Characterization of a non-mitochondrial type I phosphatidylserine decarboxylase in Plasmodium falciparum

In search of key enzymes in Plasmodium phospholipid metabolism, we demonstrate the presence of a parasite-encoded phosphatidylserine decarboxylase (PSD) in the membrane fraction of Plasmodium falciparum-infected erythrocytes. PSD cDNA, encoding phosphatidylserine decarboxylase (PfPSD), was cloned by screening a directional cDNA library derived from the trophozoite erythrocytic stage. The corresponding PfPSD gene is located on chromosome 9 of P. falciparum, contains one intron of 938 nucleotides and is transcribed into a 3.7 kb mRNA. PfPSD cDNA encodes a putative protein of 362 amino acids, with a predicted molecular mass of 42.6 kDa, which clearly belongs to the type I PSD family. Only a 35 kDa polypeptide was detected in the parasite using a specific rabbit antiserum. PfPSD has a 314VGSS317 sequence near its carboxyl-terminus that is related to the Escherichia coli, yeast and human LGST motif, which is the site of proenzyme processing. PSD enzyme was expressed in E. coli with a KM of 63 +/- 19 microM and a VMAX of 680 +/- 49 nmol of phosphatidylethanolamine formed h-1 mg-1 protein. Site-directed mutagenesis of the VGSS active site demonstrated that the PfPSD proenzyme was processed into two non-identical subunits (alpha and beta) and revealed the crucial role played by each residue in enzyme processing and activity. Using indirect immunofluorescence, PfPSD labelling was co-localized with an endoplasmic reticulum marker, but not with a mitochondrial vital dye. This P. falciparum PSD is the first type I PSD identified in the endoplasmic reticulum compartment.     

Molecular bases for sensitivity to tubulin-binding herbicides in green foxtail

We investigated the molecular bases for resistance to several classes of herbicides that bind tubulins in green foxtail (Setaria viridis L. Beauv.). We identified two alpha- and two beta-tubulin genes in green foxtail. Sequence comparison between resistant and sensitive plants revealed two mutations, a leucine-to-phenylalanine change at position 136 and a threonine-to-isoleucine change at position 239, in the gene encoding alpha2-tubulin. Association of mutation at position 239 with herbicide resistance was demonstrated using near-isogenic lines derived from interspecific pairings between green foxtail and foxtail millet (Setaria italica L. Beauv.), and herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Association of mutation at position 136 with herbicide resistance was demonstrated using herbicide sensitivity bioassays combined with allele-specific PCR-mediated genotyping. Both mutations were associated with recessive cross resistance to dinitroanilines and benzoic acids, no change in sensitivity to benzamides, and hypersensitivity to carbamates. Using three-dimensional modeling, we found that the two mutations are adjacent and located into a region involved in tubulin dimer-dimer contact. Comparison of three-dimensional alpha-tubulin models for organisms with contrasted sensitivity to tubulin-binding herbicides enabled us to propose that residue 253 and the vicinity of the side chain of residue 251 are critical determinants for the differences in herbicide sensitivity observed between organisms, and that positions 16, 24, 136, 239, 252, and 268 are involved in modulating sensitivity to these herbicides.     

A mixture model-based strategy for selecting sets of genes in multiclass response microarray experiments

MOTIVATION: Multiclass response (MCR) experiments are those in which there are more than two classes to be compared. In these experiments, though the null hypothesis is simple, there are typically many patterns of gene expression changes across the different classes that led to complex alternatives. In this paper, we propose a new strategy for selecting genes in MCR that is based on a flexible mixture model for the marginal distribution of a modified F-statistic. Using this model, false positive and negative discovery rates can be estimated and combined to produce a rule for selecting a subset of genes. Moreover, the method proposed allows calculation of these rates for any predefined subset of genes. RESULTS: We illustrate the performance our approach using simulated datasets and a real breast cancer microarray dataset. In this latter study, we investigate predefined subset of genes and point out interesting differences between three distinct biological pathways. AVAILABILITY: http://www.bgx.org.uk/software.html     

Floxed allele for conditional inactivation of the GABAB(1) gene

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter GABA. GABA(B) receptors are broadly expressed in the nervous system. Their complete absence in mice causes premature lethality or--when mice are viable--epilepsy, impaired memory, hyperalgesia, hypothermia, and hyperactivity. A spatially and temporally restricted loss of GABA(B) function would allow addressing how the absence of GABA(B) receptors leads to these diverse phenotypes. To permit a conditional gene inactivation, we flanked critical exons of the GABA(B(1)) gene with lox511 sites. GABA(B(1)) (lox511/lox511) mice exhibit normal levels of GABA(B(1)) protein, are fertile, and do not display any behavioral phenotype. We crossed GABA(B(1)) (lox511/lox511) with Cre-deleter mice to produce mice with an unrestricted GABA(B) receptor elimination. These GABA(B(1)) (-/-) mice no longer synthesize GABA(B(1)) protein and exhibit the expected behavioral abnormalities. The conditional GABA(B(1)) allele described here is therefore suitable for generating mice with a site- and time-specific loss of GABA(B) function.     

Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.     

Assessing the reversibility of the anaplerotic reactions of the propionyl-CoA pathway in heart and liver

While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH13CO3, [U-13C3]propionate, or [U-13C3]lactate + [U-13C3]pyruvate, under various concentrations of propionate. The mass isotopomer distributions of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA revealed that, in intact livers and hearts, (i) the propionyl-CoA carboxylase reaction is slightly reversible only at low propionyl-CoA flux, (ii) the methylmalonyl-CoA racemase reaction keeps the methylmalonyl-CoA enantiomers in isotopic equilibrium under all conditions tested, and (iii) the methylmalonyl-CoA mutase reaction is reversible, but its reversibility decreases as the flow of propionyl-CoA increases. The thermodynamic dis-equilibrium of the combined reactions of the propionyl-CoA pathway explains the effectiveness of anaplerosis from propionyl-CoA precursors such as heptanoate.     

Activation of the canonical beta-catenin pathway by histamine

Histamine signaling is a principal regulator in a variety of pathophysiological processes including inflammation, gastric acid secretion, neurotransmission, and tumor growth. We report that histamine stimulation causes transactivation of a T cell factor/beta-catenin-responsive construct in HeLa cells and in the SW-480 colon cell line, whereas histamine did not effect transactivation of a construct containing the mutated response construct FOP. On the protein level, histamine treatment increases phosphorylation of glycogen synthase kinase 3-beta in HeLa cells, murine macrophages, and DLD-1, HT-29, and SW-480 colon cell lines. Furthermore, histamine also decreases the phosphorylated beta-catenin content in HeLa cells and murine macrophages. Finally, pharmacological inhibitors of the histamine H1 receptor counteracted histamine-induced T cell factor/beta-catenin-responsive construct transactivation and the dephosphorylation of beta-catenin in HeLa cells and in macrophages. We conclude that the canonical beta-catenin pathway acts downstream of the histamine receptor H1 in a variety of cell types. The observation that inflammatory molecules, like histamine, activate the beta-catenin pathway may provide a molecular explanation for a possible link between inflammation and cancer.     

A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.