Binding of netrin-4 to laminin short arms regulates basement membrane assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.     

TRPA1 is differentially modulated by the amphipathic molecules trinitrophenol and chlorpromazine

TRPA1, a poorly selective Ca(2+)-permeable cation channel, is expressed in peripheral sensory neurons, where it is considered to contribute to a variety of sensory processes such as the detection of painful stimuli. Furthermore, TRPA1 was also identified in hair cells of the inner ear, but its involvement in sensing mechanical forces is still being controversially discussed. Amphipathic molecules such as trinitrophenol and chlorpromazine have been shown to provide useful tools to study mechanosensitive channels. Depending on their charge, they partition in the inner or outer sheets of the lipid bilayer, causing a curvature of the membrane, which has been demonstrated to activate or inhibit mechanosensitive ion channels. In the present study, we investigated the effect of these molecules on TRPA1 gating. TRPA1 was robustly activated by the anionic amphipathic molecule trinitrophenol. The whole-cell and single channel properties resemble those previously described for TRPA1. Moreover, we could show that the toxin GsMTx-4 acts on TRPA1. In addition to its recently described role as an inhibitor of stretch-activated ion channels, it serves as a potent activator of TRPA1 channels. On the other hand, the positively charged drug chlorpromazine modulates activated TRPA1 currents in a voltage-dependent way. The exposure of activated TRPA1 channels to chlorpromazine led to a block at positive potentials and an increased open probability at negative potentials. The variability in the shape of the I-V curve gives a first indication that native mechanically activated TRPA1 currents must not necessarily exhibit the same biophysical properties as ligand-activated TRPA1 currents.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Regulation of the muscle-specific AMP-activated protein kinase alpha2beta2gamma3 complexes by AMP and implications of the mutations in the gamma3-subunit for the AMP dependence of the enzyme

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases.     

SELDI-TOF-MS of saliva: methodology and pre-treatment effects

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.     

Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature

Plasmodium undergoes one round of multiplication in the liver prior to invading erythrocytes and initiating the symptomatic blood phase of the malaria infection. Productive hepatocyte infection by sporozoites leads to the generation of thousands of merozoites capable of erythrocyte invasion. Merozoites are released from infected hepatocytes as merosomes, packets of hundreds of parasites surrounded by host cell membrane. Intravital microscopy of green fluorescent protein-expressing P. yoelii parasites showed that the majority of merosomes exit the liver intact, adapt a relatively uniform size of 12-18 microm, and contain 100-200 merozoites. Merosomes survived the subsequent passage through the right heart undamaged and accumulated in the lungs. Merosomes were absent from blood harvested from the left ventricle and from tail vein blood, indicating that the lungs effectively cleared the blood from all large parasite aggregates. Accordingly, merosomes were not detectable in major organs such as brain, kidney, and spleen. The failure of annexin V to label merosomes collected from hepatic effluent indicates that phosphatidylserine is not exposed on the surface of the merosome membrane suggesting the infected hepatocyte did not undergo apoptosis prior to merosome release. Merosomal merozoites continued to express green fluorescent protein and did not incorporate propidium iodide or YO-PRO-1 indicating parasite viability and an intact merosome membrane. Evidence of merosomal merozoite infectivity was provided by hepatic effluent containing merosomes being significantly more infective than blood with an identical low-level parasitemia. Ex vivo analysis showed that merosomes eventually disintegrate inside pulmonary capillaries, thus liberating merozoites into the bloodstream. We conclude that merosome packaging protects hepatic merozoites from phagocytic attack by sinusoidal Kupffer cells, and that release into the lung microvasculature enhances the chance of successful erythrocyte invasion. We believe this previously unknown part of the plasmodial life cycle ensures an effective transition from the liver to the blood phase of the malaria infection.     

A molecular link between malaria and Epstein-Barr virus reactivation

Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1alpha (CIDR1alpha) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1alpha and EBV in the context of B cells. We show that CIDR1alpha binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1alpha stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1alpha can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.     

A metal-coded affinity tag approach to quantitative proteomics

The quantitative analysis of protein mixtures is pivotal for the understanding of variations in the proteome of living systems. Therefore, approaches have been recently devised that generally allow the relative quantitative analysis of peptides and proteins. Here we present proof of concept of the new metal-coded affinity tag (MeCAT) technique, which allowed the quantitative determination of peptides and proteins. A macrocyclic metal chelate complex (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) loaded with different lanthanides (metal(III) ions) was the essential part of the tag. The combination of DOTA with an affinity anchor for purification and a reactive group for reaction with amino acids constituted a reagent that allowed quantification of peptides and proteins in an absolute fashion. For the quantitative determination, the tagged peptides and proteins were analyzed using flow injection inductively coupled plasma MS, a technique that allowed detection of metals with high precision and low detection limits. The metal chelate complexes were attached to the cysteine residues, and the course of the labeling reaction was followed using SDS-PAGE and MALDI-TOF MS, ESI MS, and inductively coupled plasma MS. To limit the width in isotopic signal spread and to increase the sensitivity for ESI analysis, we used the monoisotopic lanthanide macrocycle complexes. Peptides tagged with the reagent loaded with different metals coelute in liquid chromatography. In first applications with proteins, the calculated detection limit for bovine serum albumin for example was 110 amol, and we have used MeCAT to analyze proteins of the Sus scrofa eye lens as a model system. These data showed that MeCAT allowed quantification not only of peptides but also of proteins in an absolute fashion at low concentrations and in complex mixtures.