Proton block of the pore underlies the inhibition of hERG cardiac K+ channels during acidosis

Human ether-a-go-go-related gene (hERG) potassium channels are critical determinants of cardiac repolarization. Loss of function of hERG channels is associated with Long QT Syndrome, arrhythmia, and sudden death. Acidosis occurring as a result of myocardial ischemia inhibits hERG channel function and may cause a predisposition to arrhythmias. Acidic pH inhibits hERG channel maximal conductance and accelerates deactivation, likely by different mechanisms. The mechanism underlying the loss of conductance has not been demonstrated and is the focus of the present study. The data presented demonstrate that, unlike in other voltage-gated potassium (Kv) channels, substitution of individual histidine residues did not abolish the pH dependence of hERG channel conductance. Abolition of inactivation, by the mutation S620T, also did not affect the proton sensitivity of channel conductance. Instead, voltage-dependent channel inhibition (δ = 0.18) indicative of pore block was observed. Consistent with a fast block of the pore, hERG S620T single channel data showed an apparent reduction of the single channel current amplitude at low pH. Furthermore, the effect of protons was relieved by elevating external K(+) or Na(+) and could be modified by charge introduction within the outer pore. Taken together, these data strongly suggest that extracellular protons inhibit hERG maximal conductance by blocking the external channel pore.     

Strain differences in a high response-cost daily time-place learning task

Previous research has shown that rats, unlike birds, do not readily demonstrate daily time-place learning (TPL). It has been suggested, however, that rats are more successful at these tasks if the response cost (RC) is increased. Widman et al. (2000) found that female Sprague Dawley (SD) rats learned a daily TPL task in which they were required to climb different towers depending on the time of day to find a food reward. Using a similar apparatus, we found that male SD rats learned the task, while male Long Evans rats did not. While all rats quickly learned to restrict the majority of their searching to the two towers that provided food, only the SD rats learned to go to the correct location at the correct time of day. Thus, there appears to be a strain difference in the effectiveness of a high RC task to promote learning. Tests of the timing strategies used revealed individual differences with one rat using a circadian strategy and another using an ordinal strategy. Post criterion decrements in performance did not allow sufficient testing to determine the timing strategies of the remaining rats. Possible interactions between strain, response cost, species typical behaviors and dependent measures are discussed.     

Interval time-place learning in young children

While previous research has investigated the ability of animals to learn the spatial and temporal contingencies of biologically significant events (known as time-place learning), this ability has not been studied in humans. Children ranging from 5 to 10 years old were tested on a modified interval time-place learning task using a touchscreen computer. Results demonstrate the children were able to quickly learn both the timing and the sequence of this task. Despite a lack of anticipation on baseline trials, the children continued to follow the spatio-temporal contingencies in probe sessions where these contingencies were removed. Performance on the probe sessions provide strong evidence that the children had learned the spatio-temporal contingencies. Future research is needed to determine what age-related changes in iTPL occur. Furthermore, it is argued that this procedure can be used to extend interval timing in research in children, including, but not limited to, investigation of scalar timing with longer durations than have previously been investigated.     

Surveys for Stenoma catenifer (Lepidoptera: Elachistidae) and associated parasitoids infesting avocados in Perú

Surveys for Stenoma catenifer Walsingham, the avocado seed moth, and its associated larval parasitoids were conducted in the Departments of Junín, Huánuco, Cusco, and Madre de Dios in Perú. Fruit infestation levels in some areas ranged from 0 to 58%, and parasitism of S. catenifer larvae in Junín and Huánuco was 23%. Five species of hymenopteran parasitoid in two families, Braconidae (Apanteles sp., Hypomicrogaster sp., and Chelonus sp.) and Ichneumonidae (Pristeromerus sp. and Xiphosomella sp.), were reared from larvae, and one species of tachinid fly (Chrysodoria sp.) emerged from pupae. The dominant larval parasitoid, a gregarious Apanteles sp., accounted for 55% of parasitized hosts. Branch and twig tunneling by S. catenifer larvae in a commercial Hass avocado orchard was observed in Cusco. The field attractiveness of the sex pheromone of S. catenifer was demonstrated with 73% of monitoring traps deployed in three departments (Junín, Huánuco, and Cusco) catching male moths. Approximately 55% of avocado fruit sourced from the Province of Chanchamayo (Junin) and purchased at the Mercado Modelo de Frutas in La Victoria, in central Lima were infested with larvae of S. catenifer. Infested avocado fruit sold at this market could represent a potential incursion threat to coastal Hass avocado production regions in Perú that are reportedly free of this pest.     

Zinc metalloproteinase ProA directly activates Legionella pneumophila PlaC glycerophospholipid:cholesterol acyltransferase

Enzymes secreted by Legionella pneumophila, such as phospholipases A (PLAs) and glycerophospholipid:cholesterol acyltransferases (GCATs), may target host cell lipids and therefore contribute to the establishment of Legionnaires disease. L. pneumophila possesses three proteins, PlaA, PlaC, and PlaD, belonging to the GDSL family of lipases/acyltransferases. We have shown previously that PlaC is the major GCAT secreted by L. pneumophila and that the zinc metalloproteinase ProA is essential for GCAT activity. Here we characterized the mode of PlaC GCAT activation and determined that ProA directly processes PlaC. We further found that not only cholesterol but also ergosterol present in protozoa was palmitoylated by PlaC. Such ester formations were not induced by either PlaA or PlaD. PlaD was shown here to possess lysophospholipase A activity, and interestingly, all three GDSL enzymes transferred short chain fatty acids to sterols. The three single putative catalytic amino acids (Ser-37, Asp-398, and His-401) proved essential for all PlaC-associated PLA, lysophospholipase A, and GCAT activities. A further four cysteine residues are important for the PLA/GCAT activities as well as their oxidized state, and we therefore conclude that PlaC likely forms at least one disulfide loop. Analysis of cleavage site and loop deletion mutants suggested that for GCAT activation deletion of several amino acids within the loop is necessary rather than cleavage at a single site. Our data therefore suggest a novel enzyme inhibition/activation mechanism where a disulfide loop inhibits PlaC GCAT activity until the protein is exported to the external space where it is ProA-activated.     

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.     

Leukotriene B(4) inhibits neutrophil apoptosis via NADPH oxidase activity: redox control of NF-κB pathway and mitochondrial stability

Leukotriene B(4), an arachidonic acid-derived lipid mediator, is a known proinflammatory agent that has a direct effect upon neutrophil physiology, inducing reactive oxygen species generation by the NADPH oxidase complex and impairing neutrophil spontaneous apoptosis, which in turn may corroborate to the onset of chronic inflammation. Despite those facts, a direct link between inhibition of neutrophil spontaneous apoptosis and NADPH oxidase activation by leukotriene B(4) has not been addressed so far. In this study, we aim to elucidate the putative role of NADPH oxidase-derived reactive oxygen species in leukotriene B(4)-induced anti-apoptotic effect. Our results indicate that NADPH oxidase-derived reactive oxygen species are critical to leukotriene B(4) pro-survival effect on neutrophils. This effect also relies on redox modulation of nuclear factor kappaB signaling pathway. We have also observed that LTB(4)-induced Bad degradation and mitochondrial stability require NADPH oxidase activity. All together, our results strongly suggest that LTB(4)-induced anti-apoptotic effect in neutrophils occurs in a reactive oxygen species-dependent manner. We do believe that a better knowledge of the molecular mechanisms underlying neutrophil spontaneous apoptosis may contribute to the development of more successful strategies to control chronic inflammatory conditions such as rheumatoid arthritis.     

Monocyte-to-macrophage differentiation: synthesis and secretion of a complex extracellular matrix

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.     

Unique peptide substrate binding properties of 110-kDa heat-shock protein (Hsp110) determine its distinct chaperone activity

The molecular chaperone 70-kDa heat-shock proteins (Hsp70s) play essential roles in maintaining protein homeostasis. Hsp110, an Hsp70 homolog, is highly efficient in preventing protein aggregation but lacks the hallmark folding activity seen in Hsp70s. To understand the mechanistic differences between these two chaperones, we first characterized the distinct peptide substrate binding properties of Hsp110s. In contrast to Hsp70s, Hsp110s prefer aromatic residues in their substrates, and the substrate binding and release exhibit remarkably fast kinetics. Sequence and structure comparison revealed significant differences in the two peptide-binding loops: the length and properties are switched. When we swapped these two loops in an Hsp70, the peptide binding properties of this mutant Hsp70 were converted to Hsp110-like, and more impressively, it functionally behaved like an Hsp110. Thus, the peptide substrate binding properties implemented in the peptide-binding loops may determine the chaperone activity differences between Hsp70s and Hsp110s.