Publications by authors named "Paul Sheppard"

44 Publications

Protein synthesis and actin polymerization in the rapid effects of 17β-estradiol on short-term social memory and dendritic spine dynamics in female mice.

Psychoneuroendocrinology 2021 Apr 15;128:105232. Epub 2021 Apr 15.

Department of Psychology and Neuroscience Program, University of Guelph, Guelph, ON, Canada. Electronic address:

Estrogens rapidly facilitate learning and memory, including social recognition - the ability of an animal to recognize another. In ovariectomized female mice, systemic or dorsal hippocampal administration of 17β-estradiol (E2) facilitates short-term social recognition memory within 40 min. Within the same timeframe, E2 increases dendritic spine density in CA1 dorsal hippocampal neurons of behavioural task-naïve mice and in hippocampal sections. Mechanisms underlying these effects remain unclear. Estrogens rapidly modulate actin cytoskeletal dynamics through actin polymerization and the translation of key synaptic proteins. We first determined doses of actin polymerization inhibitor latrunculin A (LAT) and protein synthesis inhibitor anisomycin (ANI) that would block short-term social recognition memory when infused into the dorsal hippocampus of ovariectomized female mice 15 min prior to testing. The highest doses that did not block social recognition prevented the facilitating effects of E2, whereas DNA transcription inhibitor, actinomycin D, could not block social recognition. As task performance may interfere with E2-facilitated increases in dendritic spine density, dendritic spine density and length were examined in task-performing and task-naïve mice. E2 increased dendritic spine density 15 but not 40 min following treatment, regardless of whether the animal had performed the social recognition task. This effect was blocked by LAT, but not ANI. Thus, both actin polymerization and protein synthesis are necessary for E2 to rapidly facilitate social recognition, whereas actin polymerization, but not protein synthesis, is required for the rapid increase in dendritic spine density brought on by E2.
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http://dx.doi.org/10.1016/j.psyneuen.2021.105232DOI Listing
April 2021

and Its Allergenic Ribotoxin I: Old Enemies but New Opportunities for Urine-Based Detection of Invasive Pulmonary Aspergillosis Using Lateral-Flow Technology.

J Fungi (Basel) 2020 Dec 31;7(1). Epub 2020 Dec 31.

ISCA Diagnostics Ltd., Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UK.

Invasive pulmonary aspergillosis (IPA) caused by is a life-threatening lung disease of immunocompromised patients. Diagnosis currently relies on non-specific chest CT, culture of the fungus from invasive lung biopsy, and detection of the cell wall carbohydrate galactomannan (GM) in serum or in BAL fluids recovered during invasive bronchoscopy. Urine provides an ideal bodily fluid for the non-invasive detection of pathogen biomarkers, with current urine-based immunodiagnostics for IPA focused on GM. Surrogate protein biomarkers might serve to improve disease detection. Here, we report the development of a monoclonal antibody (mAb), PD7, which is specific to and related species in the section , and which binds to its 18 kDa ribotoxin I. Using PD7, we show that the protein is secreted during hyphal development, and so represents an ideal candidate for detecting invasive growth. We have developed a lateral-flow device (-LFD) incorporating the mAb which has a limit of detection of ~15 ng I/mL urine. Preliminary evidence of the test's diagnostic potential is demonstrated with urine from a patient with acute lymphoid leukaemia with probable IPA. The -LFD therefore provides a potential novel opportunity for non-invasive urine-based detection of IPA caused by .
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http://dx.doi.org/10.3390/jof7010019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823411PMC
December 2020

Sex differences in cortisol and memory following acute social stress in amnestic mild cognitive impairment.

J Clin Exp Neuropsychol 2020 11 6;42(9):881-901. Epub 2020 Oct 6.

Djavad Mowafaghian Centre for Brain Health, Department of Psychology, University of British Columbia , Vancouver, BC, Canada.

Objective: Older adults with amnestic mild cognitive impairment (aMCI) develop Alzheimer's type dementia approximately 10 times faster annually than the normal population. Adrenal hormones are associated with aging and cognition. We investigated the relationship between acute stress, cortisol, and memory function in aMCI with an exploratory analysis of sex.

Method: Salivary cortisol was sampled diurnally and during two test sessions, one session with the Trier Social Stress Test (TSST), to explore differences in the relationship between cortisol and memory function in age-normal cognition (NA) and aMCI. Participants with aMCI (n = 6 women, 9 men; mean age = 75) or similarly aged NA (n = 9 women, 7 men, mean age = 75) were given tests of episodic, associative, and spatial working memory with a psychosocial stressor (TSST) in the second session.

Results: The aMCI group performed worse on the memory tests than NA as expected, and males with aMCI had elevated cortisol levels on test days. Immediate episodic memory was enhanced by social stress in NA but not in the aMCI group, indicating that stress-induced alterations in memory are different in individuals with aMCI. High cortisol was associated with impaired performance on episodic memory in aMCI males only. Cortisol in Session 1 moderated the relationship with spatial working memory, whereby higher cortisol was associated with worse performance in NA, but better spatial working memory in aMCI. In addition, effects of aMCI on perceived anxiety in response to stress exposure were moderated by stress-induced cortisol in a sex-specific manner.

Conclusions: We show effects of aMCI on Test Session cortisol levels and effects on perceived anxiety, and stress-induced impairments in memory in males with aMCI in our exploratory sample. Future studies should explore sex as a biological variable as our findings suggest that effects at the confluence of aMCI and stress can be obfuscated without sex as a consideration.
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http://dx.doi.org/10.1080/13803395.2020.1825633DOI Listing
November 2020

Origin of tungsten and geochemical controls on its occurrence and mobilization in shallow sediments from Fallon, Nevada, USA.

Chemosphere 2020 Dec 11;260:127577. Epub 2020 Jul 11.

Department of Geology, Kansas State University, Manhattan, KS, 66506, USA; Department of Geological Sciences, University of Texas at San Antonio, San Antonio, TX, 78249, USA. Electronic address:

Tungsten (W) occurrence and speciation was investigated in sediments collected from Fallon, Nevada where previous studies have linked elevated W levels in human body fluids to an unusual cluster of childhood leukemia cases. The speciation of sedimentary W was determined by μ-XRF mapping and μ-XANES. The W content of the analyzed surface sediments ranged between 81 and 25,908 mg/kg, which is significantly higher than the W content in deeper sediments which ranged from 37 to 373 mg/kg at 30 cm depth. The μ-XANES findings reveal that approximately 20-50% of the total W in the shallow sediment occurs in the metallic form (W); the rest occurs in the oxide form (WO). Because W does not occur naturally, its elevated concentrations in surface sediments point toward a possible local anthropogenic origin. The oxidation of metallic W with meteoric waters likely leads to the formation of WO. The chief water-soluble W species was identified as WO by chromatographic separation and speciation modeling. These results led us to postulate that W particles from a currently unknown but local source(s) is (are) deposited onto the soils and/or surface sediments. The W in interaction with meteoric water is oxidized to WO, and as these sediment-water interactions progress, WO is formed in the water at pH ∼7. Under pH < 7, and sufficient W concentrations, tungstate tends to polymerize, and polymerized species are less likely to adsorb onto sediments. Polymerized species have lower affinity than monomers, which leads to enhanced mobility of W.
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http://dx.doi.org/10.1016/j.chemosphere.2020.127577DOI Listing
December 2020

VISTA is an acidic pH-selective ligand for PSGL-1.

Nature 2019 10 23;574(7779):565-570. Epub 2019 Oct 23.

genOway, Lyon, France.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.
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http://dx.doi.org/10.1038/s41586-019-1674-5DOI Listing
October 2019

Multidecadal environmental pollution in a mega-industrial area in central Chile registered by tree rings.

Sci Total Environ 2019 Dec 16;696:133915. Epub 2019 Aug 16.

Laboratorio de Geo-Información y Percepción Remota, Instituto de Geografía, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile. Electronic address:

One of the most polluted areas in Chile is the Ventanas Industrial Area (VIA; 32.74°S / 71.48°W), which started in 1958 and today comprises around 16 industries in an area of ca. 4 km. A lack of consistent long-term instrumental records precludes assessing the history of contamination in the area and also limits the evaluation of mitigation actions taken since the late 1980s. Here, we use dendrochemistry as an environmental proxy to analyze environmental changes over several decades at the VIA. We present chemical measurements of tree rings from planted, exotic Cupressus macrocarpa growing near the VIA with 4-year resolution over a period of 52 years (1960-2011). These data provide unprecedented information on regional anthropogenic pollution and are compared with a tree-ring elemental record of 48 years (1964-2011) from the Isla Negra (INE) control site not exposed to VIA emissions. For the 48 years of overlap between both sites, higher concentrations of Zn, V, Co, Cd, Ag, Fe, Cr, and Al were especially registered after the year 2000 at VIA compared to INE for the periods under study. Concentrations of Pb, Cu, As, Fe, Mo, Cr, and Zn increased through time, particularly over the period 1980-1990. Decontamination plans activated in 1992 appear to have had a positive effect on the amount of some elements, but the chemical concentration in the tree rings suggest continued accumulation of pollutants in the environment. Only after several years of implementation of the mitigation measures have some elements tended to decrease in concentration, especially at the end of the evaluated period. Dendrochemistry is a useful tool to provide a long-term perspective of the dynamics of trace metal pollution and represents a powerful approach to monitor air quality variability to extend the instrumental records back in time.
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http://dx.doi.org/10.1016/j.scitotenv.2019.133915DOI Listing
December 2019

Structural plasticity of the hippocampus in response to estrogens in female rodents.

Mol Brain 2019 03 18;12(1):22. Epub 2019 Mar 18.

Department of Psychology, Graduate Program in Neuroscience, Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

It is well established that estrogens affect neuroplasticity in a number of brain regions. In particular, estrogens modulate and mediate spine and synapse formation as well as neurogenesis in the hippocampal formation. In this review, we discuss current research exploring the effects of estrogens on dendritic spine plasticity and neurogenesis with a focus on the modulating factors of sex, age, and pregnancy. Hormone levels, including those of estrogens, fluctuate widely across the lifespan from early life to puberty, through adulthood and into old age, as well as with pregnancy and parturition. Dendritic spine formation and modulation are altered both by rapid (likely non-genomic) and classical (genomic) actions of estrogens and have been suggested to play a role in the effects of estrogens on learning and memory. Neurogenesis in the hippocampus is influenced by age, the estrous cycle, pregnancy, and parity in female rodents. Furthermore, sex differences exist in hippocampal cellular and molecular responses to estrogens and are briefly discussed throughout. Understanding how structural plasticity in the hippocampus is affected by estrogens and how these effects can influence function and be influenced by other factors, such as experience and sex, is critical and can inform future treatments in conditions involving the hippocampus.
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http://dx.doi.org/10.1186/s13041-019-0442-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423800PMC
March 2019

Rapid effects of estrogens on short-term memory: Possible mechanisms.

Horm Behav 2018 08 1;104:88-99. Epub 2018 Jun 1.

Department of Psychology and Neuroscience Program, University of Guelph, Guelph, ON N1G 2W1, Canada. Electronic address:

Contribution to Special Issue on Fast effects of steroids. Estrogens affect learning and memory through rapid and delayed mechanisms. Here we review studies on rapid effects on short-term memory. Estradiol rapidly improves social and object recognition memory, spatial memory, and social learning when administered systemically. The dorsal hippocampus mediates estrogen rapid facilitation of object, social and spatial short-term memory. The medial amygdala mediates rapid facilitation of social recognition. The three estrogen receptors, α (ERα), β (ERβ) and the G-protein coupled estrogen receptor (GPER) appear to play different roles depending on the task and brain region. Both ERα and GPER agonists rapidly facilitate short-term social and object recognition and spatial memory when administered systemically or into the dorsal hippocampus and facilitate social recognition in the medial amygdala. Conversely, only GPER can facilitate social learning after systemic treatment and an ERβ agonist only rapidly improved short-term spatial memory when given systemically or into the hippocampus, but also facilitates social recognition in the medial amygdala. Investigations into the mechanisms behind estrogens' rapid effects on short term memory showed an involvement of the extracellular signal-regulated kinase (ERK) and the phosphoinositide 3-kinase (PI3K) kinase pathways. Recent evidence also showed that estrogens interact with the neuropeptide oxytocin in rapidly facilitating social recognition. Estrogens can increase the production and/or release of oxytocin and other neurotransmitters, such as dopamine and acetylcholine. Therefore, it is possible that estrogens' rapid effects on short-term memory may occur through the regulation of various neurotransmitters, although more research is need on these interactions as well as the mechanisms of estrogens' actions on short-term memory.
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http://dx.doi.org/10.1016/j.yhbeh.2018.05.019DOI Listing
August 2018

Estrogens and their receptors in the medial amygdala rapidly facilitate social recognition in female mice.

Psychoneuroendocrinology 2018 03 28;89:30-38. Epub 2017 Dec 28.

Department of Psychology and Neuroscience Program, University of Guelph, Guelph, ON, N1G 2W1, Canada. Electronic address:

Estrogens have been shown to rapidly (within 1 h) affect learning and memory processes, including social recognition. Both systemic and hippocampal administration of 17β-estradiol facilitate social recognition in female mice within 40 min of administration. These effects were likely mediated by estrogen receptor (ER) α and the G-protein coupled estrogen receptor (GPER), as administration of the respective receptor agonists (PPT and G-1) also facilitated social recognition on a rapid time scale. The medial amygdala has been shown to be necessary for social recognition and long-term manipulations in rats have implicated medial amygdalar ERα. As such, our objective was to investigate whether estrogens and different ERs within the medial amygdala play a role in the rapid facilitation of social recognition in female mice. 17β-estradiol, G-1, PPT, or ERβ agonist DPN was infused directly into the medial amygdala of ovariectomized female mice. Mice were then tested in a social recognition paradigm, which was completed within 40 min, thus allowing the assessment of rapid effects of treatments. 17β-estradiol (10, 25, 50, 100 nM), PPT (300 nM), DPN (150 nM), and G-1 (50 nM) each rapidly facilitated social recognition. Therefore, estrogens in the medial amygdala rapidly facilitate social recognition in female mice, and the three main estrogen receptors: ERα, ERβ, and the GPER all are involved in these effects. This research adds to a network of brain regions, including the medial amygdala and the dorsal hippocampus, that are involved in mediating the rapid estrogenic facilitation of social recognition in female mice.
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http://dx.doi.org/10.1016/j.psyneuen.2017.12.021DOI Listing
March 2018

Female ClockΔ19/Δ19 mice are protected from the development of age-dependent cardiomyopathy.

Cardiovasc Res 2018 02;114(2):259-271

Department of Biomedical Sciences/OVC, Centre for Cardiovascular Investigations, University of Guelph, Room 1646B, University of Guelph, Guelph, ON N1G2W1, Canada.

Aims: Circadian rhythms are important for healthy cardiovascular physiology and they are regulated by the molecular circadian mechanism. Previously, we showed that disruption of the circadian mechanism factor CLOCK in male ClockΔ19/Δ19 mice led to development of age-dependent cardiomyopathy. Here, we investigate the role of biological sex in protecting against heart disease in aging female ClockΔ19/Δ19 mice.

Methods And Results: Female ClockΔ19/Δ19 mice are protected from the development of cardiomyopathy with age, as heart structure and function are similar to 18 months of age vs. female WT mice. We show that female ClockΔ19/Δ19 mice maintain normal glucose tolerance as compared with female WT. Tissue metabolic profiling revealed that aging female ClockΔ19/Δ19 mice maintain normal cardiac glucose uptake, whereas the male ClockΔ19/Δ19 mice have increased cardiac glucose uptake consistent with pathological remodelling. Shotgun lipidomics revealed differences in phospholipids that were sex and genotype specific, including cardiolipin CL76:11 that was increased and CL72:8 that was decreased in male ClockΔ19/Δ19 mice. Additionally, female ClockΔ19/Δ19 mice show increased activation of AKT signalling and preserved cytochrome c oxidase activity compared with male ClockΔ19/Δ19 mice, which can help to explain why they are protected from heart disease. To determine how this protection occurs in females even with the Clock mutation, we examined the effects of ovarian hormones. We show that ovarian hormones protect female ClockΔ19/Δ19 mice from heart disease as ovariectomized female ClockΔ19/Δ19 mice develop cardiac dilation, glucose intolerance and reduced cardiac cytochrome c oxidase; this phenotype is consistent with the age-dependent decline observed in male ClockΔ19/Δ19 mice.

Conclusions: These data demonstrate that ovarian hormones protect female ClockΔ19/Δ19 mice from the development of age-dependent cardiomyopathy even though Clock function is disturbed. Understanding the interaction of biological sex and the circadian mechanism in cardiac growth, renewal and remodelling opens new doors for understanding and treating heart disease.
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http://dx.doi.org/10.1093/cvr/cvx185DOI Listing
February 2018

Mapping the Energetic Epitope of an Antibody/Interleukin-23 Interaction with Hydrogen/Deuterium Exchange, Fast Photochemical Oxidation of Proteins Mass Spectrometry, and Alanine Shave Mutagenesis.

Anal Chem 2017 02 9;89(4):2250-2258. Epub 2017 Feb 9.

Protein Science, Bristol-Myers Squibb , Rt. 206 & Province Line Rd., Princeton, New Jersey 08543, United States.

Epitope mapping the specific residues of an antibody/antigen interaction can be used to support mechanistic interpretation, antibody optimization, and epitope novelty assessment. Thus, there is a strong need for mapping methods, particularly integrative ones. Here, we report the identification of an energetic epitope by determining the interfacial hot-spot that dominates the binding affinity for an anti-interleukin-23 (anti-IL-23) antibody by using the complementary approaches of hydrogen/deuterium exchange mass spectrometry (HDX-MS), fast photochemical oxidation of proteins (FPOP), alanine shave mutagenesis, and binding analytics. Five peptide regions on IL-23 with reduced backbone amide solvent accessibility upon antibody binding were identified by HDX-MS, and five different peptides over the same three regions were identified by FPOP. In addition, FPOP analysis at the residue level reveals potentially key interacting residues. Mutants with 3-5 residues changed to alanine have no measurable differences from wild-type IL-23 except for binding of and signaling blockade by the 7B7 anti-IL-23 antibody. The M5 IL-23 mutant differs from wild-type by five alanine substitutions and represents the dominant energetic epitope of 7B7. M5 shows a dramatic decrease in binding to BMS-986010 (which contains the 7B7 Fab, where Fab is fragment antigen-binding region of an antibody), yet it maintains functional activity, binding to p40 and p19 specific reagents, and maintains biophysical properties similar to wild-type IL-23 (monomeric state, thermal stability, and secondary structural features).
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http://dx.doi.org/10.1021/acs.analchem.6b03058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347259PMC
February 2017

Poly-Ub-substrate-degradative activity of 26S proteasome is not impaired in the aging rat brain.

PLoS One 2013 7;8(5):e64042. Epub 2013 May 7.

Institut für Biochemie, Charité-Universitätsmedizin-Berlin, Berlin, Germany.

Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young) and 24 month old (aged) rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064042PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3646778PMC
December 2013

Comparison of size and geography of airborne tungsten particles in Fallon, Nevada, and Sweet Home, Oregon, with implications for public health.

J Environ Public Health 2012 14;2012:509458. Epub 2012 Mar 14.

Laboratory of Tree-Ring Research, University of Arizona, Tucson, AZ 85721, USA.

To improve understanding of possible connections between airborne tungsten and public health, size and geography of airborne tungsten particles collected in Fallon, Nevada, and Sweet Home, Oregon, were compared. Both towns have industrial tungsten facilities, but only Fallon has experienced a cluster of childhood leukemia. Fallon and Sweet Home are similar to one another by their particles of airborne tungsten being generally small in size. Meteorologically, much, if not most, of residential Fallon is downwind of its hard metal facility for at least some fraction of time at the annual scale, whereas little of residential Sweet Home is downwind of its tungsten facility. Geographically, most Fallon residents potentially spend time daily within an environment containing elevated levels of airborne tungsten. In contrast, few Sweet Home residents potentially spend time daily within an airborne environment with elevated levels of airborne tungsten. Although it cannot be concluded from environmental data alone that elevated airborne tungsten causes childhood leukemia, the lack of excessive cancer in Sweet Home cannot logically be used to dismiss the possibility of airborne tungsten as a factor in the cluster of childhood leukemia in Fallon. Detailed modeling of all variables affecting airborne loadings of heavy metals would be needed to legitimately compare human exposures to airborne tungsten in Fallon and Sweet Home.
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http://dx.doi.org/10.1155/2012/509458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317226PMC
January 2013

Additional analysis of dendrochemical data of Fallon, Nevada.

Chem Biol Interact 2012 Apr 27;196(3):96-101. Epub 2011 Dec 27.

Laboratory of Tree-Ring Research, University of Arizona, Tucson, Arizona 85721, USA.

Previously reported dendrochemical data showed temporal variability in concentration of tungsten (W) and cobalt (Co) in tree rings of Fallon, Nevada, US. Criticism of this work questioned the use of the Mann-Whitney test for determining change in element concentrations. Here, we demonstrate that Mann-Whitney is appropriate for comparing background element concentrations to possibly elevated concentrations in environmental media. Given that Mann-Whitney tests for differences in shapes of distributions, inter-tree variability (e.g., "coefficient of median variation") was calculated for each measured element across trees within subsites and time periods. For W and Co, the metals of highest interest in Fallon, inter-tree variability was always higher within versus outside of Fallon. For calibration purposes, this entire analysis was repeated at a different town, Sweet Home, Oregon, which has a known tungsten-powder facility, and inter-tree variability of W in tree rings confirmed the establishment date of that facility. Mann-Whitney testing of simulated data also confirmed its appropriateness for analysis of data affected by point-source contamination. This research adds important new dimensions to dendrochemistry of point-source contamination by adding analysis of inter-tree variability to analysis of central tendency. Fallon remains distinctive by a temporal increase in W beginning by the mid 1990s and by elevated Co since at least the early 1990s, as well as by high inter-tree variability for W and Co relative to comparison towns.
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http://dx.doi.org/10.1016/j.cbi.2011.12.009DOI Listing
April 2012

Tungsten toxicity.

Chem Biol Interact 2012 Apr 13;196(3):87-8. Epub 2011 Dec 13.

Odyssey Research Institute, 7032 East Rosewood Street, Tucson, AZ 85710-1236, United States.

There is emerging evidence that tungsten has toxic health effects. We summarize the recent tungsten toxicity research in this short review. Tungsten is widely used in many commercial and military applications because it has the second highest melting temperature of any element. Consequently, it is important to elucidate the potential health effects of tungsten.
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http://dx.doi.org/10.1016/j.cbi.2011.12.002DOI Listing
April 2012

Exposure to sodium tungstate and Respiratory Syncytial Virus results in hematological/immunological disease in C57BL/6J mice.

Chem Biol Interact 2012 Apr 1;196(3):89-95. Epub 2011 May 1.

Steele Children's Research Center, PO Box 245073, University of Arizona, Tucson, AZ 85724-5073, USA.

The etiology of childhood leukemia is not known. Strong evidence indicates that precursor B-cell Acute Lymphoblastic Leukemia (Pre-B ALL) is a genetic disease originating in utero. Environmental exposures in two concurrent, childhood leukemia clusters have been profiled and compared with geographically similar control communities. The unique exposures, shared in common by the leukemia clusters, have been modeled in C57BL/6 mice utilizing prenatal exposures. This previous investigation has suggested in utero exposure to sodium tungstate (Na2WO4) may result in hematological/immunological disease through genes associated with viral defense. The working hypothesis is (1) in addition to spontaneously and/or chemically generated genetic lesions forming pre-leukemic clones, in utero exposure to Na2WO4 increases genetic susceptibility to viral influence(s); (2) postnatal exposure to a virus possessing the 1FXXKXFXXA/V9 peptide motif will cause an unnatural immune response encouraging proliferation in the B-cell precursor compartment. This study reports the results of exposing C57BL/6J mice to Na2WO4 in utero via water (15 ppm, ad libetum) and inhalation (mean concentration PM5 3.33 mg/m3) and to Respiratory Syncytial Virus (RSV) within 2 weeks of weaning. Inoculation of C57BL/6J mice with RSV was associated with a neutrophil shift in 56% of 5-month old mice. When the RSV inoculation was combined with Na2WO4-exposure, significant splenomegaly resulted (p=0.0406, 0.0184, 0.0108 for control, Na2WO4-only and RSV-only, respectively) in addition to other hematological pathologies which were not significant. Exposure to Na2WO4 and RSV resulted in hematological/immunological disease, the nature of which is currently inconclusive. Further research is needed to characterize this potential leukemia mouse model.
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http://dx.doi.org/10.1016/j.cbi.2011.04.008DOI Listing
April 2012

Strategies for evaluating the environment-public health interaction of long-term latency disease: the quandary of the inconclusive case-control study.

Chem Biol Interact 2012 Apr 4;196(3):68-78. Epub 2011 Mar 4.

Methods Development and Applications Branch, HEASD/NERL/ORD (D205-05), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA.

Environmental links to disease are difficult to uncover because environmental exposures are variable in time and space, contaminants occur in complex mixtures, and many diseases have a long time delay between exposure and onset. Furthermore, individuals in a population have different activity patterns (e.g., hobbies, jobs, and interests), and different genetic susceptibilities to disease. As such, there are many potential confounding factors to obscure the reasons that one individual gets sick and another remains healthy. An important method for deducing environmental associations with disease outbreak is the retrospective case-control study wherein the affected and control subject cohorts are studied to see what is different about their previous exposure history. Despite success with infectious diseases (e.g., food poisoning, and flu), case-control studies of cancer clusters rarely have an unambiguous outcome. This is attributed to the complexity of disease progression and the long-term latency between exposure and disease onset. In this article, we consider strategies for investigating cancer clusters and make some observations for improving statistical power through broader non-parametric approaches wherein sub-populations (i.e., whole towns), rather than individuals, are treated as the cases and controls, and the associated cancer rates are treated as the dependent variable. We subsequently present some ecological data for tungsten and cobalt from studies by University of Arizona researchers who document elevated levels of tungsten and cobalt in Fallon, NV. These results serve as candidates for future hybrid ecologic case-control investigations of childhood leukemia clusters.
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http://dx.doi.org/10.1016/j.cbi.2011.02.020DOI Listing
April 2012

Novel cell- and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies.

Cell Biochem Biophys 2011 Jul;60(3):173-85

Enzo Life Sciences, 10 Executive Blvd, Farmingdale, NY 11735, USA.

Aggresomes and related inclusion bodies appear to serve as storage depots for misfolded and aggregated proteins within cells, which can potentially be degraded by the autophagy pathway. A homogenous fluorescence-based assay was devised to detect aggregated proteins inside aggresomes and inclusion bodies within an authentic cellular context. The assay employs a novel red fluorescent molecular rotor dye, which is essentially nonfluorescent until it binds to structural features associated with the aggregated protein cargo. Aggresomes and related structures were generated within cultured cells using various potent, cell permeable, proteasome inhibitors: MG-132, lactacystin, epoxomicin and bortezomib, and then selectively detected with the fluorescent probe. Employing the probe in combination with various fluorescein-labeled primary antibodies facilitated co-localization of key components of the autophagy system (ubiquitin, p62, and LC3) with aggregated protein cargo by fluorescence microscopy. Furthermore, cytoplasmic aggregates were highlighted in SK-N-SH human neuroblastoma cells incubated with exogenously supplied amyloid beta peptide 1-42. SMER28, a small molecule modulator of autophagy acting via an mTOR-independent mechanism, prevented the accumulation of amyloid beta peptide within these cells. The described assay allows assessment of the effects of protein aggregation directly in cells, without resorting to the use of non-physiological protein mutations or genetically engineered cell lines. With minor modification, the assay was also adapted to the analysis of frozen or formalin-fixed, paraffin-embedded tissue sections, with demonstration of co-localization of aggregated cargo with β-amyloid and tau proteins in brain tissue sections from Alzheimer's disease patients.
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http://dx.doi.org/10.1007/s12013-010-9138-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3112480PMC
July 2011

USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis.

Nat Commun 2010 May 4;1:13. Epub 2010 May 4.

Biotechnology Institute Thurgau, University of Konstanz, Kreuzlingen CH-8280, Switzerland.

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specific E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro, and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the first E2 enzyme but also the first known substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans.
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http://dx.doi.org/10.1038/ncomms1012DOI Listing
May 2010

Assembly, structure, and function of the 26S proteasome.

Trends Cell Biol 2010 Jul 26;20(7):391-401. Epub 2010 Apr 26.

School of Biomedical Sciences, University of Nottingham Medical School, Nottingham, UK.

The 26S proteasome is a large multiprotein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation, and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease.
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http://dx.doi.org/10.1016/j.tcb.2010.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902798PMC
July 2010

Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration.

Neurosci Lett 2009 Sep 7;460(3):205-8. Epub 2009 Jun 7.

School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.

The major human neurodegenerative diseases are characterised by ubiquitin-positive intraneuronal inclusions, however the precise nature of the ubiquitin modifications in these structures is unclear. Using a monoclonal antibody specific for Lys63-linked polyubiquitin we have performed the first immunohistochemical analysis of linkage-specific ubiquitination in vivo associated with neurodegeneration. Immunoreactivity was detected within the pathological lesions of Alzheimer's, Huntington's and Parkinson's disease brains, although staining of Lewy bodies in the substantia nigra in Parkinson's disease was rare, indicating a selective involvement of Lys63-linked polyubiquitin in inclusion biogenesis in this disorder. Immunoreactivity was also a feature in neurons of proteasome-depleted mice, suggesting a proteasomal contribution to the degradation of Lys63-linked polyubiquitinated proteins in vivo.
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http://dx.doi.org/10.1016/j.neulet.2009.05.074DOI Listing
September 2009

The role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family.

PLoS One 2009 20;4(3):e4933. Epub 2009 Mar 20.

Bioinformatics Department, ZymoGenetics, Inc, Seattle, Washington, United States of America.

Background: Type-I interferons, type-II interferons, and the IL-10 family are helical cytokines with similar three-dimensional folds. However, their homologous relationship is difficult to detect on the basis of sequence alone. We have previously described the discovery of the human type-III interferons (IFN lambda-1, -2, -3 or IL-29, IL-28A, IL-28B), which required a combination of manual and computational techniques applied to predicted protein sequences.

Principal Findings: Here we describe how the use of gene structure analysis and comparative genomics enabled a more extensive understanding of these genes early in the discovery process. More recently, additional mammalian genome sequences have shown that there are between one and potentially nine copies of interferon lambda genes in each genome, and that several species have single exon versions of the interferon lambda gene.

Significance: The variable number of single exon type-I interferons in mammals, along with recently identified genes in zebrafish homologous to interferons allows a story of interferon evolution to be proposed. This model suggests that the gene duplications and single exon retrotransposons of mammalian type-III interferons are positively selected for within a genome. These characteristics are also shared with the fish interferons and could be responsible for the generation of the IL10 family and also the single exon type-I interferons.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004933PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654155PMC
April 2009

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin.

Proc Natl Acad Sci U S A 2008 Dec 17;105(51):20197-202. Epub 2008 Dec 17.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.

Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.
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http://dx.doi.org/10.1073/pnas.0810461105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629300PMC
December 2008

Characterization of a non-UBA domain missense mutation of sequestosome 1 (SQSTM1) in Paget's disease of bone.

J Bone Miner Res 2009 Apr;24(4):632-42

School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom.

Mutations affecting the ubiquitin-associated (UBA) domain of sequestosome 1 (SQSTM1/p62) are commonly found in Paget's disease of bone (PDB) and impair SQSTM1's ability to bind ubiquitin, resulting in dysregulated NF-kappaB signaling. In contrast, non-UBA domain mutations are rarer, and little is known about how they manifest their effects. We present the first characterization at the molecular, cellular, and functional level of a non-UBA domain missense mutation (A381V) of SQSTM1. Direct sequencing of exon 7 of the SQSTM1 gene in an Italian PDB patient detected a heterozygous C to T transversion at position 1182, resulting in an alanine to valine substitution at codon 381. Pull-down assays showed the non-UBA region of SQSTM1 that contains A381 is important in mediating ubiquitin-binding affinity and that the A381V mutation exerts weak negative effects on ubiquitin binding. Structural and binding analyses of longer UBA constructs containing A381, using NMR spectroscopy and circular dichroism, showed this region of the protein to be largely unstructured and confirmed its contribution to increased ubiquitin-binding affinity. Co-transfections of U20S cells showed that the A381V mutant SQSTM1 co-localized with ubiquitin with a cellular phenotype indistinguishable from wildtype. Finally, effects of the wildtype and mutant SQSTM1 on NF-kappaB signaling were assessed in HEK293 cells co-transfected with an NF-kappaB luciferase reporter construct. A381V mutant SQSTM1 produced a level of activation of NF-kappaB signaling greater than wildtype and similar to that of UBA domain mutants, indicating that non-UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF-kappaB signaling.
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http://dx.doi.org/10.1359/jbmr.081204DOI Listing
April 2009

The cell-penetrating peptide octa-arginine is a potent inhibitor of proteasome activities.

Eur J Pharm Biopharm 2009 May 7;72(1):219-25. Epub 2008 Nov 7.

Institut für Biochemie/CCM, Charité-Universitätsmedizin-Berlin, Berlin, Germany.

Oligo-arginines are cell-penetrating peptides and find use as carriers for transportation of various membrane-impermeable biopharmaceuticals into target cells. We have found that oligo-arginines of a length of 4-10 amino acids, but especially (Arg)(8), are able to inhibit the major intracellular proteolytic system, the proteasome, with mixed-type inhibition characteristics. The IC(50) values of (Arg)(8) for the proteasomal chymotrypsin-like and caspase-like activities are approximately 100 and 200 nM, respectively. The inhibition of the trypsin-like activity never exceeds 50% even at micromolar concentrations. (Arg)(8) also inhibits 20S proteasome/PA28 complexes as well as 26S proteasomes, although with a decreased efficiency. Due to its cell membrane-penetrating capability, incubation of HeLa cells in the presence of (Arg)(8) resulted in an impaired activity of proteasomes going along with an accumulation of high-molecular mass ubiquitin-conjugated proteins, the preferred substrates of 26S proteasomes. The in vivo susceptibility of the three proteasome activities resembles that found in vitro with chymotrypsin-like>caspase-like>trypsin-like activities. Since inhibition of the proteasome system might affect fundamental basic cellular processes but on the other side might also prevent the degradation of a proteinacous cargo, we suggest that this proteasome inhibitory activity should be taken into account when oligo-arginines are being considered for use as vectors for the intracellular delivery of pharmaceuticals.
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http://dx.doi.org/10.1016/j.ejpb.2008.10.016DOI Listing
May 2009

Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies.

J Neurosci 2008 Aug;28(33):8189-98

School of Biomedical Sciences and School of Molecular Medical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.
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http://dx.doi.org/10.1523/JNEUROSCI.2218-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670564PMC
August 2008

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases.

J Biol Chem 2008 Jun 8;283(26):18177-86. Epub 2008 May 8.

The MRC Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK.

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.
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http://dx.doi.org/10.1074/jbc.M709715200DOI Listing
June 2008

Proteasome subunit proteins and neuropathology in tauopathies and synucleinopathies: Consequences for proteomic analyses.

Proteomics 2008 Mar;8(6):1221-36

Faculté des Sciences Biologiques, Université des Sciences et des Technologies Houari Boumediène, Algiers, Algeria.

Accumulation of proteins in inclusions in neurological disorders is partly due to dysfunction of the ubiquitin-proteasome system. Proteasomal dysfunction may be caused by misexpression of one or more of its subunits. A large number of antibodies reactive with proteasome subunits were screened on material from patients exhibiting tau- and synucleinopathies. Many antisera against proteasomal subunits (11S activator, 19S regulator ATPase/non-ATPase, and 20S alpha and beta resulted in a distinct nuclear and/or cytoplasmic staining of the entorhinal-hippocampal area and the temporal cortex of Alzheimer's disease (AD) patients. In particular an antibody directed against 19S regulator ATPase subunit 6b (S6b) specifically stained the neurofibrillary tangles and dystrophic neurites in AD, Down syndrome and aged nondemented controls. In other tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy and argyrophilic grain disease), neuronal and/or glial inclusions were also S6b immunoreactive. In contrast, in synucleinopathies (Lewy body disease (LBD) and multiple system atrophy) no S6b staining was seen. Real time quantitative PCR on the temporal cortex of AD patients revealed a significant increase in S6b subunit mRNA. This increase was not found in the gyrus cinguli anterior of patients with LBD. This differential expression of S6b most likely will result in different proteomic patterns. Here we present evidence to show that S6b coexists with a reporter for proteasomal dysfunction (ubiquitin(+1)), and we conclude that S6b transcript up-regulation and the dysfunction in tauopathies may be functionally related.
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http://dx.doi.org/10.1002/pmic.200700679DOI Listing
March 2008

Ubiquitin recognition by the ubiquitin-associated domain of p62 involves a novel conformational switch.

J Biol Chem 2008 Feb 14;283(9):5427-40. Epub 2007 Dec 14.

School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom.

The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-kappaB and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-kappaB signaling axis can result in human skeletal disorders, including those identified in the C-terminal ubiquitin-associated (UBA) domain of p62 in patients with Paget disease of bone. These observations suggest that the disease may involve a common mechanism related to alterations in the ubiquitin-binding properties of p62. The structural basis for ubiquitin recognition by the UBA domain of p62 has been investigated using NMR and reveals a novel binding mechanism involving a slow exchange structural reorganization of the UBA domain to a "bound" non-canonical UBA conformation that is not significantly populated in the absence of ubiquitin. The repacking of the three-helix bundle generates a binding surface localized around the conserved Xaa-Gly-Phe-Xaa loop that appears to optimize both hydrophobic and electrostatic surface complementarity with ubiquitin. NMR titration analysis shows that the p62-UBA binds to Lys 48-linked di-ubiquitin with approximately 4-fold lower affinity than to mono-ubiquitin, suggesting preferential binding of the p62-UBA to single ubiquitin units, consistent with the apparent in vivo preference of the p62 protein for Lys 63-linked polyubiquitin chains (which adopt a more open and extended structure). The conformational switch observed on binding may represent a novel mechanism that underlies specificity in regulating signalinduced protein recognition events.
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http://dx.doi.org/10.1074/jbc.M704973200DOI Listing
February 2008

Morphological and chemical characteristics of airborne tungsten particles of Fallon, Nevada.

Microsc Microanal 2007 Aug;13(4):296-303

Laboratory of Tree-Ring Research, University of Arizona, Tucson, Arizona 85721, USA.

Morphological and chemical characteristics were determined for airborne tungsten particles in Fallon, Nevada, a town that is distinguishable environmentally by elevated airborne tungsten and cobalt. From samples of airborne dust collected previously at six different places in Fallon, tungsten-rich dust particles were isolated and analyzed with automated electron microprobe and wavelength-dispersive spectrometry. Representative W particles were further analyzed using transmission electron microscopy. Morphologically, Fallon W particles are angular and small, with minimum and maximum sizes of < or = 1 microm and 5.9 microm in diameter, respectively. The number and size of tungsten-rich particles decrease in Fallon with distance from a hard-metal facility located near the center of town. Chemically, Fallon airborne W particles include mixtures of tungsten with cobalt plus other metals such as chromium, iron, and copper. No W-rich particles were identifiable as CaWO4 (scheelite) or MnWO4 (huebnerite). From d-spacings, Fallon particles are most consistent with identification as tungsten carbide. Based on these multiple lines of evidence, airborne W particles in Fallon are anthropogenic in origin, not natural. The hard-metal facility in Fallon processes finely powdered W and W-Co, and further investigation using tracer particles is recommended to definitively identify the source of Fallon's airborne tungsten.
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http://dx.doi.org/10.1017/S1431927607070237DOI Listing
August 2007