Showing papers by "David A. Pearce published in 2002"

An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder Batten disease

Abstract: Mutations in the CLN3 gene are responsible for the neurodegenerative disorder Batten disease; however, the molecular basis of this disease remains unknown. In studying a mouse model for Batten disease, we report the presence of an autoantibody to glutamic acid decarboxylase (GAD65) in cln3-knockout mice serum that associates with brain tissue but is not present in sera or brain of normal mice. The autoantibody to GAD65 has the ability to inhibit the activity of glutamic acid decarboxylase. Furthermore, brains from cln3-knockout mice have decreased activity of glutamic acid decarboxylase as a result of the inhibition of this enzyme by the autoantibody, resulting in brain samples from cln3-knockout mice having elevated levels of glutamate as compared with normal. This elevated glutamate in the brain of cln3-knockout mice co-localizes with presynaptic markers. The decreased activity of GAD65 and increased levels of glutamate may have a causative role in astrocytic hypertrophy evident in cln3-knockout mice, and in altered expression of genes involved in the synthesis and utilization of glutamate that underlie a shift from synthesis to utilization of glutamate. An autoantibody to GAD65 is also present in sera of 20 out of 20 individuals tested who have Batten disease. Postmortem tissue shows decreased reactivity to an anti-GAD65 antibody that may be due to loss of GAD65-positive neurons or due to the reactive epitope being blocked by the presence of the autoantibody. We propose that an autoimmune response to GAD65 may contribute to a preferential loss of GABAergic neurons associated with Batten disease.

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Abstract: Mineralocorticoids stimulate Na reabsorption and K secretion in principal cells of connecting tubule and col- lecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to in- crease ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein medi- ating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly in- creases K channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pKa of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K excretion.

Hypotonic induction of SGK1 and Na+ transport in A6 cells

Abstract: Serum and glucocorticoid-regulated kinase-1 (SGK1) is a serine-threonine kinase that is regulated at the transcriptional level by numerous regulatory inputs, including mineralocorticoids, glucocort...

The neuronal ceroid lipofuscinoses: mutations in different proteins result in similar disease.

Abstract: The neuronal ceroid-lipofuscinoses (NCL) are the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births. The main features of this disease are failure of psychomotor development, impaired vision, seizures, and premature death. Many biochemical and physiological studies have been initiated to determine the cellular defect underlying the disease, although only a few traits have been truly associated with the disorders. One of the paradox’s of the NCL-diseases is the characteristic accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and other cell types. However, the accumulation of this lysosomal storage material, which no doubt contributes to the neurologic disease, does not apparently lead to disease outside the CNS, and how these cellular alterations relate to the neurodegeneration in NCLs is unknown. Mutations have been identified in six distinct genes/proteins, namely CLN1, which encodes PPT1, a protein thiolesterase; CLN2, which encodes TPP1, a serine protease; and CLN3, CLN5, CLN6, and CLN8, which encode novel transmembrane proteins. Mutation in any one of these CLN-proteins results in a distinct type of NCL-disease. However, there are many shared similarities in the pathology of these diseases. The most obvious connection between PPT1, TPP1, CLN3, CLN5, CLN6, and CLN8 is their subcellular localization. To date, three of the four proteins whose subcellular localization has been confirmed, namely PPT1, TPP1, and CLN3, reside in the lysosome. We review the function of the CLN-proteins and discuss the possibility that a disruption in a common biological process leads to an NCL-disease.

Glucocorticoids Prolong Ca2+ Transients in Hippocampal-Derived H19-7 Neurons by Repressing the Plasma Membrane Ca2+-ATPase-1

Abstract: Calcium ions (Ca(2+)) play an important role in mediating an array of structural and functional responses in cells. In hippocampal neurons, elevated glucocorticoid (GC) levels, as seen during stress, perturb calcium homeostasis and result in altered neuronal excitability and viability. Ligand- and voltage-gated calcium channels have been the presumed targets of hormonal regulation; however, circumstantial evidence has suggested the possibility that calcium extrusion might be an important target of GC regulation. Here we demonstrate that GC-induced repression of the plasma membrane Ca(2+)-ATPase-1 (PMCA1) is an essential determinant of intracellular Ca(2+) levels ([Ca(2+)](i)) in cultured hippocampal H19-7 cells. In particular, GC treatment caused a prolongation of agonist-evoked elevation of [Ca(2+)](i) that was prevented by the expression of exogenous PMCA1. Furthermore, selective inhibition of PMCA1 using the RNA interference technique caused prolongation of Ca(2+) transients in the absence of GC treatment. Taken together, these observations suggest that GC-mediated repression of PMCA1 is both necessary and sufficient to increase agonist-evoked Ca(2+) transients by down-regulating Ca(2+) extrusion mechanisms in the absence of effects on calcium channels. Prolonged exposure to GCs, resulting in concomitant accumulation of [Ca(2+)](i), is likely to compromise neuronal function and viability.

Interaction with Btn2p Is Required for Localization of Rsg1p: Btn2p-Mediated Changes in Arginine Uptake in Saccharomyces cerevisiae

Abstract: Btn2p, a novel coiled-coil protein, is up-regulated in btn1Δ yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1Δ strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet. 22:55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsg1p, a down-regulator of the Can1p arginine and lysine permease. Rsg1p localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2Δ strains) fail to correctly localize Rsg1p. btn2Δ strains, like rsg1Δ strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2Δ strains, like rsg1Δ strains, demonstrate an elevated rate of uptake of [14C]arginine, which leads to increased intracellular levels of arginine. Overexpression of BTN2 results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsg1p. Our original identification of Btn2p was that it is up-regulated in the btn1Δ strain which serves as a model for the lysosomal storage disorder Batten disease. Btn1p is a vacuolar/lysosomal membrane protein, and btn1Δ suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2Δ rsg1Δ strains. We conclude that Btn2p interacts with Rsg1p and modulates arginine uptake. Up-regulation of BTN2 expression in btn1Δ strains may facilitate either a direct or indirect effect on intracellular arginine levels.

An autoantibody to GAD65 in sera of patients with juvenile neuronal ceroid lipofuscinoses.

Abstract: Juvenile neuronal ceroid lipofuscinoses (Batten disease) is a neurodegenerative disease that presents in early childhood. The main features of this disease are failure of psychomotor development, impaired vision, and seizures. Death usually occurs in late teens to early twenties. Batten disease is an autosomal-recessive disorder, resulting from mutations in the CLN3 gene product.1 Genetic testing can identify the most common mutation in CLN3 , a 1.02-kb deletion. However this accounts for only about 74% of the mutations identified thus far in CLN3 ,1,2⇓ and because identification of rarer point mutations in CLN3 is often difficult, another diagnostic tool would be useful. We have previously shown that a mouse model for Batten disease, i.e., cln3-knockout mice, and eight individuals with Batten disease have an autoimmune response to GAD65.3 In the same study we reported that a total of 20 individuals with Batten disease tested positive for the presence of autoantibodies to glutamate decarboxylase, GAD65. Furthermore, studies in the mouse revealed that the autoantibody to GAD65 is associated with astrocytic hypertrophy in the brain. Moreover, the cln3-knockout mice that have the autoimmune response to GAD65 have decreased activity of glutamic acid decarboxylase and consequently elevated levels of presynaptic glutamate in the brain.3 It has been suggested that a preferential loss of GABAergic neurons is a major factor in the progression of Batten disease.4 It is therefore plausible to predict that the GAD65 autoantibody and its resultant effects on the brain are possible contributors to the pathology associated …

Heterotrophic microbial colonization of the interior of impact-shocked rocks from Haughton impact structure, Devon Island, Nunavut, Canadian High Arctic

Abstract: The polar desert is one of the most extreme environments on Earth. Endolithic organisms can escape or mitigate the hazards of the polar desert by using the resources available in the interior of rocks. We examined endolithic communities within crystalline rocks that have undergone shock metamorphism as a result of an asteroid or comet impact. Specifically, we present a characterization of the heterotrophic endolithic community and its environment in the interior of impact-shocked gneisses and their host polymict breccia from the Haughton impact structure on Devon Island, Nunavut, Canadian High Arctic. Microbiological colonization of impact-shocked rocks is facilitated by impact-induced fissures and cavities, which occur throughout the samples, the walls of which are lined with high abundances of biologically important elements owing to the partial volatilization of minerals within the rock during the impact. 27 heterotrophic bacteria were isolated from these shocked rocks and were identified by 16S rDNA sequencing. The isolates from the shocked gneiss and the host breccia are similar to each other, and to other heterotrophic communities isolated from polar environments, suggesting that the interiors of the rocks are colonized by microorganisms from the surrounding country rocks and soils. Inductively coupled plasma–atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis were used to identify the chemical composition of the shocked materials and to document the in situ growth of microbes in their interiors. The identification of these heterotrophic communities within impact-shocked crystalline rocks extends our knowledge of the habitable biosphere on Earth. The colonization of the interiors of these samples has astrobiological applications both for considering terrestrial, microbiological contamination of meteorites from the Antarctic ice sheet and for investigating possible habitats for microbial organisms on the early Earth, and more speculatively, on Mars and other planetary bodies.

Short-term stability of the microbial community structure in a maritime Antarctic lake

Abstract: Spatial and temporal changes in the microbial community structure in a maritime Antarctic freshwater lake were investigated over a single day/night cycle in December 1999. The community structure of key microbial planktonic groups varied with depth and this was related to both physical and chemical stratification. However, in most cases, the community structure observed at specific depths did not change over the time period studied. These results suggested short-term stability in community structure, with only some minor effects of the diel changes in irradiance on the vertical distribution of planktonic organisms. This is in marked contrast to medium- and long-term studies, which show significant changes in microbial community structure with both time and depth.

Speech recognition performance comparison between DSR and AMR transcoded speech.

Abstract: In this paper the speech recognition performance obtained when using Distributed Speech Recognition (DSR) architecture is compared to that obtained when the speech is first transcoded using the Adaptive Multi-Rate (AMR) speech codec at 4.75 and 12.2 kbps. In a like-versus-like comparison, made using the Advanced DSR Front-end and the Aurora reference back-end, the DSR architecture gives substantial gains in speech recognition performance. The evaluations measure the change in Word Error Rate (WER) on the Aurora 2 and Aurora 3 databases with “perfect” endpoints. The performance with AMR 4.75 is 50% worse than DSR on Aurora 2 and 47% worse on Aurora 3. Even with the higher data rate of AMR 12.2, AMR is 17% worse than DSR on Aurora 2 and 20% worse on Aurora 3.

Altered flurothyl seizure induction latency, phenotype, and subsequent mortality in a mouse model of juvenile neuronal ceroid lipofuscinosis/batten disease.

Abstract: Summary: Purpose: Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a pediatric neurodegenerative disease characterized by vision loss, seizure activity, cognitive decline, and premature death. Discovery of the Batten disease–related gene, CLN3, led to creation of a Cln3 protein-deficient mouse model (Cln3−/−), which recapitulates some of the histopathologic characteristics of the human condition. We hypothesized that lack of Cln3 would alter seizure-related behavioral parameters. Methods: Using flurothyl gas inhalation, we examined seizure-induction latencies in Cln3−/− mice and wildtype (wt) controls at time points that represent late neonatal, immature, mature, and aged time points. We examined latency to first myoclonic jerk (LMJ), latency to loss of posture (LOP), and subsequent mortality. Results: Our results demonstrate an age-dependent alteration of seizure-induction latencies in Cln3−/−. Immature Cln−/− mice aged 35–42 days had an increased latency to both LMJ and LOP compared with age-matched wt controls. There were no significant latency differences between Cln3−/− and wt at other time points examined. Mortality after generalized seizure was high in both Cln3−/− and wt animals at late neonatal and immature developmental stages. No mortality was seen in wt mice past maturity at 6 weeks. Mature and aged Cln3−/− animals retained a vulnerability to death after seizure activity. Conclusions: These results suggest that a deficiency of Cln3 protein in the Batten model mice may result in age-dependent alteration of the neuroanatomic and biochemical substrates involved in seizure propagation and recovery. This may be important in understanding seizures, neurodegeneration, and premature death in human Batten disease.

Development of Elite Inoculant Rhizobium Strains in Southeastern Australia

Abstract: Matching rhizobial strains to host legumes is the most important factor in maximising the productivity of Australian pulse and pasture legumes. This paper reports on a system that we have developed for the selection of elite inoculant strains. This system involves the collection of Rhizobium germplasm, screening their nitrogen fixation capability and assessing their adaptation to different soil environments. Soil factors that influence the legume-Rhizobium symbiotic relationship include extremes in soil pH (highly acidic or alkaline), salinity, and high soil temperatures in crop and pastoral soils in southern Australia. These soil conditions limit the establishment of Rhizobium populations introduced as seed inoculum. Therefore, a process has been developed, and is discussed in this paper, that better aligns strain selection with plant development. FARMING in Australia is continually changing because of intensification, higher inputs and the quest for increased production as well as adapting to specific soil and climatic conditions. With intensification of cropping, the need to maximise the benefits of productive pastures and crop legumes in rotation with crops is recognised for long-term farm sustainability. To achieve these benefits, the legume must have effective root rhizosphere associations. Given the poor fertility of many Australian soils, there is a need to select appropriate plant-Rhizobium symbioses for Australian environments. Agriculturally productive legumes and their root-nodule bacteria have evolved outside of Australia. Now, more than ever, the need for legumes and root-nodule bacteria adapted to the Australian environment is critical. Rhizobial strains suitable as inoculants must have the following characteristics: able to colonise the soil and to tolerate environmental stresses, able to compete with populations of background rhizobia to form nodules, able to form effective nodules which fix N2, and to have no deleterious effects on nontarget hosts (Brockwell et al. 1995; Howieson 1999). Strains of rhizobia differ widely in their ability to survive nodulate and fix nitrogen in soil environments; thus the selection of rhizobia with specific symbiotic and competitive attributes suited to a range of soil environments must assume a high priority. Naturally occurring Rhizobium populations often occur in high numbers in soil, and can compete strongly with introduced Rhizobium inoculant. However, their ability to fix nitrogen is generally poor. The introduction of new legume genera to Australian farming systems usually requires the identification of new and specific inoculant Rhizobium strains not found in Australian soils, but necessary for optimum N fixation. Strain selection focuses on root nodule bacteria that offer a broad-host range for nodule formation and nitrogen fixation, as well as adaptation to the soil niche of the host legumes. The task of selecting rhizobial strains that match host legumes can be a long and complex process, and includes the following steps: 1. Collection, isolation and maintenance of the Rhizobium germplasm. 2. Authentication and screening of the rhizobial isolates for genetic compatibility and nitrogen fixation. 3. Assessment of the Rhizobium germplasm for edaphic adaptation and in situ performance (Howieson et al. 2000a). Much progress has been made in the selection and commercial release (or future release) of elite 1 Rutherglen Research Institute, Dept of Natural Resources and Environment, Rutherglen, Victoria 3685, Australia

Effect of immunosuppressive agents on glucocorticoid receptor function in A6 cells.

Abstract: Immunosuppressive agents such as FK-506 and rapamycin inhibit aldosterone- stimulated Na+ transport in A6 cells. Concentration dependence is consistent with the known affinities of these agents for immunophilins. The inhibition was also dependent on time, requiring preincubation with FK-506 or rapamycin before inhibition was seen. The present studies were designed to determine whether this inhibition was pretranscriptional and whether it was due to an effect on either receptor translocation or nuclear accumulation. Because transport effects of steroids in A6 cells are mediated by glucocorticoid receptors (GRs), we examined the transcriptional response of GR-regulated reporters transfected into these cells. Preincubation of cells with FK-506 and rapamycin completely blocked reporter gene activation, whereas preincubation with cyclosporin A partially inhibited this activation. A minimum of 8 h of preincubation was required before the effect was seen. Using a transiently transfected green fluorescent protein-GR construct, we examined the effect of FK-506 and rapamycin on GR translocation. GR translocation induced by dexamethasone was extremely rapid (<5 min) and was largely unaffected by FK-506 or rapamycin but was completely blocked by geldanamycin. Digital deconvolutions revealed a punctate nuclear accumulation of GR, which was still seen after preincubation with immunosuppressive agents. These agents clearly inhibit steroid action by blocking GR-stimulated gene transcription, but this effect is not mediated by altered translocation or nuclear accumulation of receptors. Inhibition of steroid-regulated gene transcription by immunosuppressive agents may explain the electrolyte abnormalities seen in patients receiving these drugs.

The impact of background rhizobial populations on inoculation response.

Abstract: Agronomic programs that target the introduction of legumes into different agro-ecosystems focus initially on the selection of legumes that have the ability to tolerate edaphic constraints that include disease resistance, water stress and tolerance to salinity, acidity and sodicity Within such selection programs the interaction of the legume with soil Rhizobium has largely been ignored In order to maximise agricultural production, specific programs must link strain selection with plant development together with an understanding of the impact of background populations PASTURE and crop legumes have been used extensively in agriculture over the past century in Australia, mainly for maintaining soil fertility These agricultural soils are often constrained in their ability to sustain productive farming systems due to factors associated with low fertility, sodicity, salinity and extremes of acidity and alkalinity These same attributes can also have a negative impact on the legume-Rhizobium symbiotic relationship reducing the ability of rhizobia to form nodules with optimal N2-fixing capacity, thus impeding the continued success of legumes in Australian agricultural systems Considerable variation in the soil populations of Rhizobium spp has been found in soils throughout Australia, with the range varying from less than 10 to in excess of 106 Rhizobium bacteria g/m soil (Gibson et al 1975; Slattery et al 1999), generally with an average soil population above 1 × 104 Rhizobium bacteria g/m soil The size of the soil population is dependent on field history, location of sampling, soil characteristics and the presence of a host plant Where there are low ( 103 Rhizobium bacteria g/m soil) introduction of new strains can be difficult and often unsuccessful (Thies et al 1991; Brockwell et al 1995) The Australian continent provides a large landmass that constitutes a wide spectrum of climatic conditions and soil environments, in which to maintain a viable agricultural industry Soil types in the pulse growing regions of southeastern Australia vary from alkaline (NW Victoria and much of South Australia), neutral acidic soils (Wimmera, central and southern Victoria) to highly acidic soils (NE Victoria and southern NSW) This wide variation in soil type has serious implications for Rhizobium survival, Rhizobium effectiveness and the need for re-inoculation of following pulse crops Techniques used for the development of elite inoculant strains have been discussed in an accompanying workshop paper (Slattery and Pearce 2001) The final criteria adopted for the selection of Rhizobium strains are the assessment of germplasm for edaphic adaptation and field performance (Howieson et al 2000; Slattery and Pearce 2001) The capacity of inoculant strains to colonise soils in sufficient quantity to provide effective nodulation is very much dependant on the soil type Thus, a thorough understanding of the presence of background populations will enable recommendations to be made regarding the need for re-inoculation of pulse legumes In this paper, we study the effect of the soil environment on the ability of rhizobia to form nodules and the need for rhizobial inoculation when introducing pulse legumes into different soil environments A comprehensive soil survey across the pulse legume growing regions of Victoria provide a critical 1 Rutherglen Research Institute, Department of Natural Resources and Environment, Rutherglen, Victoria 3685, Australia

The diversity of bacterioplankton community structures over a range of Antarctic freshwater lakes

Abstract: (2002). The diversity of bacterioplankton community structures over a range of Antarctic freshwater lakes. SIL Proceedings, 1922-2010: Vol. 28, No. 2, pp. 862-865.