bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth response of Mountain birch to air and soil temperature: is increasing leaf‐nitrogen content an acclimation to lower air temperature?
Authors: Weih M. , Karlsson P. S.
Published: March 2003

Growth and nitrogen (N) economy of mountain birch are reported here in response to temperature change. Mechanisms of temperature effects on plant growth in temperate–arctic regions are discussed in the light of decreasing growth rates and increasing leaf‐N contents along altitudinal and latitudinal temperature gradients.

Mountain birch ( Betula pubescens ssp. czerepanovii) seedlings were grown at two soil temperatures, air temperatures and nutrient concentrations in a full‐factorial experiment during one growing season in northern Sweden.

Changes in air and soil temperature affected aboveground growth more than belowground growth. An increase in air temperature increased leaf area ratio and plant‐N productivity while decreasing plant‐N concentration and leaf‐N content. A change in soil temperature affected root‐N uptake rate and plant‐N concentration, similar to the effect of a change in nutrient supply. Air and soil temperature had interactive effects on growth rate, N productivity and leaf‐N content.

The results indicate that increasing leaf‐N content with increasing altitude and latitude is not only a passive consequence of weaker N dilution by reduced growth, but also a physiological acclimation to lower air temperature.
Leaf gas exchange responses of 13 prairie grassland species to elevated CO2 and increased nitrogen supply
Authors: Lee Tali D. , Tjoelker Mark G. , Ellsworth David S. , Reich Peter B.
Published: March 2003

Leaf gas exchange responses to elevated CO2 and N are presented for 13 perennial species, representing four functional groups: C3 grasses, C4 grasses, legumes, and nonleguminous forbs. Understanding how CO2 and N effects interact is important to predict plant community response to global change.

 Plants were field‐grown in monoculture under current ambient and elevated (560 µmol mol−1) CO2 concentrations (free‐air CO2 enrichment), in combination with soil N treatments, for two growing seasons.

 All species, regardless of functional group, showed pronounced photosynthetic acclimation to elevated CO2, resulting in minimal stimulation of photosynthesis (A) averaging +15% in C3 grasses, +8% in forbs, +7% in legumes and −2% in C4 grasses. The effects of CO2 and soil N supply did not interact for any leaf traits measured. Elevated CO2 consistently decreased stomatal conductance (gs) leading to 40% increase in A/gs.

 This substantial acclimation of photosynthesis was greater in magnitude than in most field studies, and was associated with the combined effects of decreased gs and decreased leaf N concentrations in response to growth under elevated CO2.
Evaluation of a maltodextrin gel as a partial replacement for fat in a high‐ratio white‐layer cake
Authors: Conforti Frank D. , Archilla Leslie
Published: March 2003

The performance of maltodextrin gel as a replacement (25%, 50%, 75% and 100%) for shortening along with sucrose and high fructose corn syrup‐90 (HFCS‐90), adjusted for sweetness in each treatment, was evaluated in a high‐ratio white‐layer cake formulation. Two controls were used as a reference to fat‐replaced cakes: control A (100% fat and 100% sucrose) and control B (100% fat with 50% sucrose/50% HFCS‐90), which closely matched the sugar system of the fat‐replaced cakes. Volume of cakes showed that treatment F (100% fat‐substituted) was significantly (P< 0.05) different from the other treatments. Crust and crumb L‐ and b‐values indicated that control B produced a dark crust (P< 0.05) with a light crumb (P< 0.05) whereas treatment E (75% fat substituted) produced a light crust (P< 0.05) and treatment F a darker crumb (P< 0.05) with a significantly (P< 0.05) higher moisture content. Degree of staling significantly (P< 0.05) increased over time for all treatments. Sensory results indicated that treatment F produced a significantly (P< 0.05) moister, less adhesive and cohesive cake. Tenderness and sweetness scores indicated that treatments E and F were significantly (P< 0.05) tougher and less sweet, respectively, when compared with the other treatments. Results from both the physical and sensory analyses indicated that the combination of maltodextrin gel up to a 75% reduction for shortening and sucrose/HFCS‐90 resulted in satisfactory cakes.
The role of consumers in product‐oriented environmental policy: can the consumer be the driving force for environmental improvements?
Authors: Niva Mari , Timonen Päivi
Published: March 2003

In environmental policy, it is increasingly accepted that more emphasis should be placed on consumption and its implications from the point of view of the environment. Another relatively new feature is the focus on products. At the policy level, this perspective is known as product‐oriented environmental policy or, in brief, product policy. This approach is closely related to the idea of product chain thinking, which means recognizing the fact that environmentally relevant decisions are made at all stages during the products’ life cycle, from raw material extraction to consumption and beyond. Based on a Finnish study on product chain actors and environmental improvements, this article discusses the role of consumers in product policy (i) with respect to theories on consumer mecision‐making and (ii) in the light of product chain thinking. As consumers’ decision‐making models with respect to consumer products are most often based on heuristics simplifying the decision process, incorporating environmental considerations into these models is a challenging task for environmental policy.
Attitudes and consumption values of consumers of imported fruit in Guangzhou, China
Authors: Sun Ximing , Collins Ray
Published: March 2003

Research on how Chinese consumption values influence Chinese consumer behaviour is rare. First, this paper examines consumers’ attitudes towards the physical and intangible attributes of imported fruit. Then, it identifies consumers’ consumption values and the role of these values in purchasing behaviour. Data were collected through point of sale intercept surveys conducted in Guangzhou, China. Latent consumption values of consumers were identified through factor analysis. K‐means clustering revealed four natural groupings of consumers, each group demonstrating different consumption values. The results demonstrated the primary importance of symbolic values and hedonic values in the decision to purchase imported fruit. Such consumption values may derive from the intermingling of Confucian and Western cultural values. Results from this study could help to better understand interrelationships among product attributes, consumption values and cultural values, and could make a significant contribution in developing strategies to market imported fruit in China.
The physically disabled South African female consumer’s problems in purchasing clothing
Authors: Klerk Helena M. de , Ampousah Lucy
Published: March 2003

Physically disabled women today have to face the inconvenience of shopping for their own as well as their families’ clothes and the inability of finding fashionable clothes that fit any disability. A total of 40 physically disabled women between the ages of 21 years and 60 years were interviewed on the problems they experienced when shopping for fashionable clothes that fit their disabilities. A total of 40 clothing shops were observed to get information on the extent to which the shops cater for the needs of the physically disabled consumer. Results from the interviews showed that the majority of physically disabled female clothing consumers experienced major problems in finding fashionable clothes that fit their disabilities. It was also clear that these women experienced problems with unusable access routes, unsuitable parking lots, display racks that are too high, spaces between racks that are too narrow, inadequate space in fitting rooms and a lack of assistance in shops. The observations were compared with the complaints the women had and it was found that these women had not overestimated their problems.
Use of hydrogen peroxide detection strips to determine the extent of pasteurization in whole milk
Authors: Marks Nicholette E , Grandison Alistair S , Lewis Michael J
Published: March 2003

Commercially available hydrogen peroxide (H2O2) detection strips were shown to be effective in determining known levels of H2O2 in milks where the lactoperoxidase system (LPS) had been inactivated. In addition, the strips were evaluated for determination of residual H2O2 in LPS‐activated milks, following a range of heat treatments. The detection of residual H2O2 corresponded with results of zero lactoperoxidase activity. Hence, the detection strips offer a simple and accurate method for detecting the absence of lactoperoxidase, and therefore can be used to determine whether the milk has been subjected to overpasteurization. The technique provides a more convenient method than the standard procedure based on oxidation of 1,4‐phenylenediamine.
The effect of plasmin activity and cold storage of cheese milk on the composition, ripening and functionality of mozzarella‐type cheese
Authors: Somers Joanne M , Guinee Timothy P , Kelly Alan L
Published: March 2003

The effects of casein hydrolysis by plasmin on composition, ripening and functionality in mozzarella‐type cheese were studied. Milk was stored at 4°C for 0, 24 and 48 h, with and without added plasmin, before batch pasteurization and the manufacturing of mozzarella‐type cheese. In parallel experiments, the effects of incubation of milk with added plasmin for 2 h at 37°C prior to cheese manufacture and of an increase in the level of rennet added during cheesemaking were studied. Plasmin addition adversely affected the rennet coagulation properties of the milk and led to increased (two‐ to three‐fold) plasmin activity in cheese and greater plasmin‐associated hydrolysis of β‐casein to γ‐caseins during ripening. Melted cheese flow rate and stretchability increased over ripening and melting time decreased, but functional properties were not affected by either plasmin activity or cold storage. Overall, neither plasmin activity (at the levels studied) nor cold storage greatly affected the composition or functionality of mozzarella‐type cheese.
Biocontrol of Listeria monocytogenes in a model cultured milk (lben) by in situ bacteriocin production from Lactococcus lactis ssp. lactis
Authors: Benkerroum Noreddine , Ghouati Yasmine , Ghalfi Hakim , Elmejdoub Thami , Roblain Dominique , Jacques Philippe , Thonart Philippe
Published: March 2003

Bacteriocin‐producing (Bac+) Lactococcus lactis ssp. lactis CCMM/IAV/BK1 isolated from traditional lben was used in the preparation of lben from pasteurized milk to assess its potential inhibitory activity against Listeria monocytogenes ATCC 7644. Production of bacteriocin (arbitrary units, AU) in MRS broth fortified with yeast extract (MRSY) in a fermentor under controlled and uncontrolled pH conditions was also investigated. This Bac+ strain yielded about 35 times more bacteriocin when the pH was maintained constant at 6.5 than under varying pH conditions. To test the effect of in situ bacteriocin production against L. monocytogenes, lben was made from cow's milk artificially contaminated with approximately 107 cfu/mL and fermented with a mixed mesophilic starter culture consisting of the lactococcal Bac+ organism and Lc. lactis ssp. lactis biovar diacetylactis 66, a diacetyl‐producing strain, in a ratio of 1 : 1. Numbers of L. monocytogenes were monitored during fermentation and storage of lben at refrigeration temperature (c. 7°C) for up to 6 days. Performances of the Bac+ starter were compared to those of an isogenic Bac− derivative strain obtained from the Bac+ starter by curing with ethidium bromide. The results showed that the amount of L. monocytogenes decreased to below the detectable level in a 1‐mL sample within 24 h of storage at 7°C in lben fermented with the Bac+ starter culture. On the contrary, L. monocytogenes survived for 6 days of storage at 7°C in lben made with the Bac− starter. The Bac+ wild strain of the starter studied could be adequately used to produce lben or similar indigenous fermented milks of improved hygienic quality on an industrial scale. Alternatively, it could be used as an adjunct in minimally processed products or in products obtained from raw milk to add a safety factor.
Evidence that Brain‐Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA‐Induced Inactivation of Protein Kinase C in Cortical Neurons
Authors: Tremblay R , Hewitt K. , Lesiuk H. , Mealing G. , Morley P , Durkin J. P.
Published: March 2003

Abstract : Several lines of evidence indicate that a rapid loss of
neuronal protein kinase C (PKC) activity is a characteristic feature of
cerebral ischemia and is a necessary step in the NMDA‐induced death of
cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50
μM NMDA or 50 μM glutamate for 10 min caused ~80% cell
death over the next 24 h, but excitotoxic death was largely averted, i.e., by
70‐80%, in cells pretreated with brain‐derived neurotrophic factor (BDNF). An
8‐h preexposure to BDNF (50‐100 ng/ml) maximally protected cortical cells from
the effects of NMDA and glutamate, although the transient application of BDNF
between 8 and 4 h before NMDA was equally protective. These effects of BDNF
were abolished at supralethal, i.e., >100 μM, NMDA
concentrations. It is significant that BDNF pretreatment prevented the
inactivation of PKC in cortical cells normally seen 30 min to 2 h following
lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes
in NMDA channel activity because neither whole‐cell NMDA current amplitudes
nor increases in intracellular free Ca2+ concentration were altered
by the 8‐h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to
cells treated with the PKC inhibitors staurosporine (10‐20 nM),
calphostin C (1‐2.5 μM), or GF‐109203X (100 nM) at the
time of NMDA addition. These results underscore the importance of PKC
inactivation in glutamate‐induced neuronal death. They also suggest that BDNF
neuroprotection arises, at least in part, via its ability to block the
mechanism by which pathophysiological Ca2+ influx through the NMDA receptor causes membrane PKC inactivation.
β‐Amyloid Peptide Interacts Specifically with the Carboxyl‐Terminal Domain of Human Apolipoprotein E
Authors: Pillot Thierry , Goethals Marc , Najib Jamilla , Labeur Christine , Lins Laurence , Chambaz Jean , Brasseur Robert , Vandekerckhove Joel , Rosseneu Maryvonne
Published: March 2003

Abstract : Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late‐onset and sporadic forms of Alzheimer's disease, although its exact role remains unclear. We previously demonstrated that β‐amyloid peptide (Aβ) displays membrane‐destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy‐terminal lipid‐binding domain of apoE (e.g., residues 200‐299) is responsible for the Aβ‐binding activity of apoE and that this interaction involves pairs of apoE amphipathic α‐helices. We further demonstrate that Aβ is able to inhibit the association of the C‐terminal domain of apoE with lipids due to the formation of Aβ/apoE complexes resistant to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. On the contrary, the amino‐terminal receptor‐binding domain of apoE (e.g., residues 129‐169) is not able to form stable complexes with Aβ. These data extend our understanding of human apoE‐dependent binding of Aβ by involving the C‐terminal domain of apoE in the efficient formation of apoE/Aβ complex.
Interactions of the C Terminus of Metabotropic Glutamate Receptor Type 1α with Rat Brain Proteins
Authors: Ciruela F. , Robbins M. J. , Willis A. C. , McIlhinney R. A. J.
Published: March 2003

Abstract : Metabotropic glutamate receptors (mGluRs) are coupled to
G protein second messenger pathways and modulate glutamate neurotransmission
in the brain, where they are targeted to specific synaptic locations. As part
of a strategy for defining the mechanisms for the specific targeting of mGluR1
α, rat brain proteins which interact with the intracellular carboxy
terminus of mGluR1 α have been characterized, using affinity
chromatography on a glutathione S‐transferase fusion protein that
contains the last 86 amino acids of mGluR1 α. Three of the proteins
specifically eluted from the affinity column yielded protein sequences, two of
which were identified as glyceraldehyde‐3‐phosphate dehydrogenase and
β‐tubulin ; the other was an unknown protein. The identity of tubulin was
confirmed by western immunoblotting. Using a solid‐phase binding assay, the
mGluR1 α‐tubulin interaction was shown to be direct, specific, and
saturable with a KD of 2.3 ± 0.4 μM. In addition, mGluR1 α, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti‐β‐tubulin antibodies. However, mGluR1 α could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl‐terminal tail of mGluR1 α are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.
Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro
Authors: Fryer Hugh J. L. , Knox Ronald J. , Strittmatter Stephen M. , Kalb Robert
Published: March 2003

Abstract : We have used cultures of purified embryonic rat spinal
cord motor neurons to study the neurotoxic effects of prolonged ionotropic
glutamate receptor activation. NMDA and non‐NMDA glutamate receptor agonists
kill a maximum of 40% of the motor neurons in a concentration‐ and
time‐dependent manner, which can be blocked by receptor subtype‐specific
antagonists. subunit‐specific antibodies stain all of the motor neurons with
approximately the same intensity and for the same repertoire of subunits,
suggesting that the survival of the nonvulnerable population is unlikely to be
due to the lack of glutamate receptor expression. Extracellular
Ca2+ is required for excitotoxicity, and the route of entry
initiated by activation of non‐NMDA, but not NMDA, receptors is L‐type
Ca2+ channels. Ca2+ imaging of motor neurons after
application of specific glutamate receptor agonists reveals a sustained rise
in intracellular Ca2+ that is present to a similar degree in most
motor neurons, and can be blocked by appropriate receptor/channel antagonists.
Although the lethal effects of glutamate receptor agonists are seen in only a
subset of cultured motor neurons, the basis of this selectivity is unlikely to
be simply the glutamate receptor phenotype or the level/pattern of rise in
agonist‐evoked intracellular Ca2+.
Differential Effects of Glycolipid Biosynthesis Inhibitors on Ceramide‐Induced Cell Death in Neuroblastoma Cells
Authors: Bieberich Erhard , Freischütz Bettina , Suzuki Minoru , Yu Robert K
Published: March 2003

Abstract : An in vitro model of Gaucher's disease in murine
neuroblastoma X rat glioma NG108‐15 cells was used to investigate the
physiological effects of two specific inhibitors of glucosylceramide synthase,
d,l‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol
(d,l‐PDMP) and N‐butyldeoxynojirimycin (NB‐DNJ), which have
been suggested as agents for treatment of glycolipid storage disorders.
Incubation of NG108‐15 cells with conduritol‐B‐epoxide, a covalent inhibitor
of glucosylce‐ramidase, raised the intracellular concentration of
glucosylceramide (GC) by more than fourfold, indicating a glycolipid
composition equivalent to that of Gaucher's cells. The level of GC was
decreased, and the cells were depleted of gangliosides by postincubation with
d,l‐PDMP or NB‐DNJ. Treatment with d,l‐PDMP, but not with
NB‐DNJ, resulted in a dose‐dependent reduction of the growth rate and
eventually caused cell death in NG108‐15 cells on reaching confluency. An in
situ detection assay using terminal nucleotidyltransferase indicated that cell
degeneration was accompanied by apoptosis. Lipid analysis by high‐performance
TLC revealed that on incubation with d,l‐PDMP, but not with NB‐DNJ,
the concentration of endogenous ceramide was elevated by threefold. Ceramide
elevation and apoptosis were also observed when NG108‐15 cells were incubated
with daunorubicin, which was previously reported to induce programmed cell
death by stimulation of ceramide synthesis. Structural characterization by
HPLC and subsequent laser desorption mass spectrometry revealed that the
endogenous ceramide contained fatty acids with chain lengths ranging from C14
: 0 to C24 : 0. The results indicate that elevation of levels of these
ceramide species by incubation with d,l‐PDMP or daunorubicin induces
programmed cell death in NG108‐15 cells. Because ceramide accumulation and
cell death were not observed on incubation with NB‐DNJ, its use is suggested
to be less toxic than that of d,l‐PDMP for treatment of Gaucher's disease and other sphingolipid storage disorders.
Antidepressants Noncompetitively Inhibit Nicotinic Acetylcholine Receptor Function
Authors: Fryer John D , Lukas Ronald J
Published: March 2003

Abstract : Nicotinic acetylcholine receptors (nAChRs) are diverse
members of the neurotransmitter‐gated ion channel superfamily and play
critical roles in chemical signaling throughout the nervous system. The
present study establishes for the first time the acute functional effects of
sertraline (Zoloft), paroxetine (Paxil), nefazodone (Serzone), and venlafaxine
(Effexor) on two human and one chick nAChR subtype. This study also confirms
previous findings of nAChR functional block by fluoxetine (Prozac). Function
of human muscle‐type nAChR (α1βγδ) in TE671/RD cells,
human autonomic nAChR (α3β4α5 ±β2) in SH‐SY5Y
neuroblastoma cells, or chick V274T mutant α7‐nAChR heterologously
expressed in native nAChR‐null SH‐EP1 epithelial cells was measured using
86Rb+ efflux assays. Functional blockade of human muscle‐type and autonomic nAChRs is produced by each of the drugs in the low to intermediate micromolar range, and functional blockade of chick V274T‐α7‐nAChR is produced in the intermediate to high micromolar range. Functional blockade is insurmountable by increasing agonist concentrations at each nAChR subtype tested for each of these drugs, suggesting noncompetitive inhibition of nAChR function. These studies open the possibilities that nAChR subtypes in the brain could be targets for therapeutic antidepressants and could play roles in clinical depression.
A Role for Cyclic AMP in Entrainment of the Circadian Oscillator in Xenopus Retinal Photoreceptors by Dopamine but Not by Light
Authors: Hasegawa Minoru , Cahill Gregory M.
Published: March 2003

Abstract: The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2‐like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP‐sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8‐(4‐chlorophenylthio)cyclic AMP (8‐CPT‐cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8‐CPT‐cAMP alone. This indicates that there is a cyclic AMP‐sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP‐insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.
Alterations of Extracellular Calcium Elicit Selective Modes of Cell Death and Protease Activation in SH‐SY5Y Human Neuroblastoma Cells
Authors: McGinnis Kim M. , Wang Kevin K. W. , Gnegy Margaret E.
Published: March 2003

Abstract: The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH‐SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase‐3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase‐3‐like, but not calpain, activity. Pan‐caspase inhibitors protected against EGTA‐, but not CaCl2−, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase‐3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death‐mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase‐3‐like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.
GC/MS Analysis of Anandamide and Quantification of N‐Arachidonoylphosphatidylethanolamides in Various Brain Regions, Spinal Cord, Testis, and Spleen of the Rat
Authors: Yang Hsiu‐Ying T. , Karoum Farouk , Felder Christian , Badger Henry , Lin Wang Tao‐Chin , Markey Sanford P.
Published: March 2003

Abstract: Anandamide [N‐arachidonoylethanolamide (NAE)] was initially isolated from porcine brain and proposed as an endogenous ligand for cannabinoid receptors in 1992. Accumulating evidence has now suggested that, in the tissue, NAE is generated from N‐arachidonoylphosphatidylethanolamides (N‐ArPEs) by phosphodiesterase. In this study a sensitive and specific procedure was developed to quantify NAE and N‐ArPE, including organic solvent extraction, reversephase C‐18 cartridge separation, derivatization, and gas chromatography/mass spectrometry (GC/MS) analysis. NAE is converted by a two‐step derivatization procedure to a pentafluorobenzoyl ester followed by pentafluoropropionyl acylation. Quantification was performed by isotope dilution GC/MS using deuterium‐labeled NAE (NAE‐2H8) as an internal standard. The same chemical derivatization was applicable to N‐ArPE quantification. The separated N‐ArPE fractions were converted by a two‐step cleavage/derivatization procedure into the pentafluorobenzoyl ester of NAE and then to its pentafluoropropionyl amide. The derivative was quantified by GC/MS using deuterium‐labeled 1,2‐[2Ha]dioleoyl‐sn‐glycero‐3‐phospho(arachidonoyl) ethanolamide as an internal standard. Using these methods, we have found that endogenous NAE levels in rat brain, spleen, testis, liver, lung, and heart were below the level of quantification achievable (0.1 pmol/mg of protein) but that N‐ArPE is readily quantifiable and is widely distributed in the rat CNS with the highest level in the spinal cord. The striatum, hippocampus, and accumbens contain intermediate concentrations of N‐ArPE, whereas the value is lowest in the cerebellum.
Effects of Moderate Hypothermia on Constitutive and Inducible Nitric Oxide Synthase Activities After Traumatic Brain Injury in the Rat
Authors: Chatzipanteli Katina , Wada Kojiro , Busto Raul , Dietrich W. Dalton
Published: March 2003

Abstract: We investigated the effects of therapeutic hypothermia (30°C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague–Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8–2.2 atm) parasagittal fluid‐percussion (F‐P) brain injury. In normothermic rats (37°C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5 ± 68.9% of contralateral value; mean ± SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8 ± 17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30 ± 8.4 and 28.6 ± 20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30°C) preserved cNOS activity at 3 and 7 days (69.5 ± 23.3 and 78.6 ± 7.6% of contralateral value, respectively; mean ± SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021 ± 0.06 and 0.23 ± 0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature‐dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.
Contrasting Calcium Dependencies of SAPK and ERK Activations by Glutamate in Cultured Striatal Neurons
Authors: Schwarzschild Michael A. , Cole Rebecca L. , Meyers Melissa A. , Hyman Steven E.
Published: March 2003

Abstract: Stress‐activated protein kinase (SAPK) and extracellular signal‐regulated kinase (ERK), both members of the mitogen‐activated protein kinase (MAPK) family, may in some circumstances serve opposing functions with respect to cell survival. However, SAPK and ERK can also be coordinately activated in neurons in response to glutamate stimulation of NMDA receptors. To explore the mechanisms of these MAPK activations, we compared the ionic mechanisms mediating SAPK and ERK activations by glutamate. In primary cultures of striatal neurons, glutamatergic activation of ERK and one of its transcription factor targets, CREB, showed a calcium dependence typical of NMDA receptor‐mediated responses. In contrast, extracellular calcium was not required for glutamatergic, NMDA receptor‐mediated activation of SAPK and phosphorylation of its substrate, c‐Jun. Increasing extracellular calcium enhanced ERK activation but reversed SAPK activation, further distinguishing the calcium dependencies of these two NMDA receptor‐mediated effects. Finally, reducing extracellular sodium prevented the glutamatergic activation of SAPK but only partially blocked that of ERK. These contrasting ionic dependencies suggest a mechanism by which NMDA receptor activation may, under distinct conditions, differentially regulate neuronal MAPKs and their divergent functions.
Glial Cells Protect Neurons Against Oxidative Stress via Transcriptional Up‐Regulation of the Glutathione Synthesis
Authors: Iwata‐Ichikawa Emi , Kondo Yoichi , Miyazaki Ikuko , Asanuma Masato , Ogawa Norio
Published: March 2003

Abstract: We examined the effects of oxidative stress on rat
cultured mesencephalic neurons and glial cells. Glial cells were more
resistant to 6‐hydroxydopamine (6‐OHDA) and H2O2
toxicity than neurons. In glial cells, incubation with 6‐OHDA and
H2O2 induced a significant increase in the expression of
γ‐glutamylcysteine synthetase (the rate‐limiting enzyme in glutathione
synthesis) mRNA, which correlated well with increased TPA‐response element
(TRE)‐binding activity. Furthermore, a subsequent elevation in cellular total
glutathione content was also observed. In neurons, both agents decreased
TRE‐binding activity, and these cells failed to up‐regulate the glutathione
synthesis. We also examined the mechanisms of the neuroprotective effects of
glial cells using a glia conditioned medium. Neurons maintained in glia
conditioned medium up‐regulated the level of TRE‐binding activity,
γ‐glutamylcysteine synthetase mRNA expression, and total glutathione
content in response to 6‐OHDA or H2O2, and became more resistant to both agents than cells maintained in a normal medium. Neurons maintained in normal medium failed to up‐regulate the glutathione synthesis. Our results suggest that transcriptional up‐regulation of glutathione synthesis in glial cell appears to mediate brain glial cell resistance against oxidative stress, and that glial cells protect neurons via transcriptional up‐regulation of the antioxidant system.
A Novel Presenilin‐2 Splice Variant in Human Alzheimer's Disease Brain Tissue
Authors: Sato Naoya , Hori Osamu , Yamaguchi Atsushi , Lambert Jean‐Charles , Chartier‐Harlin Marie‐Christine , Robinson Philip A. , Delacourte Andre , Schmidt Ann Marie , Furuyama Tatsuo , Imaizumi Kazunori , Tohyama Masaya , Takagi Tsutomu
Published: March 2003

Abstract: Mutations in the presenilin‐1 (PS‐1) and presenilin‐2
(PS‐2) genes account for the majority of cases of early‐onset familial
Alzheimer's disease (AD). Alternative splicing forms of the PS‐1 and PS‐2 gene
products have previously been reported in fibroblast and brain tissue from
both familial and sporadic AD patients, as well as from normal tissues and
cell lines. We demonstrate here unusual alternative splicing of the PS‐2 gene
that leads to the generation of mRNA lacking exon 5 in human brain tissue.
This product was more frequently detected in brain tissue from sporadic AD
patients (70.0%; 21 of 30) than from normal age‐matched controls (17.6%; three
of 17). In cultured neuroblastoma cells, this splice variant was generated in
hypoxia but not under other forms of cellular stress. Hypoxia‐mediated
induction of this splice variant was blocked by pretreatment of neuroblastoma
cells with the protein synthesis inhibitor cycloheximide or antioxidants such
as N‐acetylcysteine and diphenyl iodonium, suggesting that hypoxia‐mediated oxidant stress might, at least in part, underlie the alternative splicing of PS‐2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N‐terminal part of PS‐2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.
Depolarization of In Situ Mitochondria Due to Hydrogen Peroxide‐Induced Oxidative Stress in Nerve Terminals
Authors: Chinopoulos Christos , Tretter Laszlo , Adam‐Vizi Vera
Published: March 2003

Abstract: Mitochondrial membrane potential (Δ?m) was determined in intact isolated nerve terminals using the membrane potential‐sensitive probe JC‐1. Oxidative stress induced by H2O2 (0.1‐1 mM) caused only a minor decrease in Δ?m. When complex I of the respiratory chain was inhibited by rotenone (2 μM), Δ?m was unaltered, but on subsequent addition of H2O2, Δ?m started to decrease and collapsed during incubation with 0.5 mM H2O2 for 12 min. The ATP level and [ATP]/[ADP] ratio were greatly reduced in the simultaneous presence of rotenone and H2O2. H2O2 also induced a marked reduction in Δ?m when added after oligomycin (10 μM), an inhibitor of F0F1‐ATPase. H2O2 (0.1 or 0.5 mM) inhibited α‐ketoglutarate dehydrogenase and decreased the steady‐state NAD(P)H level in nerve terminals. It is concluded that there are at least two factors that determine Δ?m in the presence of H2O2: (a) The NADH level reduced owing to inhibition of α‐ketoglutarate dehydrogenase is insufficient to ensure an optimal rate of respiration, which is reflected in a fall of Δ?m when the F0F1‐ATPase is not functional. (b) The greatly reduced ATP level in the presence of rotenone and H2O2 prevents maintenance of Δ?m by F0F1‐ATPase. The results indicate that to maintain Δ?m in the nerve terminal during H2O2‐induced oxidative stress, both complex I and F0F1‐ATPase must be functional. Collapse of Δ?m could be a critical event in neuronal injury in ischemia or Parkinson’s disease when H2O2 is generated in excess and complex I of the respiratory chain is simultaneously impaired.
Responses of Heat Shock Proteins hsp27, αB‐Crystallin, and hsp70 in Rat Brain After Kainic Acid‐Induced Seizure Activity
Authors: Kato Kanefusa , Katoh‐Semba Ritsuko , Takeuchi Ikuo K. , Ito Hidenori , Kamei Keiko
Published: March 2003

Abstract: We determined the changes in the levels of the mammalian small heat shock protein of 25‐28 kDa (hsp27) and the hsp αB‐crystallin in various regions of rat brain after kainic acid‐induced seizure activity by means of specific immunoassays. The levels of hsp27 in the hippocampus and entorhinal cortex were markedly increased and reached a maximum (1.5‐2 μg/mg of protein) 2‐4 days after the seizure. The levels of hsp27 in these regions were considerably high even 10 days after the seizure. A marked increase in levels of mRNA for hsp27 was also observed in the hippocampus of rats 1‐2 days after the seizure. A severalfold increase in the levels of αB‐crystallin was observed in the hippocampus and entorhinal cortex of rats 2 days after the seizure. However, the maximum levels were <50 ng/mg of protein. The levels of protein sulfhydryl group and glutathione were significantly reduced in the hippocampus of rats at 24 h after the seizure, which might have enhanced the expressions of hsp27 and αB‐crystallin. The expression of inducible mammalian hsp of 70 kDa (hsp 70) was also enhanced in the hippocampus of rats after the seizure, as detected by western and northern blotting analyses. Immunohistochemically, an intensive staining of hsp27 was observed in both glial cells and neurons in the hippocampus, piriform cortex, and entorhinal cortex of rats with kainic acid‐induced seizure. However, in the cerebellum, where the receptors for kainic acid are also rich, hsp27 was barely induced in the same rats. This might be due to high levels of the cerebellar calcium‐binding proteins parvalbumin and 28‐kDa calbindin‐D, which might have a protective effect against the kainic acid‐inducible damage.
A Globin Fragment, LVV‐Hemorphin‐7, Induces [3H]Thymidine Incorporation in a Neuronal Cell Line via the AT4 Receptor
Authors: Moeller Ingrid , Albiston Anthony L. , Lew Rebecca A. , Mendelsohn Frederick A. O. , Chai Siew‐Yeen
Published: March 2003

Abstract: The AT4 receptor was characterized initially as a specific binding site for angiotensin IV, a C‐terminal fragment of the vasoactive peptide angiotensin II. Recently, we found that LVV‐hemorphin‐7, a fragment of β globin, is an abundant peptide in the brain and binds to the AT4 receptor with high affinity and specificity. In the neuroblastoma/glioma hybrid cell line, NG108‐15, LVV‐hemorphin‐7 and angiotensin IV competed for 125I‐angiotensin IV binding in a biphasic fashion with IC50 values of 1.2 × 10‐10 and 1.1 × 10‐9M for the high‐affinity site, respectively, and 6.7 × 10‐8 and 1.5 × 10‐8M for the low‐affinity site, respectively. Both peptides were internalized rapidly by the cells. However, LVV‐hemorphin‐7, but not angiotensin IV, elicited a 1.8‐fold increase in DNA synthesis in a dose‐dependent manner. Furthermore, co‐incubation of the cells with an excess of angiotensin IV (10‐6M) inhibited LVV‐hemorphin‐7‐stimulated DNA synthesis. Therefore, whereas LVV‐hemorphin‐7 and angiotensin IV were capable of binding to the AT4 receptor, only LVV‐hemorphin‐7 elicited [3H]thymidine incorporation in NG108‐15 cells. In contrast, angiotensin IV behaved as an antagonist. The current finding suggests that LVV‐hemorphin‐7 is a functional peptide in the central nervous system and in view of its abundance in neural tissue, compared with angiotensin IV, may be of significant physiological importance.
Regulation of Transmembrane Signaling by Ganglioside GM1 :
Authors: Ravichandra B. , Joshi Preeti G.
Published: March 2003

Abstract : Interaction of antibodies to ganglioside GM1 with Neuro2a

cells was studied to investigate the role of GM1 in cell signaling. Binding of

anti‐GM1 to Neuro2a cells induced the formation of 3H‐inositol

phosphates (3H‐IPs) and elevated the intracellular Ca2+

concentration [Ca2+]i. The rise in

[Ca2+]i was due to the influx of Ca2+ from

the extracellular medium and release from intracellular Ca2+ pools.

The Ca2+ influx pathway did not allow the permeation of

Na+ or K+. The influx was inhibited by amiloride, a

specific blocker of T‐type Ca2+, channels, whereas nifedipine and

diltiazem, blockers of L‐type Ca2+ channels, did not have any

effect. Thus, anti‐GM1 appears to activate a T‐type Ca2+ channel in

Neuro2a cells. The intracellular Ca2+ release was inhibited by

pretreatment of cells with neomycin sulfate, phorbol dibutyrate, and pertussis

toxin (PTx), which also inhibited the 3H‐IP formation in Neuro2a

cells. Addition of caffeine neither elevated the [Ca2+]i

nor affected the anti‐GM1‐induced [Ca2+]i rise. The data

reveal that the binding of anti‐GM1 to Neuro2a cells activates phospholipase C

via a PTx‐sensitive G protein, which leads to formation of IPs and release of

Ca2+ from inositol trisphosphate‐sensitive pool of endoplasmic

reticulum. Anti‐GM1 also arrested the differentiation of Neuro2a cells in

culture and significantly stimulated their proliferation. This stimulatory

effect of anti‐GM1 on cell proliferation was blocked by amiloride but not by

PTx, suggesting that the influx of Ca2+ was essentially required for cell proliferation. Our data suggest a role for GM1 in the regulation of transmembrane signaling events and cell growth.
Biochemical Characterization of a Lysosomal Protease Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) and Development of an Enzyme‐Based Assay for Diagnosis and Exclusion of LINCL in HUman Specimens and Animal Models
Authors: Sohar Istvan , Sleat David E. , Jadot Michel , Lobel Peter
Published: March 2003

Abstract : Classical late‐infantile neuronal ceroid lipofuscinosis

(LINCL), a progressive and fatal neurodegenerative disease of childhood,

results from mutations in a gene (CLN2) that encodes a protein with

significant sequence similarity to prokaryotic pepstatin‐insensitive acid

proteases. We have developed a sensitive protease activity assay that allows

biochemical characterization of the CLN2 gene product in various

human biological samples, including solid tissues (brain and chorionic villi),

blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells),

and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has

a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of

fibroblasts and lymphoblasts demonstrated that lack of activity was associated

with LINCL arising from mutations in the CLN2 gene but not other

neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant

LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs'

disease). A study conducted using blood samples collected from classical LINCL

families whose affliction was confirmed by genetic analysis indicates that the

assay can distinguish homozygotes, heterozygotes, and normal controls and thus

is useful for diagnosis and carrier testing. Analysis of archival specimens

indicates that several specimens previously classified as LINCL have enzyme

activity and thus disease is unlikely to arise from mutations in

CLN2. Conversely, a specimen previously classified as juvenile NCL

lacks pepinase activity and is associated with mutations in CLN2. In

addition, several animals with NCL‐like neurodegenerative symptoms [mutant

strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6‐phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.
The ligand‐selective Domains of Corticotropin‐Releasing Factor Type 1 and Type 2 Receptor Reside in Different Extracellular Domains
Authors: Dautzenberg Frank M. , Kilpatrick Gavin J. , Wille Sandra , Hauger Richard L.
Published: March 2003

Abstract : The nonselective human corticotropin‐releasing factor

(hCRF) receptor 1 (hCRFR1) and the ligand‐selective Xenopus CRFR1

(xCRFR1), xCRFR2, and hCRFR2α were compared. To understand the

interactions of hCRF, ovine CRF (oCRF), rat urocortin (rUcn), and sauvagine,

ligands with different affinities for type 1 and type 2 CRFRs, chimeric and

mutant receptors of hCRFR1, xCRFR1, hCRFR2α, and xCRFR2 were

constructed. In cyclic AMP stimulation and CRF‐binding assays, it was

established that different extracellular regions of CRFR1 and CRFR2 conferred

their ligand selectivities. The ligand selectivity of xCRFR1 resided in five

N‐terminal amino acids, whereas the N‐terminus of both CRFR2 proteins did not

contribute to their ligand selectivities. Chimeric receptors in which the

first extracellular domain of hCRFR1 replaced that of hCRFR2α or xCRFR2

showed a similar pharmacological profile to the two parental CRFR2 molecules.

Chimeric receptors carrying the N‐terminal domain of xCRFR1 linked to

hCRFR2α or xCRFR2 displayed a novel pharmacological profile. hCRF, rUcn,

and sauvagine were bound with high affinity, whereas oCRF was bound with low

affinity. Furthermore, when three or five residues of xCRFR1

(Gln76, Gly81, Val83, His88,

Leu89 ; or Gln76, Gly81, Val83) were introduced into receptor chimeras carrying the N‐terminus of hCRFR1 linked to xCRFR2, the same novel pharmacology was observed. These data indicate a compensation mechanism of two differentially selecting regions located in different domains of both xCRFR1 and CRFR2.
Oxidative Damage to the c‐fos Gene and Reduction of Its Transcription After Focal Cerebral Ischemia
Authors: Cui Jiankun , Holmes Eric H. , Liu Philip K.
Published: March 2003

Abstract : We investigated oxidative damage to the c‐fos gene and to its transcription in the brain of Long‐Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant (p < 0.001) increase in the immunoreactivity to 8‐hydroxy‐2′‐guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0‐30 min of reperfusion in all 27 animals treated with 15‐90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3‐bromo‐7‐nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c‐fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c‐fos gene, was increased 13‐fold (p < 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR‐induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.