Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

HMGN1 and 2 remodel core and linker histone tail domains within chromatin.

The structure of the nucleosome, the basic building block of the chromatin fiber, plays a key role in epigenetic regulatory processes that affect DNA-dependent processes in the context of chromatin. Members of the HMGN family of proteins bind specifically to nucleosomes and affect chromatin structure and function, including transcription and DNA repair. To better understand the mechanisms by which HMGN 1 and 2 alter chromatin, we analyzed their effect on the organization of histone tails and linker histone H1 in nucleosomes. We find that HMGNs counteract linker histone (H1)-dependent stabilization of higher order 'tertiary' chromatin structures but do not alter the intrinsic ability of nucleosome arrays to undergo salt-induced compaction and self-association. Surprisingly, HMGNs do not displace H1s from nucleosomes; rather these proteins bind nucleosomes simultaneously with H1s without disturbing specific contacts between the H1 globular domain and nucleosomal DNA. However, HMGNs do alter the nucleosome-dependent condensation of the linker histone C-terminal domain, which is critical for stabilizing higher-order chromatin structures. Moreover, HMGNs affect the interactions of the core histone tail domains with nucleosomal DNA, redirecting the tails to more interior positions within the nucleosome. Our studies provide new insights into the molecular mechanisms whereby HMGNs affect chromatin structure.

RANAVIRUS EPIZOOTIC IN CAPTIVE EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) WITH CONCURRENT HERPESVIRUS AND MYCOPLASMA INFECTION: MANAGEMENT AND MONITORING.

Frog virus 3 (FV3) and FV3-like viruses are members of the genus Ranavirus (family Iridoviridae) and are becoming recognized as significant pathogens of eastern box turtles (Terrapene carolina carolina) in North America. In July 2011, 5 turtles from a group of 27 in Maryland, USA, presented dead or lethargic with what was later diagnosed as fibrinonecrotic stomatitis and cloacitis. The presence of FV3-like virus and herpesvirus was detected by polymerase chain reaction (PCR) in the tested index cases. The remaining 22 animals were isolated, segregated by severity of clinical signs, and treated with nutritional support, fluid therapy, ambient temperature management, antibiotics, and antiviral therapy. Oral swabs were tested serially for FV3-like virus by quantitative real-time PCR (qPCR) and tested at day 0 for herpesvirus and Mycoplasma sp. by conventional PCR. With oral swabs, 77% of the 22 turtles were FV3-like virus positive; however, qPCR on tissues taken during necropsy revealed the true prevalence was 86%. FV3-like virus prevalence and the median number of viral copies being shed significantly declined during the outbreak. The prevalence of herpesvirus and Mycoplasma sp. by PCR of oral swabs at day 0 was 55% and 68%, respectively. The 58% survival rate was higher than previously reported in captive eastern box turtles for a ranavirus epizootic. All surviving turtles brumated normally and emerged the following year with no clinical signs during subsequent monitoring. The immediate initiation of treatment and intensive supportive care were considered the most important contributing factors to the successful outcome in this outbreak.

A distinct switch in interactions of the histone H4 tail domain upon salt-dependent folding of nucleosome arrays.

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes. Likewise, H4 tail-H2A contacts stabilize array folding. However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.

Intra- and inter-nucleosome interactions of the core histone tail domains in higher-order chromatin structure.

Eukaryotic chromatin is a hierarchical collection of nucleoprotein structures that package DNA to form chromosomes. The initial levels of packaging include folding of long strings of nucleosomes into secondary structures and array-array association into higher-order tertiary chromatin structures. The core histone tail domains are required for the assembly of higher-order structures and mediate short- and long-range intra- and inter-nucleosome interactions with both DNA and protein targets to direct their assembly. However, important details of these interactions remain unclear and are a subject of much interest and recent investigations. Here, we review work defining the interactions of the histone N-terminal tails with DNA and protein targets relevant to chromatin higher-order structures, with a specific emphasis on the contributions of H3 and H4 tails to oligonucleosome folding and stabilization. We evaluate both classic and recent experiments determining tail structures, effect of tail cleavage/loss, and posttranslational modifications of the tails on nucleosomes and nucleosome arrays, as well as inter-nucleosomal and inter-array interactions of the H3 and H4 N-terminal tails.

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4.

We used a novel single-cell strategy to examine the fate of histones during G(2)-phase. Consistent with previous results, we find that in G(2)-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.

EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

Atorvastatin prevents age-related and amyloid-ß-induced microglial activation by blocking interferon-γ release from natural killer cells in the brain.

BACKGROUND: Microglial function is modulated by several factors reflecting the numerous receptors expressed on the cell surface, however endogenous factors which contribute to the age-related increase in microglial activation remain largely unknown. One possible factor which may contribute is interferon-γ (IFNγ). IFNγ has been shown to increase in the aged brain and potently activates microglia, although its endogenous cell source in the brain remains unidentified. METHODS: Male Wistar rats were used to assess the effect of age and amyloid-ß (Aß) on NK cell infiltration into the brain. The effect of the anti-inflammatory compound, atorvastatin was also assessed under these conditions. We measured cytokine and chemokine (IFNγ, IL-2, monocyte chemoattractant protein-1 (MCP-1) and IFNγ-induced protein 10 kDa (IP-10)), expression in the brain by appropriate methods. We also looked at NK cell markers, CD161, NKp30 and NKp46 using flow cytometry and western blot. RESULTS: Natural killer (NK) cells are a major source of IFNγ in the periphery and here we report the presence of CD161+ NKp30+ cells and expression of CD161 and NKp46 in the brain of aged and Aß-treated rats. Furthermore, we demonstrate that isolated CD161+ cells respond to interleukin-2 (IL-2) by releasing IFNγ. Atorvastatin, the HMG-CoA reductase inhibitor, attenuates the increase in CD161 and NKp46 observed in hippocampus of aged and Aß-treated rats. This was paralleled by a decrease in IFNγ, markers of microglial activation and the chemokines, MCP-1 and IP-10 which are chemotactic for NK cells. CONCLUSIONS: We propose that NK cells contribute to the age-related and Aß-induced neuroinflammatory changes and demonstrate that these changes can be modulated by atorvastatin treatment.

Influence of oviposition-inducing hormone on spawning and mortality in the endangered Panamanian golden frog (Atelopus zeteki).

BACKGROUND: With Panamanian golden frogs (Atelopus zeteki; PGFs) likely extirpated from the wild, ensuring long-term sustainability of captive populations is crucial in order to conserve this critically endangered species. Unfortunately, PGFs display a unique reproductive behavior involving a prolonged period of amplexus leading to challenges in their successful captive propagation. The Maryland Zoo in Baltimore has observed high levels of mortality during the breeding season and suboptimal reproductive success leading to the use of hormone stimulation to aid in reproduction and health management. METHODS: This project aimed to develop induced ovulation and health management protocols by (1) evaluating different doses of gonadotropin releasing hormone analogue (GnRHa), (2) comparing the efficacy of GnRHa and GnRHa + metoclopramide, (3) determining latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Female PGFs (n = 174) were given GnRHa either in various concentrations (Experiment 1) or combined with metoclopramide (Experiment 2), and oviposition success, latency, and mortality were measured as binary response variables. RESULTS: Overall, the use of exogenous hormones significantly decreased mortality when compared to the control data of natural egg-laying females. GnRHa doses of 0.05 µg/g body weight produced similar ovulation rates compared to higher doses, and the addition of metoclopramide did not increase oviposition success compared to GnRHa alone. Lastly, results indicate the majority of female PGFs will release eggs within 48 h following the initial pulse of hormones with a small percentage ovipositing after a second pulse. CONCLUSION: Findings from this study will benefit captive management of PGFs by documenting the increased survival of females when given hormone stimulation and defining appropriate GnRHa doses and expected latency to spawning.

Photon-counting computed tomography of lanthanide contrast agents with a high-flux 330-µm-pitch cadmium zinc telluride detector in a table-top system.

Purpose: We present photon-counting computed tomography (PCCT) imaging of contrast agent triplets similar in atomic number ( Z ) achieved with a high-flux cadmium zinc telluride (CZT) detector. Approach: The table-top PCCT imaging system included a 330 - µ m -pitch CZT detector of size 8 mm × 24 mm 2 capable of using six energy bins. Four 3D-printed 3-cm-diameter phantoms each contained seven 6-mm-diameter vials with water and low and high concentration solutions of various contrast agents. Lanthanum ( Z = 57 ), gadolinium (Gd) ( Z = 64 ), and lutetium ( Z = 71 ) were imaged together and so were iodine ( Z = 53 ), Gd, and holmium ( Z = 67 ). Each phantom was imaged with 1-mm aluminum-filtered 120-kVp cone beam x rays to produce six energy-binned computed tomography (CT) images. Results: K -edge images were reconstructed using a weighted sum of six CT images, which distinguished each contrast agent with a root-mean-square error (RMSE) of < 0.29 % and 0.51% for the 0.5% and 5% concentrations, respectively. Minimal cross-contamination in each K -edge image was seen, with RMSE values < 0.27 % in vials with no contrast. Conclusion: This is the first preliminary demonstration of simultaneously imaging three similar Z contrast agents with a difference in Z as low as 3.

No evidence in support of a prodromal respiratory control signature in the TgF344-AD rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative condition disturbing major brain networks, including those pivotal to the motor control of breathing. The aim of this study was to examine respiratory control in the TgF344-AD transgenic rat model of AD. At 8-11 months of age, basal minute ventilation and ventilatory responsiveness to chemostimulation were equivalent in conscious wild-type (WT) and TgF344-AD rats. Under urethane anesthesia, basal diaphragm and genioglossus EMG activities were similar in WT and TgF344-AD rats. The duration of phenylbiguanide-induced apnoea was significantly shorter in TgF344-AD rats compared with WT. Following bilateral cervical vagotomy, diaphragm and genioglossus EMG responsiveness to chemostimulation were intact in TgF344-AD rats. Amyloid precursor protein C-terminal fragments were elevated in the TgF344-AD brainstem, in the absence of amyloid-ß accumulation or alterations in tau phosphorylation. Brainstem pro-inflammatory cytokine concentrations were not increased in TgF344-AD rats. We conclude that neural control of breathing is preserved in TgF344-AD rats at this stage of the disease.

Structural studies of heparan sulfate hexasaccharides: new insights into iduronate conformational behavior.

There is a growing opinion that the conformational dynamics within HS chains is critical to their observed biological activities. Investigations into HS conformational dynamics are problematic, given the structural complexity and heterogeneity of HS chains. However, this goal will be more obtainable once we understand the important roles HS sequence/sulfation patterns play in determining the conformational dynamics of iduronate units. This is the first study to compare isomers of N-sulfated oligosaccharides, with respect to the conformational versatility of their internal iduronates. Characterization by NMR spectroscopy of two HS oligosaccharides derived from porcine mucosal HS enabled the measurement of iduronate coupling constants, while under the influence of different flanking saccharide sequences. By fitting our coupling constant data to a new set of theoretical coupling constants, calculated using explicit water molecular dynamic simulations, we are able to offer new insights into the role sequence/sulfation patterns play in influencing iduronate conformational behavior. Fitting of experimental data, using our new theoretically derived coupling constants, suggests that replacement of the N-sulfate group to the reducing side of IdoUA by an N-acetyl group has little effect on the balance of IdoUA conformational equilibrium. Fitting of coupling constants for sequences GlcNS-IdoUA(2S)-GlcNS and GlcNS(6S)-IdoUA(2S)-GlcNS suggests that the flanking 6-O-sulfate group alters the balance of the IdoUA(2S) equilibrium more toward the (2)S0 conformation. There is also the suggestion that a cooperative effect may exist for N- and 6-O sulfation. These observations could be the key to understanding the important regulatory function attributed to 6-O-sulfation within HS chains.

Differential phosphorylation of Smad1 integrates BMP and neurotrophin pathways through Erk/Dusp in axon development.

Sensory axon development requires concerted actions of growth factors for the precise control of axonal outgrowth and target innervation. How developing sensory neurons integrate different cues is poorly understood. We demonstrate here that Smad1 activation is required for neurotrophin-mediated sensory axon growth in vitro and in vivo. Through differential phosphorylation, Smad1 exerts transcriptional selectivity to regulate the expression and activity of Erk1 and Erk2-two key neurotrophin effectors. Specifically, bone morphogenetic proteins (BMPs) signal through carboxy-terminal phosphorylation of Smad1 (pSmad1C) to induce Erk1/2 transcription for enhanced neurotrophin responsiveness. Meanwhile, neurotrophin signaling results in linker phosphorylation of Smad1 (pSmad1L), which in turn upregulates an Erk-specific dual-specificity phosphatase, Dusp6, leading to reduced pErk1/2 and constituting a negative-feedback loop for the prevention of axon overgrowth. Together, the BMP and neurotrophin pathways form a tightly regulated signaling network with a balanced ratio of Erk1/2 and pErk1/2 to direct the precise connections between sensory neurons and peripheral targets.

The age- and amyloid-ß-related increases in Nogo B contribute to microglial activation.

The family of reticulons include three isoforms of the Nogo protein, Nogo A, Nogo B and Nogo C. Nogo A is expressed on neuronal tissue and its primary effect is widely acknowledged to be inhibition of neurite outgrowth. Although both Nogo B and Nogo C are also expressed in neuronal tissue, their roles in the CNS remain to be identified. In this study, we set out to assess whether expression of Nogo A or Nogo B was altered in tissue prepared from aged rats in which increased microglial activation is accompanied by decreased synaptic plasticity. The data indicate that Nogo B, but not Nogo A, was markedly increased in hippocampal tissue prepared from aged rats and that, at least in vitro, Nogo B increased several markers of microglial activation. In a striking parallel with the age-related changes, we demonstrate that intracerebroventricular delivery of amyloid-ß (Aß)(1-40)+Aß(1-42) for 8 days was associated with a depression of long-term potentiation (LTP) and an increase in markers of microglial activation and Nogo B. In both models, evidence of cell stress was identified by increased activity of caspases 8 and 3 and importantly, incubation of cultured neurons in the presence of Nogo B increased activity of both enzymes. The data identify, for the first time, an effect of Nogo B in the brain and specifically show that its expression is increased in conditions where synaptic plasticity is compromised.

The impact of glial activation in the aging brain.

The past decade or so has witnessed a rekindling of interest in glia requiring a re-evaluation of the early descriptions of astrocytes as merely support cells, and microglia as adopting either a resting state or an activated state in a binary fashion. We now know that both cell types contribute to the optimal functioning of neurons in the healthy brain, and that altered function of either cell impacts on neuronal function and consequently cognitive function. The evidence indicates that both astrocytic and microglial phenotype change with age and that the shift from the resting state is associated with deterioration in synaptic function. In this review, we consider the rapidly-expanding array of functions attributed to these cells and focus on evaluating the changes in cell activation that accompany ageing.

Evidence that heparin saccharides promote FGF2 mitogenesis through two distinct mechanisms.

Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.

A new model for the domain structure of heparan sulfate based on the novel specificity of K5 lyase.

Elucidation of the molecular structure of heparan sulfate (HS) is the key to understanding its functional versatility as a co-receptor for growth factors and morphogens. We have identified and exploited the novel substrate specificity of the coliphage K5 lyase in studies of the domain organization of HS. We show that K5 lyase cleaves HS principally within non-sulfated sequences of four or more N-acetylated disaccharides. Uniquely, sections comprising alternating N-acetylated and N-sulfated units are resistant to the enzyme, as are the highly sulfated S domains. Spacing of the K5 lyase cleavage sites ( approximately 7-8 kDa) is similar to that of the S domains. On the basis of these findings, we propose a refined model of the structure of HS in which N-acetylated sequences of four to five disaccharide units (GlcNAc-GlcUA)(4-5) are positioned centrally between the S domains. The latter are embedded within N-acetylated and N-sulfated sequences, forming extended regions of hypervariable sulfation distributed at regular intervals along the polymer chain. K5 lyase provides a means of excision of these composite sulfated regions for structural and functional analyses.