Allosteric regulation of pentameric ligand-gated ion channels: An emerging mechanistic perspective

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies.     

Role of extracellular calcium and mitochondrial oxygen species in psychosine-induced oligodendrocyte cell death

Globoid cell leukodystrophy (GLD) is a metabolic disease caused by mutations in the galactocerebrosidase (GALC) gene. GALC is a lysosomal enzyme whose function is to degrade galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive intracellular accumulation of PSY. It is widely held that PSY is the main trigger for the degeneration of myelinating cells and progressive white-matter loss. However, still little is known about the molecular mechanisms by which PSY imparts toxicity. Here, we address the role of calcium dynamics during PSY-induced cell death. Using the human oligodendrocyte cell line MO3.13, we report that cell death by PSY is accompanied by robust cytosolic and mitochondrial calcium (Ca²⁺) elevations, and by mitochondrial reactive oxygen species (ROS) production. Importantly, we demonstrate that the reduction of extracellular calcium content by the chelating agent ethylenediaminetetraacetic acid can decrease intra-mitochondrial ROS production and enhance cell viability. Antioxidant administration also reduces mitochondrial ROS production and cell loss, but this treatment does not synergize with Ca²⁺ chelation. Our results disclose novel intracellular pathways involved in PSY-induced death that may be exploited for therapeutic purposes to delay GLD onset and/or slow down its progression.Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a childhood leukodystrophy triggered by mutations in the galactocerebrosidase (GALC) gene; the physio-pathological hallmarks of GLD are progressive demyelination, reactive astrocytosis and microgliosis.¹ GALC is a lysosomal enzyme essential for the normal catabolism of galacto-lipids, including galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of function causes progressive accumulation of PSY, a cytotoxic metabolite that has been assumed as the main cause for GLD pathogenesis.² PSY leads to Schwann cell and oligodendrocyte death, but still little is known about the molecular mechanisms by which PSY imparts toxicity. It has been demonstrated that PSY accumulates in cell membrane raft micro-domains, disrupting their architecture³ and inhibiting protein kinase C translocation to the plasma membrane.⁴ Recently, increased raft clustering was also reported in cultured dorsal root ganglion neurons prepared from the GLD murine model (i.e., the Twitcher mouse), and this was associated with the dysregulation of tyrosine kinase receptor A membrane recruitment and ligand-tyrosine kinase receptor A activated endocytosis.⁵ PSY induces p53-mediated apoptotic cell death,⁶ tumor necrosis factor-related apoptosis,^{6, 7} activation of secretory phospholipase A2,⁸ cytochrome C release from mitochondria and apoptosis activation via the caspase-9 pathway.⁹ Moreover, several authors found that peroxisomal β-oxidation was significantly inhibited and very long-chain fatty acid levels and reactive oxygen species (ROS) production were increased in PSY-treated cells.^{10, 11}Calcium (Ca²⁺) is an essential ion for cell life, acting as a key second messenger in almost all cellular functions. It is well established that Ca²⁺ is one of the main second messengers involved in apoptotic cell death in neurons and in other cell types; sustained cytosolic Ca²⁺ increase can activate apoptosis.¹² This can originate from extracellular influx or by release from intracellular stores like the endoplasmic reticulum.¹³ Importantly, mitochondria are also involved in Ca²⁺ homeostasis.¹⁴ Mitochondrial Ca²⁺ in basal conditions is maintained at low concentrations, but mitochondria are organelles that can take up high Ca²⁺ concentrations; indeed, different stimuli, such as nutrients, hormones or neurotransmitters that increase the cytoplasmic Ca²⁺ content also induce intra-mitochondrial Ca²⁺ increase.¹⁵ If this increase is relevant, ROS production increases and this is associated with mitochondrial membrane destruction, release of cytochrome C and apoptosis induction.¹⁶ During this process, pro-apoptotic Bcl2 family of proteins plays a crucial role by regulating the intracellular/mitochondrial Ca²⁺ content, and by inducing mitochondrial permeabilization, the essential step for cytochrome C release and caspase activation.^{12, 17}It has been reported that some sphingolipid metabolites, such as ceramides and sphingosine, can play a crucial role in many steps of apoptosis induction as regulators of some Bcl2 family proteins, by increasing intracellular Ca²⁺ levels and inducing mitochondrial stress.¹⁸ However, these mechanisms have never been explored during PSY-induced cell death.In this article, we report on the role of intracellular Ca²⁺ dynamics during PSY-induced cell death in vitro. Using the human oligodendrocyte cell line MO3.13 and fluorescent probes, we measured Ca²⁺ variations in cytoplasm and mitochondria upon PSY administration until cell death. Moreover, we studied oxidative stress production in mitochondria by flow cytometry and time-lapse confocal fluorescence microscopy. Finally, in order to rescue cell viability in presence of PSY, we investigated the use of Ca²⁺ chelation in the extracellular medium, and its possible synergic effect with antioxidant treatment.     

Fertility and uterine hemodynamic in cows after artificial insemination with semen assessed by fluorescent probes

Fluorescent probes (propidium iodide, Hoechst 33342, fluorescein isothiocyanate–conjugated Pisum sativum agglutinin, and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) were used to simultaneously evaluate the integrity of plasma and acrosomal membranes as well as mitochondrial membrane function in cryopreserved bovine semen and to verify its influence on fertility and postinsemination uterine vascularization. One hundred eighty-two Nellore cows were distributed for artificial insemination (AI) using semen batches separated according to the cell percentage presenting intact plasma membrane, intact acrosome, and high mitochondrial function (IPIAH): group G (44.5% IPIAH, n = 68), group M (23.0% IPIAH, n = 56), and group R (8.5% IPIAH, n = 58). The uterine hemodynamic was evaluated by Doppler sonogram in three periods: 30 hours before AI, 4 and 24 hours after AI were considered the resistance index and the uterine vascularization score. The pregnancy rate of group G (64.7%) was greater (P > 0.05) compared with group R (36.2%), but both did not differ from group M (50.0%). There was no effect (P > 0.05) of semen quality on uterine vascularization. Greater vascularization was noticed 4 hours after AI than 30 hours before and 24 hours after AI. Semen evaluation using fluorescent probes contributes to predicting fertilizing potential of semen. The use of semen with less percentage of IPIAH sperm does not alter uterine hemodynamic in cows.     

Beta-diversity in seasonally dry tropical forests (SDTF) in the Caatinga Biogeographic Domain, Brazil, and its implications for conservation

Tropical biomes are species rich, but some biomes such as seasonally dry tropical forests (SDTFs) are still inadequately studied compared to their co-occurring rain forest and savanna. SDTFs occur in areas of high environmental heterogeneity, resulting in high beta (β)-diversity or species turnover, but this has so far only been accessed using a single β-diversity measure, and at a spatial scale that is of limited applicability for reserve planning. The Caatinga Biogeographic Domain in Brazil contains the largest known extent of SDTF which are poorly studied and inadequately reserved. We therefore studied the variation in species richness and species turnover among SDTF between localities and between known floristic communities. From six localities within the Caatinga Biogeographic Domain we recorded all tree species with a circumference at breast height equaling or exceeding 10 cm within 106 400 m² survey plots. From the species presence/absence data we calculated three measures of β-diversity between pairs of study localities and between different floristic communities representing: (i) species similarity, (ii) differences between species richness, and (iii) species gain and loss. Our results confirm the high β-diversity of SDTFs and species turnover between localities and also between floristic communities. The three indices were also complementary to each other and can be used to maximize accuracy in β-diversity studies. The implications of our study for conservation and reserve planning of SDTFs are discussed.     

Seed storage hemicelluloses as wet-end additives in papermaking

Xyloglucans and galactomannans are examples of hemicelluloses that can be accumulated in seeds of many plants, being extensively studied and used for industrial applications. Guar gum and starch are polysaccharides currently used as wet-end additives in papermaking, whereas xyloglucans have never been reported to improve paper quality. In this work we show that different types of xyloglucans improved the mechanical properties of paper sheets without affecting the optical ones. Addition of 1% (w/w) of hemicelluloses to cellulosic pulp was able to increase by about 30% the mechanical properties such as burst and tear indexes. Seeds of several species could be used as source for the production of wet-end additives, since the results did not vary with the source of polysaccharides. Even if the utilisation of these hemicelluloses will not cost less than starch or guar gum, it might represent an important strategy for sustainable use of rainforest species.     

Chemical Composition and Antimicrobial Activity of the Essential Oils from Several Hypericum Taxa (Guttiferae) Growing in Central Italy (Appennino Umbro‐Marchigiano)

The chemical composition of the essential oils of nine taxa from seven sections of Hypericum L. (Guttiferae; H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. montanum, H. richeri subsp. richeri, H. hyssopifolium, H. hirsutum, H. hircinum subsp. majus, and H. tetrapterum) occurring in central Italy (Appennino Umbro‐Marchigiano) was analyzed by GC/FID and GC/MS. A total of 186 compounds were identified in the different species and subspecies, accounting for 86.9–92.8% of the total oils. The major fraction of the oil was always represented by sesquiterpene hydrocarbons (30.3–77.2%), while quantitative differences occurred between the other classes of volatiles depending on the taxa considered. Chemical composition of the nine Hypericum entities with respect to the taxonomical classification was discussed. Essential oils obtained from six taxa, i.e., H. perforatum subsp. perforatum, H. perforatum subsp. veronense, H. calycinum, H. richeri subsp. richeri, H. hirsutum and H. tetrapterum, were also tested for their antimicrobial properties against five different microbial strains by the broth‐microdilution method, and they were found to have significant activity (expressed as MIC) on B. subtilis, moderate activity on C. albicans and S. aureus, and weak activity on E. coli and E. faecalis, the most active being those from H. hirsutum, H. richeri subsp. richeri, and H. tetrapterum.     

Differences in protonation of ubiquinone and menaquinone in fumarate reductase from Escherichia coli

Escherichia coli quinol-fumarate reductase operates with both natural quinones, ubiquinone (UQ) and menaquinone (MQ), at a single quinone binding site. We have utilized a combination of mutagenesis, kinetic, EPR, and Fourier transform infrared methods to study the role of two residues, Lys-B228 and Glu-C29, at the quinol-fumarate reductase quinone binding site in reactions with MQ and UQ. The data demonstrate that Lys-B228 provides a strong hydrogen bond to MQ and is essential for reactions with both quinone types. Substitution of Glu-C29 with Leu and Phe caused a dramatic decrease in enzymatic reactions with MQ in agreement with previous studies, however, the succinate-UQ reductase reaction remains unaffected. Elimination of a negative charge in Glu-C29 mutant enzymes resulted in significantly increased stabilization of both UQ-* and MQ-* semiquinones. The data presented here suggest similar hydrogen bonding of the C1 carbonyl of both MQ and UQ, whereas there is different hydrogen bonding for their C4 carbonyls. The differences are shown by a single point mutation of Glu-C29, which transforms the enzyme from one that is predominantly a menaquinol-fumarate reductase to one that is essentially only functional as a succinate-ubiquinone reductase. These findings represent an example of how enzymes that are designed to accommodate either UQ or MQ at a single Q binding site may nevertheless develop sufficient plasticity at the binding pocket to react differently with MQ and UQ.     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

Tree dominance and diversity in Minas Gerais,Brazil

Quantifying diversity is an old challenge for ecologists and is also a social demand given the increasing threats to natural areas. We sought to work on these issues by using data from 158 vegetation remnants (over 350,000 trees) in southeastern Brazil (Minas Gerais State—MG, nearly 600,000 km²). Specifically, we sought to answer the following questions. (1) How many trees and tree species currently exist in MG vegetation remnants? (2) How much of such biodiversity is present in each vegetation domain (Atlantic Forest, Cerrado and Caatinga) and vegetation type (Seasonally Dry tropical forest “semideciduous”, Seasonally Dry tropical forest “deciduous”, Rain Forest, Swamp, Cerrado and Cerradão) in MG? (3) How much has been lost in regards to tree amount and tree species? We built a 0.1-degree cell grid to estimate the number of trees via spatial regression and used Fisher’s alpha and Fisher’s log series to provide estimates on how many tree species there are and were in MG. We found the number of trees in Minas Gerais to be approximately 24.5 × 10⁹, and the number of tree species to be between 3592 and 3743. The most abundant species distribution among the vegetation domains and vegetation types followed inversely the environmental heterogeneity of the classes. Consequently, the most abundant species in MG belonged to the Cerrado domain, where there was less environmental heterogeneity. The numbers of trees and tree species lost were estimated at 68.54 and 4.03–8.42% of the original values, respectively. We discuss that due to the consequences of human impacts that go back in the region for over a thousand years, other processes not considered in this study, such as habitat degradation by isolation, alterations of food webs, unsustainable use, and climate change, might have caused local extinctions and potentially increased the number of species lost. We believe our results may guide conservation initiatives by providing a base for future environmental laws, parks planning, and the development of more appropriate vegetation management techniques in MG. In addition, our results may inspire future quantitative ecological studies in the tropics.