Calmodulin Is a Functional Regulator of Cav1.4 L-type Ca2+ Channels

Cav1.4 channels are unique among the high voltage-activated Ca²⁺ channel family because they completely lack Ca²⁺-dependent inactivation and display very slow voltage-dependent inactivation. Both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained Ca²⁺ influx through Cav1.4 channels is required to couple slow graded changes of the membrane potential with tonic glutamate release. Loss of Cav1.4 function causes severe impairment of retinal circuitry function and has been linked to night blindness in humans and mice. Recently, an inhibitory domain (ICDI: inhibitor of Ca²⁺-dependent inactivation) in the C-terminal tail of Cav1.4 has been discovered that eliminates Ca²⁺-dependent inactivation by binding to upstream regulatory motifs within the proximal C terminus. The mechanism underlying the action of ICDI is unclear. It was proposed that ICDI competitively displaces the Ca²⁺ sensor calmodulin. Alternatively, the ICDI domain and calmodulin may bind to different portions of the C terminus and act independently of each other. In the present study, we used fluorescence resonance energy transfer experiments with genetically engineered cyan fluorescent protein variants to address this issue. Our data indicate that calmodulin is preassociated with the C terminus of Cav1.4 but may be tethered in a different steric orientation as compared with other Ca²⁺ channels. We also find that calmodulin is important for Cav1.4 function because it increases current density and slows down voltage-dependent inactivation. Our data show that the ICDI domain selectively abolishes Ca²⁺-dependent inactivation, whereas it does not interfere with other calmodulin effects.Retinal photoreceptors and bipolar cells contain a highly specialized type of synapse designated ribbon synapses. Glutamate release in these synapses is controlled via graded and sustained changes in membrane potential that are maintained throughout the duration of a light stimulus (1, 2). In recent years, it became clear that Cav1.4 L-type Ca²⁺ channels are the main channel subtype converting these analog input signals into corresponding permanent glutamate release (1, 3–5). In support of this mechanism, mutations in the Cav1.4 gene have been identified in patients suffering from congenital stationary night blindness type 2 and X-linked cone rod dystrophy (6–8). Individuals displaying congenital stationary night blindness type 2 as well as mice deficient in Cav1.4 typically have abnormal electroretinograms that indicate a loss of neurotransmission from the rods to second order bipolar cells, which is attributable to a loss of Cav1.4 (3).Retinal Cav1.4 channels are set apart from other high voltage-activated (HVA)³ Ca²⁺ channels by their total lack of Ca²⁺-dependent inactivation (CDI) and their very slow voltage-dependent inactivation (VDI). Recently, we and others discovered an inhibitory domain (ICDI: inhibitor of CDI) in the C-terminal tail of the Cav1.4 channel that eliminates Ca²⁺-dependent inactivation in this channel by binding to upstream regulatory motifs (9, 10). Importantly, introducing the ICDI into the backbone of Cav1.2 or Cav1.3 almost completely abolishes the CDI of these channels. Contrasting with the clear cut function, the underlying mechanism by which ICDI abolishes CDI remains controversial. It was suggested that ICDI displaces the Ca²⁺ sensor calmodulin (CaM) from binding to the proximal C terminus (10), suggesting that the binding sites of CaM and ICDI are largely overlapping or allosterically coupled to each other. Alternatively, our own data rather suggested that CaM and the ICDI domain bind to different portions of the proximal C terminus (9). We proposed that the interaction between the ICDI domain and the EF-hand, a motif with a central role for transducing CDI (11–16), switches off CDI without impairing binding of CaM to the channel. In this study, we designed experiments to differentiate between these two models. Here, using FRET in HEK293 cells, we provide evidence that in living cells, CaM is bound to the full-length C terminus of Cav1.4 (i.e. in the presence of ICDI). Furthermore, our data suggest that the steric orientation of the CaM/Cav channel complex differs between Cav1.2 and Cav1.4 channels. We show that CaM preassociation with Cav1.4 controls current density and also affects VDI. Thus, although CaM does not trigger CDI in Cav1.4 as it does in other HVA Ca²⁺ channels, it is still an important regulator of this channel.     

Predictive Mapping of Plant Species and Communities Using GIS and Landsat Data in a Southern Mongolian Mountain Range

We assessed presence/absence prediction of plant species and communities in a southern Mongolian mountain range from geospatial data using a randomized sampling approach. One hundred randomized vegetation samples (3?×?3 m) were collected within the 2?×?2 km summit region of the Dund Saykhan range, which forms part of the core zone of the Gobi Gurvan Saykhan National Park in arid southern Mongolia. Using logistic regression, habitat preference models for all abundant species (n?=?52) and communities (n?=?5) were constructed; predictors were derived from Landsat 5 imagery and a digital elevation model. Nagelkerkes r ² was used for an initial data mining, and all significant models were validated by splitting the data and using one half for accuracy assessment based on the AUC (Area Under the receiver operating characteristic Curve)-values. Significant models could be built for half of the species. Altitude proved to be the most important predictor followed by variables derived from Landsat data. The clear altitudinal distribution patterns most definitely reflect precipitation; overall biodiversity in this arid environment is widely controlled by moisture availability. The chosen approach may prove valuable for applied studies wherever spatial data on species distributions are required for conservation efforts.     

The tolerance of the lucerne flea, Sminthurus viridis (Collembola: Sminthuridae), to currently registered pesticides in Australia

Sminthurus viridis (lucerne flea) is a major crop and pasture pest throughout southern regions of Australia that experience a Mediterranean type climate. It is particularly problematic at the establishment phase, and chemicals are currently the main option available to farmers for their control. Few studies have assessed the currently registered pesticides for the control of S. viridis , and rates of application are generally similar to the frequently co-occurring red-legged earth mite, Halotydeus destructor , despite few comparisons between species for their susceptibility being made. Here, we examine the response of S. viridis to six pesticides (α-cypermethrin, bifenthrin, omethoate, methidathion, chlorpyrifos and phosmet) and then directly compare them with H. destructor . S. viridis displayed similar dose–response curves for bifenthrin, omethoate, chlorpyrifos and phosmet. It had greater sensitivity to methidathion and greater tolerance to α-cypermethrin. S. viridis was found to be significantly more tolerant than H. destructor to all pesticides tested, with the exception of chlorpyrifos. This indicates that rates of application for control of S. viridis should be greater than H. destructor and that the organophosphates are likely to provide the most effective control of this pest.     

Pisonia grandis monocultures limit the spread of an invasive ant—a case of carbohydrate quality?

The mechanisms by which invasive species are able to spread into and dominate natural communities are poorly understood and remain a focus of invasion research. In this quest, studying invasions that are limited by a controlling factor will be more informative than will studies documenting unabated spread and impacts. Some ant species are very successful invaders, and research demonstrating abiotic and biotic factors limiting their success has aided the understanding of invasion ecology. We report here a study showing the highly invasive African big headed ant Pheidole megacephala having a novel distribution on coral cays within Australia’s Great Barrier Reef. These patterns displayed a clear limitation of its distribution with monocultures of the tree Pisonia grandis. This distribution was contrary to the known environmental limitations of the ant, and the limitation could not be associated with an underlying abiotic determinant of the vegetation type. We present these distributional patterns, and following consideration of all known biotic and abiotic limitations of ant invasions we discuss the potential that the peculiar ecophysiology of P. grandis is the causal factor. Specifically, we suggest that the quality of carbohydrate supply to ants is a limitation to invasive spread in much the same way that carbohydrate quantity is known to affect ant population densities in other ecosystems.     

ER Export of KAT1 is Correlated to the Number of Acidic Residues within a Triacidic Motif

For a number of ion channels, including the potassium (K⁺) inward rectifying channel from Arabidopsis thaliana (KAT1), diacidic endoplasmic reticulum (ER) export motifs have been identified. These motifs consist of two acidic amino acids (aspartate (D) and/or glutamate (E)) separated by any amino acid. To specify the role of single acidic amino acids for efficiency of ER export, we analysed a sequence of KAT1 that included the originally identified diacidic ER export motif (DxE) plus an additional D just upstream of the diacidic motif. Analysis of single, double and triple mutations of the acidic amino acids of the DxDxE motif revealed a gradual reduction of ER export depending on the number of mutated acidic residues. The amount of reduction in ER export was not related to the position, but only to the number of mutated acidic amino acids. These results show that a triacidic motif is essential for efficient ER export of KAT1. Function of the triacidic motif probably involves cooperative binding to Sec24.     

The relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease

Changes in the balance of cholesterol absorption and synthesis and moderately elevated plasma plant sterols have been suggested to be atherogenic. Measuring cholestanol, lathosterol, campesterol, and sitosterol, we investigated the relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease (CAD) in 2,440 participants of the Ludwigshafen Risk and Cardiovascular health (LURIC) study. The coronary status was determined by angiography, and the severity of CAD was assessed by the Friesinger Score (FS). An increase in the ratio of cholestanol to cholesterol was associated with high FS (P = 0.006). In contrast, a high ratio of lathosterol to cholesterol went in parallel with low FS (P < 0.001). Whereas the campesterol to cholesterol ratio significantly correlated with the FS (P = 0.026), the relationship of the sitosterol to cholesterol ratio with the FS did not reach statistical significance in the whole group. Increased campesterol, sitosterol, and cholestanol to lathosterol ratios were associated high FS (P < 0.001). To conclude, there is a modest association of high cholesterol absorption and low cholesterol synthesis with an increased severity of CAD. An atherogenic role of plasma plant sterols themselves, however, seems unlikely in subjects without sitosterolaemia.     

Linkage Analysis Reveals the Independent Origin of Poeciliid Sex Chromosomes and a Case of Atypical Sex Inheritance in the Guppy (Poecilia reticulata)

Among different teleost fish species, diverse sex-determining mechanisms exist, including environmental and genetic sex determination, yet chromosomal sex determination with male heterogamety (XY) prevails. Different pairs of autosomes have evolved as sex chromosomes among species in the same genus without evidence for a master sex-determining locus being identical. Models for evolution of Y chromosomes predict that male-advantageous genes become linked to a sex-determining locus and suppressed recombination ensures their co-inheritance. In the guppy, Poecilia reticulata, a set of genes responsible for adult male ornaments are linked to the sex-determining locus on the incipient Y chromosome. We have identified >60 sex-linked molecular markers to generate a detailed map for the sex linkage group of the guppy and compared it with the syntenic autosome 12 of medaka. We mapped the sex-determining locus to the distal end of the sex chromosome. We report a sex-biased distribution of recombination events in female and male meiosis on sex chromosomes. In one mapping cross, we observed sex ratio and male phenotype deviations and propose an atypical mode of genetic sex inheritance as its basis.     

Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3–11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-α and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4⁺ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88^−/− mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-γ-dependent CD4⁺ T cell polarization and antibody switching.Human filariasis is a major neglected tropical disease. More than 150 million individuals are infected with the filarial worms responsible for lymphatic filariasis (LF)⁴ (Wuchereria bancrofti and Brugia malayi) and onchocerciasis (Onchocerca volvulus). Over 40 million suffer from disfiguring and incapacitating disease with an estimated 1.5 billion people at risk of infection, ranking filariasis as one of the major causes of global morbidity (1).A feature of filarial pathogenesis is a host inflammatory response provoked by the death of larvae and adult stages within parasitized tissues (2). All causative agents of LF and O. volvulus harbor an intracellular symbiotic bacterium, Wolbachia, and are reliant on this endosymbiont for embryogenesis, growth, and survival (3). Previous studies have determined that the inflammatory potential of B. malayi and O. volvulus is dependent on the presence of Wolbachia. For example, Wolbachia-containing filarial extracts induce activation and tolerance in murine macrophages (4, 5), activate human monocytes (6), and activate human and murine neutrophils (7, 8). In addition, O. volvulus and B. malayi extracts containing Wolbachia stimulate neutrophil recruitment to the corneal stroma and development of corneal haze in a murine model of ocular onchocerciasis, in contrast with an aposymbiotic filaria (9). Moreover, isolated Wolbachia from filaria or from insect cells can replicate these effects (8, 10). The activation of neutrophils results in further neutrophil recruitment leading to the disruption of normal corneal clarity and development of stromal haze (11).Activation and subsequent desensitization of macrophages by Wolbachia molecules has been shown to be dependent on TLR2 and the adaptor molecule MyD88 (5, 10). Further studies have established that Wolbachia-induced inflammation is dependent on TLR2 and TLR6 recognition and signaling through the MyD88/Mal pathway and are independent of TRIF and TRAM (12). However, Wolbachia ligands for TLR2/TLR6 have not been characterized. To address this, we used the TLR receptor recognition profile to identify TLR2/6 ligands in the Wolbachia genome. In this study, we demonstrate that Wolbachia-derived diacyl-lipoproteins are candidate stimulatory molecules required for TLR2/6 ligation and production of pro-inflammatory cytokine and chemokine responses. Furthermore, we show that a synthetic Wolbachia lipopeptide (Diacyl WoLP) induces TLR2/6-dependent corneal inflammation, and TLR2-dependent TNFα responses in filarial disease models and up-regulates surface markers of human lymphatic endothelium. Diacyl WoLP also induced activation and maturation of dendritic cells and generated type 1 CD4⁺ T cell and antibody responses to filarial antigens.     

Immuno‐MALDI‐TOF MS: New perspectives for clinical applications of mass spectrometry

The discovery of novel biomarkers by means of advanced detection tools based on proteomic analysis technologies necessitates the development of improved diagnostic methods for application in clinical routine. On the basis of three different application examples, this review presents the limitations of conventional routine diagnostic assays and illustrates the advantages of immunoaffinity enrichment combined with MALDI‐TOF MS. Applying this approach increases the specificity of the analysis supporting a better diagnostic recognition, sensitivity, and differentiation of certain diseases. The use of MALDI‐TOF MS as detection method facilitates the identification of modified peptides and proteins providing additional information. Further, employing respective internal standard peptides allows for relative and absolute quantitation which is mandatory in the clinical context. Although MALDI‐TOF MS is not yet established for clinical routine diagnostics this technology has a high potential for improvement of clinical diagnostics and monitoring therapeutic efficacy.