The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants

The photosynthetic gene rbcL has been lost or dramatically altered in some lineages of nonphotosynthetic parasitic plants, but the dynamics of these events following loss of photosynthesis and whether rbcL has sustained functionally significant changes in photosynthetic parasitic plants are unknown. To assess the changes to rbcL associated with the loss of functional constraints for photosynthesis, nucleotide sequences from nonparasitic and parasitic plants of Scrophulariales were used for phylogeny reconstruction and character analysis. Plants in this group display a broad range of parasitic abilities, from photosynthetic ("hemiparasites") to nonphotosynthetic ("holoparasites"). With the exception of Conopholis (Orobanchaceae), the rbcL locus is present in all parasitic plants of Scrophulariales examined. Several holoparasitic genera included in this study, including Boschniakia, Epifagus, Orobanche, and Hyobanche, have rbcL pseudogenes. However, the holoparasites Alectra orobanchoides, Harveya capensis, Harveya purpurea, Lathraea clandestina, Orobanche corymbosa, O. fasciculata, and Striga gesnerioides have intact open reading frames (ORFs) for the rbcL gene. Phylogenetic hypotheses based on rbcL are largely in agreement with those based on sequences of the nonphotosynthetic genes rps2 and matK and show a single origin of parasitism, and loss of photosynthesis and pseudogene formation have been independently derived several times in Scrophulariales. The mutations in rbcL in nonparasitic and hemiparasitic plants would result in largely conservative amino acid substitutions, supporting the hypothesis that functional proteins can experience only a limited range of changes, even in minimally photosynthetic plants. In contrast, ORFs in some holoparasites had many previously unobserved missense substitutions at functionally important amino acid residues, suggesting that rbcL genes in these plants have evolved under relaxed or altered functional constraints.      

Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1

Background

Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington''s disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.

Methodology/Principal Findings

We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked.

Conclusions/Significance

These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.     

Poor quality vital anti-malarials in Africa - an urgent neglected public health priority

Background

The most common pesticide products for controlling malaria-transmitting mosquitoes combine two distinct modes of action: 1) conventional insecticidal activity which kills mosquitoes exposed to the pesticide and 2) deterrence of mosquitoes away from protected humans. While deterrence enhances personal or household protection of long-lasting insecticidal nets and indoor residual sprays, it may also attenuate or even reverse communal protection if it diverts mosquitoes to non-users rather than killing them outright.

Methods

A process-explicit model of malaria transmission is described which captures the sequential interaction between deterrent and toxic actions of vector control pesticides and accounts for the distinctive impacts of toxic activities which kill mosquitoes before or after they have fed upon the occupant of a covered house or sleeping space.

Results

Increasing deterrency increases personal protection but consistently reduces communal protection because deterrent sub-lethal exposure inevitably reduces the proportion subsequently exposed to higher lethal doses. If the high coverage targets of the World Health Organization are achieved, purely toxic products with no deterrence are predicted to generally provide superior protection to non-users and even users, especially where vectors feed exclusively on humans and a substantial amount of transmission occurs outdoors. Remarkably, this is even the case if that product confers no personal protection and only kills mosquitoes after they have fed.

Conclusions

Products with purely mosquito-toxic profiles may, therefore, be preferable for programmes with universal coverage targets, rather than those with equivalent toxicity but which also have higher deterrence. However, if purely mosquito-toxic products confer little personal protection because they do not deter mosquitoes and only kill them after they have fed, then they will require aggressive "catch up" campaigns, with behaviour change communication strategies that emphasize the communal nature of protection, to achieve high coverage rapidly.     

Sodium binding to d-glucuronate residues: Crystal structure of sodium d-glucuronate monohydrate

Three-dimensional X-ray diffraction data were used to determine the crystal structure of sodium β-d-glucuronate monohydrate, a model system for investigating the factors involved in the binding of sodium ions to d-glucuronate residues of glycosaminoglycans. Crystals of the salt are monoclinic, space group P2₁, with a = 9.206(3) Å, b = 7.007(2) Å, c = 7.378(3) Å, β = 96.84(3)°, and Z = 2. Intensity data for 858 reflections were measured with an automated diffractometer. A trial structure, obtained by direct methods, was refined by least squares to R = 0.035. An outstanding feature of the crystal packing is the interaction of d-glucuronate anions with sodium ions. The sodium ion is coordinated to three symmetry-related $\text{d}$-glucuronate anions and to one water molecule. The d-glucuronate anion binds sodium cations through the three following sites: one that involves a carboxyl oxygen atom combined with ring oxygen O-5; one that includes a single carboxyl oxygen atom, and one composed of the O-3–O-4 pair of hydroxyl groups.     

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.     

High-throughput bioinformatics with the Cyrille2 pipeline system

Background  

Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses are often interdependent and chained together to form complex workflows or pipelines. Given the volume of the data used and the multitude of computational resources available, specialized pipeline software is required to make high-throughput analysis of large-scale omics datasets feasible.     

Stereochemistry of nucleic acids and their constituents. X. Solid-state base-stacking patterns in nucleic acid constituents and polynucleotides

The base-stacking patterns in over 70 published crystal structures of nucleic acid constituents and polynucleotides were examined. Several recurring stacking patterns were found. Base stacking in the solid state apparently is very specific, with particular modes of interaction persisting in various crystalline environments. The vertical stacking of purities and pyrimidines in polynucleotides is similar to that observed in crystals of nucleic acid constituents. Only partial base overlap was found in the majority of the structures examined. Usually, the base overlap is accomplished by positioning polar substituents over the ring system of an adjacent base. The stacking interactions are similar to those found in the crystal structures of other polar aromatic compounds, but are considerably different from the ring–ring interactions in nonpolar aromatic compounds. Apparently, dipole-induced dipole forces are largely responsible for solid-state base stacking. It is found that halogen substituents affect base-stacking patterns. In general, the presence of a halogen substituent results in a stacking pattern which permits intimate contact between the halogen atom and adjacent purine or pyrimidine rings. Considering differences in the stacking patterns found for halogenated and nonhalogenated pyrimidines, a model is proposed to account for the mutagenic effects of halogenated pyrimidines.     

Patch-exploitation patterns in an egg parasite of elm leaf beetle

Eggs of elm beetle,Pyrrhalta luteola (Muller), normally occur in masses which can be viewed as discrete host patches in space and time. Analysis of>800 egg masses from 3 field sites in northern California revealed that the number of eggs/mass varied from 2 to>40 and that the imported egg parasiteTetrastichus gallerucae (Fonsc.) exploited a relatively large proported of the egg masses at certain times. The spatial relationship between parasitization and number of eggs/egg mass was assessed in 2 ways-i.e., for exploited masses only and for all masses combined (exploited+nonexploited). Percent parasitization was density independent in most cases for both data sets. It is suggested that both methods of analysis can provide patterns which are relevant to biological control of insect pests.