A high-affinity,radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

A new radioiodinated photoaffinity compound, [¹²⁵I]YE(Bpa)WSLAAPQRFNH₂, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF₂ receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (K_D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF₂ receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [¹²⁵I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF₂ receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF₂ receptor is observed. [¹²⁵I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF₂ receptors in vitro.     

The Toxoplasma gondii calcium‐dependent protein kinase 7 is involved in early steps of parasite division and is crucial for parasite survival

Apicomplexan parasites express various calcium‐dependent protein kinases (CDPKs), and some of them play essential roles in invasion and egress. Five of the six CDPKs conserved in most Apicomplexa have been studied at the molecular and cellular levels in Plasmodium species and/or in Toxoplasma gondii parasites, but the function of CDPK7 was so far uncharacterized. In T. gondii, during intracellular replication, two parasites are formed within a mother cell through a unique process called endodyogeny. Here we demonstrate that the knock‐down of CDPK7 protein in T. gondii results in pronounced defects in parasite division and a major growth deficiency, while it is dispensable for motility, egress and microneme exocytosis. In cdpk7‐depleted parasites, the overall DNA content was not impaired, but the polarity of daughter cells budding and the fate of several subcellular structures or proteins involved in cell division were affected, such as the centrosomes and the kinetochore. Overall, our data suggest that CDPK7 is crucial for proper maintenance of centrosome integrity required for the initiation of endodyogeny. Our findings provide a first insight into the probable role of calcium‐dependent signalling in parasite multiplication, in addition to its more widely explored role in invasion and egress.     

The <Emphasis Type="Italic">Vitis vinifera</Emphasis> sugar transporter gene family: phylogenetic overview and macroarray expression profiling

Background  

In higher plants, sugars are not only nutrients but also important signal molecules. They are distributed through the plant via sugar transporters, which are involved not only in sugar long-distance transport via the loading and the unloading of the conducting complex, but also in sugar allocation into source and sink cells. The availability of the recently released grapevine genome sequence offers the opportunity to identify sucrose and monosaccharide transporter gene families in a woody species and to compare them with those of the herbaceous Arabidopsis thaliana using a phylogenetic analysis.     

Regulation of actin dynamics by annexin 2

Annexin 2 is a ubiquitous Ca(2+)-binding protein that is essential for actin-dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin-rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co-expressing annexin 2-CFP and actin-YFP exhibit Ca(2+)-dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose-dependent manner. By measuring actin polymerisation rates in the presence of barbed-end and pointed-end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin.     

The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling

Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.     

Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.     

Association between Repeated Unpredictable Chronic Mild Stress (UCMS) Procedures with a High Fat Diet: A Model of Fluoxetine Resistance in Mice

Major depressive disorder is a debilitating disease. Unfortunately, treatment with antidepressants (ADs) has limited therapeutic efficacy since resistance to AD is common. Research in this field is hampered by the lack of a reliable natural animal model of AD resistance. Depression resistance is related to various factors, including the attendance of cardiovascular risk factors and past depressive episodes. We aimed to design a rodent model of depression resistance to ADs, associating cardiovascular risk factors with repeated unpredicted chronic mild stress (UCMS). Male BALB/c mice were given either a regular (4% fat) or a high fat diet (45% fat) and subjected to two 7-week periods of UCMS separated by 6 weeks. From the second week of each UCMS procedure, vehicle or fluoxetine (10 mg/kg, i.p.) was administrated daily. The effects of the UCMS and fluoxetine in both diet conditions were assessed using physical (coat state and body weight) and behavioural tests (the reward maze test and the splash test). The results demonstrate that during the second procedure, UCMS induced behavioural changes, including coat state degradation, disturbances in self-care behaviour (splash test) and anhedonia (reward maze test) and these were reversed by fluoxetine in the regular diet condition. In contrast, the high-fat diet regimen prevented the AD fluoxetine from abolishing the UCMS-induced changes. In conclusion, by associating UCMS—an already validated animal model of depression—with high-fat diet regimen, we designed a naturalistic animal model of AD resistance related to a sub-nosographic clinical entity of depression.