Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca⁺⁺ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca⁺⁺ could bind the Nav1.1 C-terminal region with micromolar affinity.     

The enzymes of the transsulfuration pathways: active-site characterizations

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.     

Solid character of membrane ceramides: a surface rheology study of their mixtures with sphingomyelin

The compression and shear viscoelasticities of egg-ceramide and its mixtures with sphingomyelin were investigated using oscillatory surface rheology performed on Langmuir monolayers. We found high values for the compression and shear moduli for ceramide, compatible with a solid-state membrane, and extremely high surface viscosities when compared to typical fluid lipids. A fluidlike rheological behavior was found for sphingomyelin. Lateral mobilities, measured from particle tracking experiments, were correlated with the monolayer viscosities through the usual hydrodynamic relationships. In conclusion, ceramide increases the solid character of sphingomyelin-based membranes and decreases their fluidity, thus drastically decreasing the lateral mobilities of embedded objects. This mechanical behavior may involve important physiological consequences in biological membranes containing ceramides.     

α-Tocopherol impairs 7-ketocholesterol-induced caspase-3-dependent apoptosis involving GSK-3 activation and Mcl-1 degradation on 158N murine oligodendrocytes

In important and severe neurodegenerative pathologies, 7-ketocholesterol, mainly resulting from cholesterol autoxidation, may contribute to dys- or demyelination processes. On various cell types, 7-ketocholesterol has often been shown to induce a complex mode of cell death by apoptosis associated with phospholipidosis. On 158N murine oligodendrocytes treated with 7-ketocholesterol (20 μg/mL corresponding to 50 μM, 24–48 h), the induction of a mode of cell death by apoptosis characterised by the occurrence of cells with condensed and/or fragmented nuclei, caspase activation (including caspase-3) and internucleosomal DNA fragmentation was observed. It was associated with a loss of transmembrane mitochondrial potential (ΔΨm) measured with JC-1, with a dephosphorylation of Akt and GSK3 (especially GSK3β), and with degradation of Mcl-1. With α-tocopherol (400 μM), which was capable of counteracting 7-ketocholesterol-induced apoptosis, Akt and GSK3β dephosphorylation were inhibited as well as Mcl-1 degradation. These data underline that the potential protective effects of α-tocopherol against 7-ketocholesterol-induced apoptosis do not depend on the cell line considered, and that the cascade of events (Akt/GSK3β/Mcl-1) constitutes a link between 7-ketocholesterol-induced cytoplasmic membrane dysfunctions and mitochondrial depolarisation leading to apoptosis.     

Transactivation of the epidermal growth factor receptor by heat shock protein 90 via Toll-like receptor 4 contributes to the migration of glioblastoma cells

Extracellular heat shock protein HSP90α was reported to participate in tumor cell growth, invasion, and metastasis formation through poorly understood signaling pathways. Herein, we show that extracellular HSP90α favors cell migration of glioblastoma U87 cells. More specifically, externally applied HSP90α rapidly induced endocytosis of EGFR. This response was accompanied by a transient increase in cytosolic Ca(2+) appearing after 1-3 min of treatment. In the presence of EGF, U87 cells showed HSP90α-induced Ca(2+) oscillations, which were reduced by the ATP/ADPase, apyrase, and inhibited by the purinergic P(2) inhibitor, suramin, suggesting that ATP release is requested. Disruption of lipid rafts with methyl β-cyclodextrin impaired the Ca(2+) rise induced by extracellular HSP90α combined with EGF. Specific inhibition of TLR4 expression by blocking antibodies suppressed extracellular HSP90α-induced Ca(2+) signaling and the associated cell migration. HSPs are known to bind lipopolysaccharides (LPSs). Preincubating cells with Polymyxin B, a potent LPS inhibitor, partially abrogated the effects of HSP90α without affecting Ca(2+) oscillations observed with EGF. Extracellular HSP90α induced EGFR phosphorylation at Tyr-1068, and this event was prevented by both the protein kinase Cδ inhibitor, rottlerin, and the c-Src inhibitor, PP2. Altogether, our results suggest that extracellular HSP90α transactivates EGFR/ErbB1 through TLR4 and a PKCδ/c-Src pathway, which induces ATP release and cytosolic Ca(2+) increase and finally favors cell migration. This mechanism could account for the deleterious effects of HSPs on high grade glioma when released into the tumor cell microenvironment.     

Dietary adaptations of temperate primates: comparisons of Japanese and Barbary macaques

Habitat, diet and leaf chemistry are compared between Japanese and Barbary macaques to reveal the similarities and differences in dietary adaptations of temperate primates living at the eastern and western extremes of the genus Macaca. Tree species diversity and proportion of fleshy-fruited species are much higher in Japan than in North Africa. Both species spend considerable annual feeding time on leaves. Japanese macaques prefer fruits and seeds over leaves, and Barbary macaques prefer seeds. These characteristics are adaptive in temperate regions where fruit availability varies considerably with season, since animals can survive during the lean period by relying on leaf and other vegetative foods. The two species are different with respect to the higher consumption of herbs by Barbary macaques, and the leaves consumed contain high condensed and hydrolysable tannin for Barbary but not for Japanese macaques. Barbary macaques supplement less diverse tree foods with herbs. Because of the low species diversity and high tannin content of the dominant tree species, Barbary macaques may have developed the capacity to cope with tannin. This supports the idea that digestion of leaves is indispensable to survive in temperate regions where fruit and seed foods are not available for a prolonged period during each year.     

Phase resetting analysis of high potassium epileptiform activity in CA3 region of the rat hippocampus

The theory of phase resetting can reveal important information about the dynamic behavior of a periodic system when a single brief stimulus is applied to that system at the appropriate time. Phase resetting studies have revealed the existence in some biological systems of a vulnerable stimulus window generating desynchronization and suppression of the activity. The objective of this study was to test the hypothesis that a "singular" stimulus could annihilate this activity. Perfusion with the high-K solution produced synchronous, quasi-periodic population bursts with inter-burst interval of ~0.8-1.5 seconds. A single 0.1 ms duration anodic pulse of programmable delay and magnitude was applied to the somatic layer of the CA3 pyramidal cells. Three types of phase-resetting behavior were observed: (1) Weak resetting with little or no effect on the timing of the subsequent burst, (2) Strong resetting where the applied current pulse delayed the next event by one time period, (3) Singular behavior where the applied pulse partially or completely suppressed the subsequent bursting. The singular stimulus parameter window, however, was very narrow making it difficult to generate the singular behavior reliably. Nevertheless, the results indicate that singularities exist for high potassium neural activity and that a well timed pulse applied with the right amplitude can suppress this activity. This study suggests that phase resetting of a population of neurons is possible for quasi-periodic interictal activity and similar techniques could be applied to the control of epileptic seizures.