bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of issues in administering library services to nursing studies students at Glasgow Caledonian University
Authors: Crawford John
Published: March 2003

Glasgow Caledonian University has had a Scottish Office pre‐registration nursing and midwifery contract since 1996. Nursing studies students seemed dissatisfied with the library service and there were frequent complaints. A major study was undertaken during 2000 consisting of: an initial lis‐link enquiry, separate analysis of returns from nursing studies students of the Library’s annual general satisfaction survey (conducted every February), separate analysis of returns from nursing studies students of the Library’s opening hours planning survey, and four focus groups held in October 2000. These studies showed the concerns of nursing studies students to be similar to other students but more strongly felt. The four main issues were textbook availability, journal availability, opening hours and staff helpfulness. Working conditions, placement requirements, study requirements and domestic circumstances were all found to be important factors. IT skill levels tended to be low but there is a growing appreciation of the need for training in this area.

Concluded that:

Library’s services to nursing studies students have become enmeshed with the problems of delivery and assessment of education for nurses.

Greatly extended opening hours are essential including evening opening during vacations.

The problem of access to textbooks is so severe that conventional solutions are not going to work. Programmes of core text digitization and the promotion of e‐books are needed.

Reciprocal access programmes with local hospital libraries is essential.
A first class knowledge service: developing the National electronic Library for Health
Authors: Turner Alison , Fraser Veronica , Muir Gray J. A. , Toth Ben
Published: March 2003

With the introduction of clinical governance and the move towards evidence‐based practice, there is a growing need to provide health professionals and patients, and the public, with high quality information. Developments in the health service have added weight and urgency to this need. The National electronic Library for Health (NeLH) has a key role to play in providing health professionals with a core knowledge base of accredited and evaluated information. The Pilot NeLH was launched in November and this article outlines the progress and achievements made during this time. The NeLH is based around a central website featuring core resources and links to commissioned specialist collections. Over 70 information resources, including bibliographic databases and full text publications, are accessible via the NeLH, which aims to act as a one‐stop shop to support evidence‐based decision‐making. Much work has been undertaken on national procurement and licensing, particularly in partnership with National Health Service (NHS) libraries. Partnerships as a whole are crucial to ensure true seamless access for health professionals. Key partners include NHS libraries, NHS Direct Online and the electronic Library for Social Care. A short glossary is included for those readers less familiar with current health service developments in the UK.
Decreased Hippocampal Expression, but Not Functionality, of GABAB Receptors After Transient Cerebral Ischemia in Rats
Authors: Francis Joseph , Zhang Yu , Ho Warren , Wallace M. Christopher , Zhang Liang , Eubanks James H.
Published: March 2003

Abstract : We have examined the effects of transient global ischemia
on both the gene expression levels and the functionality of GABAB
receptors in rat brain, using antisense in situ hybridization and
electrophysiological evaluations. At the level of gene expression, no
significant change in GABAB receptor expression was observed in any
hippocampal subfield at either 6 or 12 h after challenge. At 24 h
postchallenge, however, a significant decrease in GABAB receptor
expression was observed in both the CA1 and CA3 subfields, whereas no change
was observed in the dentate granule cell layer. Although expression in both
the vulnerable CA1 and less vulnerable CA3 subfields was diminished at this
time postchallenge, there was no significant difference in the degree of the
diminished expression between these subfields. At the functional level, the
dose‐dependent ability of baclofen (1‐100 μM) to inhibit an evoked
excitatory postsynaptic potential (f‐EPSP) in the CA1 subfield was evaluated
at 24 h postischemia, in comparison with the dose‐response observed in
sham‐operated subjects. No significant differences were observed in the
efficacy of GABAB receptor‐mediated inhibition of the elicited
f‐EPSP at any of the baclofen concentrations examined. These data demonstrate
that although the mRNA expression levels for the GABAB receptor are
diminished in both vulnerable and less vulnerable neurons of Ammon's horn at
24 h following transient global ischemia, the functionality of the
GABAB receptor system is maintained at this time postchallenge.
Hierarchical Phosphorylation of Recombinant Tau by the Paired‐Helical Filament‐Associated Protein Kinase Is Dependent on Cyclic AMP‐Dependent Protein Kinase
Authors: Jicha Gregory A. , Donnell Audrey O' , Weaver Charles , Angeletti Ruth , Davies Peter
Published: March 2003

Abstract : Immunoaffinity‐purified paired helical filaments (PHFs)
from Alzheimer's disease (AD) brain homogenates contain an associated protein
kinase activity that is able to induce the phosphorylation of PHF proteins on
addition of exogenous MgCl2 and ATP. PHF kinase activity is shown
to be present in immunoaffinity‐purified PHFs from both sporadic and familial
AD, Down's syndrome, and Pick's disease but not from normal brain homogenates.
Although initial studies failed to show that the kinase was able to induce the
phosphorylation of tau, additional studies presented in this article show that
only cyclic AMP‐dependent protein kinase‐pretreated recombinant tau is a
substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide
mapping, and site‐directed mutagenesis have identified the PHF kinase
phosphorylation sites as amino acids Thr361 and Ser412
in htau40. In addition, the cyclic AMP‐dependent protein kinase
phosphorylation sites that direct the PHF kinase have been mapped to amino
acids Ser356 and Ser409 in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD‐derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.
Adenovirus‐Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons
Authors: Simons M. , Beinroth S. , Gleichmann M. , Liston P. , Korneluk R. G. , MacKenzie A. E. , Bähr M. , Klockgether T. , Robertson G. S. , Weller M. , Schulz J. B.
Published: March 2003

Abstract : The inhibitor of apoptosis (IAP) family of anti‐apoptotic
genes, originally discovered in baculovirus, exists in animals ranging from
insects to humans. Here, we investigated the ability of IAPs to suppress cell
death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar
granule neurons undergo apoptosis when switched from 25 to 5 mM
potassium, and excitotoxic cell death in response to glutamate. We examined
the endogenous expression of four members of the IAP family, X
chromosome‐linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis
inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot
analysis in cultured cerebellar granule neurons. Cerebellar granule neurons
express significant levels of RIAP2 mRNA and protein, but expression of RIAP1,
NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not
change when cells were switched from 25 to 5 mM potassium. To
determine whether ectopic expression of IAP influenced neuronal survival after
potassium withdrawal or glutamate exposure, we used recombinant adenoviral
vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar
granule neurons. We demonstrate that forced expression of IAPs efficiently
blocked potassium withdrawal‐induced
N‐acetly‐Asp‐Glu‐Val‐Asp‐specific caspase activity and reduced DNA
fragmentation. However, neurons were only protected from apoptosis up to 24 h
after potassium withdrawal, not at later time points suggesting that IAPS
delay but do not block apoptosis in cerebellar granule neurons. In contrast,
treatment with 100 μM or 1 mM glutamate did not induce caspase activity and adenoviral‐mediated expression of IAPs had no influence on subsequent excitotoxic cell death.
Peroxynitrite‐Induced Alterations in Synaptosomal Membrane Proteins
Authors: Koppal Tanuja , Drake Jennifer , Yatin Servet , Jordan Brad , Varadarajan Sridhar , Bettenhausen Lori , Butterfield D. Allan
Published: March 2003

Abstract : Peroxynitrite (ONOO‐) is a highly reactive,
oxidizing anion with a half‐life of <1 s that is formed by reaction of
superoxide radical anion with nitric oxide. Several reports of
ONOO‐ ‐induced oxidation of lipids, proteins, DNA, sulfhydryls, and
inactivation of key enzymes have appeared. ONOO‐ has also been
implicated as playing a role in the pathology of several neurodegenerative
disorders, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis,
among others. Continuing our laboratory's interest in free radical oxidative
stress in brain cells in AD, the present study was designed to investigate the
damage to brain neocortical synaptosomal membrane proteins and the
oxidation‐sensitive enzyme glutamine synthetase (GS) caused by exposure to
ONOO‐. These synaptosomal proteins and GS have previously been
shown by us and others to have been oxidatively damaged in AD brain and also
following treatment of synaptosomes with amyloid β‐peptide. The results
of the current study showed that exposure to physiological levels of
ONOO‐ induced significant protein conformational changes,
demonstrated using electron paramagnetic resonance in conjunction with a
protein‐specific spin label, and caused oxidation of proteins, measured by the
increase in protein carbonyls. ONOO‐ also caused inactivation of GS
and led to neuronal cell death examined in a hippocampal cell culture system.
All these detrimental effects of ONOO‐ were successfully attenuated
by the thiol‐containing antioxidant tripeptide glutathione. This research
shows that ONOO‐ can oxidatively modify both membranous and
cytosolic proteins, affecting both their physical and chemical nature. These
findings are discussed with reference to the potential involvement of
ONOO‐ in AD neurodegeneration.
The pH‐Sensitive Dye Acridine Orange as a Tool to MonitorExocytosis/Endocytosis in Synaptosomes
Authors: Zoccarato Franco , Cavallini Lucia , Alexandre Adolfo
Published: March 2003

Abstract : We introduce the use of the pH‐sensitive dye acridine
orange (AO) to monitor exo/endocytosis of acidic neurotransmitter‐containing
vesicles in synaptosomes. AO is accumulated exclusively in acidic
v‐ATPase‐dependent bafilomycin (Baf)‐sensitive compartments. A fraction of the
accumulated AO is rapidly released (fluorescence increase) upon depolarization
with KCl in the presence of Ca2+. The release (completed in 5‐6 s)
is followed by reuptake to values below the predepolarization baseline. The
reuptake, but not the release, is inhibited by Baf added 5 s prior to KCl. In
a similar protocol, Baf does not affect the initial fast phase of glutamate
release measured enzymatically, but it abolishes the subsequent slow phase.
Thus, the fast AO release corresponds to the rapid phase of glutamate release
and the slow phase depends on vesicle cycling. AO reuptake depends in part on
the progressive accumulation of acid‐loaded vesicles during cycling. Stopping
exocytosis at selected times after KCl by Ca2+ removal with EGTA
evidences endocytosis : Its T1/2 was 12 ± 0.6 s.
The KA+, channel inhibitors 4‐aminopyridine (100
μM) and α‐dendrotoxin (10‐100 nM) are known to
induce glutamate release by inducing the firing of Na+ channels ;
their action is potentiated by the activation of protein kinase C. Also these
agents promote a Ca2+‐dependent AO release, which is prevented by
the Na+ channel inhibitor tetrodotoxin and potentiated by
4β‐phorbol 12‐myristate 13‐acetate (PMA). With α‐dendrotoxin,
endocytosis was monitored by stopping exocytosis at selected times with EGTA
or alternatively with Cd2+ or tetrodotoxin. The
T1/2 of endocytosis, which was unaffected by PMA, was 12
± 0.4 s with EGTA and Cd2+ and 9.5 ± 0.5 s with tetrodotoxin. Protein kinase C activation appeared to facilitate vesicle turnover.
Structure and Functional Characterization of a Novel Human Low‐Voltage Activated Calcium Channel
Authors: Williams Mark E. , Washburn Mark S. , Hans Michael , Urrutia Arturo , Brust Paul F. , Prodanovich Patricia , Harpold Michael M. , Stauderman Kenneth A.
Published: March 2003

Abstract : We have isolated and characterized overlapping cDNAs
encoding a novel, voltage‐gated Ca2+ channel α1
subunit, α1H, from a human medullary thyroid carcinoma cell
line. The α1H subunit is structurally similar to previously
described α1 subunits. Northern blot analysis indicates that
α1H mRNA is expressed throughout the brain, primarily in the
amygdala, caudate nucleus, and putamen, as well as in several nonneuronal
tissues, with relatively high levels in the liver, kidney, and heart.
Ba2+ currents recorded from human embryonic kidney 293 cells
transiently expressing α1H activated at relatively
hyperpolarized potentials (‐50 mV), rapidly inactivated (τ = 17 ms), and
slowly deactivated. Similar results were observed in Xenopus oocytes
expressing α1H. Singlechannel measurements in human embryonic
kidney 293 cells revealed a single‐channel conductance of ~9 pS. These
channels are blocked by Ni2+ (IC50 = 6.6 μM)
and the T‐type channel antagonists mibefradil (~50% block at 1 μM)
and amiloride (IC50 = 167 μM). Thus,
α1H‐containing channels exhibit biophysical and
pharmacological properties characteristic of low voltage‐activated, or T‐type,
Ca2+ channels.
Contribution of Metabotropic Glutamate Receptor mGluR4 to L‐2‐[3H]Amino‐4‐Phosphonobutyrate Binding in Mouse Brain
Authors: Thomsen Christian , Hampson David R.
Published: March 2003

Abstract : The binding of
L‐2‐[3H]amino‐4‐phosphonobutyrate ([3H]L‐AP4) was
examined in brain sections of wild‐type mice and mice lacking the mGluR4
subtype of metabotropic glutamate receptors (mGluRs). Very high relative
densities of [3H]L‐AP4 binding were observed in the molecular layer
of the cerebellar cortex, the nucleus basalis, the outer layer of the superior
colliculus, and the substantia nigra. In mGluR4 knock‐out mice, very low
levels of binding were observed in these regions. The moderate levels of
binding observed with wild‐type mice in the molecular layer of the hippocampal
dentate gyrus and in the thalamus were absent in mGluR4 knock‐out mice. In
contrast, the moderate levels observed in most of the cerebral cortex, caudate
putamen, and globus pallidus were not different in mGluR4 knock‐out mice
compared with wild‐type. In these regions, mGluR8 is likely to be labeled by
[3H]L‐AP4 because mGluR8 is expressed in such brain regions and,
like mGluR4, has high affinity for L‐AP4. We conclude that mGluR4 contributes
substantially to the high‐affinity binding site for [3H]L‐AP4 in several regions of mouse brain, including cerebellar cortex, nucleus basalis, thalamus, superior colliculus, substantia nigra, and hippocampal dentate gyrus.
Clostridium Neurotoxins Influence Serotonin Uptake and Release Differently in Rat Brain Synaptosomes
Authors: Najib Abderrahim , Pelliccioni Patricia , Gil Carles , Aguilera José
Published: March 2003

Abstract: Clostridium neurotoxins produce inhibition of both basal and K+‐evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37°C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2+‐metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium‐dependent, high‐affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2+‐metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C‐terminal portion of the TeTx heavy chain (Hc‐TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose‐dependent manner. The IC50 of HC‐TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high‐affinity site at the serotonin transporter or at other serotonin high‐affinity sites. These results could indicate that TeTx and HC‐TeTx bind to different targets than BoNT/A in the plasma membrane.
Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes
Authors: Aleu Jordi , Barat Ana , Burgos Javier S. , Solsona Carles , Marsal Jordi , Ramírez Galo
Published: March 2003

Abstract: Injection of chick cerebellar membranes, rich in kainate binding sites, into Xenopus oocytes resulted in the structural integration of chick membrane patches into the oocyte plasma membrane that could be easily identified by specific immunofluorescent staining. Application of kainate to the oocyte perfusion medium, under voltage‐clamp conditions, induced dose‐dependent (EC50= 87 ± 14 μM) inward currents, confirming the functional incorporation to the oocyte of kainate‐driven channels. Responses to kainate were consistently nondesensitizing and strongly potentiated by cyclothiazide, suggesting the selective involvement of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)‐preferring receptors. Binding experiments with (S)‐[3H]AMPA confirmed the presence in the chick membrane preparation of low‐affinity AMPA receptors (KD= 278 nM) amounting to <2% of the total population of kainate binding sites. A tenfold concentration of guanine nucleotides, with different degrees of phosphorylation, blocked the responses to 100 μM kainate by ∼90%. In the case of GMP, additional concentration‐inhibition studies yielded an IC50 of 180 ± 11 μM. Our results illustrate the apparent failure of kainate‐binding proteins to form functional channels, even when maintaining their own native membrane environment, and confirm the antagonistic behavior of guanine nucleotides, including GMP, toward glutamate receptors, in agreement with previous results of ligand‐binding experiments and, more interestingly, with the marked neuroprotective effects of some guanine nucleotides in different excitotoxicity experimental paradigms.
Inhibition of Cholesterol Production but Not of Nonsterol Isoprenoid Products Induces Neuronal Cell Death
Authors: Michikawa Makoto , Yanagisawa Katsuhiko
Published: March 2003

Abstract: Deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, has been known to cause an inhibition of DNA synthesis and cell growth, and to induce apoptosis in nonneuronal cells. To investigate whether this is also the case in neurons, we examined the effect of a 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor on the viability of neuronal cultures prepared from fetal rat brains. Treatment with compactin, a competitive inhibitor of HMG‐CoA reductase, induced neuronal death in a dose‐dependent manner. Concurrent treatment with cholesterol, β‐migrating very low density lipoprotein, mevalonate, or squalene substantially inhibited the induction of neuronal death by compactin. Cell death was also induced by treatment with squalestatin, which specifically inhibits cholesterol biosynthesis at a site down‐stream from the generation of nonsterol metabolites. Furthermore, squalestatin‐induced neuronal death was inhibited by concurrent incubation with squalene but not mevalonate. In contrast, cell growth of proliferating cells such as NIH 3T3 and PC12 cells was exclusively dependent on the level of nonsterol isoprenoid products and not that of cholesterol. The results of this study clearly indicate that the viability of neurons, different from that of nonneuronal cells, depends on the intracellular cholesterol content and not on the intermediate nonsterol isoprenoid products.
Prostate Apoptosis Response‐4 Production in Synaptic Compartments Following Apoptotic and Excitotoxic Insults
Authors: Duan Wenzhen , Rangnekar Vivek M. , Mattson Mark P.
Published: March 2003

Abstract: Synapses are often located at great distances from the cell body and so must be capable of transducing signals into both local and distant responses. Although progress has been made in understanding biochemical cascades involved in neuronal death during development of the nervous system and in various neurodegenerative disorders, it is not known whether such cascades function locally in synaptic compartments. Prostate apoptosis response‐4 (Par‐4) is a leucine zipper and death domain‐containing protein that plays a role in neuronal apoptosis. We now report that Par‐4 levels are rapidly increased in cortical synaptosomes and in dendrites of hippocampal neurons in culture and in vivo, following exposure to apoptotic or excitotoxic insults. Par‐4 expression is regulated at the translational level within synaptic compartments. Par‐4 antisense treatment suppressed mitochondrial dysfunction and caspase activation in synaptosomes and prevented death of cultured hippocampal neurons following exposure to excitotoxic and apoptotic insults. Local translational regulation of death‐related proteins in synaptic compartments may play a role in programmed cell death, adaptive remodeling of synapses, and neurodegenerative disorders.
A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase
Authors: Ezaki Junji , Tanida Isei , Kanehagi Nobuo , Kominami Eiki
Published: March 2003

Abstract: The specific accumulation of the hydrophobic protein,
subunit c of ATP synthase, in lysosomes from the cells of patients with the
late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by
lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2
gene) has been identified recently. To elucidate the mechanism of lysosomal
storage of subunit c, antibodies against the human CLN2 gene product
(Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46‐kDa
protein in normal control skin fibroblasts and carrier heterozygote cells,
whereas it was absent in cells from four patients with LINCL. RT‐PCR analysis
indicated the presence of mRNA for CLN2 in cells from the four
different patients tested, suggesting a low efficiency of translation of mRNA
or the production of the unstable translation products in these patient cells.
Pulse‐chase analysis showed that Cln2p was synthesized as a 67‐kDa precursor
and processed to a 46‐kDa mature protein (t1/2 = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high‐density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the β subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.
Differential Coupling of Serotonin 5‐HT1A and 5‐HT1B Receptors to Activation of ERK2 and Inhibition of Adenylyl Cyclase in Transfected CHO Cells
Authors: Mendez Jorge , Kadia Tapan M. , Somayazula Ravi K. , El‐Badawi Khaled I. , Cowen Daniel S.
Published: March 2003

Abstract: Although the subtypes of serotonin 5‐HT1 receptors have distinct structure and pharmacology, it has not been clear if they also exhibit differences in coupling to cellular signals. We have sought to compare directly the coupling of 5‐HT1A and 5‐HT1B receptors to adenylyl cyclase and to the mitogen‐activated protein kinase ERK2 (extracellular signal‐regulated kinase‐2). We found that 5‐HT1B receptors couple better to activation of ERK2 and inhibition of adenylyl cyclase than do 5‐HT1A receptors. 5‐HT stimulated a maximal fourfold increase in ERK2 activity in nontransfected cells that express endogenous 5‐HT1B receptors at a very low density and a maximal 13‐fold increase in transfected cells expressing 230 fmol of 5‐HT1B receptor/mg of membrane protein. In contrast, activation of 5‐HT1A receptors stimulated only a 2.8‐fold maximal activation of ERK2 in transfected cells expressing receptors at 300 fmol/mg of membrane protein but did stimulate a 12‐fold increase in activity in cells expressing receptors at 3,000 fmol/mg of membrane protein. Similarly, 5‐HT1A, but not 5‐HT1B, receptors were found to cause significant inhibition of forskolin‐stimulated cyclic AMP accumulation only when expressed at high densities. These findings demonstrate that although both 5‐HT1A and 5‐HT1B receptors have been shown to couple to G proteins of the Gi class, they exhibit differences in coupling to ERK2 and adenylyl cyclase.
Inhibition of Peroxynitrite‐Mediated Oxidation of Dopamine by Flavonoid and Phenolic Antioxidants and Their Structural Relationships
Authors: Kerry Nicole , Rice‐Evans Catherine
Published: March 2003

Abstract: The interaction between peroxynitrite and dopamine and the inhibition of this reaction by plant‐derived antioxidants have been investigated. Peroxynitrite promoted the oxidation of dopamine to 6‐hydroxyindole‐5‐one as characterised by HPLC and photodiode array spectra, akin to the products of the tyrosinase‐dopamine reaction, but no evidence of dopamine nitration was obtained. Although peroxynitrite did not cause nitration of dopamine in vitro, the catecholamine is capable of inhibiting the formation of 3‐nitrotyrosine from peroxynitrite‐mediated nitration of tyrosine. The plant‐derived phenolic compounds, caffeic acid and catechin, inhibited peroxynitrite‐mediated oxidation of dopamine. This effect is attributed to the ability of catechol‐containing antioxidants to reduce peroxynitrite through electron donation, resulting in their oxidation to the corresponding o‐quinones. The antioxidant effect of caffeic acid and catechin was comparable to that of the endogenous antioxidant, glutathione. In contrast, the structurally related monohydroxylated hydroxycinnamates, p‐coumaric acid and ferulic acid, which inhibit tyrosine nitration through a mechanism of competitive nitration, did not inhibit peroxynitrite‐induced dopamine oxidation. The findings of the present study suggest that certain plant‐derived phenolics can inhibit dopamine oxidation.
Internalization and Proteolytic Action of Botulinum Toxins in CNS Neurons and Astrocytes
Authors: Verderio Claudia , Coco Silvia , Rossetto Ornella , Montecucco Cesare , Matteoli Michela
Published: March 2003

Abstract: Tetanus and botulinum toxins bind and are internalized at the neuromuscular junction. Botulinum neurotoxins (BoNTs) enter the cytosol at the motor nerve terminal; tetanus neurotoxin (TeNT) proceeds retroaxonally inside the motor axon to reach the spinal cord inhibitory interneurons. Although the major target of BoNTs is the peripheral cholinergic terminals, CNS neurons are susceptible to intoxication as well. We investigated the route of entry and the proteolytic activity of BoNT/B and BoNT/F in cultured hippocampal neurons and astrocytes. We show that, differently from TeNT, which enters hippocampal neurons via the process of synaptic vesicle (SV) recycling, BoNTs are internalized and cleave the substrate synaptobrevin/VAMP2 via a process independent of synaptic activity. Labeling of living neurons with Texas Red‐conjugated BoNTs and fluoresceinated dextran revealed that these toxins enter hippocampal neurons via endocytic processes not mediated by SV recycling. Botulinum toxins also exploit endocytosis to enter cultured astrocytes, where they partially cleave cellubrevin, a ubiquitous synaptobrevin/VAMP isoform. These results indicate that, in spite of their closely related protein structure, TeNT and BoNTs use different routes to penetrate hippocampal neurons. These findings bear important implications for the identification of the protein receptors of clostridial toxins.
Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins
Authors: Stasi A. M. Michela Di , Mallozzi Cinzia , Macchia Gianfranco , Petrucci Tamara C. , Minetti Maurizio
Published: March 2003

Abstract : Peroxynitrite, the product of the radical‐radical

reaction between nitric oxide and superoxide anion, is a potent oxidant

involved in tissue damage in neurodegenerative disorders. We investigated the

modifications induced by peroxynitrite in tyrosine residues of proteins from

synaptosomes. Peroxynitrite treatment (≥50 μM) induced tyrosine

nitration and increased tyrosine phosphorylation. Synaptophysin was identified

as one of the major nitrated proteins and pp60src kinase

as one of the major phosphorylated substrates. Further fractionation of

synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas

phosphorylated pp60src was enriched in the postsynaptic

density fraction. Tyrosine phosphorylation was increased by treatment with

50‐500 μM peroxynitrite and decreased by higher concentrations,

suggesting a possible activation/inactivation of kinases. Immunocomplex kinase

assay proved that peroxynitrite treatment of synaptosomes modulated the

pp60src autophosphorylation activity. The addition of

bicarbonate (CO2 1.3 mM) produced a moderate enhancing

effect on some nitrate proteins but significantly protected the activity of

pp60src against peroxynitrite‐mediated inhibition so that

at 1 mM peroxynitrite, the kinase was still more active than in

untreated synaptosomes. The phosphotyrosine phosphatase activity of

synaptosomes was inhibited by peroxynitrite (≥50 μM) but

significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrite‐mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre‐ and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.
The Amino Terminus with a Conserved Glutamic Acid of G Protein‐Coupled Receptor Kinases Is Indispensable for Their Ability to Phosphorylate Photoactivated Rhodopsin
Authors: Yu Qing‐Ming , Cheng Zhi‐Jie , Gan Xiao‐Qing , Bao Guo‐Bin , Li Lin , Pei Gang
Published: March 2003

Abstract : To investigate functions of the consensus amino terminus of G protein‐coupled receptor kinases (GRKs), two amino terminus‐truncated mutants (▵30 or ▵15) and two single‐amino‐acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single‐point mutation (E7A) greatly decreased GRK1’s activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (▵30 or ▵15) of GRK2, representative of another GRK subfamily, also abolished the kinase’s activity toward Rho*. The similar single‐point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329‐rhodopsin‐augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein‐coupled receptors.
The Detoxification of Cumene Hydroperoxide by the Glutathione System of Cultured Astroglial Cells Hinges on Hexose Availability for the Regeneration of NADPH
Authors: Kussmaul Lothar , Hamprecht Bernd , Dringen Ralf
Published: March 2003

Abstract : The ability of astroglia‐rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione‐mediated peroxide detoxification by astrocytes. Under the conditions used, 200 μM CHP disappeared from the incubation buffer with a half‐time of ~ 10 min. The half‐time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinae, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocirate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.
Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury
Authors: Franz G. , Reindl M. , Patel S. C. , Beer R. , Unterrichter I. , Berger T. , Schmutzhard E. , Poewe W. , Kampfl A.
Published: March 2003

Abstract : Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague‐Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively : Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.
High Expression of the γ5 Isoform of G Protein in Neuroepithelial Cells and Its Replacement of the γ2 Isoform During Neuronal Differentiation in the Rat Brain
Authors: Morishita Rika , Shinohara Haruo , Ueda Hiroshi , Kato Kanefusa , Asano Tomiko
Published: March 2003

Abstract : High concentrations of G proteins, which include multiple

isoforms of each subunit, α, β, and γ, are expressed in the

adult brain. In this study, we concentrated attention on changes of these

isoforms during embryonic development in the rat brain. Concentrations of

γ2 as well as GoAα, GoBα,

and β2 were low in early embryogenesis and then increased,

whereas expression of γ5, in contrast, was initially high

followed by a drop, with only very low levels observed throughout postnatal

development. Among the other isoforms, Gi1α,

Gsα‐short, G12α, G13α,

β4, γ3, γ7, and

γ12 were present in the embryonic brain at low levels, but

their levels markedly increased after birth. In contrast, the levels of

Gi2α, Gsα‐long, Gq/11α, and

β1 were essentially constant throughout. Immunohistochemical

staining of the brain vesicles in the embryos showed γ5 to be

specifically expressed in the proliferative region of the ventricular zone,

whereas γ2 was mainly present in differentiated neuronal

cells of the marginal zone. Furthermore, differentiation of P19 mouse

embryonal carcinoma cells to neuronal cells with retinoic acid induced the

expression of γ2 and a decrease of γ5, the

major isoform in the undifferentiated state. These results suggest that

neuronal differentiation is responsible for the on/off switch of the

expression of γ2 and γ5 subunits.
Bax and Bcl‐2 Interaction in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis
Authors: Vukosavic Slobodanka , Dubois‐Dauphin Michel , Romero Norma , Przedborski Serge
Published: March 2003

Abstract : It has been proposed that mutations in copper/zinc‐superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl‐2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS‐linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl‐2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl‐2, Bcl‐XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl‐2 and Bcl‐XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl‐2 in transgenic mSOD1 mice, overexpression of Bcl‐2 increases the formation of Bcl‐2 : Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl‐2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl‐2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl‐2 : Bax heterodimers. Based on these findings, it may be concluded that Bcl‐2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.
Activation of the Endothelin Receptor Inhibits the G Protein‐Coupled Inwardly Rectifying Potassium Channel by a Phospholipase A2‐Mediated Mechanism
Authors: Rogalski Sherri L. , Cyr Curt , Chavkin Charles
Published: March 2003

Abstract: To develop a malleable system to model the well‐described, physiological interactions between Gq/11‐coupled receptor and Gi/o‐coupled receptor signaling, we coexpressed the endothelin A receptor, the μ‐opioid receptor, and the G protein‐coupled inwardly rectifying potassium channel (Kir 3) heteromultimers in Xenopus laevis oocytes. Activation of the Gi/o‐coupled μ‐opioid receptor strongly increased Kir 3 channel current, whereas activation of the Gq/11‐coupled endothelin A receptor inhibited the Kir 3 response evoked by μ‐opioid receptor activation. The magnitude of the inhibition of Kir 3 was channel subtype specific; heteromultimers composed of Kir 3.1 and Kir 3.2 or Kir 3.1 and Kir 3.4 were significantly more sensitive to the effects of endothelin‐1 than heteromultimers composed of Kir 3.1 and Kir 3.5. The difference in sensitivity of the heteromultimers suggests that the endothelin‐induced inhibition of the opioid‐activated current was caused by an effect at the channel rather than at the apioid receptor. The endothelin‐1‐mediated inhibition was mimicked by arachidonic acid and blocked by the phospholipase A2 inhibitor arachidonoyl trifluoromethyl ketone. Consistent with a possible phospholipase A2‐mediated mechanism, the endothelin‐1 effect was blocked by calcium chelation with BAPTA‐AM and was not affected by kinase inhibition by either staurosporine or genistein. The data suggest the hypothesis that Gq/11‐coupled receptor activation may interfere with Gi/o‐coupled receptor signaling by the activation of phospholipase A2 and subsequent inhibition of effector function by a direct effect of an eicosanoid on the channel.
Distinct Secretases, a Cysteine Protease and a Serine Protease, Generate the C Termini of Amyloid β‐Proteins Aβ1‐40 and Aβ1‐42, Respectively
Authors: Figueiredo‐Pereira Maria E. , Efthimiopoulos Spiros , Tezapsidis Nikolaos , Buku Angeliki , Ghiso Jorge , Mehta Pankaj , Robakis Nikolaos K.
Published: March 2003

Abstract: The carboxy‐terminal ends of the 40‐ and 42‐amino acids amyloid β‐protein (Aβ) may be generated by the action of at least two different proteases termed γ(40)‐ and γ(42)‐secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)‐transfected cell cultures with several dipeptidyl aldehydes including N‐benzyloxycarbonyl‐Leu‐leucinal (Z‐LL‐CHO) and the newly synthesized N‐benzyloxycarbonyl‐Val‐leucinal (Z‐VL‐CHO). All dipeptidyl aldehydes tested inhibited production of both Aβ1‐40 and Aβ1‐42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of γ‐secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two γ‐secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E‐64d. This agent inhibited production of secreted Aβ1‐40, with a concomitant accumulation of its cellular precursor indicating that γ(40)‐secretase is a cysteine protease. In contrast, this treatment increased production of secreted Aβ1‐42. No inhibition of Aβ production was observed with the potent calpain inhibitor I (acetyl‐Leu‐Leu‐norleucinal), suggesting that calpain is not involved. Together, these results indicate that γ(40)‐secretase is a cysteine protease distinct from calpain, whereas γ(42)‐secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the β‐secretase C‐terminal APP fragment and a decrease of the α‐secretase C‐terminal APP fragment, indicating that this mutation shifts APP cleavage from the α‐secretase site to the β‐secretase site.
Characterization of Detergent‐Insoluble Complexes Containing the Familial Alzheimer’s Disease‐Associated Presenilins
Authors: Parkin Edward T. , Hussain Ishrut , Karran Eric H. , Turner Anthony J. , Hooper Nigel M.
Published: March 2003

Abstract: Many cases of early‐onset familial Alzheimer’s disease have been linked to mutations within two genes encoding the proteins presenilin‐1 and presenilin‐2. The presenilins are 48‐56‐kDa proteins that can be proteolytically cleaved to generate an N‐terminal fragment (∼25‐35 kDa) and a C‐terminal fragment (∼17‐20 kDa). The N‐ and C‐terminal fragments of presenilin‐1, but not full‐length presenilin‐1, were readily detected in both human and mouse cerebral cortex and in neuronal and glioma cell lines. In contrast, presenilin‐2 was detected almost exclusively in cerebral cortex as the full‐length molecule with a molecular mass of 56 kDa. The association of the presenilins with detergent‐insoluble, low‐density membrane microdomains, following the isolation of these structures from cerebral cortex by solubilization in Triton X‐100 and subsequent sucrose density gradient centrifugation, was also examined. A minor fraction (10%) of both the N‐ and C‐terminal fragments of presenilin‐1 was associated with the detergent‐insoluble, low‐density membrane microdomains, whereas a considerably larger proportion of full‐length presenilin‐2 was present in the same membrane microdomains. In addition, a significant proportion of full‐length presenilin‐2 was present in a high‐density, detergent‐insoluble cytoskeletal pellet enriched in β‐actin. The presence of the presenilins in detergent‐insoluble, low‐density membrane microdomains indicates a possible role for these specialized regions of the membrane in the lateral separation of Alzheimer’s disease‐associated proteins within the lipid bilayer and/or in the distinct functions of these proteins.
Pyrrolidine Dithiocarbamate Induces Bovine Cerebral Endothelial Cell Death by Increasing the Intracellular Zinc Level
Authors: Kim Chul Hoon , Kim Joo Hee , Xu Jan , Hsu Chung Y. , Ahn Young Soo
Published: March 2003

Abstract: The antioxidant and metal‐chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serumdepleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o‐phenanthroline, bathophenanthroline disulfonic acid, and N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum‐deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as cooper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC‐induced endothelial cell death was greater than that of copper. Zn‐EDTA did not block PDTC‐induced cell death, whereas Ca‐EDTA and Cu‐EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N‐(6‐methoxy‐8‐quinolyl)‐p‐toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn‐EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC‐induced cerebral endothelial cell death.