Structural characterization of the transmembrane and cytoplasmic domains of human CD4

Cluster determinant 4 (CD4) is a type I transmembrane glycoprotein of 58 kDa. It consists of an extracellular domain of 370 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids at the C-terminal end. We investigated the structure of the 62 C-terminal residues of CD4, comprising its transmembrane and cytoplasmic domains. The five cysteine residues of this region have been replaced with serine and histidine residues in the polypeptide CD4mut. Uniformly 15N and 13C labeled protein was recombinantly expressed in E. coli and purified. Functional binding activity of CD4mut to protein VpU of the human immunodeficiency virus type 1 (HIV-1) was verified. Close to complete NMR resonance assignment of the 1H, 13C, and 15N spins of CD4mut was accomplished. The secondary structure of CD4mut in membrane simulating dodecylphosphocholine (DPC) micelles was characterized based on secondary chemical shift analysis, NOE-based proton-proton distances, and circular dichroism spectroscopy. A stable transmembrane helix and a short amphipathic helix in the cytoplasmic region were identified. The fractional helicity of the cytoplasmic helix appears to be stabilized in the presence of DPC micelles, although the extension of this helix is reduced in comparison to previous studies on synthetic peptides in aqueous solution. The role of the amphipathic helix and its potentially variable length is discussed with respect to the biological functions of CD4.     

Membrane-anchored inhibitory peptides capture human immunodeficiency virus type 1 gp41 conformations that engage the target membrane prior to fusion

Soluble peptides derived from the C-terminal heptad repeat domain of human immunodeficiency virus type 1 (HIV-1) gp41 are potent inhibitors of HIV-1 entry and gp41-induced fusion. Target membrane-anchored variants of these peptides have been shown to retain inhibitory activity. Both soluble and membrane-anchored C peptides (MACs) are thought to block fusion by binding to the N-terminal coiled coil domain of gp41 and preventing formation of the final six-helix bundle structure. However, interactions of target MACs with gp41 must be restricted to a subset of trimers that have their hydrophobic fusion peptides inserted into the target membrane. This unique feature of MACs was used to identify the intermediate step of fusion at which gp41 engaged the target membrane. Fusion between HIV envelope-expressing effector cells and target cells was measured by fluorescence microscopy. Expression of MACs in target cells led to less than twofold reduction in the extent of fusion. However, when reaction was first arrested by adding lysolipids that disfavored membrane merger, and the lipids were subsequently removed by washing, control cells supported fusion, whereas those that expressed MACs did not. The drastically improved potency of MACs implies that, at lipid-arrested stage, gp41 bridges the viral and target cell membranes and therefore more optimally binds the membrane-anchored peptides. Experimental demonstration of this intermediate shows that, similar to fusion induced by many other viral glycoproteins, engaging the target membrane by HIV-1 gp41 permits coupling between six-helix bundle formation and membrane merger.     

Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-down experiments confirmed the interaction, and indicated that it depends on the PDZ-domain binding motif of Na(v)1.5. Co-expression experiments in HEK293 cells showed that PTPH1 shifts the Na(v)1.5 availability relationship toward hyperpolarized potentials, whereas an inactive PTPH1 or the tyrosine kinase Fyn does the opposite. The results of this study suggest that tyrosine phosphorylation destabilizes the inactivated state of Na(v)1.5.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

Genetic population structure of the endemic fourline wrasse (Larabicus quadrilineatus) suggests limited larval dispersal distances in the Red Sea

The connectivity among marine populations is determined by the dispersal capabilities of adults as well as their eggs and larvae. Dispersal distances and directions have a profound effect on gene flow and genetic differentiation within species. Genetic homogeneity over large areas is a common feature of coral reef fishes and can reflect high dispersal capability resulting in high levels of gene flow. If fish larvae return to their parental reef, gene flow would be restricted and genetic differentiation could occur. Larabicus quadrilineatus (Labridae) is considered as an endemic fish species of the Red Sea and Gulf of Aden. The juveniles of this species are cleaner fish that feed on ectoparasites of other fishes. Here, we investigated the genetic population structure and gene flow in L. quadrilineatus among five locations in the Red Sea to infer connectivity among them. To estimate genetic diversity, we analysed 369 bp of 237 mitochondrial DNA control region sequences. Haplotype and nucleotide diversities were higher in the southern than in the northern Red Sea. Analysis of molecular variance (amova) detected the highest significant genetic variation between northern and central/southern populations (Phi(CT) = 0.01; P < 0.001). Migration analysis revealed a several fold higher northward than southward migration, which could be explained by oceanographic conditions and spawning season. Even though the Phi(ST) value of 0.01 is rather low and implies a long larval dispersal distance, estimates based on the isolation-by-distance model show a very low mean larval dispersal distance (0.44-5.1 km) compared to other studies. In order to enable a sustainable ornamental fishery on the fourline wrasse, the results of this study suggest that populations in the northern and southern Red Sea should be managed separately as two different stocks. The rather low larval dispersal distance of about 5 km needs to be considered in the design of marine protected areas to enable connectivity and self-seeding.     

Within-Host and Population Transmission of bla OXA-48 in K. pneumoniae and E. coli

During a large hospital outbreak of OXA-48 producing bacteria, most K. pneumoniae _OXA-48 isolates were phenotypically resistant to meropenem or imipenem, whereas most E. coli _OXA-48 isolates were phenotypically susceptible to these antibiotics. In the absence of molecular gene-detection E. coli _OXA-48 could remain undetected, facilitating cross-transmission and horizontal gene transfer of bla _OXA-48. Based on 868 longitudinal molecular microbiological screening results from patients carrying K. pneumoniae _OXA-48 (n = 24), E. coli _OXA-48 (n = 17), or both (n = 40) and mathematical modelling we determined mean durations of colonisation (278 and 225 days for K. pneumoniae _OXA-48 and E. coli _OXA-48, respectively), and horizontal gene transfer rates (0.0091/day from K. pneumoniae to E. coli and 0.0015/day vice versa). Based on these findings the maximum effect of horizontal gene transfer of bla _OXA-48 originating from E. coli _OXA-48 on the basic reproduction number (R ₀) is 1.9%, and it is, therefore, unlikely that phenotypically susceptible E. coli _OXA-48 will contribute significantly to the spread of bla _OXA-48.     

Expression of SR-BI receptor and StAR protein in rat ocular tissues

The class-B type-I scavenger receptor (SR-BI) plays a key role in cholesterol homeostasis; it mediates the selective uptake of lipoprotein cholesterol to steroidogenic tissues. We show by RT-PCR, western blot, in situ hybridization and immunohistochemistry analysis that SR-BI is highly expressed in different neuro-retinal and non-neuronal cells types on rat eye. Immunohistochemistry of the steroidogenic acute regulatory protein (StAR) involved in neurosteroid production showed the same expression pattern than SR-BI in rat eye. Our results may suggest a key role of these genes in the ocular cholesterol metabolism for membranes biosynthesis and neurosteroidogenesis.     

Interaction of 14-3-3 protein with regulator of G protein signaling 7 is dynamically regulated by tumor necrosis factor-alpha

Regulators of G protein signaling (RGS) constitute a family of proteins with a conserved RGS domain of approximately 120 amino acids that accelerate the intrinsic GTP hydrolysis of activated Galpha(i) and Galpha(q) subunits. The phosphorylation-dependent interaction of 14-3-3 proteins with a subset of RGS proteins inhibits their GTPase-accelerating activity in vitro. The inhibitory interaction between 14-3-3 and RGS7 requires phosphorylation of serine 434 of RGS7. We now show that phosphorylation of serine 434 is dynamically regulated by TNF-alpha. Cellular stimulation by TNF-alpha transiently decreased the phosphorylation of serine 434 of RGS7, abrogating the inhibitory interaction with 14-3-3. We examined the effect of 14-3-3 on RGS-mediated deactivation kinetics of G protein-coupled inwardly rectifying K(+) channels (GIRKs) in Xenopus oocytes. 14-3-3 inhibited the function of wild-type RGS7, but not that of either RSG7(P436R) or RGS4, two proteins that do not bind 14-3-3. Our findings are the first evidence that extracellular signals can modulate the activity of RGS proteins by regulating their interaction with 14-3-3.