Nucleolipids as potential organogelators

Four different series of nucleolipids or bola-nucleolipids were synthesized or re-synthesized. Most of the compounds were studied with respect to their gelation properties toward either water or aromatic, hetero-aromatic, and aliphatic hydrocarbons. Bola-nucleolipids 6 and 7 do not gelate any solvent tested, neither as sole additive nor by adding up to 10 wt% of a 1:1 mixture. The nucleolipid 22 carrying the antiviral acyclovir as a head group proved to be a potent organogelator for aromatic hydrocarbons such as toluene, but not for hetarenes, aliphatic hydrocarbons or water. The mono-tailed nucleolipid 24 exhibits excellent organogelator properties for both aromatic and aliphatic hydrocarbons. These were studied as a function of concentration and temperature.     

The crystal structure of the BAR domain from human Bin1/amphiphysin II and its implications for molecular recognition

BAR domains are found in proteins that bind and remodel membranes and participate in cytoskeletal and nuclear processes. Here, we report the crystal structure of the BAR domain from the human Bin1 protein at 2.0 A resolution. Both the quaternary and tertiary architectures of the homodimeric Bin1BAR domain are built upon "knobs-into-holes" packing of side chains, like those found in conventional left-handed coiled-coils, and this packing governs the curvature of a putative membrane-engaging concave face. Our calculations indicate that the Bin1BAR domain contains two potential sites for protein-protein interactions on the convex face of the dimer. Comparative analysis of structural features reveals that at least three architectural subtypes of the BAR domain are encoded in the human genome, represented by the Arfaptin, Bin1/Amphiphysin, and IRSp53 BAR domains. We discuss how these principal groups may differ in their potential to form regulatory heterotypic interactions.     

Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau(RD)) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation DeltaK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with DeltaK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau(RD) is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for beta-structure. (c) Soluble mutants of tau(RD) that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau(RD) in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau(RD) (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau(RD) disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau(RD) that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.     

Toxicity and metabolism of the chloral-derived mammalian alkaloid 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) in PC12 cells

Chloral-derived beta-carbolines, which are structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 5), are discussed to contribute to neuronal cell death in idiopathic Parkinson's disease. The cytotoxicity of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo, 4) to neuronal-like clonal pheochromocytoma PC12 cells was examined by the determination of lactate dehydrogenase (LDH) release. After incubation for 48 h, 4 showed a strong dose-dependent cytotoxic activity towards PC12 cells with an ED50 value of 230 microM. In PC12 cells reductive dehalogenation of 4 was observed giving rise to the formation of 1-dichloromethyl-1,2,3,4-tetrahydro-beta-carboline (6) as a main TaClo metabolite exhibiting a cytotoxic potential comparable to that of TaClo. An X-ray structure analysis, performed for the trifluoroacetyl derivative of 6, revealed the N-substituent of such a highly chlorinated agent to be dramatically pushed out of the beta-carboline ring 'plane' due to the high steric demand of the huge dichloromethyl group at C(1).     

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

A database search of the Paramecium genome reveals 34 genes related to Ca²⁺-release channels of the inositol-1,4,5-trisphosphate (IP₃) or ryanodine receptor type (IP₃R, RyR). Phylogenetic analyses show that these Ca²⁺ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP₃Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP₃Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca²⁺ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP₃ channel, a group II member (P. tetraurelia IP₃R_N-1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP₃R_N affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca²⁺ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca²⁺ as signaling molecule.Ca²⁺ is an important component of cell activity in all organisms, from protozoa to mammals. Thereby Ca²⁺ may originate from the outside medium and/or from internal stores (7, 18). Ca²⁺ release from internal stores is mediated by various Ca²⁺ release channels (CRCs), of which the inositol-1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) families have been studied most extensively (8, 9, 29, 63). IP₃Rs and RyRs have been identified in various metazoan organisms (reviewed in references 9, 28, and 104). According to these reviews, there exist three genetically distinct isoforms of each receptor type in mammals and orthologues have been identified in various nonmammalian vertebrates, e.g., frogs, chickens, and fish. RyRs and IP₃Rs were also cloned and sequenced in the invertebrates Drosophila melanogaster and Caenorhabditis elegans, which possess one copy of each receptor type.Functional evidence for Ca²⁺ release in response to ryanodine or IP₃ receptor agonists has been described in several unicellular systems. Treatment of permeabilized Plasmodium chabaudi parasites with IP₃ results in Ca²⁺ release, which is inhibited by the IP₃ receptor antagonist heparin (69). Another apicomplexan parasite, Toxoplasma gondii, responds to agonists and antagonists of both, ryanodine and IP₃ receptors, by mediating increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (56). Stimulation of Trypanosoma cruzi with carbachol results in increased [Ca²⁺]_i and IP₃ (59). IP₃ and cyclic ADP-ribose induces Ca²⁺ release in Euglena gracilis microsome fractions in a dose-dependent manner (61). In the giant algae Chara corallina and Nitrella translucens, IP₃ produces action potentials involving increased [Ca²⁺]_i (93). Treatment of vacuolar membrane vesicles from Candida albicans with IP₃ results in Ca²⁺ release, blocked by heparin and ruthenium red (14). IP₃ generates and maintains a Ca²⁺ gradient in the hyphal tip of Neurospora crassa and the IP₃-sensitive channels have been reconstituted and characterized with the planar bilayer method (87). In summary, these publications suggest that IP₃-dependent signaling pathways are conserved among unicellular organisms, including protozoa.Despite these data, the molecular characterization of IP₃ or ryanodine receptors in low eukaryotes is currently a challenge since the identification of orthologues has not been possible thus far, probably because of evolutionary sequence divergence (66). Traynor et al. (96) identified an IP₃ receptor-like protein, IplA, in Dictyostelium discoideum, which possesses regions related to IP₃R sequences, but thus far no evidence for IP₃ interaction exists. We have recently described an IP₃R in the ciliated protozoa Paramecium tetraurelia (referred to here as P. tetraurelia IP₃R_N) (53), with features characteristic of mammalian IP₃Rs in terms of topology and ability for IP₃ binding. The expression level of P. tetraurelia IP₃R_N is modulated by extracellular Ca²⁺ concentrations ([Ca²⁺]_o) and immunofluorescence studies reveal an unexpected localization to the contractile vacuole complex (CVC), the major organelle involved in osmoregulation (2). The ionic composition of the contractile vacuole fluid by ion-selective microelectrodes (91) suggests that the organelle plays a major role in expelling an excess of cytosolic Ca²⁺. Therefore, these IP₃Rs may here mediate a latent, graded reflux of Ca²⁺ for fine-tuning of [Ca²⁺]_i and thus serve [Ca²⁺] homeostasis (53).Besides [Ca²⁺] homeostasis, the Paramecium cell has to regulate a variety of well-characterized processes (75). This includes exocytosis of dense-core secretory vesicles (trichocysts) (71, 74, 99). Each cell possesses up to 1,000 trichocysts attached to the cell membrane. Their contents can be extruded synchronously in response to natural stimuli, i.e., predators (34, as confirmed by Knoll et al. [49]), to artificial polyamine secretagogues such as aminoethyldextran (AED) (78), to caffeine (48) or to the ryanodine substitute, 4-chloro-meta-cresol (4-CmC) (46). Their expulsion strictly depends on Ca²⁺ (10) and is accompanied by an increase of intracellular [Ca²⁺]_i (24, 47). This Ca²⁺ signal originates from rapid mobilization of cortical stores, the alveolar sacs (33, 64, 74), superimposed by Ca²⁺ influx (46, 72). It thus represents a SOC-type mechanism (SOC, store-operated Ca²⁺ entry) known from mammalian systems (81).Upon exocytosis stimulation ∼60% of their total Ca²⁺ is released from alveolar sacs (33). These are Ca²⁺ stores (90) represented by flat membrane compartments tightly attached at the cell membrane surrounding each trichocyst docking site. They possess a SERCA-type pump located at the membrane facing the cell center (36, 37) and a luminal high-capacity/low-affinity CaBP of the calsequestrin type (73). Thus far, Ca²⁺ release channels of these stores were identified only indirectly as cells respond by exocytosis to the RyR activators caffeine (54, 48) and 4-CmC (46). However, an involvement of conserved RyRs has remained questionable as ryanodine is not able to activate Ca²⁺ release from alveolar sacs, as is the case with IP₃ (54). Therefore, one of the most intriguing questions is the elucidation of the molecular nature of the channels mediating Ca²⁺ release from alveolar sacs upon stimulated exocytosis.In the present work we describe a novel family of CRCs (P. tetraurelia CRC-IV-1), whose members display several properties of the channels postulated above. In detail, the identified CRC-IV-1 channels localize to the alveolar sacs. Functional and fluorochrome analyses after gene silencing reveal that they are essential for mediating Ca²⁺ release and exocytosis in response to AED, caffeine, or 4-CmC. Their classification as “novel” CRC type is based on a restricted relationship to the C-terminal channel domains of IP₃Rs and RyRs. The overall size and the number of putative transmembrane domains resemble IP₃Rs, but N-terminal parts of CRC-IV-1 channels do not show any conservation, such as an IP₃-binding domain. Therefore, CRC-IV-1 channels represent distant relatives of IP₃Rs and RyRs and may belong to an ancestral Ca²⁺ signaling pathway.     

The Interaction between Coagulation Factor 2 Receptor and Interleukin 6 Haplotypes Increases the Risk of Myocardial Infarction in Men

The aim of the study was to investigate if the interaction between the coagulation factor 2 receptor (F2R) and the interleukin 6 (IL6) haplotypes modulates the risk of myocardial infarction (MI) in the Stockholm Heart Epidemiology Program (SHEEP). Seven SNPs at the F2R locus and three SNPs at the IL6 locus were genotyped. Haplotypes and haplotype pairs (IL6*F2R) were generated. A logistic regression analysis was performed to analyze the association of the haplotypes and haplotype pairs with the MI risk. Presence of an interaction between the two haplotypes in each haplotype pair was calculated using two different methods: the statistical, on a multiplicative scale, which includes the cross product of the two factors into the logistic regression model; the biological, on an additive scale, which evaluates the relative risk associated with the joint presence of both factors. The ratio between the observed and the predicted effect of the joint exposure, the synergy index (S), indicates the presence of a synergy (S>1) or of an antagonism (S<1). None of the haplotypes within the two loci was associated with the risk of MI. Out of 22 different haplotype pairs, the haplotype pair 17 GGG*ADGTCCT was associated with an increased risk of MI with an OR (95%CI) of 1.58 (1.05–2.41) (p = 0.02) in the crude and an OR of 1.72 (1.11–2.67) (p = 0.01) in the adjusted analysis. We observed the presence of an interaction on a multiplicative scale with an OR (95%CI) of 2.24 (1.27–3.95) (p = 0.005) and a slight interactive effect between the two haplotypes on an additive scale with an OR (95%CI) of 1.56 (1.02–2.37) (p = 0.03) and S of 1.66 (0.89–31). In conclusion, our results support the hypothesis that the interaction between these two functionally related genes may influence the risk of MI and suggest new mechanisms involved in the genetic susceptibility to MI.     

Inhibition of APP trafficking by tau protein does not increase the generation of amyloid-beta peptides

Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.     

The ontogeny of hemodynamic responses to prolonged umbilical cord occlusion in fetal sheep.

There is evidence that preterm fetuses have blunted chemoreflex-mediated responses to hypoxia. However, the preterm fetus has much lower aerobic requirements than at term, and so moderate hypoxia may not be sufficient to elicit maximal chemoreflex responses; there are only limited quantitative data on the ontogeny of chemoreflex and hemodynamic responses to severe asphyxia. Chronically instrumented fetal sheep at 0.6 (n = 12), 0.7 (n = 12), and 0.85 (n = 8) of gestational age (GA; term = 147 days) were exposed to 30, 25, or 15 min of complete umbilical cord occlusion, respectively. At all ages, occlusion was associated with early onset of bradycardia, profoundly reduced femoral blood flow and conductance, and hypertension. The 0.6-GA fetuses showed a significantly slower and lesser fall in femoral blood flow and conductance compared with the 0.85-GA group, with a correspondingly reduced relative rise in mean arterial blood pressure. As occlusion continued, the initial adaptation was followed by loss of peripheral vasoconstriction and progressive development of hypotension in all groups. The 0.85-GA fetuses showed significantly more sustained reduction in femoral conductance but also more rapid onset of hypotension than either of the younger groups. Electroencephalographic (EEG) activity was suppressed during occlusion in all groups, but the degree of suppression was less at 0.6 GA than at term. In conclusion, the near-midgestation fetus shows attenuated initial (chemoreflex) peripheral vasomotor responses to severe asphyxia compared with more mature fetuses but more sustained hemodynamic adaptation and reduced suppression of EEG activity during continued occlusion of the umbilical cord.