Epigenetic regulation of neuronal dendrite and dendritic spine development

Dendrites and the dendritic spines of neurons play key roles in the connectivity of the brain and have been recognized as the locus of long-term synaptic plasticity, which is correlated with learning and memory. The development of dendrites and spines in the mammalian central nervous system is a complex process that requires specific molecular events over a period of time. It has been shown that specific molecules are needed not only at the spine’s point of contact, but also at a distance, providing signals that initiate a cascade of events leading to synapse formation. The specific molecules that act to signal neuronal differentiation, dendritic morphology, and synaptogenesis are tightly regulated by genetic and epigenetic programs. It has been shown that the dendritic spine structure and distribution are altered in many diseases, including many forms of mental retardation (MR), and can also be potentiated by neuronal activities and an enriched environment. Because dendritic spine pathologies are found in many types of MR, it has been proposed that an inability to form normal spines leads to the cognitive and motor deficits that are characteristic of MR. Epigenetic mechanisms, including DNA methylation, chromatin remodeling, and the noncoding RNA-mediated process, have profound regulatory roles in mammalian gene expression. The study of epigenetics focuses on cellular effects that result in a heritable pattern of gene expression without changes to genomic encoding. Despite extensive efforts to understand the molecular regulation of dendrite and spine development, epigenetic mechanisms have only recently been considered. In this review, we will focus on epigenetic mechanisms that regulate the development and maturation of dendrites and spines. We will discuss how epigenetic alterations could result in spine abnormalities that lead to MR, such as is seen in fragile X and Rett syndromes. We will also discuss both general methodology and recent technological advances in the study of neuronal dendrites and spines.     

Fickean and Non-Fickean Water Desorption During Vacuum Drying of <Emphasis Type=Italic>L. bulgaricus</Emphasis>

The recovery of Lactobacillus bulgaricus was studied in correlation to the kinetics of cell drying. When bacteria were dehydrated at 30 °C, either in the presence or the absence of sucrose, the drying kinetics corresponds to a Fickean diffusion in correspondence with a short lag time. In contrast, when the bacteria were dehydrated at 70 °C in the absence of sugar, the kinetics corresponds to an anomalous diffusion, and the lag time is four to five times higher than that at 30 °C. However, when drying at 70 °C was carried out in the presence of sucrose, drying kinetics turned into a Fickean process parallel to a substantial decrease in the lag time. The pattern of water desorption was correlated with the critical water activity. When the drying kinetics corresponds to a Fickean diffusion, the lag time started to increase at 0.7 water activity, but when the cells were dried at 70 °C, the damage started at 0.5 water activity. This observation indicates that the drying rate affects the pattern of water desorption, and it can change the value of critical water activity. These results put into relevance that the cell recovery is due to the drying history and that the recovery increase produced by sucrose can be related to the maintenance of kinetic barriers for water desorption.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Low diversity and high levels of population genetic structuring in introduced eastern mosquitofish (<Emphasis Type="Italic">Gambusia holbrooki</Emphasis>) in the greater Melbourne area,Australia

Eastern mosquitofish (Gambusia holbrooki) were introduced into Australia in 1925 and released to control mosquitoes. Gambusia holbrooki rapidly became invasive in recipient environments and now threaten native fauna. In this study, we used five polymorphic microsatellite loci and sequence from two mitochondrial genes, cytochrome b and cytochrome oxidase I, to evaluate genetic variation, colonisation and movement patterns of introduced G. holbrooki in the greater Melbourne area, and to assist in identifying the feasibility of local eradication. Microsatellite variation was consistently low within populations and there was evidence of bottleneck events for several populations. Populations displayed significant structuring associated with river basins rather than geographic distance, suggesting that habitat connectivity is important for dispersal. However, a few populations within river basins were more closely related to populations in other river basins than within their own basin, most likely reflecting a role of human-assisted dispersal in population establishment. Mitochondrial sequencing revealed only a single haplotype and suggested all populations were founded by individuals from a common source. These genetic data help delineate boundaries for local management strategies.     

Mechanism of the Anticoagulant Activity of Thrombin Mutant W215A/E217A

The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and in vivo. Previous x-ray structural studies have shown that WE assumes a partially collapsed conformation that is similar to the inactive E* form, which explains its drastically reduced activity toward substrate. Whether this collapsed conformation is genuine, rather than the result of crystal packing or the mutation introduced in the critical 215–217 β-strand, and whether binding of thrombomodulin to exosite I can allosterically shift the E* form to the active E form to restore activity toward protein C are issues of considerable mechanistic importance to improve the design of an anticoagulant thrombin mutant for therapeutic applications. Here we present four crystal structures of WE in the human and murine forms that confirm the collapsed conformation reported previously under different experimental conditions and crystal packing. We also present structures of human and murine WE bound to exosite I with a fragment of the platelet receptor PAR1, which is unable to shift WE to the E form. These structural findings, along with kinetic and calorimetry data, indicate that WE is strongly stabilized in the E* form and explain why binding of ligands to exosite I has only a modest effect on the E*-E equilibrium for this mutant. The E* → E transition requires the combined binding of thrombomodulin and protein C and restores activity of the mutant WE in the anticoagulant pathway.Thrombin is the pivotal protease of blood coagulation and is endowed with both procoagulant and anticoagulant roles in vivo (1). Thrombin acts as a procoagulant when it converts fibrinogen into an insoluble fibrin clot, activates clotting factors V, VIII, XI, and XIII, and cleaves PAR1² and PAR4 on the surface of human platelets thereby promoting platelet aggregation (2). Upon binding to thrombomodulin, a receptor present on the membrane of endothelial cells, thrombin becomes unable to interact with fibrinogen and PAR1 but increases >1,000-fold its activity toward the zymogen protein C (3). Activated protein C generated from the thrombin-thrombomodulin complex down-regulates both the amplification and progression of the coagulation cascade (3) and acts as a potent cytoprotective agent upon engagement of EPCR and PAR1 (4).The dual nature of thrombin has long motivated interest in dissociating its procoagulant and anticoagulant activities (5–12). Thrombin mutants with anticoagulant activity help rationalize the bleeding phenotypes of several naturally occurring mutations and could eventually provide new tools for pharmacological intervention (13) by exploiting the natural protein C pathway (3, 14, 15). Previous mutagenesis studies have led to the identification of the E217A and E217K mutations that significantly shift thrombin specificity from fibrinogen toward protein C relative to the wild type (10–12). Both constructs were found to display anticoagulant activity in vivo (10, 12). The subsequent discovery of the role of Trp-215 in controlling the balance between pro- and anti-coagulant activities of thrombin (16) made it possible to construct the double mutant W215A/E217A (WE) featuring >19,000-fold reduced activity toward fibrinogen but only 7-fold loss of activity toward protein C (7). These properties make WE the most potent anticoagulant thrombin mutant engineered to date and a prototype for a new class of anticoagulants (13). In vivo studies have revealed an extraordinary potency, efficacy, and safety profile of WE when compared with direct administration of activated protein C or heparin (17–19). Importantly, WE elicits cytoprotective effects (20) and acts as an antithrombotic by antagonizing the platelet receptor GpIb in its interaction with von Willebrand factor (21).What is the molecular mechanism underscoring the remarkable functional properties of WE? The mutant features very low activity toward synthetic and physiological substrates, including protein C. However, in the presence of thrombomodulin, protein C is activated efficiently (7). A possible explanation is that WE assumes an inactive conformation when free but is converted into an active form in the presence of thrombomodulin. The ability of WE to switch from inactive to active forms is consistent with recent kinetic (22) and structural (23, 24) evidence of the significant plasticity of the trypsin fold. The active form of the protease, E, coexists with an inactive form, E*, that is distinct from the zymogen conformation (25). Biological activity of the protease depends on the equilibrium distribution of E* and E, which is obviously different for different proteases depending on their physiological role and environmental conditions (25). The E* form features a collapse of the 215–217 β-strand into the active site and a flip of the peptide bond between residues Glu-192 and Gly-193, which disrupts the oxyanion hole. These changes have been documented crystallographically in thrombin and other trypsin-like proteases such as αI-tryptase (26), the high temperature requirement-like protease (27), complement factor D (28), granzyme K (29), hepatocyte growth factor activator (30), prostate kallikrein (31), prostasin (32, 33), complement factor B (34), and the arterivirus protease nsp4 (35). Hence, the questions that arise about the molecular mechanism of WE function are whether the mutant is indeed stabilized in the inactive E* form and whether it can be converted to the active E form upon thrombomodulin binding.Structural studies of the anticoagulant mutants E217K (36) and WE (37) show a partial collapse of the 215–217 β-strand into the active site that abrogates substrate binding. The collapse is similar to, but less pronounced than, that observed in the structure of the inactive E* form of thrombin where Trp-215 relinquishes its hydrophobic interaction with Phe-227 to engage the catalytic His-57 and residues of the 60-loop after a 10 Å shift in its position (24). These more substantial changes have been observed recently in the structure of the anticoagulant mutant Δ146–149e (38), which has proved that stabilization of E* is indeed a molecular mechanism capable of switching thrombin into an anticoagulant. It would be simple to assume that both E217K and WE, like Δ146–149e, are stabilized in the E* form. However, unlike Δ146–149e, both E217K and WE carry substitutions in the critical 215–217 β-strand that could result into additional functional effects overlapping with or mimicking a perturbation of the E*-E equilibrium. A significant concern is that both structures suffer from crystal packing interactions that may have biased the conformation of side chains and loops near the active site (24). The collapsed structures of E217K and WE may be artifactual unless validated by additional structural studies where crystal packing is substantially different.To address the second question, kinetic measurements of chromogenic substrate hydrolysis by WE in the presence of saturating amounts of thrombomodulin have been carried out (37), but these show only a modest improvement of the k_cat/K_m as opposed to >57,000-fold increase observed when protein C is used as a substrate (7, 37). The modest effect of thrombomodulin on the hydrolysis of chromogenic substrates is practically identical to that seen upon binding of hirugen to exosite I (37) and echoes the results obtained with the wild type (39) and other anticoagulant thrombin mutants (7, 9, 10, 12, 38). That argues against the ability of thrombomodulin alone to significantly shift the E*-E equilibrium in favor of the E form. Binding of a fragment of the platelet receptor PAR1 to exosite I in the D102N mutant stabilized in the E* form (24) does trigger the transition to the E form (23), but evidence that a similar long-range effect exists for the E217K or WE mutants has not been presented.In this study we have addressed the two unresolved questions about the mechanism of action of the anticoagulant thrombin mutant WE. Here we present new structures of the mutant in its human and murine versions, free and bound to a fragment of the thrombin receptor PAR1 at exosite I. The structures are complemented by direct energetic assessment of the binding of ligands to exosite I and its effect on the E*-E equilibrium.     

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.     

H2AX: functional roles and potential applications

Upon DNA double-strand break (DSB) induction in mammals, the histone H2A variant, H2AX, becomes rapidly phosphorylated at serine 139. This modified form, termed γ-H2AX, is easily identified with antibodies and serves as a sensitive indicator of DNA DSB formation. This review focuses on the potential clinical applications of γ-H2AX detection in cancer and in response to other cellular stresses. In addition, the role of H2AX in homeostasis and disease will be discussed. Recent work indicates that γ-H2AX detection may become a powerful tool for monitoring genotoxic events associated with cancer development and tumor progression.     

Reply to Hussey et al.: The requirement for accurate diet-tissue discrimination factors for interpreting stable isotopes in sharks

Carbon and nitrogen stable isotope analyses have improved our understanding of food webs and movement patterns of aquatic organisms. These techniques have recently been applied to diet studies of elasmobranch fishes, but isotope turnover rates and isotope diet–tissue discrimination are still poorly understood for this group. We performed a diet switch experiment on captive sandbar sharks (Carcharhinus plumbeus) as a model shark species to determine tissue turnover rates for liver, whole blood, and white muscle. In a second experiment, we subjected captive coastal skates (Leucoraja spp.) to serial salinity reductions to measure possible impacts of tissue urea content on nitrogen stable isotope values. We extracted urea from spiny dogfish (Squalus acanthias) white muscle to test for effects on nitrogen stable isotopes. Isotope turnover was slow for shark tissues and similar to previously published estimates for stingrays and teleost fishes with low growth rates. Muscle isotope data would likely fail to capture seasonal migrations or diet switches in sharks, while liver and whole blood would more closely reflect shorter term movement or shifts in diet. Nitrogen stable isotope values of skate blood and skate and dogfish white muscle were not affected by tissue urea content, suggesting that available diet–tissue discrimination estimates for teleost fishes with similar physiologies would provide accurate estimates for elasmobranchs.     

Defining Life: The Virus Viewpoint

Are viruses alive? Until very recently, answering this question was often negative and viruses were not considered in discussions on the origin and definition of life. This situation is rapidly changing, following several discoveries that have modified our vision of viruses. It has been recognized that viruses have played (and still play) a major innovative role in the evolution of cellular organisms. New definitions of viruses have been proposed and their position in the universal tree of life is actively discussed. Viruses are no more confused with their virions, but can be viewed as complex living entities that transform the infected cell into a novel organism—the virus—producing virions. I suggest here to define life (an historical process) as the mode of existence of ribosome encoding organisms (cells) and capsid encoding organisms (viruses) and their ancestors. I propose to define an organism as an ensemble of integrated organs (molecular or cellular) producing individuals evolving through natural selection. The origin of life on our planet would correspond to the establishment of the first organism corresponding to this definition.