Anti-chemorepulsive effects of vascular endothelial growth factor and placental growth factor-2 in dorsal root ganglion neurons are mediated via neuropilin-1 and cyclooxygenase-derived prostanoid production

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion.     

Modulation of plasma membrane H+-ATPase from oat roots by lysophosphatidylcholine, free fatty acids and phospholipase A2

Plasma membrane vesicles were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots in an aqueous polymer two-phase system. The plasma membranes possessed high specific ATPase activity [ca 4 μmol P₁ (mg protein)⁻¹ min⁻¹ at 37°C]. Addition of lysophosphatidylcholine (lyso-PC) produced a 2–3 fold activation of the plasma membrane ATPase, an effect due both to exposure of latent ATP binding sites and to a true activation of the enzyme. Lipid activation increased the affinity for ATP and caused a shift of the pH optimum of the H⁺ -ATPase activity to 6.75 as compared to pH 6.45 for the negative H⁺-ATPase. Activation was dependent on the chain length of the acyl group of the lyso-PC, with maximal activition obtained by palmitoyl lyso-PC. Free fatty acids also activated the membrane-bound H⁺-ATPase. This activation was also dependent on chain length and to the degree of unsaturation, with linolenic and arachidonic acid as the most efficient fatty acids. Exogenously added PC was hydrolyzed to lyso-PC and free fatty acids by an enzyme in the plasma membrane preparation, presumably of the phospholipase A type. Both lyso-PC and free fatty acids are products of phospholipase A₂ (EC 3.1.1.4) action, and addition of phospholipase A₂ from animal sources increased the H⁺-ATPase activity within seconds. Interaction with lipids and fatty acids could thus be part of the regulatory system for H⁺-ATPase activity in vivo, and the endogenous phospholipase may be involved in the regulation of the H⁺-ATPase activity in the plasma membranne.     

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target <Emphasis Type="Italic">lox</Emphasis>-Sites

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.     

Interaction between a 54-kilodalton mammalian cell surface protein and cowpea mosaic virus

Cowpea mosaic virus (CPMV), a plant virus that is a member of the picornavirus superfamily, is increasingly being used for nanotechnology applications, including material science, vascular imaging, vaccine development, and targeted drug delivery. For these applications, it is critical to understand the in vivo interactions of CPMV within the mammalian system. Although the bioavailability of CPMV in the mouse has been demonstrated, the specific interactions between CPMV and mammalian cells need to be characterized further. Here we demonstrate that although the host range for replication of CPMV is confined to plants, mammalian cells nevertheless bind and internalize CPMV in significant amounts. This binding is mediated by a conserved 54-kDa protein found on the plasma membranes of both human and murine cell lines. Studies using a deficient cell line, deglycosidases, and glycosylation inhibitors showed that the CPMV binding protein (CPMV-BP) is not glycosylated. A possible 47-kDa isoform of the CPMV-BP was also detected in the organelle and nuclear subcellular fraction prepared from murine fibroblasts. Further characterization of CPMV-BP is important to understand how CPMV is trafficked through the mammalian system and may shed light on how picornaviruses may have evolved between plant and animal hosts.     

Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c(3) isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.     

C-terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling

A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 microM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wt(low) to less than 20% within 1 h, whereas the chimera B2eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.     

Estimating the Hidden Burden of Bovine Tuberculosis in Great Britain

The number of cattle herds placed under movement restrictions in Great Britain (GB) due to the suspected presence of bovine tuberculosis (bTB) has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown) within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC) to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI) where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR). Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26–4.9; 95% CI) in a herd of 30 cattle up to 4.9 (0.99–14.0) in a herd of 400. Under this model we estimate that 50% (33–67) of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11–42) of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence unless this extrinsic infectious pressure is simultaneously addressed.     

Dissolved and particulate organic matter in contrasting Zostera marina (eelgrass) sediments

The influence of seagrass Zostera marina on sediment characteristics was examined in two contrasting sediments, one organic-rich and one organic-poor. The presence of plants leads to reduced sediment redox potential in both sediment types compared to bare sediment with the largest effects in the organic-poor sediment. Z. marina stimulated the sulfate reduction rates in organic-poor sediment with ∼50% and higher pools of dissolved organic carbon (DOC) were found. In contrast, sulfate reduction rates were lower in vegetated compared to bare sites in the organic-rich sediment. Despite a low contribution of dissolved carbohydrate (DCHO) to the DOC pool (<5%), the seagrass vegetation was responsible for an increase of ∼50% in DCHO pools with a peak in the root zone suggesting that Z. marina supplied DCHO to the pore waters. The Z. marina meadows also enhanced the contribution of particulate carbohydrate (PCHO) to sedimentary particulate organic carbon (POC) pools by 6-14% compared to bare sediment. Although the PCHO pools were higher in organic-rich than organic-poor sediments, the analyses of carbohydrate composition revealed that three groups of neutral sugars including glucose, galactose and mannose+xylose were the major compounds of PCHO and contributed with >60% to sedimentary carbohydrate pools at both sites. Only glucose showed depletion with depth in the vegetated sediments, whereas the percentage of ribose and rhamnose increased indicating a selective degradation of labile carbohydrates in the meadows. Galactose and mannose+xylose appeared to represent a refractory part of carbohydrate that remained after degradation of the more labile components. The sugar content was rather constant with depth at the bare organic-rich sediment indicating that only recalcitrant carbohydrate pools were buried. There was less difference in the PCHO composition profiles between vegetated and bare organic-poor sediments.