Ascorbate uptake is decreased in the hippocampus of ageing rats

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.     

Immunization with a vaccine combining herpes simplex virus 2 (HSV-2) glycoprotein C (gC) and gD subunits improves the protection of dorsal root ganglia in mice and reduces the frequency of recurrent vaginal shedding of HSV-2 DNA in guinea pigs compared to immunization with gD alone

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation.     

Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G₃-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo⁻ (Kf⁻) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.     

Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing

Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC₅₀ = 4.5 nM) compared with vorinostat (VOR; EC₅₀ = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC₅₀ = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.     

Genomic versatility and functional variation between two dominant heterotrophic symbionts of deep-sea Osedax worms

An unusual symbiosis, first observed at ∼3000 m depth in the Monterey Submarine Canyon, involves gutless marine polychaetes of the genus Osedax and intracellular endosymbionts belonging to the order Oceanospirillales. Ecologically, these worms and their microbial symbionts have a substantial role in the cycling of carbon from deep-sea whale fall carcasses. Microheterogeneity exists among the Osedax symbionts examined so far, and in the present study the genomes of the two dominant symbionts, Rs1 and Rs2, were sequenced. The genomes revealed heterotrophic versatility in carbon, phosphate and iron uptake, strategies for intracellular survival, evidence for an independent existence, and numerous potential virulence capabilities. The presence of specific permeases and peptidases (of glycine, proline and hydroxyproline), and numerous peptide transporters, suggests the use of degraded proteins, likely originating from collagenous bone matter, by the Osedax symbionts. ¹³C tracer experiments confirmed the assimilation of glycine/proline, as well as monosaccharides, by Osedax. The Rs1 and Rs2 symbionts are genomically distinct in carbon and sulfur metabolism, respiration, and cell wall composition, among others. Differences between Rs1 and Rs2 and phylogenetic analysis of chemotaxis-related genes within individuals of symbiont Rs1 revealed the influence of the relative age of the whale fall environment and support possible local niche adaptation of ‘free-living'' lifestages. Future genomic examinations of other horizontally-propogated intracellular symbionts will likely enhance our understanding of the contribution of intraspecific symbiont diversity to the ecological diversification of the intact association, as well as the maintenance of host diversity.     

Adducin Is Required for Desmosomal Cohesion in Keratinocytes

Adducin is a protein organizing the cortical actin cytoskeleton and a target of RhoA and PKC signaling. However, the role for intercellular cohesion is unknown. We found that adducin silencing induced disruption of the actin cytoskeleton, reduced intercellular adhesion of human keratinocytes, and decreased the levels of the desmosomal adhesion molecule desmoglein (Dsg)3 by reducing its membrane incorporation. Because loss of cell cohesion and Dsg3 depletion is observed in the autoantibody-mediated blistering skin disease pemphigus vulgaris (PV), we applied antibody fractions of PV patients. A rapid phosphorylation of adducin at serine 726 was detected in response to these autoantibodies. To mechanistically link autoantibody binding and adducin phosphorylation, we evaluated the role of several disease-relevant signaling molecules. Adducin phosphorylation at serine 726 was dependent on Ca²⁺ influx and PKC but occurred independent of p38 MAPK and PKA. Adducin phosphorylation is protective, because phosphorylation-deficient mutants resulted in loss of cell cohesion and Dsg3 fragmentation. Thus, PKC elicits both positive and negative effects on cell adhesion, since its contribution to cell dissociation in pemphigus is well established. We additionally evaluated the effect of RhoA on adducin phosphorylation because RhoA activation was shown to block pemphigus autoantibody-induced cell dissociation. Our data demonstrate that the protective effect of RhoA activation was dependent on the presence of adducin and its phosphorylation at serine 726. These experiments provide novel mechanisms for regulation of desmosomal adhesion by RhoA- and PKC-mediated adducin phosphorylation in keratinocytes.     

Detecting spatial regimes in ecosystems

Research on early warning indicators has generally focused on assessing temporal transitions with limited application of these methods to detecting spatial regimes. Traditional spatial boundary detection procedures that result in ecoregion maps are typically based on ecological potential (i.e. potential vegetation), and often fail to account for ongoing changes due to stressors such as land use change and climate change and their effects on plant and animal communities. We use Fisher information, an information theory‐based method, on both terrestrial and aquatic animal data (U.S. Breeding Bird Survey and marine zooplankton) to identify ecological boundaries, and compare our results to traditional early warning indicators, conventional ecoregion maps and multivariate analyses such as nMDS and cluster analysis. We successfully detected spatial regimes and transitions in both terrestrial and aquatic systems using Fisher information. Furthermore, Fisher information provided explicit spatial information about community change that is absent from other multivariate approaches. Our results suggest that defining spatial regimes based on animal communities may better reflect ecological reality than do traditional ecoregion maps, especially in our current era of rapid and unpredictable ecological change.     

Occurrence and settlement of the common shipworm Teredo navalis (Bivalvia: Teredinidae) in Bremerhaven harbours, northern Germany

The shipworm Teredo navalis L. is a xylophagous bivalve mollusc (Bivalvia: Teredinidae) with a long record of being very destructive to wooden ships and harbour buildings. It has been reported from numerous sites at the coasts of both the North and Baltic Seas since the eighteenth century. Here, we document for the first time the occurrence of live adult T. navalis in the harbours of Bremerhaven (Weser estuary, northern Germany). From August to December 1998, various wooden structures (fir floating fenders and pier posts, oak piles) from seven stations in different docks of two harbours (überseehafen, Fischereihafen) were investigated for the presence and density of live specimens and burrows of T. navalis. The settlement of larval shipworms was studied by exposing experimental fir panels 0.06 m² in size at 20 stations at water depths between 1 and 2 m for periods of 4 months between July and November. In addition, hydrographic profiles (0–8 m water depth) were obtained at 17 stations in five docks once every month from August to December. Live adult shipworms were found in both fir floating fenders and oak piles at four stations. The largest specimen found was 250 mm long. Shipworm burrows were detected at five stations in almost every wooden structure investigated but their abundances differed significantly: Maximum values were >10,000 m^–2 in fir floating fenders, 4,600 m^–2 in oak piles and 200 m^–2 in fir pier posts. Actual shipworm infestation was detected at three of 16 stations in the exposed fir panels (1–3 burrow holes per panel). Water temperatures and salinities varied considerably during the 4-month investigation period. Temperatures decreased from 19.9°C in August to 0.7°C in December. Salinities ranged from 17.6 in August to 1.1 in November, but only at two lock stations during November and December did value drop below 5, which is regarded as the lethal limit for the larvae of this euryhaline teredinid species. We conclude that T. navalis encounters favourable conditions for growth and reproduction in the harbours of Bremerhaven, at least during summer and autumn, and is a common element of the harbour ecosystem. Therefore, a persistent infestation of all wooden structures after a relatively short period of time seems to be highly probable. Electronic Publication     

Desmoglein 2 Compensates for Desmoglein 3 but Does Not Control Cell Adhesion via Regulation of p38 Mitogen-activated Protein Kinase in Keratinocytes

Desmosomal cadherins are transmembrane adhesion molecules that provide cell adhesion by interacting in the intercellular space of adjacent cells. In keratinocytes, several desmoglein (Dsg1–4) and desmocollin (Dsc1–3) isoforms are coexpressed. We have shown previously that Dsg2 is less important for keratinocyte cohesion compared with Dsg3 and that the latter forms a complex with p38 MAPK. In this study, we compared the involvement of Dsg2 and Dsg3 in the p38 MAPK-dependent regulation of keratinocyte cohesion. We show that loss of cell adhesion and keratin filament retraction induced by Dsg3 depletion is ameliorated by specific p38 MAPK inhibition. Furthermore, in contrast to depletion of Dsg2, siRNA-mediated silencing of Dsg3 induced p38 MAPK activation, which is in line with immunoprecipitation experiments demonstrating the interaction of activated p38 MAPK with Dsg3 but not with Dsg2. Cell fractionation into a cytoskeleton-unbound and a cytoskeleton-anchored desmosome-containing pool revealed that Dsg3, in contrast to Dsg2, is present in relevant amounts in the unbound pool in which activated p38 MAPK is predominantly detectable. Moreover, because loss of cell adhesion by Dsg3 depletion was partially rescued by p38 MAPK inhibition, we conclude that, besides its function as an adhesion molecule, Dsg3 is strengthening cell cohesion via modulation of p38 MAPK-dependent keratin filament reorganization. Nevertheless, because subsequent targeting of Dsg3 in Dsg2-depleted cells led to drastically enhanced keratinocyte dissociation and Dsg2 was enhanced at the membrane in Dsg3 knockout cells, we conclude that Dsg2 compensates for Dsg3 loss of function.