bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycorrhizal vs saprotrophic status of fungi: the isotopic evidence
Authors: Hobbie Erik A. , Weber Nancy S. , Trappe James M.
Published: March 2003

Relative abundances of carbon and nitrogen isotopes in fungal sporocarps are useful in assessing mycorrhizal or saprotrophic status, and might provide insights into the evolutionary history of these traits.

 Sporocarps of known mycorrhizal or saprotrophic genera were collected at Woods Creek, OR, USA, and isotopically compared with foliage, litter, soils and wood collected from the same site. Possible trophic strategies were then isotopically assessed in archived specimens of the Pezizales of known molecular phylogeny from the western United States.

 At Woods Creek, mycorrhizal fungi were 3.5‰ ± 0.6‰ depleted in δ13C and 5.7‰ ± 0.4‰ enriched in δ15N compared with saprotrophic fungi. By contrast, fungi from four genera of uncertain mycorrhizal status (Clavulina,Helvella,Otidia, andRamaria) were only 0.4‰ ± 0.4‰ enriched in δ13C and 1.2‰ ± 1.1‰ depleted in δ15N relative to mycorrhizal fungi.

 In archived samples, the δ13C measurements appeared to be a better indicator of trophic strategy than δ15N measurements. The δ13C measurements suggested that mycorrhizal or saprotrophic status was conserved within families of the Pezizales (as determined by molecular phylogeny), with the Helvellaceae and Tuberaceae mycorrhizal and Discinaceae and Morchellaceae being largely saprotrophic.
Analysis of arbuscular mycorrhizas using symbiosis‐defective plant mutants
Authors: Marsh John F. , Schultze Michael
Published: March 2003

Arbuscular mycorrhizas (AM), mutualistic plant–fungus symbioses formed on the majority of land plants, appear to play an important role in plant mineral nutrition, plant health, and might influence the structure of plant communities. Progress in understanding the genetic and molecular basis of this important symbiotic association has been hampered by the obligate biotrophy of the fungal partner, and by the lack of mycorrhiza formation on the plant model species Arabidopsis thaliana. Recently, legume plants, such as Medicago truncatula and Lotus japonicus, have been chosen as experimental systems for research of plant–microbe symbioses. The application of both reverse‐ and traditional forward‐genetic approaches, in conjunction with genomics initiatives in model legumes, will rapidly increase our understanding of the plant‐encoded genetic determinants of the arbuscular mycorrhiza. An essential step in this process has been the generation, isolation and analysis of mycorrhizal mutants. This review provides an introduction to existing plant mutants affected in AM formation.
UV and blue light signalling: pathways regulating chalcone synthase gene expression in Arabidopsis
Authors: Jenkins Gareth I. , Long Joanne C. , Wade Helena K. , Shenton Matthew R. , Bibikova Tatiana N.
Published: March 2003

UV‐B, UV‐A and blue light control a variety of aspects of plant development via distinct photoreceptors and signalling pathways. The known photoreceptors for UV‐A/blue light are cryptochrome (cry)1 and cry2, and the phototropism photoreceptor, phototropin. Redox processes are important in cry and phototropin signal transduction. A specific photoreceptor for UV‐B has not been identified and there appear to be several possible UV‐B signalling pathways. We are investigating the UV and blue light regulation of transcription of the chalcone synthase gene (CHS) in Arabidopsis. Experiments with photoreceptor mutants show that distinct UV‐A/blue (cry mediated) and UV‐B photoreception systems control CHS expression. Experiments with an Arabidopsis cell suspension culture show that the UV‐B and cry1 signalling pathways differ kinetically and pharmacologically. In contrast to some other UV‐B responses, the UV‐B induction of CHS does not appear to involve oxidative stress signalling. Promoter elements and candidate transcription factors that effect CHS induction have been identified. Interactions within a network of UV‐B, cry and phytochrome signalling pathways regulate CHS expression. Synergistic interactions between the UV‐B pathway and distinct UV‐A and blue‐light pathways maximize the response. In addition, specific phytochromes positively control the cry1 pathway via distinct potentiation and coaction effects, and negatively regulate the UV‐B pathway.
Calcium‐based signalling systems in guard cells
Authors: Ng CarL. K.‐Y. , Mcainsh Martin R. , Gray Julie E. , Hunt LeE. , Leckie Calum P. , Mills Lewis , Hetherington Alistair M.
Published: March 2003

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular processes in both plants and animals. As an example, Ca2+ has been shown to be a second messenger in the signal transduction pathways by which stomatal guard cells respond to external stimuli. Regulated increases in the cytosolic concentration of free calcium ions ([Ca2+]cyt) in guard cells have been observed to be a common intermediate in many of the pathways leading to either opening or closing of the stomatal pore. This observation has prompted investigations into how specificity is encoded in the Ca2+ signal. It has been suggested that the key to generating stimulus‐specific calcium signatures lies in the ability to access differentially the cellular machinery controlling calcium influx and release from intracellular stores. Several important components of the calcium‐based signalling pathways have been identified in guard cells including cADPR, phospholipase C–InsP3, InsP6 and H2O2. These data suggest that the pathways for intracellular mobilization of Ca2+ are evolutionarily conserved between plants and animals.

Abbreviations

ABA, abscisic acid; [Ca2+]cyt, cytosolic free calcium concentration; [Ca2+]ext, external calcium concentration; IK,in; inward‐rectifying K+ currents; InsP3, inositol‐1,4,5‐trisphosphate; InsP6, inositol hexakisphosphate; PLC, phospholipase C; PLD, phospholipase D; PA, phosphatidic acid; H2O2, hydrogen peroxide; AAPK, ABA‐activated serine‐threonine protein kinase; cADPR, cyclic adenosine 5′‐diphosphoribose; U73122, 1‐(6‐{[17â‐3‐methoxyestra‐1,3,5(10)‐trien‐17‐yl]amino}hexyl)‐1H‐pyrrole‐2, 5‐dione; RyR; ryanodine receptor; CICR; calcium‐induced calcium‐release; ICa, inward calcium current.
Composted recycled organic matter suppresses soil‐borne diseases of field crops
Authors: Tilston E. L. , Pitt D. , Groenhof A. C.
Published: March 2003

• Suppression of soil‐borne diseases of horticultural crops by composts has been attributed to the activities of antagonistic microorganisms. The potential for abiotic disease suppression in field‐grown crops was explored using glasshouse‐based bio‐assays for five pathogens of agricultural crops and eight formulations of recycled organic matter compost.

• The effects of compost formulation and postmaturation processing were investigated by comparing disease severity on recycled organic matter composts with a commercial seedling compost. Comparisons were made within native and autoclaved series of composts. Autoclaving perturbed compost chemistry without the addition of other materials.

• The severity of autoclaving‐induced changes to compost chemistry were related to formulation and processing issues. Extractable carbon content, nitrate‐N content and pH all contributed to the suppressive capacity of recycled organic matter composts. There may also be a biotic component to the suppression observed.

• For each pathogen the influential chemical characteristics of suppressive composts were the same as those identified for soils in other studies. There is potential for the suppressive capacity of recycled organic matter composts to be improved.
Developmental shifts in watermelon growth and reproduction caused by the squash bug, Anasa tristis
Authors: Biernacki Maciej , Lovett‐Doust Jon
Published: March 2003

Compared with leaf‐feeding herbivores, little is known about how sap‐feeding herbivores affect plant growth, morphology and reproduction. This study examines effects of the sap‐feeding squash bug ( Anasa tristis
) on watermelon (
Citrullus lanatus ) root, leaf and reproductive structures.

Plants at the four‐leaf stage were exposed to different densities of caged squash bugs for 67 d (to plant maturity).

Initial effects were on roots. Herbivory was associated with a significant reduction in mean total root length, root surface area and number of root tips, as well as an increase in root diameters. Herbivore‐exposed plants had significantly more leaves, although leaf lifespan was decreased. Both total plant dry mass and fruit dry mass per unit leaf area were significantly greater in controls. Significant effects of herbivores on plant reproductive traits included delayed flower formation (by 7–12 d), change in floral sex ratio (in favor of femaleness), increased fruit abortion, and smaller fruit size.

Developmental consequences were related to changes in plant water relations, including decreased water‐use efficiency. Water use in treated plants was three to nine times greater per unit of plant dry mass than in controls.
Ecology and evolution of light‐dependent and light‐independent phytogenic volatile organic carbon
Authors: Lerdau Manuel , Gray Dennis
Published: March 2003

The low molecular weight hydrocarbons produced by plants form a uniquely exciting group of compounds. Produced by a common biosynthetic route, they play multiple and complex roles in organismal, ecological, and atmospheric processes. While some of these compounds have clearly identified functions within plants, others are made for reasons not yet fully understood. Here, both light‐dependent and light‐independent emissions are reviewed, together with regulation of production and possible functions of light‐dependent volatile organic carbon (VOC). In addition to issues regarding the phylogenetic origins of VOC emissions, the origins of the pivotal enzymes that give rise to the observed emission phenotypes are discussed. Studies on the evolution and regulation of their production and emission provide an amazing opportunity for scientists working from the molecular to the tropospheric scales to interact.

Contents

Summary
199

I.
Introduction
199

II.
Light‐independent emissions
200

III.
Light‐dependent emissions
201

IV.
Regulation of production
202

V.
Possible functions of light‐dependent VOCs
204

VI.
Evolutionary aspects of phytogenic VOC
206

Acknowledgements
206

References
209
Source–sink balance and carbon allocation below ground in plants exposed to ozone
Authors: Andersen Christian P.
Published: March 2003

The role of tropospheric ozone in altering plant growth and development has been the subject of thousands of publications over the last several decades. Still, there is limited understanding regarding the possible effects of ozone on soil processes. In this review, the effects of ozone are discussed using the flow of carbon from the atmosphere, through the plant to soils, and back to the atmosphere as a framework. A conceptual model based on carbohydrate signaling is used to illustrate physiological changes in response to ozone, and to discuss possible feedbacks that may occur. Despite past emphasis on above‐ground effects, ozone has the potential to alter below‐ground processes and hence ecosystem characteristics in ways that are not currently being considered.ContentsSummary213I.Introduction213II.Source–sink model: carbohydrate signaling214III.Effect of ozone on above‐ground sources and sinks216IV.Decreased allocation below ground218V.Carbon flux to soils220VI.Soil food web223VII.Summary, conclusions and future research223Acknowledgements223References223
Phytochrome‐hormonal signalling networks
Authors: Halliday Karen J. , Fankhauser Christian
Published: March 2003

Through time, plants have evolved an extraordinary ability to interpret environmental cues. One of the most reliable of these cues is light, and plants are particularly adept at sensing and translating environmental light signals. The phytochrome family of photoreceptors monitor cues such as daylength or vegetative shade and adjust development to reflect change in these parameters. Indeed, it is their ability to coordinate these complex developmental changes that underpins the remarkable success of plants. Evidence is mounting that hormones control many of these light‐mediated changes. Therefore, if we are to understand how light manipulates development we need to explore the interplay between light and hormonal signalling. Toward this goal, this review highlights the known convergence points of the phytochrome and the hormonal networks and explores their interactions.

Contents

Summary
449

I.
Introduction
449

II.
The phytochrome protein
450

III.
Bacteriophytochromes
450

IV.
IBacteriophytochrome signalling
450

V.
Plant phytochrome signalling
451

VI.
Ethylene perception and signalling
451

VII.
Cytokinin perception and signalling
452

VIII.
Brassinosteroid perception and signalling
453

IX.
Gibberellin signalling
455

X.
Auxin signalling
456

XI.
Proteolysis in light and hormonal signalling
458

XII.
Conclusion
459

Acknowledgements
459

References
459
Anti‐microbial hand washes for domestic use – their effectiveness in vitro and in normal use
Authors: Sharp Kay , Haysom Iain , Parkinson Rosamund
Published: March 2003

The killing or removal of microbes from the hands is a critical factor in food safety as many studies have shown the hands to be both an important source of microbes and powerful agents of cross‐contamination in hospital and domestic situations. In response to this concern, a number of novel hand‐washing products have appeared on the market. These products contain anti‐microbial agents and claim to be more effective at removing bacteria than soap bars and conventional liquid soaps. This study attempts to test these claims by comparing the effectiveness of a conventional soap bar, a conventional liquid soap and an anti‐microbial liquid soap containing triclosan. In vitro tests demonstrate that the anti‐microbial liquid soap is more effective than conventional liquid soaps in reducing the viability of six bacterial species and that this effect is both time and dose dependent. However, when the three soaps were compared for their ability to reduce microbial counts on the hands no differences were observed between the three products. For all three soaps, counts after washing sometimes went up and sometimes down when compared with pre‐wash counts. This was the case both when the soaps were used ‘normally’– that is, with great variation in the time taken, water and soap volumes used and method of washing and after a standardized, rigorous wash recommended in clinical situations. Furthermore, reduction in microbial counts from hands contaminated by handling raw meat was no greater for the anti‐microbial than for the conventional liquid soap.
Incommensurate paradigms? Bridging the divide between technocrats and the lay public in health care planning: recent experience from Wales
Authors: Longley Marcus
Published: March 2003

The pressure on the UK National Health Service (NHS) to increase public involvement in the planning of future services has never been greater. Politicians and others argue that ‘lay wisdom’ has much to contribute, and that such involvement is essential if public confidence in the service is to be maintained. Many managers and clinicians, however, operating within an essentially technocratic paradigm, remain unconvinced. Their views are often reinforced by a lack of experience of methods for public involvement that can meet the many practical difficulties involved. Using Arnstein’s ‘ladder of citizen participation’, this paper describes the principal nuances of these two paradigms, explores the nature of the conflict between them, and assesses the extent to which they are really incommensurate. It then briefly describes some of the key methodological principles emerging from public involvement work in parts of the NHS in Wales, and assesses their potential to bridge the divide between the paradigms.
Mycobacterium aviumssp.paratuberculosis: its incidence, heat resistance and detection in milk and dairy products
Authors: Grant Irene R , Rowe Michael T , Dundee Louise , Hitchings Edward
Published: March 2003

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne’s disease in cattle and other ruminants and has been implicated as a possible cause of Crohn’s disease in humans. The organism gains access to raw milk directly through excretion into the milk within the udder, and indirectly through faecal contamination during milking. MAP has been shown to survive commercial pasteurization in naturally infected milk, even at the extended holding time of 25 s. Pasteurized milk must therefore be considered a vehicle of transmission of MAP to humans. Isolation methods for MAP from milk are problematical, chiefly because of the absence of a suitable selective medium. This makes food surveillance programs and research on this topic difficult. The MAP problem can be addressed in two main ways: by devising a milk‐processing strategy that ensures the death of the organism; and/or strategies at farm level to prevent access of the organism into raw milk. Much of the research to date has been devoted to determining if a problem exists and, if so, the extent of the problem. Little has been directed at possible solutions. Given the current state of information on this topic and the potential consequences for the dairy industry, research is urgently needed so that a better understanding of the risks and the efficacy of possible processing solutions can be determined.
Building bridges: towards integrated library and information services for mental health and social care
Authors: Blackburn Norma
Published: March 2003

This research focuses on identifying the information needs of health professionals, social workers and staff from voluntary agencies, who work together in the provision of services for people with a mental illness. The aim is to provide a fully integrated library and information service for mental health and social care. A number of recent government reports have prompted this research. These have culminated in the publication of the National Service Framework for Mental Health. It was decided to conduct a qualitative study using 17 semi‐structured interviews with individuals and multi‐professional groups. A thematic analysis highlighted a number of key themes including: information need, information seeking behaviour and information flows. Results have indicated that because of the dispersed nature of mental health services, across primary and secondary care, and from statutory and non‐statutory organisations, information technology is viewed as the only realistic vehicle to provide the required information. As specialist mental health trusts are configured, covering wider geographical areas, this can only increase the reliance on information technology, for information access and information sharing.
Transient Inhibition of Glutamate Uptake In Vivo Induces Neurodegeneration when Energy Metabolism Is Impaired
Authors: Del Rayo Sánchez‐Carbente Ma. , Massieu Lourdes
Published: March 2003

Abstract : Impairment of glutamate transport during ischemia might
be related to the elevation of the extracellular concentration of glutamate
and ischemic neuronal damage. Additionally, impairment of energy metabolism in
vivo leads to neurodegeneration apparently mediated by a secondary excitotoxic
mechanism. In vitro observations show that glucose deprivation and inhibition
of energy metabolism exacerbate the toxic effects of glutamate. We have
previously shown that glutamate uptake inhibition in vivo by
l‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) leads to a substantial elevation in the extracellular concentration of excitatory amino acids that is not associated with cell death. These observations suggest that energy depletion during ischemia might be determinant of ischemic neuronal damage. To investigate whether impairment of energy metabolism in vivo increases neuronal susceptibility to glutamate uptake inhibition, we studied the effect of glutamate accumulation induced by the intrahippocampal or intrastriatal administration of PDC in energy‐deficient rats chronically treated with 3‐nitropropionic acid (3‐NP), which irreversibly inhibits the tricarboxylic acid cycle and electron transport chain. Extracellular glutamate levels were monitored by HPLC from fractions collected from microdialysis probes, and neuronal damage was evaluated by histological analysis. Our results show that glutamate uptake inhibition leads to marked neuronal damage in energy‐deficient rats but not in intact animals, which apparently is not related to an additional elevation of glutamate levels induced by 3‐NP.
Neuroadaptations in Ionotropic and Metabotropic Glutamate Receptor mRNA Produced by Cocaine Treatment
Authors: Ghasemzadeh M. Behnam , Nelson Linda C. , Lu Xin‐Yun , Kalivas Peter W.
Published: March 2003

Abstract : The expression of glutamate receptor/subunit mRNAs was examined 3 weeks after discontinuing 1 week of daily injections of saline or cocaine. The level of mRNA for GluR1‐4, NMDAR1, and mGluR5 receptors was measured with in situ hybridization and RT‐PCR. In nucleus accumbens, acute cocaine treatment significantly reduced the mRNA level for GluR3, GluR4, and NMDAR1 subunits, whereas repeated cocaine reduced the level for GluR3 mRNA. Acute cocaine treatment also reduced the NMDAR1 mRNA level in dorsolateral striatum and ventral tegmental area. In prefrontal cortex, repeated cocaine treatment significantly increased the level of GluR2 mRNA. The GluR2 mRNA level was not changed by acute or repeated cocaine in any other brain regions examined. Repeated cocaine treatment also significantly increased mGluR5 mRNA levels in nucleus accumbens shell and dorsolateral striatum. Functional properties of the ionotropic glutamate receptors are determined by subunit composition. In addition, metabotropic glutamate receptors can modulate synaptic transmission and the response to stimulation of ionotropic receptors. Thus, the observed changes in levels of AMPA and NMDA receptor subunits and the mGluR5 metabotropic receptor may alter excitatory neurotransmission in the mesocorticolimbic dopamine system, which could play a significant role in the enduring biochemical and behavioral effects of cocaine.
The Copper Chaperone CCS Is Abundant in Neurons and Astrocytes in Human and Rodent Brain
Authors: Rothstein Jeffrey D. , Dykes‐Hoberg Margaret , Corson Laura B. , Becker Mark , Cleveland Don W. , Price Donald L. , Culotta Valeria Cizewski , Wong Phillip C.
Published: March 2003

Abstract : Copper trafficking in mammalian cells is highly regulated. CCS is a copper chaperone that donates copper to the antioxidant enzyme copper/zinc superoxide dismutase 1 (SOD 1). Mutations of SOD1 are responsible for ~20% of familial amyotrophic lateral sclerosis (FALS). Monospecific antibodies were generated to evaluate the localization and cellular distribution of this copper chaperone in human and mouse brain as well as other organs. CCS is found to be ubiquitously expressed by multiple tissues and is present in particularly high concentrations in kidney and liver. In brain and spinal cord, CCS was found throughout the neuropil, with expression largely confined to neurons and some astrocytes. Like SOD1, CCS immunoreactivity was intense in Purkinje cells, deep cerebellar neurons, and pyramidal cortical neurons, whereas in spinal cord, CCS was highly expressed in motor neurons. In cortical neurons, CCS was present in the soma and proximal dendrites, as well as some axons. Although the distribution of CCS paralleled that of SOD1, there was a 12‐30‐fold molar excess of SOD1 over CCS. That both SOD1 and CCS are present, together, in cells that degenerate in ALS also emphasizes the potential role of CCS in mutant SOD1‐mediated toxicity.
Cytokine Induction of Inducible Nitric Oxide Synthase in an Oligodendrocyte Cell Line
Authors: Bhat Narayan R. , Zhang Peisheng , Bhat Aruna N.
Published: March 2003

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen‐activated protein kinase (MAPK) activation and cytokine‐mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor‐α (TNFα), interleukin‐1 (IL‐1), and interferon‐γ (IFNγ), had either minimal (TNFα) or no (IL‐1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT‐PCR analysis, the expression of iNOS. TNFα and IL‐1 were able to activate p38 MAPK in a time‐ and dose‐dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL‐1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination‐treated cells in a dose‐dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine‐treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro‐L‐arginine methyl ester, had a minimal protective effect against cytokine‐mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.
Insulin Transiently Increases Tau Phosphorylation
Authors: Lesort Mathieu , Jope Richard S. , Johnson Gail V. W.
Published: March 2003

Abstract : The modulation of tau phosphorylation in response to insulin was examined in human neuroblastoma SH‐SY5Y cells. Insulin treatment resulted in a transient increase in tau phosphorylation followed by a decrease in tau phosphorylation that correlated directly with a sequential activation and deactivation of glycogen synthese kinase‐3β (GSK‐3β). The insulin‐induced increase in tau phosphorylation and concurrent activation of GSK‐3β was rapid (<2 min) and transient, and was associated with increased tyrosine phosphorylation of GSK‐3β. The increase in GSK‐3β tyrosine phosphorylation corresponded directly to an increase in the association of Fyn tyrosine kinase with GSK‐3β, and Fyn immunoprecipitated from cells treated with insulin for 1 min phosphorylated GSK‐3β to a significantly greater extent than Fyn immunoprecipitated from control cells. Subsequent to the increase in GSK‐3β activation and tau phosphorylation, treatment of cells with insulin for 60 min resulted in a dephosphorylation of tau and a decrease in GSK‐3β activity. Thus, insulin rapidly and transiently activated GSK‐3β and modulated tau phosphorylation, alterations that may contribute to neuronal plasticity.
Up‐Regulation of Protein Chaperones Preserves Viability of Cells Expressing Toxic Cu/Zn‐Superoxide Dismutase Mutants Associated with Amyotrophic Lateral Sclerosis
Authors: Bruening Wendy , Roy Josée , Giasson Benoit , Figlewicz Denise A , Mushynski Walter E , Durham Heather D
Published: March 2003

Abstract : Mutations in the Cu/Zn‐superoxidedismutase (SOD‐1) gene underlie some familial cases of amytotrophic lateral sclerosis, a neurodegenerative disorder charactreized by loss of cortical, brainstem, and spinal motor nrurons. We present evidence that SOD‐1 mutants alter the activity of molecular chaperones that aid in proper protein folding and targeting of abnormal proteins for degradation. In a cultured cell line (NOH 3T3), resistance to mutant SOD‐1 toxicity correlated with increased overall chaperoning activity (measured by the ability of cytosolic extracts to prevent heat denaturation of catalase) as well as with up‐regulation of individual chaperones/stress proteins. In transgenic mice expressing human SOD‐1 with the G93A mutation, chaperoning activity was decreased in lumbar spinal cord but increased or unchanged in clinically unaffected tissues. Increasing the level of the stress‐inducible chaperone 70‐kDa heat shock protein by gene transfer reduced formation of mutant SOD‐containing proteinaceous aggregates in cultured primary motor neurons expressing G93A SOG‐1 and prolonged their survival. We propose that insufficiency of molecular chaperones may be directly involved in loss of motor neurons in this disease.
Dependence of Neurones on Astrocytes in a Coculture System Renders Neurones Sensitive to Transforming Growth Factor \gb\1‐Induced Glutamate Toxicity
Authors: Brown David R
Published: March 2003

Abstract : Transforming growth factor β1 (TGF‐β1) has been implicated in formation of astrocyte scars, which prevents axonal regeneration. A coculture system of astrocytes and cerebellar cells was used to investigate possible neurotoxic effects of TGF‐β1. Although not directly neurotoxic, TGF‐β1 was toxic to cerebellar cells in the presence of astrocytes. This toxicity is based on an effect of the cytokine on astrocytes, as conditioned medium from astrocyte cultures treated with TGF‐β1 was more toxic by a similar mechanism. This neurotoxicity was mediated by glutamate present in the culture medium as demonstrated by inhibition by MK‐801. Astrocytic ability to metabolise glutamate was compromised by TGF‐β1, as this cytokine increased glutamate concentration. The astrocytes in the coculture system responded to the presence of neurones by secreting neuroprotective interleukin‐6, which was partly protective against the TGF‐β1‐induced toxicity. In the coculture system, neurones responded to the presence of astrocytes by a reduction in resistance to glutamate toxicity. On addition of TGF‐β1, which compromised astrocytic clearance of glutamate, this reduction in resistance to glutamate toxicity led to a reduction in neuronal survival. These results suggest that when neurones are cocultured with astrocytes they become dependent on astrocytes for survival. This dependence makes neurones susceptible to damage when astrocytes are activated by substances such as TGF‐β1.
Structure and Function of the Third Intracellular Loop of the 5‐Hydroxytryptamine2A Receptor: The Third Intracellular Loop Is α‐Helical and Binds Purified Arrestins
Authors: Gelber Edward I. , Kroeze Wesley K. , Roth Bryan L. , Gray John A. , Sinar Christine A. , Hyde Edward G. , Gurevich Vsevolod , Benovic Jeffrey , Roth Bryan L.
Published: March 2003

Abstract: Understanding the precise structure and function of the intracellular domains of G protein‐coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5‐hydroxytryptamine2A (5‐HT2A) receptor, a model Gαq‐coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5‐HT2A receptor, a region previously implicated in G‐protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with α‐helical and β‐loop, ‐turn, and ‐sheet structure. Using random peptide phage libraries, we identified several arrestin‐like sequences as i3‐interacting peptides. We subsequently found that all three known arrestins (β‐arrestin, arrestin‐3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5‐HT2A receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3‐arrestin interactions. Dual‐label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (β‐arrestin or arrestin‐3) and 5‐HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5‐HT2A receptor represents a structurally ordered domain composed of α‐helical and β‐loop, ‐turn, and ‐sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5‐HT2A receptors in vivo.
Cyclic AMP‐Responsive Element Binding Protein in Brain Mitochondria
Authors: Cammarota Martín , Paratcha Gustavo , Bevilaqua Lia R. M. , De Stein Miguelina Levi , Lopez Margarita , De Iraldi Amanda Pellegrino , Izquierdo Iván , Medina Jorge H.
Published: March 2003

Abstract: Cyclic AMP‐responsive element binding protein (CREB) is critically involved in many important brain functions, including the formation of long‐term memory. CREB is the best characterized member of a family of transcription factors (CREB/ATF family) recognized to be important nuclear targets for intracellular signal transduction systems. Here we show, by using different approaches, that CREB is unexpectedly localized to mitochondria of the rat brain. Controlled subcellular fractionation of hippocampus and cerebral cortex showed that both synaptic and nonsynaptic mitochondria exhibited immunoreactivity to the phosphorylated form of CREB (pCREB). Moreover, CREB extracted from synaptic mitochondria is able to be phosphorylated by the catalytic subunit of protein kinase A and dephosphorylated by protein phosphatase 1 or 2B. DNA mobility shift assays showed the presence of binding activity to the calcium—cyclic AMP‐responsive element in mitochondrial extracts from hippocampus; this binding complex was specifically supershifted by an anti‐CREB antibody. Immunoelectron microscopic analysis of hippocampal subcellular fractions revealed that pCREB immunoreactivity is localized in close association with the inner mitochondrial membrane. These results, together with recent findings describing the presence and phosphorylation of CREB in developing dendrites, suggest that CREB may participate in different mechanisms involved in the communication between extracellular signals and the expression of genes.
Truncation Releases Olfactory Receptors from the Endoplasmic Reticulum of Heterologous Cells
Authors: Gimelbrant Alexander A. , Stoss Thomas D. , Landers Teresa M. , McClintock Timothy S.
Published: March 2003

Abstract: Olfactory receptors are difficult to express functionally
in heterologous cells. We found that olfactory receptors traffic poorly to the
plasma membrane even in cells with neuronal phenotypes, including cell lines
derived from the olfactory epithelium. Other than mature olfactory receptor
neurons, few cells appear able to traffic olfactory receptors to the plasma
membrane. In human embryonic kidney 293 cells and Xenopus fibroblasts, olfactory receptor immunoreactivity overlapped with a marker for the endoplasmic reticulum (ER) but not with markers for the Golgi apparatus or endosomes. Except for the ER, olfactory receptors were therefore absent from organelles normally involved in the plasma membrane trafficking of receptors. Olfactory receptors truncated prior to transmembrane domain VI were expressed in the plasma membrane, however. Co‐expression of the missing C‐terminal fragment with these truncated receptors prvented their expression in the plasma membrane. Intramolecular interactions between N‐ and C‐terminal domains joined by the third cytoplasmic loop appear to be responsible for retention of olfactory receptors in the ER of heterologous cells. Our results are consistent with misfolding of the receptors but could also be explained by altered trafficking of the receptors.
[1‐13C]Glucose Metabolism in the Tumoral and Nontumoral Cerebral Tissue of a Glioma‐Bearing Rat
Authors: Bouzier Anne‐Karine , Quesson Bruno , Valeins Henri , Canioni Paul , Merle Michel
Published: March 2003

Abstract: C6 cells were used to establish a glioma‐bearing rat model
by stereotaxic injection in the left caudate nucleus. The tumor status was
evaluated by magnetic resonance imaging and conventional histology. The
glioma‐bearing rats were infused for 1 h with a [1‐13C]glucose
solution. Perchloric acid extracts of the tumor and the ipsilateral and
contralateral hemispheres were analyzed by 13C‐NMR spectroscopy.
The 13C‐labeling patterns in compounds, mainly amino acids, indicated no drastic modification of carbon metabolism in both ipsilateral and contralateral hemispheres, as compared with control rats, whereas profound metabolic differences between brain tissue and tumor were observed. Glutamine C4 enrichment was lower in the glioma than in the brain [mean ± SD values, 5.4 ± 2.3 (n = 5) and 15.0 ± 0.8% (n = 10), respectively] and also lower than the glutamate C4 enrichment in the glioma (mean ± SD value, 22.6 ± 4.2%; n = 5), indicating that tumor glutamine was neither synthesized inside the glioma nor taken up from the surrounding brain. The glutamine C4 enrichment in the serum (6.7 ± 0.5%; n = 10) suggested that the glioma imported glutamine from the blood, a process probably connected with angiogenesis.
Role of Conserved Serine Residues in the Interaction of Agonists with D3 Dopamine Receptors
Authors: Sartania Nana , Strange Philip G.
Published: March 2003

Abstract: To understand the role of conserved serine residues in the
fifth transmembrane domain (Ser192, Ser193, and
Ser196) of the D3 dopamine receptor, these have been
mutated individually to alanine, and the ligand binding properties of the
mutant receptors have been evaluated. The mutations had little or no effect on
the binding of the antagonist spiperone and the agonist quinpirole, indicating
that the overall conformation of the receptor was unaffected. The binding of
dopamine and 7‐hydroxydipropylaminotetralin, agonists containing hydroxyl
groups, was, however, of lower affinity for the Ser192 mutation but
unaffected by the other mutations (Ser193 and Ser196).
Therefore, for the agonists tested, the hydroxyl groups interact exclusively
with Ser192.
Molecular Cloning of Testican‐2
Authors: Vannahme Christian , Schübel Sabine , Herud Marcus , Gösling Silke , Hülsmann Hanni , Paulsson Mats , Hartmann Ursula , Maurer Patrik
Published: March 2003

Abstract: We have screened a human cDNA library using an expressed sequence tag related to the BM‐40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin‐like domain, a Ca2+‐binding domain, a thyroglobulin‐like domain, and a C‐terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican‐1 and was termed testican‐2. Testican‐1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican‐2 showed a 6.1‐kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+‐binding EF‐hand domain and the thyroglobulin‐like domain of testican‐2 showed a reversible Ca2+‐dependent conformational change in circular dichroism studies. Testican‐1 and ‐2 form a novel Ca2+‐binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.
Brain‐Derived Neurotrophic Factor Stimulates Interactions of Shp2 with Phosphatidylinositol 3‐Kinase and Grb2 in Cultured Cerebral Cortical Neurons
Authors: Yamada Masashi , Ohnishi Hiroshi , Sano Shin‐ichiro , Araki Toshiyuki , Nakatani Atsushi , Ikeuchi Toshihiko , Hatanaka Hiroshi
Published: March 2003

Abstract: Shp2, a protein tyrosine phosphatase possessing SH2 domains, is utilized in the intracellular signaling of various growth factors. Shp2 is highly expressed in the CNS. Brain‐derived neurotrophic factor (BDNF), a member of the neurotrophin family, which also shows high levels of expression in the CNS, exerts neurotrophic and neuromodulatory effects in CNS neurons. We examined how BDNF utilizes Shp2 in its signaling pathway in cultured cerebral cortical neurons. We found that BDNF stimulated coprecipitation of several tyrosinephosphorylated proteins with anti‐Shp2 antibody and that Grb2 and phosphatidylinositol 3‐kinase (PI3‐K) were coprecipitated with anti‐Shp2 antibody in response to BDNF. In addition, both anti‐Grb2 and anti‐PI3‐K antibodies coprecipitated Shp2 in response to BDNF. The BDNF‐stimulated coprecipitation of the tyrosine‐phosphorylated proteins, Grb2, and PI3‐K with anti‐Shp2 antibody was completely inhibited by K252a, an inhibitor of TrkB receptor tyrosine kinase. This BDNF‐stimulated Shp2 signaling was markedly sustained as well as BDNF‐induced phosphorylation of TrkB and mitogen‐activated protein kinases. In PC12 cells stably expressing TrkB, both BDNF and nerve growth factor stimulated Shp2 signaling similarly to that by BDNF in cultured cortical neurons. These results indicated that Shp2 shows cross‐talk with various signaling molecules including Grb2 and PI3‐K in BDNF‐induced signaling and that Shp2 may be involved in the regulation of various actions of BDNF in CNS neurons.
Leukemia Inhibitory Factor Is an Autocrine Survival Factor for Schwann Cells
Authors: Dowsing Bruce J. , Morrison Wayne A. , Nicola Nicos A. , Starkey Graham P. , Bucci Tamara , Kilpatrick Trevor J.
Published: March 2003

Abstract: Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up‐regulated by Schwann cells at the injury site. LIF receptors are also up‐regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit β and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti‐LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.
Activation of Nuclear Factor κB by Nitric Oxide in Rat Striatal Neurones
Authors: Simpson C. S. , Morris B. J.
Published: March 2003

Abstract:Nitric oxide (NO), an intercellular messenger in the brain, has been implicated in both neuronal plasticity and neurotoxicity. It has been suggested that NO can activate the DNA binding activity of nuclear factor κB (NF‐κB) family proteins in some cell types while having an inhibitory effect in others. In this study we have investigated the effect of acute NO in primary neuronal cultures of rat striatum using immunohistochemistry. Exposure of neurones to the NO‐mimeticS‐nitroso‐n‐acetylpenicillamine (SNAP; 200 μM) and to bacterial lipopolysaccharide (LPS; 10 μg/ml) for 30 min increased nuclear protein expression of the p50 subunit of NF‐κB. SNAP also enhanced nuclear protein expression of the p65 subunit of NF‐κB. Simultaneously, the cytoplasmic expression of phosphorylated inhibitory protein IκBα was dramatically increased by SNAP (200 μM), LPS (10 μg/ml), and kainate (50 μM) treatment. In the adult rat, stimulation with NOR‐3 (2 mg/kg), a NO donor, increased NF‐κB DNA binding activity in the striatum after 45 min. Because glucocorticoids inhibit NF‐κB activity, primary cultures were pretreated with dexamethasone (50 μM) before SNAP, LPS, and kainate treatment, and the effect on the protein expression level of the individual subunits p50 and p65 present in the classical form of the transcription factor NF‐κB was assessed. Dexamethasone pretreatment resulted in a marked reduction of p65 protein in striatal neurones after SNAP, LPS, and kainate, whereas p50 expression was reduced by dexamethasone pretreatment only after an LPS stimulus. This study indicates that NO‐releasing compounds can directly induce nuclear NF‐κB subunit expression in rat striatum and that glucocorticoids selectively inhibit p65 subunit expression following exposure to NO.
Caspase‐3 Expression by Cerebellar Granule Neurons Is Regulated by Calcium and Cyclic AMP
Authors: Moran Julio , Itoh Takayuki , Reddy Usha R. , Chen Mei , Alnemri Emad S. , Pleasure David
Published: March 2003

Abstract : Caspase‐3 enzyme activity is induced, and cell death

follows, when cerebellar granule neurons (CGNs) from 8‐day‐old rats are

transferred from an extracellular concentration of 25 mM

K+ (25 mM [K+]e) to 5 mM

[K+]e. Death of these neurons is diminished by an

inhibitor of caspase‐3 but not by an inhibitor of caspase‐1. Actinomycin D and

cycloheximide inhibit induction of caspase‐3 and prevent death. Experiments in

which CGN intracellular Ca2+ concentration

([Ca2+]i) was manipulated by either changing

[K+]e or adding a voltage‐gated Ca2+ channel

antagonist or a Ca2+ ionophore to the medium showed that caspase‐3

mRNA rises 2.5‐fold when [Ca2+]i is diminished from 300

to 150 nM, with a corresponding rise in peak caspase enzyme activity.

Whereas the caspase‐3 mRNA level does not rise further with a still greater

diminution in [Ca2+]i, peak caspase enzyme activity

continues to increase, reaching sevenfold induction when

[Ca2+]i is reduced to 55 nM. In CGNs in which

[Ca2+]i is set at 55 nM by incubation in 5

mM [K+]e, treatment with forskolin or dibutyryl

3′,5′‐cyclic adenosine‐5′‐monophosphate delays caspase‐3

induction and diminishes death but does not alter

[Ca2+]i. We conclude that, in immature CGNs, both

caspase‐3 transcription and the subsequent processing of caspase‐3 are induced

by a fall in [Ca2+]i. Elevating cyclic AMP content

delays caspase‐3 induction by a mechanism that does not require an increase in

[Ca2+]i.
p53 Induction by Tumor Necrosis Factor‐α and Involvement of p53 in Cell Death of Human Oligodendrocytes
Authors: Ladiwala Uma , Li Hewei , Antel Jack P. , Nalbantoglu Josephine
Published: March 2003

Abstract : Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor‐α (TNF‐α) has been implicated as a mediator of OL cell injury. TNF‐α is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF‐α mediates cell death is through the activation of c‐jun N‐terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF‐α leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF‐α‐induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF‐α treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus‐mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF‐α‐mediated cell death of human adult OLs.