Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures

Proline racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline racemases may have therapeutic potential.     

Neuropsychopharmacology and Neurogenetic Aspects of Executive Functioning: Should Reward Gene Polymorphisms Constitute a Diagnostic Tool to Identify Individuals at Risk for Impaired Judgment?

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual's genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.     

An Image-Based Drug Susceptibility Assay Targeting the Placental Sequestration of Plasmodium falciparum-Infected Erythrocytes

Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.     

Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.     

A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes

Bacterial plasmids introduced into the human malaria parasite Plasmodium falciparum replicate well but are poorly segregated during mitosis. In this paper, we screened a random P.falciparum genomic library in order to identify sequences that overcome this segregation defect. Using this approach, we selected for parasites that harbor a unique 21 bp repeat sequence known as Rep20. Rep20 is one of six different repeats found in the subtelomeric regions of all P.falciparum chromosomes but which is not found in other eukaryotes or in other plasmodia. Using a number of approaches, we demonstrate that Rep20 sequences lead to dramatically improved episomal maintenance by promoting plasmid segregation between daughter merozoites. We show that Rep20(+), but not Rep20(-), plasmids co-localize with terminal chromosomal clusters, indicating that Rep20 mediates plasmid tethering to chromosomes, a mechanism that explains the improved segregation phenotype. This study implicates a direct role for Rep20 in the physical association of chromosome ends, which is a process that facilitates the generation of diversity in the terminally located P.falciparum virulence genes.     

Elimination of Mange Mites <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> var. <Emphasis Type="Italic">suis</Emphasis>from Two Naturally Infested Danish Sow Herds Using a Single Injection Regime with Doramectin

Attempts to eliminate Sarcoptes scabiei var. suis were made in 2 naturally infested sow herds, by intramuscular (IM) injection of doramectin (Dectomax^®, Pfizer, New York, USA). A single injection strategy was used. In one of the herds, the environment was treated with an acaricide following dry cleaning of floors, walls and equipment. In the second herd, no environmental treatment was performed. Results were measured by skin lesion scoring, ear scrapings to show Sarcoptes scabiei var. suis, and calculating rubbing index throughout the observation period of 20 months following treatment. Skin lesion scores decreased and stayed low following treatment for the entire observation period. Live Sarcoptes scabiei var. suis mites were isolated prior to treatment from both herds, but not following treatment. Rubbing index decreased following treatment, but was occasionally at or above 0.4. The results of these studies indicate that elimination of Sarcoptes scabiei var. suis from 2 naturally infested herds was successful, using doramectin in a single injection strategy. Precautions must be taken to ensure adequate dosing of every pig, and to avoid reinfestation due to poor biosecurity.     

Spatio-temporal distribution of sperm whales (Physeter macrocephalus) off Kaikoura,New Zealand,in relation to bathymetric features

Using a shore-based station we monitored the position of sperm whales (Physeter macrocephalus) within the Kaikoura submarine canyon from 2010 to 2012. We tracked sperm whales using a theodolite station for a total of 290 days. We extracted the distance from the nearest coast, the depth and the bathymetric slope using ArcGIS 10.1. We estimated the seasonal spatial distribution of sperm whales using general additive models. The distribution varied significantly between seasons; individuals were found in deeper water and further offshore in the spring than in winter. This study improved our understanding of the variability of sperm whale distribution patterns off Kaikoura. We determined that the distribution was linked to the bathymetric features and we hypothesized that whales adapted their use of the submarine canyon in relation to food aggregation. We would encourage further studies to evaluate the sperm whale relationship with oceanographic variables off Kaikoura.