Improvement of protein secondary structure prediction by combination of statistical algorithms and circular dichroism.

Three different approaches (propensity curve shifting, hydropathy index evaluation, and iterative attribution/cancellation of secondary structure) to the use of secondary structure percentages derived from circular dichroism measurements to improve the success rate of a protein secondary structure prediction method, without using decision constants, are described and compared. Propensity-curve shifting appears to be the best-performing approach, bearing an increase of 5.3% in the success rate of single-residue structural prediction when exact information on the secondary structure, obtained by X-ray crystallography, is employed; with information of an accuracy comparable to that obtainable by circular dichroism, the improvement stays between 3.5 and 4.9%, for a three-state prediction. Although developed with circular dichroism in mind, the method can use percentages of secondary structure obtained by any other experimental methodology from which they can be inferred, for instance Raman spectroscopy and infrared spectroscopy.     

The ecology of the banded civet (Hemigalus derbyanus) in Southeast Asia with implications for mesopredator release,zoonotic diseases,and conservation

Habitat loss and degradation threaten forest specialist wildlife species, but some generalist mesopredators exploit disturbed areas and human‐derived food, which brings them into closer contact with humans. Mesopredator release is also important for human health for known zoonotic disease reservoirs, such as Asian civets (Viverridae family), since this group includes the intermediator species for the SARS‐CoV‐1 outbreak. Here we use camera trapping to evaluate the habitat associations of the widespread banded civet (Hemigalus derbyanus) across its range in Southeast Asia. At the regional scale, banded civet detections among published studies were positively associated with forest cover and negatively associated with human population. At the local scale (within a landscape), hierarchical modeling of new camera trapping showed that abundance was negatively associated with forest loss and positively associated with distance to rivers. These results do not support mesopredator release and suggest a low likelihood overlap with humans in degraded habitats and, therefore, a low risk of zoonotic disease transmission from this species in the wild. We also estimate that banded civet distribution has contracted to under 21% of its currently recognized IUCN Red List range, only 12% of which falls within protected areas, and a precipitous recent decline in population size. Accordingly, we suggest the banded civet''s Red List status should be re‐evaluated in light of our findings.     

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     

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.