Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gα_q, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP₂ that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gα_q or Gβ₁γ₂ did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP₂ in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.     

Differential Expression of microRNAs in Francisella tularensis-Infected Human Macrophages: miR-155-Dependent Downregulation of MyD88 Inhibits the Inflammatory Response

Francisella tularensis is a Gram-negative, facultative intracellular pathogen that replicates in the cytosol of macrophages and is the causative agent of the potentially fatal disease tularemia. A characteristic feature of F. tularensis is its limited proinflammatory capacity, but the mechanisms that underlie the diminished host response to this organism are only partially defined. Recently, microRNAs have emerged as important regulators of immunity and inflammation. In the present study we investigated the microRNA response of primary human monocyte-derived macrophages (MDMs) to F. tularensis and identified 10 microRNAs that were significantly differentially expressed after infection with the live vaccine strain (LVS), as judged by Taqman Low Density Array profiling. Among the microRNAs identified, miR-155 is of particular interest as its established direct targets include components of the Toll-like receptor (TLR) pathway, which is essential for innate defense and proinflammatory cytokine production. Additional studies demonstrated that miR-155 acted by translational repression to downregulate the TLR adapter protein MyD88 and the inositol 5′-phosphatase SHIP-1 in MDMs infected with F. tularensis LVS or the fully virulent strain Schu S4. Kinetic analyses indicated that miR-155 increased progressively 3-18 hours after infection with LVS or Schu S4, and target proteins disappeared after 12–18 hours. Dynamic modulation of MyD88 and SHIP-1 was confirmed using specific pre-miRs and anti-miRs to increase and decrease miR-155 levels, respectively. Of note, miR-155 did not contribute to the attenuated cytokine response triggered by F. tularensis phagocytosis. Instead, this microRNA was required for the ability of LVS-infected cells to inhibit endotoxin-stimulated TNFα secretion 18–24 hours after infection. Thus, our data are consistent with the ability of miR-155 to act as a global negative regulator of the inflammatory response in F. tularensis-infected human macrophages.     

Role of cranberry juice on molecular-scale surface characteristics and adhesion behavior of Escherichia coli

Cranberry juice has long been believed to benefit the prevention and treatment of urinary tract infections (UTIs). As the first step in the development of infection, bacterial adhesion is of great research interest, yet few studies have addressed molecular level adhesion in this context. P-fimbriated Escherichia coli play a major role in the development of a serious type of UTI, acute pyelonephritis. Experiments were conducted to investigate the molecular-scale effects of cranberry juice on two E. coli strains: HB101, which has no fimbriae, and the mutant HB101pDC1 which expresses P-fimbriae. Atomic force microscopy (AFM) was used to investigate both bacterial surface characteristics and adhesion forces between a probe surface (silicon nitride) and the bacteria, providing a direct evaluation of bacterial adhesion and interaction forces. Cranberry juice affected bacterial surface polymer and adhesion behavior after a short exposure period (<3 h). Cranberry juice affected the P-fimbriated bacteria by decreasing the adhesion forces between the bacterium and tip and by altering the conformation of the surface macromolecules on E. coli HB101pDC1. The equilibrium length of polymer (P-fimbriae) on this bacterium decreased from approximately 148 to approximately 48 nm upon being exposed to cranberry juice. Highly acidic conditions were not necessary for the prevention of bacterial adhesion, since neutralization of cranberry juice solutions to pH = 7.0 allowed us to observe differences in adhesion between the E. coli strains. Our results demonstrate molecular-level changes in the surfaces of P-fimbriated E. coli upon exposure to neutralized cranberry juice.     

Ossicular density in golden moles (Chrysochloridae)

The densities of middle ear ossicles of golden moles (family Chrysochloridae, order Afrosoricida) were measured using the buoyancy method. The internal structure of the malleus was examined by high-resolution computed tomography, and solid-state NMR was used to determine relative phosphorus content. The malleus density of the desert golden mole Eremitalpa granti (2.44 g/cm³) was found to be higher than that reported in the literature for any other terrestrial mammal, whereas the ossicles of other golden mole species are not unusually dense. The increased density in Eremitalpa mallei is apparently related both to a relative paucity of internal vascularization and to a high level of mineralization. This high density is expected to augment inertial bone conduction, used for the detection of seismic vibrations, while limiting the skull modifications needed to accommodate the disproportionately large malleus. The mallei of the two subspecies of E. granti, E. g. granti and E. g. namibensis, were found to differ considerably from one another in both size and shape.     

T-cell specific defect in expression of the NTPDase CD39 as a biomarker for lupus

Regulatory T cells (T_regs) are critical for maintenance of peripheral tolerance via suppression of T-cell responses, and absence of T_regs results in autoimmunity. The role of aberrations in the T_reg pool for the development of systemic lupus erythematosus (SLE, lupus) remains uncertain. T_reg-mediated generation of adenosine, dependent on the ectonucleotidase CD39, is an important mechanism for suppression of T-cell responses. We tested whether decreases in numbers of T_regs, and specifically CD39-expressing T_regs, are associated with human lupus. We studied 15 SLE patients, six patients with rheumatoid arthritis (RA) and 24 healthy controls. T_reg phenotypic markers, including CD39 expression, were studied by flow cytometry. Varying numbers of sorted T_regs cells were co-cultured with responder T (T_resp) cells, with proliferation assessed by ³H-thymidine incorporation. The proportion of T_regs as defined by Foxp3⁺ CD25^+high CD127^−/low was similar in lupus and control populations. CD39-expressing T_regs comprised 37 ± 13% of the T_reg population in healthy controls and 36 ± 21% in lupus subjects using nonsteroidal immunosuppressants to control active disease, but was nearly absent in five of six lupus subjects with minimally active disease. In contrast to healthy controls and lupus subjects without the CD39 defect, in SLE subjects with the CD39 defect, adenosine-dependent T_reg-mediated suppression was nearly absent. These results suggest that functional defects in T_regs, rather than reduced T_reg numbers, are important for the loss of peripheral tolerance in lupus. Presentation of this defect may serve as a biomarker for untreated disease.     

Soldier caste influences on candidate primer pheromone levels and juvenile hormone-dependent caste differentiation in workers of the termite Reticulitermes flavipes

Caste systems and the division of labor they make possible are common underlying features of all social insects. Multiple extrinsic factors have been shown to impact caste composition in social insect colonies. Primer pheromones are one type of extrinsic caste-regulatory factor; they are chemical signaling molecules produced by certain colony members to impact developmental physiology of recipient nestmates. However, only limited evidence exists regarding primer pheromones and their actions in eusocial termites. In previous research we identified two soldier-produced terpenes, γ-cadinene (CAD) and γ-cadinenal (ALD), as candidate primer pheromones of the lower termite Reticulitermes flavipes. In the present study we tested hypotheses related to CAD and ALD action in recipient individuals. We examined the influences of terminally developed soldier termites on (1) CAD and ALD levels and (2) caste differentiation in developmentally totipotent workers. Our findings show CAD and ALD (respectively) are caste stimulatory and inhibitory components of chemical blends present in soldier heads, ALD levels increase significantly (10.9×) in workers only in the presence of soldiers, and soldiers can reduce developmental-hormone response thresholds of workers, presumably via ALD action. These findings provide novel evidence supporting that CAD and ALD are authentic caste-regulatory primer pheromones in Reticulitermes.     

Specific inhibition of the aspartate aminotransferase of Plasmodium falciparum

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria.     

Global and endemic Asian lineages of the emerging pathogenic fungus Batrachochytrium dendrobatidis widely infect amphibians in China

Aim Panzootic chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) is the proximate cause of rapid amphibian declines across diverse biomes. While the origin of Bd remains unclear, increasingly the global trade in amphibians is associated with the spread of the infection. Global samples of Bd genotypes from previously unsampled regions are essential to test this hypothesis. In this paper, we present a study of the prevalence and phylogeny of Bd in both invasive and native amphibian species in markets and in the wild in ten provinces of China. Location China. Method We used a nested PCR assay to amplify the ribosomal internal transcribed spacer region of Bd followed by sequencing. Result Our results showed 246 of 2734 amphibians testing positive for Bd, with 157 positive samples in the wild (7.6%) and 89 in markets (13.5%). 30 haplotypes of Bd were identified, including 20 first detections. Introduced Lithobates catesbeianus had the highest prevalence of infection and the largest number of Bd haplotypes in both the wild and markets. Phylogenetic analysis based on 73 haplotypes (57 from Asia and 16 from other continents) showed that a unique, well‐supported, basal haplotype is present in Asia. Phylogeographical analyses revealed that some geographical structure exists amongst a subset of global haplotypes. Main conclusions Strains of the basal haplotype infected Babina pleuraden, an amphibian that is endemic to China, and Andrias japonicus, endemic to Japan, showing that Southeast Asia harbours a novel endemic lineage of amphibian‐associated Bd. Our data suggest that Bd in Asia pre‐dates the expansion of a globalized lineage of Bd, a finding that is indicative of a broader association of amphibians and chytrids than has previously been recognized. More genetic data from Bd isolates are needed to reveal the phylogenetic relationship of Bd in China compared to that found elsewhere.     

Diversity of active aerobic methanotrophs along depth profiles of arctic and subarctic lake water column and sediments

Methane (CH₄) emitted from high-latitude lakes accounts for 2–6% of the global atmospheric CH₄ budget. Methanotrophs in lake sediments and water columns mitigate the amount of CH₄ that enters the atmosphere, yet their identity and activity in arctic and subarctic lakes are poorly understood. We used stable isotope probing (SIP), quantitative PCR (Q-PCR), pyrosequencing and enrichment cultures to determine the identity and diversity of active aerobic methanotrophs in the water columns and sediments (0–25 cm) from an arctic tundra lake (Lake Qalluuraq) on the north slope of Alaska and a subarctic taiga lake (Lake Killarney) in Alaska''s interior. The water column CH₄ oxidation potential for these shallow (∼2 m deep) lakes was greatest in hypoxic bottom water from the subarctic lake. The type II methanotroph, Methylocystis, was prevalent in enrichment cultures of planktonic methanotrophs from the water columns. In the sediments, type I methanotrophs (Methylobacter, Methylosoma and Methylomonas) at the sediment-water interface (0–1 cm) were most active in assimilating CH₄, whereas the type I methanotroph Methylobacter and/or type II methanotroph Methylocystis contributed substantially to carbon acquisition in the deeper (15–20 cm) sediments. In addition to methanotrophs, an unexpectedly high abundance of methylotrophs also actively utilized CH₄-derived carbon. This study provides new insight into the identity and activity of methanotrophs in the sediments and water from high-latitude lakes.