Modo-UG, a marsupial nonclassical MHC class I locus

Modo-UG is a class I gene located in the MHC of the marsupial Monodelphis domestica, the gray, short-tailed opossum. Modo-UG is expressed as three alternatively spliced mRNA forms, all of which encode a transmembrane form with a short cytoplasmic tail that lacks phosphorylation sites typically found in classical class I molecules. The three alternative mRNAs would encode a full-length form, an isoform lacking the α2 domain, and one lacking both α2 and α3 domains. Genotyping both captive-bred and wild M. domestica from different geographic regions revealed no variation in the residues that make up Modo-UG’s peptide-binding groove. Modo-UG’s low polymorphism is contrasting to that of a nearby class I locus, Modo-UA1, which has a highly polymorphic peptide-binding region. Absence of functional polymorphism in Modo-UG is therefore not a general feature of opossum class I genes but the result of negative selection. Modo-UG is the first MHC linked marsupial class I to be described that appears to clearly have nonclassical features.Electronic Supplementary Material Supplementary material is available for this article at     

Cerebral malaria: role of microparticles and platelets in alterations of the blood-brain barrier

Brain lesions of cerebral malaria (CM) are characterised by a sequestration of Plasmodium falciparum-parasitised red blood cells (PRBC), leucocytes and platelets within brain microvessels, by an excessive release of pro-inflammatory cytokines as well as by disruption of the blood-brain barrier (BBB). We evaluated the possibility that PRBC and platelets interact and induce functional alterations in brain endothelium. Using an in vitro model of endothelial lesion, we showed that platelets can act as bridges between PRBC and endothelial cells (EC) allowing the binding of PRBC to endothelium devoid of cytoadherence receptors. Furthermore, platelets potentiated the cytotoxicity of PRBC for brain EC by inducing an alteration of the integrity of their monolayer and increasing their apoptosis. These findings provide insights into the mechanisms by which platelets can be deleterious to the brain endothelium during CM. Another aspect of inflammatory and infectious diseases is that they often lead to activation of vascular and blood cells. Such activation results in an enhanced vesiculation, i.e. the release of circulating microparticles (MP). We thus explored plasma levels of endothelial MP in Malawian children with malaria. Plasma MP numbers were markedly increased on admission only in patients with severe malaria complicated with coma. Using the experimental mouse model of CM, we evaluated the pathogenic implications of MP using genetically deficient mice in which the capacity to vesiculate is impaired. Such mice, lacking the ABCA-1 gene, upon infection by Plasmodium berghei ANKA, showed complete resistance to CM. When purified from infected susceptible animals, MP were able to reduce normal plasma clotting time and to significantly enhance tumour necrosis factor release from na?ve macrophages. Altogether these data provide a novel insight into the pathogenic mechanisms leading to the neurological syndrome. The finding that ABCA-1 gene deletion confers complete protection against cerebral pathology, linked to an impaired MP production, provides new potential targets for therapeutic amelioration of severe malaria.     

Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.     

Limitation of cell adhesion by the elasticity of the extracellular matrix

Cell/matrix adhesions are modulated by cytoskeletal or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity arises from the activation of a mechanosensor located within the adhesion itself. We show that this mechanism accounts for the observed directional growth of focal adhesions and the reduction or even cessation of their growth when cells adhere to a soft extracellular matrix. We predict quantitatively that both the elasticity and the thickness of the matrix play a key role in the dynamics of focal adhesions. Two different types of dynamics are expected depending on whether the thickness of the matrix is of order of or much larger than the adhesion size. In the latter situation, we predict that the adhesion region reaches a saturation size that can be tuned by the mechanical properties of the matrix.     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

The plasticins: membrane adsorption, lipid disorders, and biological activity

The present study investigates the relationships between structural polymorphism, adsorption onto membrane mimetic support, lipid disturbance, and biological activity of bactericidal 23-residue, glycine-leucine-rich dermaseptin orthologues from the Phyllomedusinae frog skin, the "plasticins". Biological activities were evaluated using the membrane models DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) for prokaryotic membranes and DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) for eukaryotic membranes. We performed a conformational analysis of plasticins by molecular simulations and spectroscopic methods and analyzed phospholipid perturbations by infrared spectroscopy. Adsorption onto synthetic model membranes was quantified by surface plasmon resonance. Biological assays including antimicrobial and membrane potential-dissipating activities, together with hemolytic tests and imaging analysis of cytotoxicity, were carried out to clarify the peptide-membrane interactions. Two major groups were distinguished: (i) Neutral plasticins revealed the presence of strong beta-structures with the zwitterionic or anionic phospholipid vesicles. They were weakly adsorbed in the range of antibacterial activity concentrations (micromolar). Nevertheless, for millimolar concentrations, they caused perturbations at the interface peptide-DMPG vesicles and in the bilayer alkyl chains, suggesting insertion into bacterial membranes. (ii) Cationic plasticins revealed multiple conformational transitions, including destabilized helix states, beta-structures, and disordered states. Peptide-lipid complex densities depended on hydrophobic bond strengths. The most soluble cationic plasticins were strongly adsorbed, with stable peptide-lipid interactions inducing noticeable perturbations of bilayer alkyl chains, pointing out possible insertion into bacterial membranes. In contrast, cytotoxic plasticins were less adsorbed, with less stable peptide-lipid interactions causing membrane dehydration, formation of peptide-membrane hydrogen bonds, and little disturbances of lipid alkyl chains. These characteristics could be compatible with their putative action on intracellular targets leading to apoptosis.     

Metabolism of arachidonic acid to eicosanoids within the nucleus

The eicosanoids are a diverse family of molecules that have powerful effects on cell function. They are best known as intercellular messengers, having autocrine and paracrine effects following their secretion from the cells that synthesize them. Many of the eicosanoids are produced from one polyunsaturated fatty acid, arachidonic acid. The diversity of possible products that can be synthesized from arachidonic acid is due, in part to the variety of enzymes that can act on it. Over the past 15 years, studies have placed many, but not all, of these enzymes at or inside the nucleus. In some cases, the nuclear import or export of arachidonic acid-processing enzymes is highly regulated. Furthermore, nuclear receptors that are activated by specific eicosanoids are known to exist. Taken together, these findings indicate that the enzymatic conversion of arachidonic acid to specific signaling molecules can occur in the nucleus, that it is regulated, and that the synthesized products may act within the nucleus. The objectives of this commentary are to review what is known about the metabolism of arachidonic acid to eicosanoids within the nucleus and to point to important areas for future discovery.     

Structural insight into poplar glutaredoxin C1 with a bridging iron-sulfur cluster at the active site

Glutaredoxins are glutathione-dependent enzymes that function to reduce disulfide bonds in vivo. Interestingly, a recent discovery indicates that some glutaredoxins can also exist in another form, an iron-sulfur protein [Lillig, C. H., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 8168-8173]. This provides a direct connection between glutaredoxins and iron-sulfur proteins, suggesting a possible new regulatory role of iron-sulfur clusters along with the new functional switch of glutaredoxins. Biochemical studies have indicated that poplar glutaredoxin C1 (Grx-C1) is also such a biform protein. The apo form (monomer) of Grx-C1 is a regular glutaredoxin, and the holo form (dimer) is an iron-sulfur protein with a bridging [2Fe-2S] cluster. Here, we report the structural characterizations of poplar Grx-C1 in both the apo and holo forms by NMR spectroscopy. The solution structure of the reduced apo Grx-C1, which is the first plant Grx structure, shows a typical Grx fold. When poplar Grx-C1 forms a dimer with an iron-sulfur cluster, each subunit of the holo form still retains the overall fold of the apo form. The bridging iron-sulfur cluster in holo Grx-C1 is coordinated near the active site. In addition to the iron-sulfur cluster linker, helix alpha3 of each subunit is probably involved in the direct contact between the two subunits. Moreover, two glutathione molecules are identified in the vicinity of the iron-sulfur cluster and very likely participate in cluster coordination. Taken together, we propose that the bridging [2Fe-2S] cluster is coordinated by the first cysteine at the glutaredoxin active site from each subunit of holo Grx-C1, along with two cysteines from two glutathione molecules. Our studies reveal that holo Grx-C1 has a novel structural and iron-sulfur cluster coordination pattern for an iron-sulfur protein.