Tet1 Isoforms Differentially Regulate Gene Expression, Synaptic Transmission, and Memory in the Mammalian Brain.

The dynamic regulation of DNA methylation in postmitotic neurons is necessary for memory formation and other adaptive behaviors. Ten-eleven translocation 1 (TET1) plays a part in these processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating active DNA demethylation. However, attempts to pinpoint its exact role in the nervous system have been hindered by contradictory findings, perhaps due in part, to a recent discovery that two isoforms of the Tet1 gene are differentially expressed from early development into adulthood. Here, we demonstrate that both the shorter transcript (Tet1S ) encoding an N-terminally truncated TET1 protein and a full-length Tet1 (Tet1FL ) transcript encoding canonical TET1 are co-expressed in the adult mouse brain. We show that Tet1S is the predominantly expressed isoform and is highly enriched in neurons, whereas Tet1FL is generally expressed at lower levels and more abundant in glia, suggesting their roles are at least partially cell type-specific. Using viral-mediated, isoform and neuron-specific molecular tools, we find that the individual repression of each transcript leads to the dysregulation of unique gene ensembles and contrasting changes in basal synaptic transmission. In addition, Tet1S repression enhances, while Tet1FL impairs, hippocampal-dependent memory in male mice. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the mammalian brain.SIGNIFICANCE STATEMENT In the brain, activity-dependent changes in gene expression are required for the formation of long-term memories. DNA methylation plays an essential role in orchestrating these learning-induced transcriptional programs by influencing chromatin accessibility and transcription factor binding. Once thought of as a stable epigenetic mark, DNA methylation is now known to be impermanent and dynamically regulated, driving neuroplasticity in the brain. We found that Tet1, a member of the ten-eleven translocation (TET) family of enzymes that mediates removal of DNA methyl marks, is expressed as two separate isoforms in the adult mouse brain and that each differentially regulates gene expression, synaptic transmission and memory formation. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the CNS.

Comparison of on-line detectors for field flow fractionation analysis of nanomaterials.

Characterization of nanomaterials must include analysis of both size and chemical composition. Many analytical techniques, such as dynamic light scattering (DLS), are capable of measuring the size of suspended nanometer-sized particles, yet provide no information on the composition of the particle. While field flow fractionation (FFF) is a powerful nanoparticle sizing technique, common detectors used in conjunction with the size separation, including UV, light-scattering, and fluorescence spectroscopy, do not provide the needed particle compositional information. Further, these detectors do not respond directly to the mass concentration of nanoparticles. The present work describes the advantages achieved when interfacing sensitive and elemental specific detectors, such as inductively coupled plasma atomic emission spectroscopy and mass spectrometry, to FFF separation analysis to provide high resolution nanoparticle sizing and compositional analysis at the µg/L concentration level, a detection at least 10-100-fold lower than DLS or FFF-UV techniques. The full benefits are only achieved by utilization of all detector capabilities, such as dynamic reaction cell (DRC) ICP-MS. Such low-level detection and characterization capability is critical to nanomaterial investigations at biologically and environmentally relevant concentrations. The techniques have been modified and applied to characterization of all four elemental constituents of cadmium selenide-zinc sulfide core-shell quantum dots, and silver nanoparticles with gold seed cores. Additionally, sulfide coatings on silver nanoparticles can be detected as a potential means to determine environmental aging of nanoparticles.

Mechanical properties and structure of the biological multilayered material system, Atractosteus spatula scales.

During recent decades, research on biological systems such as abalone shell and fish armor has revealed that these biological systems employ carefully arranged hierarchical multilayered structures to achieve properties of high strength, high ductility and light weight. Knowledge of such structures may enable pathways to design bio-inspired materials for various applications. This study was conducted to investigate the spatial distribution of structure, chemical composition and mechanical properties in mineralized fish scales of the species Atractosteus spatula. Microindentation tests were conducted, and cracking patterns and damage sites in the scales were examined to investigate the underlying protective mechanisms of fish scales under impact and penetration loads. A difference in nanomechanical properties was observed, with a thinner, stiffer and harder outer layer (indentation modulus ∼69 GPa and hardness ∼3.3 GPa) on a more compliant and thicker inner layer (indentation modulus ∼14.3 GPa and hardness ∼0.5 GPa). High-resolution scanning electron microscopy imaging of a fracture surface revealed that the outer layer contained oriented nanorods embedded in a matrix, and that the nanostructure of the inner layer contained fiber-like structures organized in a complex layered pattern. Damage patterns formed during microindentation show complex deformation mechanisms. Images of cracks identify growth through the outer layer, then deflection along the interface before growing and arresting in the inner layer. High-magnification images of the crack tip in the inner layer show void-linking and fiber-bridging exhibiting inelastic behavior. The observed difference in mechanical properties and unique nanostructures of different layers may have contributed to the resistance of fish scales to failure by impact and penetration loading.

Characterization of silver nanoparticles using flow-field flow fractionation interfaced to inductively coupled plasma mass spectrometry.

The ability to detect and identify the physiochemical form of contaminants in the environment is important for degradation, fate and transport, and toxicity studies. This is particularly true of nanomaterials that exist as discrete particles rather than dissolved or sorbed contaminant molecules in the environment. Nanoparticles will tend to agglomerate or dissolve, based on solution chemistry, which will drastically affect their environmental properties. The current study investigates the use of field flow fractionation (FFF) interfaced to inductively coupled plasma-mass spectrometry (ICP-MS) as a sensitive and selective method for detection and characterization of silver nanoparticles. Transmission electron microscopy (TEM) is used to verify the morphology and primary particle size and size distribution of precisely engineered silver nanoparticles. Subsequently, the hydrodynamic size measurements by FFF are compared to dynamic light scattering (DLS) to verify the accuracy of the size determination. Additionally, the sensitivity of the ICP-MS detector is demonstrated by fractionation of µg/L concentrations of mixed silver nanoparticle standards. The technique has been applied to nanoparticle suspensions prior to use in toxicity studies, and post-exposure biological tissue analysis. Silver nanoparticles extracted from tissues of the sediment-dwelling, freshwater oligochaete Lumbriculus variegatus increased in size from approximately 31-46nm, indicating a significant change in the nanoparticle characteristics during exposure.

Geochemical investigations of metals release from submerged coal fly ash using extended elutriate tests.

A storage pond dike failure occurred at the Tennessee Valley Authority Kingston Fossil Plant that resulted in the release of over 3.8 million cubic meters (5 million cubic yards) of fly ash. Approximately half of this material deposited in the main channel of the Emory River, 3.5 km upstream of the confluence of the Emory and Clinch Rivers, Tennessee, USA. Remediation efforts to date have focused on targeted removal of material from the channel through hydraulic dredging, as well as mechanical excavation in some areas. The agitation of the submerged fly ash during hydraulic dredging introduces river water into the fly ash material, which could alter the redox state of metals present in the fly ash and thereby change their sorption and mobility properties. A series of extended elutriate tests were used to determine the concentration and speciation of metals released from fly ash. Results indicated that arsenic and selenium species released from the fly ash materials during elutriate preparation were redox stable over the course of 10d, with dissolved arsenic being present as arsenate, and dissolved selenium being present as selenite. Concentrations of certain metals, such as arsenic, selenium, vanadium, and barium, increased in the elutriate waters over the 10d study, whereas manganese concentrations decreased, likely due to oxidation and precipitation reactions.

Evaluation of ionic contribution to the toxicity of a coal-mine effluent using Ceriodaphnia dubia.

The United States Environmental Protection Agency has defined national in-stream water-quality criteria (WQC) for 157 pollutants. No WQC to protect aquatic life exist for total dissolved solids (TDS). Some water-treatment processes (e.g., pH modifications) discharge wastewaters of potentially adverse TDS into freshwater systems. Strong correlations between specific conductivity, a TDS surrogate, and several biotic indices in a previous study suggested that TDS caused by a coal-mine effluent was the primary stressor. Further acute and chronic testing in the current study with Ceriodaphnia dubia in laboratory-manipulated media indicated that the majority of the effluent toxicity could be attributed to the most abundant ions in the discharge, sodium (1952 mg/L) and/or sulfate (3672 mg/L), although the hardness of the effluent (792 +/- 43 mg/L as CaCO3) ameliorated some toxicity. Based on laboratory testing of several effluent-mimicking media, sodium- and sulfate-dominated TDS was acutely toxic at approximately 7000 microS/cm (5143 mg TDS/L), and chronic toxicity occurred at approximately 3200 microS/cm (2331 mg TDS/L). At a lower hardness (88 mg/L as CaCO3), acute and chronic toxicity end-points were decreased to approximately 5000 microS/cm (3663 mg TDS/L) and approximately 2000 microS/cm (1443 mg TDS/L), respectively. Point-source discharges causing in-stream TDS concentrations to exceed these levels may risk impairment to aquatic life.

Evaluation of ecologically relevant bioassays for a lotic system impacted by a coal-mine effluent, using Isonychia.

Many studies investigating the ecotoxicological impacts of industrial effluents on fresh-water biota utilize standardized test species such as the daphnids, Ceriodaphnia dubia, Daphnia magna, and the fathead minnow, Pimephales promelas. Such species may not be the most predictive or ecologically relevant gauges of the responses of instream benthic macroinvertebrates to certain stressors, such as total dissolved solids. An indigenous species approach should be adopted, using a sensitive benthic collector-filterer following development of practical laboratory bioassays. In the Leading Creek Watershed (southeast Ohio), an aggregated approximately 99% reduction in mean mayfly abundance for all impacted sites was observed below a coal-mine effluent with mean specific conductivity (SC) of 8,109 (7,750-8,750) microS cm(-1). The mayfly, Isonychia, was exposed for 7-days to a simulation of this effluent, in lotic microcosms. Based on lowest observable adverse effect concentrations, Isonychia survival was a more sensitive endpoint to SC (1,562 microS cm(-1)) than were 7-day C. dubia survival and fecundity (3,730 microS cm(-1)). Isonychia molting, a potentially more sensitive endpoint, was also examined. Using traditional test species to assess discharges to surface water alone may not adequately protect benthic macroinvertebrate assemblages in systems impaired by discharges high in SC.

Field and laboratory assessment of a coal processing effluent in the Leading Creek Watershed, Meigs County, Ohio.

The U.S. Environmental Protection Agency has not recommended water quality criteria (WQC) to protect aquatic life from elevated sodium and sulfate concentrations, such as those associated with the coal-processing effluent of Meigs County Mine #31. This discharge, received by a tributary of the Leading Creek Watershed (SE Ohio), had a mean specific conductivity (SC) of 8,109 (7,750-8,750) microS/cm and total metal concentrations below acute WQC. The mean 48-h LC(50) for Ceriodaphnia dubia in the effluent was 6,713 +/- 99 microS/cm; mean 48-h survival was 44% for study sites downstream of the effluent. The best indicators of impairment used in this study were Ceriodaphnia fecundity, in situ Corbicula fluminea growth, EPT minus Hydropsychidae (richness and relative abundance), and relative Ephemeroptera abundance. Mayflies, reduced by more than 99% below the effluent, were absent from all but the furthest downstream study site. SC was strongly correlated with Corbicula growth (r = -0.9755, p = 0.0009) and EPT minus Hydropsychidae richness (r = -0.8756, p < 0.0001), suggesting the effluent was primarily responsible for biotic impairment. Our results indicated that SC levels, a measure of dissolved solids, in the Leading Creek Watershed that exceeded approximately 3,700 microS/cm impaired sensitive aquatic fauna.

Neurotrophin-3 is increased in skin in human diabetic neuropathy.

Neurotrophin-3 (NT-3), a member of the neurotrophin family, has been shown to be necessary for the development of muscle spindle and Merkel cell afferent nerve fibres in animal models. The presence of NT-3 in the suprabasal epidermis, where many unmyelinated sensory fibres terminate, has been shown for the first time. As these fibres are affected in early diabetic neuropathy and a clinical trial of recombinant human NT-3 in diabetic neuropathy is in progress, the concentrations of endogenous NT-3 in skin of 24 patients at different stages of diabetic polyneuropathy have been investigated. NT-3 concentrations, measured with a specific immunoassay, were significantly higher in affected skin biopsies from patients with diabetic neuropathy than matched control skin (diabetic skin 6.32 (1.18) pg/mg v control skin 1.28 (0.05) (mean (SEM)); p<0.004, Mann-Whitney U test), particularly in the later stages. The optical density of NT-3-immunostaining was also significantly greater in the epidermis in diabetic patients (diabetic epidermis 0.30 (0.06) v controls 0.24 (0.01); p<0.02). No correlation was found between individual quantitative sensory tests and the increase of NT-3 concentration. The increase of NT-3 seems to reflect the degree of skin denervation in diabetic neuropathy, and may represent a compensatory mechanism. The concentrations of NT-3 in other peripheral targets deserve study in diabetic neuropathy.

GDNF and its receptor component Ret in injured human nerves and dorsal root ganglia.

Glial cell line-derived neurotrophic factor (GDNF) is trophic to motor and sensory neurones in animal models. GDNF mRNA is up-regulated in Schwann cells after peripheral nerve injury in rats. We have quantified and localized GDNF and its receptor component Ret, for the first time in any species, in injured human peripheral nerves and dorsal root ganglia (DRG) avulsed from the spinal cord. Significantly higher levels of GDNF were found in nerve distal to the site of the injury than in proximal or intact nerve, and in avulsed DRG than in post-mortem control DRG. GDNF immunostaining was seen in Schwann cells and in DRG neurones, especially of small and medium size, with significantly increased numbers of medium sized sensory neurones immunoreactive for GDNF after avulsion. Ret immunoreactivity was restricted to DRG neurones and axons, with no significant changes in numbers of positive DRG cells after injury. Our findings suggest that GDNF may play a role in injured human nerves and sensory ganglia, particularly in medium sized sensory neurones.

Nonexpression of CD15 by neoplastic glia: a barrier to metastasis?

Cluster of differentiation 15 (CD15) monoclonal antibodies recognise cell adhesion molecules on the surface of many cells including normal astrocytes and metastatic carcinoma cells. The CD15 epitope (fucosyl-N-acetyl-lactosamine), an adhesive oligosaccharide, functions as a ligand for the selectin family of membrane receptors. These include CD62, a cytokineinducible glycoprotein found in platelets and endothelial cells. CD15 is one of a series of putative adhesion molecules expressed in nervous tissue. Selectin-carbohydrate interactions have been implicated in the metastatic spread of cancer cells. We have immunostained a variety of cultured human brain tumours, three cell lines derived from experimental rat gliomas, two specimens of cultured human foetal astrocytes, two metastatic carcinoma cell lines and human umbilical vein endothelial cells (HUVEC) using two monoclonal antibodies which recognise CD15. While all of the animal glioma cells were positive for CD15, only two human glioma cell lines, derived from an anaplastic astrocytoma and a glioblastoma multiforme, respectively, displayed limited reactivity. Chromium radiolabel binding assays of CD15-positive and -negative cell lines including glioma and carcinoma-derived cells, using HUVEC as an attachment substrate, were carried out in the presence and absence of CD15 monoclonal antibody. The level of adhesion of neoplastic cells to HUVEC not only corresponded to CD 15 expression but application of anti-CD 15 monoclonal antibodies considerably reduced adhesion. We postulate that the absence of CD15 on human glioma cells may explain, to some extent, the general failure of intrinsic brain tumours to metastasis by precluding the adhesion of circulating neoplastic glia to 'target' organ endothelium.

d-Fenfluramine- and d-norfenfluramine-induced hypophagia: differential mechanisms and involvement of postsynaptic 5-HT receptors.

Severe depletion of 5-hydroxytryptamine (5-HT) by para-chlorophenylalanine (pCPA, 150 mg/kg per day x3) did not alter the hypophagic effect of d-fenfluramine (1-3 mg/kg i.p.) 1 h after food presentation in 24-h food-deprived rats, and moderately and comparably increased the hypophagic effects of its metabolite, d-norfenfluramine (0.35-1.0 mg/kg i.p.), and of the 5-HT1C receptor agonist, 1-(3-chlorophenyl)piperazine (mCPP; 1.5, 2.0 mg/kg i.p.). Chronic treatment with mCPP (2.5 mg/kg i.p. x 14) attenuated the hypophagia induced by d-norfenfluramine (1, 1.5 mg/kg) but not d-fenfluramine (1, 3 mg/kg). 1-(1-Naphthyl)piperazine (3, 8 mumol/kg s.c.), which has greater affinity for 5-HT1C than for 5-HT2 receptors, had no effect on the hypophagia induced by d-fenfluramine (1.25, 2.0 mg/kg), but 1.3 and 3 mumol/kg 1-(1-naphthyl)piperazine largely and comparably attenuated the substantial hypophagic effect of d-norfenfluramine (0.75 mg/kg). The essentially complete hypophagic action of d-norfenfluramine (1.25 mg/kg) was inhibited by 1-(1-naphthyl)piperazine with ID50 = 2.13 mumol/kg. Ketanserin, which binds more weakly than 1-(1-naphthyl)piperazine to 5-HT1C receptors and more strongly to 5-HT2 receptors, attenuated weaker but not stronger hypophagic effects of d-fenfluramine (1.25, 2.0 mg/kg) when given at high dosage (8, 16 mumol/kg s.c.). Ketanserin (16 mumol/kg) also weakly attenuated the hypophagia due to d-norfenfluramine (0.75 mg/kg), but not the essentially complete hypophagia due to d-norfenfluramine (1.25 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of housing, restraint and chronic treatments with mCPP and sertraline on behavioural responses to mCPP.

The effects of pretreatments on behavioural responses to activation of 5-HT1C receptors by m-chlorophenylpiperazine (mCPP) were investigated. The hypo locomotor and anxiogenic effects of mCPP (social interaction test) were influenced neither by previous housing (single versus grouped) nor by restraint (2 h, 24 h previously). In the absence of mCPP, 24 h group housing led to decreased social interaction and the restraint procedure led to significant decreases of feeding and locomotion. The hypophagic effect of mCPP was unaffected by previous restraint. However, chronic pretreatment with mCPP (2.5 mg/kg per day IP x 14) or with the antidepressant 5-HT reuptake inhibitor sertraline (5 mg/kg per day SC x 14) attenuated all three behaviours. The above findings are discussed with respect to published data on effects of pretreatments on responses to the activation of 5-HT1C receptors.

The effect of a peptide aldehyde reversible inhibitor of elastase on a human leucocyte elastase-induced model of emphysema in the hamster.

The peptide aldehyde Ro 31-3537, N alpha-(1-adamantanesulphonyl)-N epsilon-(4-carboxybenzoyl)-L-lysyl-L-alanyl-L- valinal, is a reversible competitive, hydrophilic, specific inhibitor of elastase. Its Ki against human leucocyte elastase is 6 x 10(-8) M. The effect of this compound has been studied on a model of emphysema in the hamster induced by multiple sequential intratracheal doses of human leucocyte elastase. Concomitant intratracheal dosing of 200 micrograms of inhibitor with the enzyme significantly reduces lung damage as measured by quasi-static lung compliance and by histological assessment of the emphysema.

A new class of inhibitors of human leucocyte elastase.

Studies of the inhibition of elastases at a molecular level have resulted in the identification of protected dipeptides which are reversible and highly specific inhibitors of human leucocyte elastase (HLE). These have been further developed by increasing their hydrophilicity and potency to give a new family of elastase inhibitors, typically N alpha-(1-adamantanesulphonyl)-N epsilon-(4-carboxybenzoyl)-L-lysyl-L-alanyl-L-valinal. These compounds are active in pharmacological models designed to detect compounds of potential therapeutic value in the treatment of emphysema.

Pharmacological and biochemical activities of tenoxicam (Ro 12-0068), a new non-steroidal anti-inflammatory drug.

Tenoxicam, a new non-steroidal anti-inflammatory drug has been compared with piroxicam and indomethacin in a range of pharmacological and biochemical inflammation test systems. In a chronic (17-day) adjuvant arthritis in the rat, tenoxicam and piroxicam were equally effective in reducing several indices of inflammation and were less ulcerogenic and better tolerated than indomethacin. The oxicams reduced the oedematous and cellular components of a carrageenan pleurisy at 4 hours while at 24 hours they increased exudate volume and selectively inhibited the accumulation of mononuclear cells. These agents also reduced the inflammatory component of a delayed hypersensitivity response to methylated bovine serum albumin in the mouse. The oxicams were about 100-fold less active than indomethacin as inhibitors of prostaglandin synthetase but all three compounds reduced about equally the release of prostaglandin E2 from phagocytosing rat PMN and interleukin 1-stimulated human rheumatoid synovial cells. The compounds had no effect on the release of superoxide anion, lysosomal enzymes or collagenase from cultured cells, neither did they inhibit isolated collagenase. Only indomethacin stabilized albumin against heat denaturation.

Assessment of drugs for activity in established type II collagen arthritis.

Arthritis was induced in a proportion of rats sensitized with type II collagen and Freund's incomplete adjuvant. Rats which developed arthritis had significantly higher antibody titres and significantly greater delayed hypersensitivity responses to type II collagen than rats which did not develop arthritis. Anti-inflammatory and anti-rheumatic drugs were evaluated against type II collagen arthritis. Dexamethasone reduced inflammatory swelling, reduced both the antibody titre and delayed hypersensitivity to type II collagen and exerted joint protection. Indomethacin reduced inflammatory swelling. Azathioprine, Clozic, Levamisole, D-Penicilamine and Sulphapyridine did not exert significant beneficial activity in this test.

An analysis of mineral uptake in apple rootstock seedlings.

Eight families from biparental crosses of apple rootstocks and 12 families from open pollinated Malus spp. were analysed in 2 years for N, P, K, Ca and Mg content of the foliage. Highly significant differences were found between the families for all elements. There were no significant differences between the means of the biparental group and the open pollinated group. Ca and K content were significantly more variable in the open pollinated families compared with the biparental families. It is suggested that this increased variation could prove useful in breeding for efficiency of mineral uptake by apple rootstocks.