Showing papers by "David A. Pearce published in 2010"

The structure, biological activity and biogeochemistry of cryoconite aggregates upon an Arctic valley glacier: Longyearbreen, Svalbard

Abstract: Glacier surfaces support unique microbial food webs dominated by organic and inorganic debris called ‘cryoconite’. Observations from Longyearbreen, Spitsbergen, show how these aggregate particles can develop an internal structure following the cementation of mineral grains (mostly quartz and dolomite) by filamentous microorganisms. Measurements of carbon and dissolved O2 show that these microorganisms, mostly cyanobacteria, promote significant rates of photosynthesis (average 17 μgC g−1 d−1) which assist aggregate growth by increasing the biomass and producing glue-like extracellular polymeric substances. The primary production takes place not only upon the surface of the aggregates but also just beneath, due to the translucence of the quartz particles. However, since total photosynthesis is matched by respiration (average 19 μgC g−1 d−1), primary production does not contribute directly to cryoconite accumulation upon the glacier surface. The microorganisms therefore influence the surface albedo most by cementing dark particles and organic debris together, rather than simply growing over it. Time-lapse photographs show that cryoconite is likely to reside upon the glacier for years as a result of this aggregation. These observations therefore show that a better understanding of the relationship between supraglacial debris and ablation upon glaciers requires an appreciation of the biological processes that take place during summer.

mTOR Complex-2 Activates ENaC by Phosphorylating SGK1

Abstract: The serum- and glucocorticoid-induced kinase 1 (SGK1) plays a central role in hormone regulation of epithelial sodium (Na+) channel (ENaC)-dependent Na+ transport in the distal nephron. Phosphorylation within a carboxy-terminal domain, designated the hydrophobic motif (HM), determines the activity of SGK1, but the identity of the HM kinase is unknown. Here, we show that the highly conserved serine-threonine kinase mammalian target of rapamycin (mTOR) is essential for the phosphorylation of the HM of SGK1 and the activation of ENaC. We observed that mTOR, in conjunction with rictor (mTORC2), phosphorylated SGK1 and stimulated ENaC. In contrast, when mTOR assembled with raptor in the rapamycin-inhibited complex (mTORC1), it did not phosphorylate SGK1 or stimulate ENaC. Inhibition of mTOR blocked both SGK1 phosphorylation and ENaC-mediated Na+ transport, whereas specific inhibition of mTORC1 had no effect. Similarly, small hairpin RNA–mediated knockdown of rictor inhibited SGK1 phosphorylation and Na+ current, whereas knockdown of raptor had no effect. Finally, in co-immunoprecipitation experiments, SGK1 interacted selectively with rictor but not with raptor, suggesting selective recruitment of SGK1 to mTORC2. We conclude that mTOR, specifically mTORC2, is the HM kinase for SGK1 and is required for ENaC-mediated Na+ transport, thereby extending our understanding of the molecular mechanisms underlying Na+ balance.

High levels of spatial heterogeneity in the biodiversity of soil prokaryotes on Signy Island, Antarctica

Abstract: In a previous study, soil bacterial diversity at environmentally distinct locations on Signy Island was examined using denaturing gradient gel electrophoresis (DGGE) profiling, and a range of chemical variables in soils was determined in order to describe variations between them. The dominant bacterial communities of all locations were found to be significantly different, although higher levels of similarity were observed between locations with similar physico-chemical characteristics, such as at penguin rookeries, seal wallows and vegetated soils. Extending this study, here soil prokaryote biodiversity was compared between 15 distinct locations in order to elucidate any interaction between four general habitat types on Signy Island (South Orkney Islands, maritime Antarctic) and any influence of previous human impacts at these sites. Specific sites were selected to represent the range of different soil environments present and to cover a range of environmental factors present in the maritime Antarctic which are known to influence bacterial community composition in soils elsewhere. A diverse prokaryote community is described, again with the majority of excised and sequenced bands belonging to the Bacteroidetes. Although DGGE profiling identified significant differences in prokaryotic biodiversity between all sampling sites, aggregations of banding patterns were also apparent across the different soil environments examined. Correlations between specific DGGE profiles and 10 selected soil parameters suggested that much of this variation could be explained by differences in the levels of environmental disturbance and soil pH. In particular, a greater proportion of variation in soil bacterial diversity was explained by differences in soil properties at human-disturbed locations than at undisturbed locations, with higher explanatory values by edaphic factors in the former and soil metal content in the later. In general, our data indicate that small-scale variation is an important factor in understanding patterns of prokaryotic distributions in soil habitats in the maritime Antarctic environment.

Biodiversity of air-borne microorganisms at Halley station, Antarctica

Abstract: A study of air-borne microbial biodiversity over an isolated scientific research station on an ice-shelf in continental Antarctica was undertaken to establish the potential source of microbial colonists. The study aimed to assess: (1) whether microorganisms were likely to have a local (research station) or distant (marine or terrestrial) origin, (2) the effect of changes in sea ice extent on microbial biodiversity and (3) the potential human impact on the environment. Air samples were taken above Halley Research Station during the austral summer and austral winter over a 2-week period. Overall, a low microbial biodiversity was detected, which included many sequence replicates. No significant patterns were detected in the aerial biodiversity between the austral summer and the austral winter. In common with other environmental studies, particularly in the polar regions, many of the sequences obtained were from as yet uncultivated organisms. Very few marine sequences were detected irrespective of the distance to open water, and around one-third of sequences detected were similar to those identified in human studies, though both of these might reflect prevailing wind conditions. The detected aerial microorganisms were markedly different from those obtained in earlier studies over the Antarctic Peninsula in the maritime Antarctic.

Expression and role of serum and glucocorticoid-regulated kinase 2 in the regulation of Na+/H+ exchanger 3 in the mammalian kidney

Abstract: Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium (Na+) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with Na+/H+ exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgk1, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgk1, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured Na+/H+ exchange activity by Na+-dependent cell pH recovery. Constitutively active sgk2, but not sgk1, stimulated Na+/H+ exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgk1 and that sgk2 may play a previously unrecognized role in the control of transtubular Na+ transport through NHE3 in the proximal tubule.

Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for Batten disease

Abstract: Juvenile Batten disease is an autosomal recessive pediatric neurodegenerative disorder caused by mutations in the CLN3 gene. The CLN3 protein primarily resides in the lysosomal membrane, but its function is unknown. We demonstrate that CLN3 interacts with SBDS, the protein mutated in Shwachman―Bodian― Diamond syndrome patients. We demonstrate that this protein―protein interaction is conserved between Btn1 p and Sdo1 p, the respective yeast Saccharomyces cerevisiae orthologs of CLN3 and SBDS. It was previously shown that deletion of BTN1 results in alterations in vacuolar pH and vacuolar (H + )-ATPase (V-ATPase)-dependent H + transport and ATP hydrolysis. Here, we report that an SDO1 deletion strain has decreased vacuolar pH and V-ATPase-dependent H + transport and ATP hydrolysis. These alterations result from decreased V-ATPase subunit expression. Overexpression of BTN1 or the presence of ionophore carbonyl cyanide m-chlorophenil hydrazone (CCCP) causes decreased growth in yeast lacking SDO1. In fact, in normal cells, overexpression of BTN1 mirrors the effect of CCCP, with both resulting in increased vacuolar pH due to alterations in the coupling of V-ATPase-dependent H + transport and ATP hydrolysis. Thus, we propose that Sdo1p and SBDS work to regulate Btn1 p and CLN3, respectively. This report highlights a novel mechanism for controlling vacuole/lysosome homeostasis by the ribosome maturation pathway that may contribute to the cellular abnormalities associated with juvenile Batten disease and Shwachman―Bodian― Diamond syndrome.

Genotype does not predict severity of behavioural phenotype in juvenile neuronal ceroid lipofuscinosis (Batten disease)

Abstract: Aim The primary aim of this investigation was to examine genotype and clinical phenotype differences in individuals with juvenile neuronal ceroid lipofuscinosis (JNCL) who were homozygous for a common disease-causing deletion or compound heterozygous. The secondary aim was to cross-validate the Child Behavior Checklist (CBCL) and the Unified Batten Disease Rating Scale (UBDRS), a disease-specific JNCL rating scale. Method Sixty individuals (28 males, 32 females; mean age 15y 1mo, SD 4y 9mo, range 5y 8mo–31y 1mo) with JNCL completed the UBDRS. Results No significant genotype and clinical phenotype differences were identified when comparing individuals homozygous for the deletion with a heterogeneous group of compound heterozygous individuals. There were significant correlations among related behaviour items and scales on the CBCL and UBDRS (Spearman’s rho ranging from 0.39 [p<0.05] to 0.72 [p<0.01]). Behaviour and physical function ratings were uncorrelated, supporting divergent validity of these two constructs in JNCL. Interpretation Previous reports of genotype and clinical phenotype differences were unsupported in this investigation, which did not find differences between individuals homozygous or heterozygous for the CLN3 deletion. The CBCL, an already validated measure of behaviour problems, appears valid for use in JNCL and cross-validates well with the UBDRS.

Akt2/PKBbeta-sensitive regulation of renal phosphate transport

Abstract: AIM: The protein kinase B (PKB)/Akt is known to stimulate the cellular uptake of glucose and amino acids. The kinase is expressed in proximal renal tubules. The present study explored the influence of Akt/PKB on renal tubular phosphate transport. METHODS: The renal phosphate transporter NaPi-IIa was expressed in Xenopus oocytes with or without PKB/Akt and Na(+) phosphate cotransport determined using dual electrode voltage clamp. Renal phosphate excretion was determined in Akt2/PKBbeta knockout mice (akt2(-/-)) and corresponding wild-type mice (akt2(+/+)). Transporter protein abundance was determined using Western blotting and phosphate transport by (32)P uptake into brush border membrane vesicles. RESULTS: The phosphate-induced current in NaPi-IIa-expressing Xenopus oocytes was significantly increased by the coexpression of Akt/PKB. Phosphate excretion [micromol per 24 h per g BW] was higher by 91% in akt2(-/-) than in akt2(+/+) mice. The phosphaturia of akt2(-/-) mice occurred despite normal transport activity and expression of the renal phosphate transporters NaPi-IIa, NaPi-IIc and Pit2 in the brush border membrane, a significantly decreased plasma PTH concentration (by 46%) and a significantly enhanced plasma 1,25-dihydroxyvitamin D(3) concentration (by 46%). Moreover, fractional renal Ca(2+) excretion was significantly enhanced (by 53%) and bone density significantly reduced (by 11%) in akt2(-/-) mice. CONCLUSIONS: Akt2/PKBbeta plays a role in the acute regulation of renal phosphate transport and thus contributes to the maintenance of phosphate balance and adequate mineralization of bone.

Altered sensitivity to excitotoxic cell death and glutamate receptor expression between two commonly studied mouse strains.

Abstract: Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders, including ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, and Huntington's disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to N-methyl-D-aspartate (NMDA)-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent on the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity likely are due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/S(v)E(v) mouse backgrounds.

Regulation of renal tubular glucose reabsorption by Akt2/PKBβ

Abstract: Akt/PKB is known to regulate the facilitative glucose carrier GLUT4. Nothing is known, however, of the role of Akt/PKB in the regulation of renal epithelial transport. To explore whether Akt2/PKBβ ...

Regulation of gastric acid secretion by PKB/Akt2.

Abstract: Pharmacological inhibition of phosphoinositol 3 kinase (PI3K) and partial deficiency of phosphoinositide dependent kinase PDK1 have previously been shown to enhance basal gastric acid secretion. PI3K/PDK1 dependent signaling involves activation of protein kinase B/Akt, which may thus be similarly involved in the regulation of gastric acid secretion. To test that hypothesis, gastric acid secretion was determined in isolated glands from gene targeted mice lacking functional Akt2 (akt2(-/-)) or from their wild type littermates (akt2(+/+)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in akt2(-/-) and akt2(+/+) mice. Na(+)-independent pH recovery (DeltapH/min) following an ammonium pulse, a measure of H(+)/K(+) ATPase activity, was, however, significantly faster in akt2(-/-) than in akt2(+/+) mice. In both genotypes, DeltapH/min was virtually abolished by H(+)/K(+) ATPase inhibitor omeprazole (100 muM). Increase of extracellular K(+) concentrations to 35 mM (replacing Na(+)) increased DeltapH/min to a significantly larger extent in akt2(+/+) than in akt2(-/-) mice and dissipated the differences between the genotypes. Similarly, treatment with 5 muM forskolin enhanced DeltapH/min significantly only in akt2(+/+) mice and abolished the differences between the genotypes. Conversely, protein kinase A inhibitor H89 (50 nM) decreased DeltapH/min to similarly low values in both genotypes. In conclusion, Akt2 suppresses gastric acid secretion and contributes to or even accounts for the inhibition of gastric acid secretion by PI3K.

High frequency measurements of dissolved inorganic and organic nutrients using instrumented moorings in the southern and central North Sea

Abstract: The aim of this study was to investigate the cycling of dissolved inorganic and organic nutrients using moored instrumented buoys (SmartBuoys) during the spring bloom in the North Sea. The instrumentation on the buoys enabled high frequency measurements of water-column integrated irradiance and in situ chlorophyll to be made, and also preserved water sample collection which were used for dissolved inorganic and organic nutrient analyses. The SmartBuoys were located in the year-round well-mixed plume zone associated with the River Thames and in the summer stratified central North Sea. These site locations allowed comparison of nutrient concentrations and cycling, and spring bloom development at two contrasting sites. The spring bloom was expected to be initiated at both stations due to increasing insolation and decreasing suspended load leading to higher water-column integrated irradiance. Due to differences in suspended load between the sites, the spring bloom started ∼2 months earlier in the central North Sea. The spring bloom in the Thames plume also resulted in higher maximum phytoplankton biomass due to the higher pre-bloom nutrient concentrations associated with riverine input. The use of SmartBuoys is also shown to allow the cycling of dissolved organic nutrients to be examined over the critical, and often undersampled, spring bloom period. Dissolved Organic Nitrogen (DON) clearly increased during the spring bloom in the central North Sea compared to winter concentrations. DON also increased in the Thames plume although showing greater winter variability related to higher riverine and sedimentary dissolved organic matter input at this shallow (∼18 m) coastal site. DON increase during the spring bloom was therefore related to primary production at both sites probably due to active release by phytoplankton. At both stations DON decreased to pre-bloom concentrations as the bloom declined suggesting the released DON was bioavailable and removed due to heterotrophic uptake and production. The preserved nutrient samples from the central North Sea site were also suitable for Dissolved Organic Phosphorus (DOP) analysis due to their low suspended load with similar trends and cycling to DON, albeit at lower concentrations. This suggested similar processes controlling both DON and DOP. The variable timing of short term events such as the spring bloom makes sampling away from coastal regions difficult without the use of autonomous technology. This study demonstrates for the first time the applicability of using preserved samples from automated buoys for the measurement of dissolved organic nutrients.

Blunted IgE-mediated activation of mast cells in mice lacking the serum- and glucocorticoid-inducible kinase SGK3.

Abstract: Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase a...

Low-latency mac-layer handoff for a high-altitude platform delivering broadband communications

Abstract: This paper presents a low-latency handoff algorithm that exploits the unique characteristics of a high-altitude platform (HAP) multi-cell system payload to significantly reduce the need for payload stabilization The handoff algorithm incorporates a novel time reuse Time-Division Multiplex/Time-Division Multiple Access (TDM/TDMA) frame structure, similar to that available with IEEE 80216 Single-and multiple-frequency variants have been developed, with the intention of keeping the handoff process transparent to the user The multiple-frequency variant is designed to increase system capacity by using a sequence of frequencies in different parts of the frame Performance of the single-frequency-band handoff scheme is assessed using a generic pitch model It has been shown to perform robustly, offering low-latency handoff, even with very short cell dwell times Using a number of developed HAP mobility models — such as bounce, random walk, and rotation — the worst-case handoff performance is also quantified for a distribution of fixed users within a coverage area It is shown that local user traffic hotspots are the dominant factor affecting performance, but that mitigation strategies are available