Critical role for hyperpolarization-activated cyclic nucleotide-gated channel 2 in the AIF-mediated apoptosis

Cellular calcium uptake is a controlled physiological process mediated by multiple ion channels. The exposure of cells to either one of the protein kinase C (PKC) inhibitors, staurosporine (STS) or PKC412, can trigger Ca²⁺ influx leading to cell death. The precise molecular mechanisms regulating these events remain elusive. In this study, we report that the PKC inhibitors induce a prolonged Ca²⁺ import through hyperpolarization‐activated cyclic nucleotide‐gated channel 2 (HCN2) in lung carcinoma cells and in primary culture of cortical neurons, sufficient to trigger apoptosis‐inducing factor (AIF)‐mediated apoptosis. Downregulation of HCN2 prevented the drug‐induced Ca²⁺ increase and subsequent apoptosis. Importantly, the PKC inhibitors did not cause Ca²⁺ entry into HEK293 cells, which do not express the HCN channels. However, introduction of HCN2 sensitized them to STS/PKC412‐induced apoptosis. Mutagenesis of putative PKC phosphorylation sites within the C‐terminal domain of HCN2 revealed that dephosphorylation of Thr⁵⁴⁹ was critical for the prolonged Ca²⁺ entry required for AIF‐mediated apoptosis. Our findings demonstrate a novel role for the HCN2 channel by providing evidence that it can act as an upstream regulator of cell death triggered by PKC inhibitors.     

The steroid glycoside H.g.-12 from Hoodia gordonii activates the human bitter receptor TAS2R14 and induces CCK release from HuTu-80 cells

Steroid glycosides extracted from the succulent plant Hoodia gordonii are suggested to have appetite-suppressant effects both in animals and humans. Yet, the mechanisms underlying the putative satiety action of Hoodia steroid glycosides are not fully understood. We found that H.g.-12, a steroid glycoside purified from H. gordonii extract, initiated cholecystokinin (CCK) secretion both ex vivo in rat intestine and in vitro in the human enteroendocrine (EC) cell line HuTu-80. CCK is known to exert central effects on appetite suppression via the vagus nerve which afferents terminate in the gut wall. Recent data show that G protein-coupled receptors signaling bitter taste (T2Rs) are expressed in both rodent and human gastrointestinal tract. It was further demonstrated that bitter sensing is functional in mouse STC-1 EC cells and leads to CCK secretion via increased intracellular Ca2(+) concentrations. Based on the bitter taste of H. gordonii purified extracts, we assessed whether H.g.-12 could activate human bitter receptors. The steroid glycoside activated selectively TAS2R7 and TAS2R14, both heterologously expressed in HEK 293 cells. Removing an essential structural feature from the steroid glycoside inhibited H.g.-12-induced Ca2(+) increase in TAS2R14-expressing HEK cells and abolished H.g.-12-induced CCK secretion from human EC cells. Similarly, a nonspecific bitter receptor antagonist abolished H.g.-12-induced CCK secretion in HuTu-80 cells. These results point to a potential route of action by which components of Hoodia might influence appetite control. Our data also provide additional evidence that bitter taste-sensing mechanisms are coupled to hormone release from EC cells in the intestine. Moreover, we identified a natural agonist of TAS2R7 and TAS2R14 for further studies on the role of bitter receptors in satiety control and food intake.     

Evaluating the sampling bias in pattern of subterranean species richness: combining approaches

We investigated the pattern of species richness of obligate subterranean (troglobiotic) beetles in caves in the northwestern Balkans, given unequal and biased sampling. On the regional scale, we modeled the relationship between species numbers and sampling intensity using an asymptotic Clench (Michaelis–Menten) function. On the local scale, we calculated Chao 2 species richness estimates for 20 × 20 km grid cells, and investigated the distribution of uniques, species found in only one cave within the grid cell. Cells having high positive residuals, those with above average species richness than expected according to the Clench function, can be considered true hotspots. They were nearly identical to the observed areas of highest species richness. As sampling intensity in a grid cell increases the expected number of uniques decreases for any fixed number of species in the grid cell. High positive residuals show above average species richness for a certain level of sampling intensity within a cell, so further sampling has the most potential for additional species. In some cells this was supported by high numbers of uniques, also indicating insufficient sampling. Cells with low negative residuals have fewer species than would be expected, and some of them also had a low number of uniques, both indicating sufficient sampling. By combining different analyses in a novel way we were able to evaluate observed species richness pattern as well as identify, where further sampling would be most beneficial. Approach we demonstrate is of broad interest to study of biota with high levels of endemism, small distribution ranges and low catchability.     

Inflammation in fetal sheep from intra-amniotic injection of Ureaplasma parvum

Bronchopulmonary dysplasia is associated with chorioamnionitis and fetal lung inflammation. Ureaplasma species are the bacteria most frequently isolated from chorioamnionitis. Very chronic ureaplasma colonization of amniotic fluid causes low-grade lung inflammation and functional lung maturation in fetal sheep. Less is known about shorter exposures of the fetal lung. Therefore, we hypothesized that ureaplasmas would cause an acute inflammatory response that would alter lung development. Singleton ovine fetuses received intra-amniotic Ureaplasma parvum serovar 3 or control media at 110, 117, or 121 days and were delivered at 124 days gestational age (term = 150 days). Inflammation was assessed by 1) cell counts in bronchoalveolar lavage fluid (BALF), and 2) cytokine mRNA measurements, immunohistochemistry, and flow cytometry for inflammatory cells and elastin and α-smooth muscle actin (α-SMA) staining in lung tissue. Neutrophils were increased in BALF 3 days after exposure to ureaplasmas (P = 0.01). Myeloperoxidase-positive cells increased after 3 days (P = 0.03), and major histocompatibility complex (MHC) class II-positive cells increased after 14 days of ureaplasma exposure (P = 0.001). PU.1 (macrophage marker)- or CD3 (T lymphocyte marker)-positive cells were not induced by ureaplasmas. CD3-positive cells in the posterior mediastinal lymph node increased in ureaplasma-exposed animals at 3, 7, and 14 days (P = 0.002). Focal elastin depositions decreased in alveolar septa at 14 days (P = 0.002), whereas α-SMA increased in arteries and bronchioli. U. parvum induced a mild acute inflammatory response and changed elastin and α-SMA deposition in the lung, which may affect lung structure and subsequent development.     

Old museum samples and recent taxonomy: A taxonomic,biogeographic and conservation perspective of the <Emphasis Type="Italic">Niphargus tatrensis</Emphasis> species complex (Crustacea: Amphipoda)

Natural history museum collections harbour valuable information on species. The usefulness of such data critically depends on the accurate identification of species, which has been challenged by introduction of molecular techniques into taxonomy. However, most collections may suffer from DNA degradation, due to age and/or improper preservation; hence the identification of specimens depends solely on morphological features. This study explores how and to what extent morphological data can help to solve ambiguous taxonomic cases based on selected species concepts and with the use of operational criteria in a species-hypothesis testing procedure. The studied taxon, the Niphargus tatrensis species complex, comprises freshwater subterranean amphipods, distributed across Central Europe, the taxonomic status of which was debated extensively between 1930 and 1960. Using the general species concept, character- and tree-based operational criteria reveal northern and southern diagnosable and exclusive lineages identified here as N. tatrensis Wrześniowski, 1888 and N. scopicauda sp. n., respectively. The remaining populations represent the non-exclusive N. aggtelekiensis Dudich, 1932, which occurs from the eastern Alps to Hungary. In the entire complex, altitudinal distribution is largely limited to areas above 400 m, where the mean annual temperature never exceeds 9°C. Seemingly well-defined distributional ranges of N. tatrensis and N. aggtelekiensis are fragmented in an ecological sense, which raises the question whether two of the three species recognised here actually consist of several unidentified taxa. Morphological similarity between the species, numerous polymorphic features, and the association with cool temperatures lead to a hypothesis in which fragmentation of the ancestral range occurred during post-Pleistocene climate warming, reducing gene flow across lowland populations due to niche conservatism of the ancestral species and/or to invasion of competitive species along the Danube and Drava rivers. The results are discussed regarding how old museum samples are conducive to more detailed molecular-taxonomic and conservation studies.     

A statistical model for multidimensional irreversible electroporation cell death in tissue

Background  

Irreversible electroporation (IRE) is a minimally invasive tissue ablation technique which utilizes electric pulses delivered by electrodes to a targeted area of tissue to produce high amplitude electric fields, thus inducing irreversible damage to the cell membrane lipid bilayer. An important application of this technique is for cancer tissue ablation. Mathematical modelling is considered important in IRE treatment planning. In the past, IRE mathematical modelling used a deterministic single value for the amplitude of the electric field required for causing cell death. However, tissue, particularly cancerous tissue, is comprised of a population of different cells of different sizes and orientations, which in conventional IRE are exposed to complex electric fields; therefore, using a deterministic single value is overly simplistic.     

Vaccination-induced IgG response to Galα1-3GalNAc glycan epitopes in lambs protected against Haemonchus contortus challenge infection

Lambs vaccinated with Haemonchus contortus excretory/secretory (ES) glycoproteins in combination with the adjuvant Alhydrogel are protected against H. contortus challenge infection. Using glycan micro-array analysis we showed that serum from such vaccinated lambs contains IgG antibodies that recognise the glycan antigen Galα1-3GalNAc-R and GalNAcβ1-4(Fucα1-3)GlcNAc-R. Our studies revealed that H. contortus glycoproteins contain Galα1-3Gal-R as well as significant levels of Galα1-3GalNAc-R, which has not been previously reported. Extracts from H. contortus adult worms contain a galactosyltransferase acting on glycan substrates with a terminal GalNAc, indicating that the worms possess the enzymatic potential to synthesise terminal Gal-GalNAc moieties. These data illustrate that glycan micro-arrays constitute a promising technology for fast and specific analysis of serum anti-glycan antibodies in vaccination studies. In addition, this approach facilitates the discovery of novel, antigenic parasite glycan antigens that may have potential for developing glycoconjugate vaccines or utilization in diagnostics.     

Similarity in ectoparasite faunas of Palaearctic rodents as a function of host phylogenetic, geographic or environmental distances: Which matters the most?

Different host species harbour parasite faunas that are anywhere from very similar to very different in species composition. A priori, the similarity in the parasite faunas of any two host species should decrease with increases in either the phylogenetic distance, the distinctness of the environments occupied or the geographical distance between these hosts. We tested these predictions using extensive data on the faunas of fleas (Insecta: Siphonaptera) and gamasid mites (Acari: Parasitiformes) parasitic on rodents across the Palaearctic. For each pair of host species, we computed the similarity in parasite faunas based on both species composition as well as the phylogenetic and/or taxonomic distinctness of parasite species. Phylogenetic distances between hosts were based on patristic distances through a rodent phylogeny, geographic distances were computed from geographic range data, and environmental dissimilarity was measured from the average climatic and vegetation scores of each host range. Using multiple regressions on distance matrices to assess the separate explanatory power of each of the three dependent variables, environmental dissimilarity between the ranges of host species emerged as the best predictor of dissimilarity between parasite faunas, especially for fleas; in the case of mites, phylogenetic distance between host species was also important. A closer look at the data indicates that the flea and mite faunas of two hosts inhabiting different environments are always different, whilst hosts living in similar environments can have either very similar or dissimilar parasite faunas. Additional tests showed that dissimilarity in flea or mite faunas between host geographic ranges was best explained by dissimilarity in vegetation, followed by dissimilarity in climatic conditions. Thus, external environmental factors may play greater roles than commonly thought in the evolution of host-parasite associations.     

Protein Localization in Escherichia coli Cells: Comparison of the Cytoplasmic Membrane Proteins ProP,LacY, ProW,AqpZ, MscS,and MscL

Fluorescence microscopy has revealed that the phospholipid cardiolipin (CL) and FlAsH-labeled transporters ProP and LacY are concentrated at the poles of Escherichia coli cells. The proportion of CL among E. coli phospholipids can be varied in vivo as it is decreased by cls mutations and it increases with the osmolality of the growth medium. In this report we compare the localization of CL, ProP, and LacY with that of other cytoplasmic membrane proteins. The proportion of cells in which FlAsH-labeled membrane proteins were concentrated at the cell poles was determined as a function of protein expression level and CL content. Each tagged protein was expressed from a pBAD24-derived plasmid; tagged ProP was also expressed from the chromosome. The osmosensory transporter ProP and the mechanosensitive channel MscS concentrated at the poles at frequencies correlated with the cellular CL content. The lactose transporter LacY was found at the poles at a high and CL-independent frequency. ProW (a component of the osmoregulatory transporter ProU), AqpZ (an aquaporin), and MscL (a mechanosensitive channel) were concentrated at the poles in a minority of cells, and this polar localization was CL independent. The frequency of polar localization was independent of induction (at arabinose concentrations up to 1 mM) for proteins encoded by pBAD24-derived plasmids. Complementation studies showed that ProW, AqpZ, MscS, and MscL remained functional after introduction of the FlAsH tag (CCPGCC). These data suggest that CL-dependent polar localization in E. coli cells is not a general characteristic of transporters, channels, or osmoregulatory proteins. Polar localization can be frequent and CL independent (as observed for LacY), frequent and CL dependent (as observed for ProP and MscS), or infrequent (as observed for AqpZ, ProW, and MscL).Modern developments in fluorescence microscopy have led to a new understanding of the organization of bacterial cells, particularly protein and lipid localization (21, 56). Analysis of the subcellular localization of diverse proteins and lipids has shown that they are not uniformly distributed. The phospholipid cardiolipin (CL) localizes at the poles and septal regions (36), and there is evidence for segregation of phosphatidylethanolamine (PE) from phosphatidylglycerol (PG) in the membranes of living Escherichia coli cells (69). Localization of many proteins that are integral or peripheral to the cytoplasmic membrane has been studied by fusing them to green fluorescent protein (GFP) (or its derivatives), and it is possible to classify the fusion proteins according to their subcellular localization. The first group, comprised of proteins that are concentrated at the cell poles, includes chemoreceptors (31, 62), the lactose permease LacY (43), and the metabolic sensor kinases DcuS and CitA (55). Members of the second group form helices that extend from pole to pole and include MreB (25), MinD (57), the Sec protein export system (58), and RNase E, which is the main component of the RNA degradosome in E. coli (67). Other proteins may appear to be similarly distributed due to their association with the Sec system (58). Members of the third group are uniformly distributed and include the mechanosensitive channel MscL (45) and the sensor kinase KdpD (32).The polar localization of proteins appears to be a critical feature of the complicated internal localization of bacteria. For example, it is important for temporally and spatially accurate placement of the septum during cell division (15). However, the mechanism of protein organization at bacterial cell poles is still unclear, and in many cases its functional role has not been determined. Do the poles merely serve as a receptacle for proteins, superstructures, or membrane domains with no functional effects, or is this location functionally important for membrane proteins and lipids?Recent evidence indicates that the subcellular localization of the transporter ProP in E. coli is related to membrane phospholipid composition, cardiolipin localization, and ProP function (51, 52). E. coli cells from cultures grown to exponential phase contain mostly the zwitterionic phospholipid PE (approximately 75 mol%) and the anionic phospholipids PG (approximately 20 mol%) and CL (approximately 5 mol%) (8). (Note that cardiolipin is diphosphatidylglycerol.) However, the phospholipid composition depends on the bacterial growth conditions. We found that the proportion of CL among E. coli lipids varies directly with growth medium osmolality (68), and increased CL synthesis was at least partially attributed to regulation of the cls locus encoding cardiolipin synthase (52). There is residual CL in cls bacteria, indicating that there is an alternative pathway for CL synthesis (51). The CL-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to show that CL clusters at the poles and septa in growing E. coli cells (36, 52). This result was corroborated by analyzing the phospholipid composition of E. coli minicells (DNA-free cells resulting from asymmetric cell division) (24, 51).ProP is an osmosensory transporter that senses increasing osmolality and responds by mediating the cytoplasmic accumulation of organic osmolytes (e.g., proline, glycine betaine, and ectoine). Biochemical regulation of the ProP protein ensures that ProP activity increases with increasing assay medium osmolality (49). We showed that ProP and CL colocalize at the poles and near the septa of dividing E. coli cells and that the polar concentration of ProP correlates with the polar concentration of CL (52). Moreover, we showed that the osmolality required to activate ProP increased in parallel to the CL content when E. coli was cultivated in media with increasing osmolality (51, 52, 68). The osmolality required to activate ProP was also a direct function of CL content in proteoliposomes reconstituted with purified ProP (51). We concluded that concentration at the cell poles controlled the osmoregulatory function of ProP by placing the transporter in a cardiolipin-rich environment.To determine whether CL-dependent membrane protein localization is a general phenomenon in E. coli, we compared the subcellular localization of ProP with that of its paralogue LacY, a well-characterized lactose transporter (16). LacY and ProP are both members of the major facilitator superfamily and H⁺ symporters. LacY transports the nutrient lactose, and LacY activity decreases while ProP activity increases with increasing osmolality (9). Nagamori et al. reported polar localization of a LacY-GFP fusion protein in E. coli (43). We confirmed this observation and demonstrated that, in contrast to the behavior of ProP, the polar concentration of LacY did not correlate with the polar concentration of CL (51).In this work we further explored the relationship between CL and protein localization in E. coli. We compared ProP with other proteins related to cellular osmoregulation. Bacteria use arrays of osmoregulatory mechanisms to survive and function when the osmotic pressure of their environment changes. In E. coli, the aquaporin AqpZ mediates transmembrane water flux, the transporters ProP, ProU, BetT, and BetU mediate organic osmolyte accumulation at high osmotic pressure, and the mechanosensitive (MS) channels MscL and MscS mediate solute efflux in response to osmotic downshock (71). Localization of these proteins might be expected since AqpZ might influence cell morphology changes by accelerating water flux at particular positions on the cell surface and the pressure sensitivities of MscL and MscS are known to depend on membrane curvature in vitro (18).For ProP and LacY, we labeled the inserted peptide tag CCPGCC with the biarsenical fluorescein reagent FlAsH-EDT₂ (fluorescein arsenical helix binder, bis-EDT adduct) (1, 2) to examine the subcellular localization of AqpZ, the integral membrane component ProW of the osmoregulatory ATP-binding cassette (ABC) transporter ProU, and the MS channel proteins MscS and MscL in cls⁺ and cls bacteria. Fluorescence microscopy was used to determine the proportion of cells with labeled protein concentrated at the poles as a function of bacterial CL content and protein expression level. For ProP, the frequency with which MscS was concentrated at cell poles was proportional to the level and polar concentration of CL. LacY concentrated at the cell poles at a high and CL-independent frequency. The frequencies with which AqpZ, MscL, and ProW concentrated at the cell poles and septa were low (up to 12%) and CL independent.     

Oligomeric Amyloid-β Inhibits the Proteolytic Conversion of Brain-derived Neurotrophic Factor (BDNF), AMPA Receptor Trafficking,and Classical Conditioning

Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.