pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Recombinant human SMOCs produced by in vitro refolding: calcium-binding properties and interactions with serum proteins

Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K(d) values in the 0.7-1 microM range), while the other had lower affinity (K(d) values 20-25 microM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins.     

A presynaptically toxic secreted phospholipase A2 is internalized into motoneuron-like cells where it is rapidly translocated into the cytosol

The molecular mechanism of the presynaptic toxicity of secreted phospholipase A2 (sPLA2) neurotoxins, including that of ammodytoxin A (AtxA), has not been resolved. Here we report the action of AtxA on mouse motoneuron-like cells, on which it induced characteristic neurotoxic effects on synaptic vesicles and on the reorganization of F-actin. AtxA also released fatty acids from the plasmalemma. Its significantly less neurotoxic V31W mutant showed similar effects on cells but with a much higher rate of hydrolysis than the wild-type, indicating that high enzymatic activity alone is not sufficient for the observed effects. The neurotoxic action was observed by confocal microscopy of a fluorescently labelled AtxA and by electron microscopy of a nanogold-labelled toxin. The Atx-binding proteins were tagged by a photo-cross-linking reagent conjugated to the toxin. AtxA was taken up rapidly by the cells, where it interacted within minutes with calmodulin and 14-3-3 proteins in the cytosol. These data demonstrate, for the first time, the translocation of an sPLA2 from the extracellular space into the cytosol of a cell. Such an event may thus be important in explaining the action of a range of homologous endogenous sPLA2 enzymes in mammals whose roles in various cellular processes are not yet completely understood.     

Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.     

The relationship between dietary specialism and availability of food and water on cannibalistic interactions among predatory mites in protected crops

The interplay between dietary specialism, the tolerance of food and water stress and level of cannibalism is likely to be important in determining the outcome of biological control using inundative releases of multiple natural enemies, such as phytoseiid mites in protected crops. The dietary specialist, Phytoseiulus persimilis, with a short immature development time (4-5 days) when plentiful food was available had a low ability to survive without food (5 days), even with access to water. The dietary generalists, Neoseiulus californicus, N. cucumeris and lphiseius degenerans, had longer immature development times (by up to 2 days) than P. persimilis. Survival ability differed amongst the generalist species when they were starved but provided with constant access to water. Both N. californicus and N. cucumeris survived the longest (8-10 days) and I. degenerans survived the shortest period (4 days). No negative intra-specific interaction between immatures was observed with P. persimilis when food was available and in the absence of food this species tended to starve rather than act cannibalistically. Both N. californicus and N. cucumeris showed a low degree of cannibalism between immatures, either when food was available, or when starved but given access to water. Even when food was available survival of I. degenerans fell by 30% in 4 days and remained at 60-70% for 3 further days; survival continued to decline rapidly when they were starved but provided with water. This indicates that immatures of I. degenerans could either feed on dead conspecifics or that they were capable of a degree of cannibalism. Adult females of P. persimilis did not feed on conspecific eggs even when deprived of food but provided with water. Adult female N. californicus and N. cucumeris did feed on conspecific eggs but at a low level (<1 egg per day), which occurred only after 48 h starvation. Although egg cannibalism occurred more consistently with adult female I. degenerans than with other mite species it was at a low level (<1 egg per day). If the tendency to cannibalism, not just of eggs but with more susceptible life stages such as larvae, is reduced when water is available freely this could be important in determining the interactions that occur under natural conditions.     

Synthesis and reactivity of the monosaccharide esters of amino acids as models of teichoic acid fragment

The increasing prevalence of sepsis from Gram-positive bacterial pathogens necessitates further evaluation of the basic assumptions about the molecular pathogenesis of septic shock. Since diverse physiological functions of Gram-positive bacteria are controlled by the degree of esterification of teichoic acids with D-alanine, we examined the reactivity of monosaccharide esters in which anomerically free or protected D-glucose is linked through its C-6 hydroxy group to either phenylalanyl or tyrosyl residues as models for teichoic acid fragment. We show that the attached sugar moiety induces activation of the amino acid residue. Due to the enhanced reactivity of the NH₂ group in the monosaccharide esters studied, the formation of products generated by intramolecular and intermolecular glycation reactions is accelerated resulting in heterogeneous mixture of compounds. These findings suggest that, if similar adducts are formed by glycation of D-alanine in teichoic acid of Gram-positive bacteria, they should be examined as potential bioactive ligands or chemical message for infection.     

Atmospheric conditions during high ragweed pollen concentrations in Zagreb,Croatia

We examined the atmospheric conditions favourable to the occurrence of maximum concentrations of ragweed pollen with an extremely high risk of producing allergy. Over the 2002–2009 period, daily pollen data collected in Zagreb were used to identify two periods of high pollen concentration (> 600 grains/m³) for our analysis: period A (3–4 September 2002) and period B (6–7 September 2003). Synoptic conditions in both periods were very similar: Croatia was under the influence of a lower sector high pressure system moving slowly eastward over Eastern Europe. During the 2002–2009 period, this type of weather pattern (on ~ 70% of days), in conjunction with almost non-gradient surface pressure conditions in the area (on ~ 30% of days) characterised days when the daily pollen concentrations were higher than 400 grains/m³. Numerical experiments using a mesoscale model at fine resolution showed successful multi-day simulations reproducing the local topographic influence on wind flow and in reasonable agreement with available observations. According to the model, the relatively weak synoptic flow (predominantly from the eastern direction) allowed local thermal circulations to develop over Zagreb during both high pollen episodes. Two-hour pollen concentrations and 48-h back-trajectories indicated that regional-range transport of pollen grains from the central Pannonian Plain was the cause of the high pollen concentrations during period A. During period B, the north-westward regional-range transport in Zagreb was supplemented significantly by pronounced horizontal recirculation of pollen grains. This recirculation happened within the diurnal local circulation over the city, causing a late-evening increase in pollen concentration.