bluuebunny/crossref_metadata_2025 huggingface dataset:
Emodin – a secondary metabolite with multiple ecological functions in higher plants
Authors: Izhaki Ido
Published: March 2003

The anthraquinone emodin, identified in 17 plant families distributed worldwide, has numerous biological activities, some of which exhibit a wide spectrum of ecological impacts by mediating biotic or abiotic interactions of plants with their environment. Here the evidence for direct and indirect effects of emodin on plant survival and reproduction is reviewed. Emodin in vegetative organs may help protect plants against herbivores, pathogens, competitors and extrinsic abiotic factors (e.g. high light intensities). In unripe fruit pulp, emodin may facilitate seed dispersal by protecting the immature fruit against predispersal seed predation whereas in ripe pulp it may deter frugivores and thus reduce the chances that seeds will be defecated beneath the parent plant. It also accelerates the passage of seeds through the digestive tract, potentially reducing dispersal distance and increasing seed viability upon dispersal. In certain circumstances both of the last two effects could also have negative fitness consequences for plants. Natural selection should favor secondary metabolites with multiple functions because they protect the plants against a variety of unpredictable biotic and abiotic environments. Such metabolites also enhance plant defenses by using different molecular targets of specific enemies through a variety of mechanisms of action. Emodin illustrates the wide and often overlooked potential for chemical multifunctionality in plant secondary metabolites.
The effects of commercial starter culture and storage temperature on the oxidative stability and diacetyl production in butter
Authors: Bakirci Ihsan , Çelik Serafettin , Özdemir Cihat
Published: March 2003

In this study, the effects of four different direct‐vat‐set (DVS) commercial starter cultures (CHN11, CHN22, EZAL BT001 and MM100) and storage temperatures (4 ± 1 and −18 ± 2°C) on the oxidative stability and diacetyl production in butter during a 4‐month period were investigated. Samples were taken from butter on days 3, 30, 60, 90 and 120. Analyses of titration acidity, peroxide value, free fatty acids and diacetyl contents of the samples taken were carried out. The effects of the starter cultures on the peroxide value and diacetyl contents were found to be significant (P < 0.01) while the storage temperatures significantly affected all parameters tested. Interaction between the type of culture and storage temperature was significant for free fatty acids (P < 0.05) and diacetyl content (P < 0.01).
Dopamine D2 Receptor‐Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum
Authors: Dickinson Shelly D. , Sabeti Jilla , Larson Gaynor A. , Giardina Karen , Rubinstein Marcelo , Kelly Michele A. , Grandy David K. , Low Malcolm J. , Gerhardt Greg A. , Zahniser Nancy R.
Published: March 2003

Abstract : Presynaptic D2 dopamine (DA) autoreceptors,
which are well known to modulate DA release, have recently been shown to
regulate DA transporter (DAT) activity. To examine the effects of
D2 DA receptor deficiency on DA release and DAT activity in dorsal
striatum, we used mice genetically engineered to have two
(D2+/+), one (D2+/‐), or no
(D2‐/‐) functional copies of the gene coding for the
D2 DA receptor. In vivo microdialysis studies demonstrated that
basal and K+‐evoked extracellular DA concentrations were similar in
all three genotypes. However, using in vivo electrochemistry, the
D2‐/‐ mice were found to have decreased DAT function,
i.e., clearance of locally applied DA was decreased by 50% relative to that in
D2+/+ mice. In D2+/+ mice, but not
D2‐/‐ mice, local application of the D2‐like
receptor antagonist raclopride increased DA signal amplitude, indicating
decreased DA clearance. Binding assays with the cocaine analogue
[3H]WIN 35,428 showed no genotypic differences in either density or
affinity of DAT binding sites in striatum or substantia nigra, indicating that
the differences seen in DAT activity were not a result of decreased DAT
expression. These results further strengthen the idea that the D2 DA receptor subtype modulates activity of the striatal DAT.
Serine‐23 Is a Major Protein Kinase A Phosphorylation Site on the Amino‐Terminal Head Domain of the Middle Molecular Mass Subunit of Neurofilament Proteins
Authors: Sihag Ram K. , Jaffe Howard , Nixon Ralph A. , Rong Xianhui
Published: March 2003

Abstract : We have shown previously that phosphate groups on the
amino‐terminal head domain region of the middle molecular mass subunit of
neurofilament proteins (NF‐M) are added by second messenger‐dependent protein
kinases. Here, we have identified Ser23 as a specific protein
kinase A phosphorylation site on the native NF‐M subunit and on two synthetic
peptides, S1 (14RRVPTETRSSF24) and
S2 (21RSSFSRVSGSPSSGFRSQSWS41), localized
within the amino‐terminal head domain region. Ser23 was identified
as a phosphorylation site on the 32P‐labeled α‐chymotryptic
peptide that carried >80% of the 32P‐phosphates incorporated
into the NF‐M subunit by protein kinase A. The synthetic peptides
S1 and S2 were phosphorylated 18 and two times more
efficiently by protein kinase A than protein kinase C, respectively. Neither
of the peptides was phosphorylated by casein kinase II. The sequence analyses
of the chemically modified phosphorylated serine residues showed that
Ser23 was the major site of phosphorylation for protein kinase A on
both S1 and S2 peptides. Low levels of incorporation of
32P‐phosphates into Ser22, Ser28, and
Ser32 by protein kinase A were also observed. Protein kinase C
incorporated 32P‐phosphates into Ser22,
Ser23, Ser25, Ser28, Ser32, and a
threonine residue, but none of these sites could be assigned as a major site
of phosphorylation. Analyses of the phosphorylated synthetic peptides by
liquid chromatography‐tandem mass spectrometry also showed that protein kinase
A phosphorylated only one site on peptide S1 and that ions with up
to four phosphates were detected on peptide S2. Analysis of the
data from the tandem ion trap mass spectrometry by using the computer program
PEPSEARCH did not unequivocally identify the specific sites of phosphorylation
on these serine‐rich peptides. Our data suggest that Ser23 is a
major protein kinase A‐specific phosphorylation site on the amino‐terminal
head region of the NF‐M subunit. Phosphorylation of Ser23 on the NF‐M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon.
Effects of Pretreatment with N‐(2‐Chloroethyl)‐N‐Ethyl‐2‐Bromobenzylamine (DSP‐4) on Methamphetamine Pharmacokinetics and Striatal Dopamine Losses
Authors: Fornai Francesco , Giorgi Filippo S. , Alessandrì Maria G. , Giusiani Mario , Corsini Giovanni U.
Published: March 2003

Abstract : We recently demonstrated that pretreatment with
N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) exacerbates experimental parkinsonism induced by methamphetamine. The mechanism responsible for this effect remains to be elucidated. In this study, we investigated whether the exacerbation of chronic dopamine loss in DSP‐4‐pretreated animals is due to an impairment in the recovery of dopamine levels once the neurotoxic insult is generated or to an increased efficacy of the effects induced by methamphetamine. We administered different doses of methamphetamine either to DSP‐4‐pretreated or to intact Swiss‐Webster mice and evaluated the methamphetamine‐induced striatal dopamine loss at early and prolonged intervals. As a further step, we evaluated the striatal pharmacokinetics of methamphetamine, together with its early biochemical effects. We found that previous damage to norepinephrine terminals produced by DSP‐4 did not modify the recovery of striatal dopamine levels occurring during several weeks after methamphetamine. By contrast, pretreatment with DSP‐4 exacerbated early biochemical effects of methamphetamine, which were already detectable 1 h after methamphetamine administration. In addition, in norepinephrine‐depleted animals, the clearance of striatal methamphetamine is prolonged, although the striatal concentration peak observed at 1 h is unmodified. These findings, together with the lack of a methamphetamine enhancement when DSP‐4 was injected 12 h after methamphetamine administration, suggest that in norepinephrine‐depleted animals, a more pronounced acute neuronal sensitivity to methamphetamine occurs.
Role of Endoplasmic Reticulum, Endosomal–Lysosomal Compartments, and Microtubules in Amyloid Precursor Protein Metabolism of Human Neurons
Authors: LeBlanc Andréa C. , Goodyer Cynthia G.
Published: March 2003

Abstract: A wide interest in amyloid precursor protein (APP) metabolism stems from the fact that increased amounts of amyloid β peptide (Aβ), arising through proteolytic processing of APP, likely play a significant role in Alzheimer's disease. As Alzheimer's disease pathology is limited almost exclusively to the human species, we established human primary neuron cultures to address the possibility of distinctive APP processing in human CNS neurons. In the present study, we investigate the role of organelles and protein trafficking in APP metabolism. Using brefeldin A, we failed to detect APP processing into Aβ in the endoplasmic reticulum. Monensin and the lysomotropic agents, NH4Cl and chloroquine, revealed a bypass pH‐dependent secretory pathway in a compartment between the endoplasmic reticulum and the medial Golgi, resulting in the secretion of full‐length APP. Colchicine treatment resulting in the loss of neurites inhibited processing of APP through the secretory, but not the endosomal–lysosomal, pathway of APP metabolism. The serine protease inhibitor, leupeptin, indicates a role for lysosomes in APP, Aβ, and APP C‐terminal fragment turnover. These results demonstrate that the regulation of APP metabolism in human neurons differs considerably from those reported in rodent CNS primary neuron cultures or continuously dividing cell types.
Relationships Among Seizures, Extracellular Amino Acid Changes, and Neurodegeneration Induced by 4‐Aminopyridine in Rat Hippocampus: A Microdialysis and Electroencephalographic Study
Authors: Peña Fernando , Tapia Ricardo
Published: March 2003

Abstract: 4‐Aminopyridine is a powerful convulsant that induces the release of neurotransmitters, including glutamate. We report the effect of intrahippocampal administration of 4‐aminopyridine at six different concentrations through microdialysis probes on EEG activity and on concentrations of extracellular amino acids and correlate this effect with histological changes in the hippocampus. 4‐Aminopyridine induced in a concentration‐dependent manner intense and frequent epileptic discharges in both the hippocampus and the cerebral cortex. The three highest concentrations used induced also a dose‐dependent enhancement of extracellular glutamate, aspartate, and GABA levels and profound hippocampal damage. Neurodegenerative changes occurred in CA1, CA3, and CA4 subfields, whereas CA2 was spared. In contrast, microdialysis administration of a depolarizing K+ concentration and of tetraethylammonium resulted in increased amino acid levels but no epileptic activity and no or moderate neuronal damage. These results suggest that seizure activity induced by 4‐aminopyridine is due to a combined action of excitatory amino acid release and direct stimulation of neuronal firing, whereas neuronal death is related to the increased glutamate release but is independent of seizure activity. In addition, it is concluded that the glutamate release‐inducing effect of 4‐aminopyridine results in excitotoxicity because it occurs at the level of nerve endings, thus permitting the interaction of glutamate with its postsynaptic receptors, which is probably not the case after K+ depolarization.
Relationships Between Ligand Affinities for the Cerebellar Cannabinoid Receptor CB1 and the Induction of GDP/GTP Exchange
Authors: Kearn Christopher S. , Greenberg Marcie J. , DiCamelli Ralph , Kurzawa Kristen , Hillard Cecilia J.
Published: March 2003

Abstract: The hypothesis of these studies is that ligand efficacy at
the neuronal CB1 receptor is dependent on the ratio of ligand affinities for
the active and inactive states of the receptor. Agonist efficacy was
determined in rat cerebellar membranes using agonist‐induced guanosine
5′‐O‐(3‐[35S]thiotriphosphate) binding; efficacy was
variable among the CB1 agonists examined. Ligand affinities for the active and
inactive state of the CB1 receptor were determined by competition with
[3H]CP55940 and [3H]SR141716A in the presence of 5′‐guanylylimidodiphosphate, respectively. All of the agonists investigated had a higher affinity for the active state than the inactive state. The fraction of CB1 receptors in the active state at a maximally effective concentration was calculated for each agonist and was found to correlate significantly with agonist efficacy. These studies demonstrate that the CB1 receptor of the cerebellum can assume an active conformation in the absence of agonist and that the variability in efficacy among CB1 receptor agonists can be explained by the relative affinities of these ligands for the CB1 receptor in the active and inactive states.
Inducible Nitric Oxide Synthase Up‐Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis
Authors: Almer Gabriele , Vukosavic Slobodanka , Romero Norma , Przedborski Serge
Published: March 2003

Abstract: Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS‐like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age‐matched transgenic mice expressing normal SOD1 and of wild‐type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end‐stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS‐immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region un‐affected by mSOD1. Similarly, no up‐regulation of iNOS is detected in the spinal cord of age‐matched transgenic mice expressing normal SOD1 or of wild‐type mice. The time course of the spinal cord gliosis and iNOS up‐regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age‐matched transgenic mice expressing normal SOD1 and wild‐type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up‐regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.
Intracellular Accumulation of Detergent‐Soluble Amyloidogenic Aβ Fragment of Alzheimer's Disease Precursor Protein in the Hippocampus of Aged Transgenic Mice
Authors: Li Qiao‐Xin , Maynard Christa , Cappai Roberto , McLean Catriona A. , Cherny Robert A. , Lynch Toni , Culvenor Janetta G. , Trevaskis Jim , Tanner Jane E. , Bailey Karen A. , Czech Christian , Bush Ashley I. , Beyreuther Konrad , Masters Colin L.
Published: March 2003

Abstract: To study amyloid β‐protein (Aβ) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C‐terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP‐C100 and Aβ were produced in brain and some peripheral tissues and Aβ was produced in serum. Using antibodies specific for the Aβ C terminus we found that Tg C100.V717F produced a 1.6‐fold increase in Aβ42/Aβ40 compared with Tg C100.WT. Approximately 30% of total brain Aβ (∼122 ng/g of wet tissue) was water‐soluble. The remaining 70% of Aβ partitioned into the particulate fraction and was completely sodium dodecyl sulfate‐soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate‐insoluble Aβ. Immunohistochemistry with an Aβ(5‐8) antibody showed that Aβ or Aβ‐containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble Aβ levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of Aβ or Aβ‐containing fragments and to screen for compounds directed at the γ‐secretase activity.
A Novel Subtype of Prostacyclin Receptor in the Central Nervous System
Authors: Watanabe Yumiko , Matsumura Kiyoshi , Takechi Hajime , Kato Koichi , Morii Hiroshi , Björkman Margareta , Langstrom Bengt , Noyori Ryoji , Suzuki Masaaki , Watanabe Yasuyoshi
Published: March 2003

Abstract: Recently, in the course of our search for the prostacyclin
receptor in the brain, we found a novel subtype, designated as IP2,
which was finely discriminated by use of the specific ligand
(15R)‐16‐m‐tolyl‐17,18,19,20‐tetranorisocarbacyclin
(15R‐TIC) and specifically localized in the rostral part of the
brain. In the present study, the tritiated compound
15R‐[15‐3H]TIC was synthesized and utilized for more
specific research on IP2. The specificity of binding to rat brain
regions was confirmed by use of several prostacyclin derivatives including
15S‐TIC. Mapping of 15R‐ and 15S‐[3H]TIC
binding in adjacent pairs of frozen sections of rat brain demonstrated a quite
similar pattern of distribution in almost all rostral brain regions,
indicating that the regions may contain only the IP2 subtype. On
the other hand, 15R‐[3H]TIC binding was very faint as
compared with 15S‐[3H]TIC binding in the caudal medullary
region. High densities of 15R‐[3H]TIC binding sites were
shown in the dorsal part of the lateral septal nucleus, thalamic nuclei,
limbic structures, and some of the cortical regions. Scatchard plot analysis
showed two components of high‐affinity 15R‐[3H]TIC binding
in the rostral regions, one with a KD value at ∼1
nM and the other with ∼30 nM. These results strengthen
our previous finding that a different subtype of prostacyclin receptor is
expressed in the CNS, and the map with 15R‐[3H]TIC
obtained here could guide further studies on the molecular and functional
properties of the IP2.
Internalization and Recycling of the CB1 Cannabinoid Receptor
Authors: Hsieh C. , Brown S. , Derleth C. , Mackie K.
Published: March 2003

Abstract : Tolerance develops rapidly to cannabis, cannabinoids, and

related drugs acting at the CB1 cannabinoid receptor. However, little is known

about what happens to the receptor as tolerance is developing. In this study,

we have found that CB1 receptors are rapidly internalized following agonist

binding and receptor activation. Efficacious cannabinoid agonists (WIN

55,212‐2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide

(an analogue of an endogenous cannabinoid, anandamide) was less effective,

causing internalization only at high concentration, whereas

Δ9‐tetrahydrocannabinol caused little internalization, even

at 3 μM. CB1 internalized via clathrin‐coated pits as

sequestration was inhibited by hypertonic sucrose. Internalization did not

require activated G protein αi, αo, or

αs subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.
G Protein‐Coupled Cyclic AMP Signaling in Postmortem Brain of Subjects with Mood Disorders
Authors: Dowlatshahi Dar , MacQueen Glenda M. , Wang Jun‐Feng , Reiach James S. , Young L. Trevor
Published: March 2003

Abstract : Components of cyclic AMP (cAMP) signaling were examined in postmortem cerebral cortex of a well characterized group of patients with mood disorders and nonpsychiatric control subjects. We measured G protein levels, adenylyl cyclase (AC) activity, and CREB levels in cerebral cortex of the subjects with respect to diagnosis, treatment, and suicide. There was no effect of diagnosis on any measure, except for a trend toward decreased stimulated AC activity in subjects with mood disorders relative to control subjects. We also detected a significant effect of suicide on temporal cortex CREB levels in subjects that died as a result of suicide relative to those that did not, which was more evident in patients with major depressive disorder. Bipolar disorder (BD) subjects treated with anticonvulsants at the time of death had decreased temporal cortex CREB levels relative to those not receiving anticonvulsants. Furthermore, we found a trend toward decreased occipital cortex GαS (short) levels in BD subjects treated with lithium. These results support the hypothesis of altered cAMP signaling in mood disorders and raise the possibility that factors other than diagnosis, such as treatment and suicide, may be relevant to cell‐signaling abnormalities reported in the literature.
Insulin Modulation of Cloned Mouse NMDA Receptor Currents in Xenopus Oocytes
Authors: Liao Guey‐Ying , Leonard John P.
Published: March 2003

Abstract : Modulation of recombinant

N‐methyl‐d‐aspartate receptor (NMDAR) currents by insulin

was studied using the Xenopus oocyte expression system. Insulin (0.8

μM, 10 min) regulated NMDAR currents in a subunit‐specific manner. Currents from ε1/ξ1, ε2/ξ1, and ε4/ξ1 receptors were variably potentiated, whereas currents from ε3/ξ1 receptors were not. Protein tyrosine kinases (PTKs) and protein kinase C were found to be involved in insulin‐mediated modulation in an NMDAR subtype‐specific way. Pretreatment with a specific PTK inhibitor, lavendustin A, attenuated and blocked the insulin effect on ε2/ξ1 and ε4/ξ1, respectively. Preincubation with selective protein kinase C inhibitors, staurosporine or calphostin C, depressed the response of ε1/ξ1 and ε2/ξ1 receptors to insulin. Basal regulation of NMDAR currents by endogenous PTKs and protein tyrosine phosphatases (PTPs) was also investigated. Of the four receptor subtypes, only ε1/ξ1 receptor currents were affected by basal PTK inhibition via lavendustin A, whereas PTP inhibition by phenylarsine oxide or orthovanadate enhanced currents from ε1/ξ1 and ε2/ξ1 receptors. Surprisingly, a stimulatory PTP modulation was observed for ε4/ξ1. As NMDAR subunits are differentially expressed in the brain, the observed subtype‐specific modulations of NMDAR currents by insulin, PTKs, and PTPs may provide important insights into certain NMDAR‐dependent physiological and pathological processes.
The Phosphatidylinositol 3‐Kinase Inhibitor Wortmannin Alters the Metabolism of the Alzheimer's Amyloid Precursor Protein
Authors: Petanceska Suzana S. , Gandy Sam
Published: March 2003

Abstract : One of the hallmarks of Alzheimer's disease is the

accumulation of senile plaques in brain, extracellular lesions comprised

mostly of aggregates of the amyloid β‐peptide (Aβ). Aβ is

proteolytically derived from the Alzheimer's amyloid precursor protein (APP).

The generation of Aβ and nonamyloidogenic derivatives of APP involves

utilization of alternative processing pathways and multiple subcellular

compartments. To improve our understanding of the regulation of APP

processing, we investigated the effects of wortmannin, a phosphatidyl‐inositol

3‐kinase (PI3‐kinase) inhibitor, on APP processing. PI3‐kinases form a

multifaceted family of enzymes that represent converging points for multiple

signal transduction pathways and also act as key regulators of vesicular

trafficking. In N2a neuroblastoma cells expressing either wild‐type APP or the

“Swedish” familial Alzheimer's disease‐associated mutant variant

of APP, wortmannin treatment resulted in decreased release of both Aβ and

soluble APPα. In parallel, full‐length APP and both processed

derivatives accumulated inside the cells. These effects were not present at

nanomolar concentrations of wortmannin, but only at micromolar concentrations,

implying the possible involvement of a recently described trans‐Golgi

network (TGN)‐associated PI3‐kinase that is resistant to nanomolar

concentrations of the inhibitor, but sensitive to micromolar concentrations.

All effects were reversible when the drug was removed from the cell culture

medium. Given the suspected site of action of this novel PI3‐kinase activity

at the TGN, it is tempting to speculate that the unexpected increase in the

levels of both intracellular soluble APPα and intracellular Aβ might be due to wortmannin‐induced covesiculation of APP together with its respective secretase enzymes within the TGN, leading to the execution of α‐, β‐, and γ‐secretase reactions.
Association of the Tyrosine Phosphatase SHP‐2 with Transducin‐α and a 97‐kDa Tyrosine‐Phosphorylated Protein in Photoreceptor Rod Outer Segments
Authors: Bell Michael W. , Alvarez Kathy , Ghalayini Abboud J.
Published: March 2003

Abstract : Increasing evidence indicates that tyrosine

phosphorylation, controlled by the concerted action of tyrosine kinases and

protein tyrosine phosphatases (PTPs), plays important roles in retinal

photoreceptor rod outer segments (ROS). We characterized PTP activity in

isolated bovine ROS that is significantly inhibited by orthovanadate.

Incubating ROS in the presence of exogenous Mg2+, ATP, and orthovanadate dramatically enhanced the tyrosine phosphorylation of several endogenous proteins. SHP‐2, a PTP with two SH2 domains, was identified in ROS by immunoblot analysis and was found to associate with ROS membranes. Immunocytochemisry showed localization of SHP‐2 in photoreceptor outer segments and possibly in the outer plexiform, inner nuclear, and inner plexiform cell layers of the retina as well. SHP‐2 associated with transducin‐α and a 97‐kDa tyrosine‐phosphorylated protein in ROS, suggesting the formation of a multimeric signaling complex. Based on its association with transducin‐α and a 97‐kDa protein, SHP‐2 may regulate the tyrosine phosphorylation of endogenous proteins, including transducin‐α, and may play a significant role in a novel signaling pathway in photoreceptors.
Tumor Necrosis Factor‐α Increases Lactoferrin Transcytosis Through the Blood‐Brain Barrier
Authors: Fillebeen Carine , Dehouck Bénédicte , Benaïssa Monique , Dhennin‐Duthille Isabelle , Cecchelli Roméo , Pierce Annick
Published: March 2003

Abstract : Lactoferrin (Lf) is an iron‐binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood‐brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor‐α (TNF‐α). As TNF‐α can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF‐α, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune‐mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up‐regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.
Proteolytic Fragments of Alzheimer’s Disease‐Associated Presenilin 1 Are Present in Synaptic Organelles and Growth Cone Membranes of Rat Brain
Authors: Beher Dirk , Elle Christine , Underwood John , Davis John B. , Ward Robin , Karran Eric , Masters Colin L. , Beyreuther Konrad , Multhaup Gerd
Published: March 2003

Abstract: Previous studies have demonstrated the molecular linkage of three causative genes for early‐onset Alzheimer’s disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the ∼20‐kDa C‐terminal and ∼30‐kDa N‐terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N‐ and C‐terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.
Differential Involvement of Central and Peripheral Norepinephrine in the Central Lipopolysaccharide‐Induced Interleukin‐6 Responses in Mice
Authors: Song Dong‐Keun , Im Yeong‐Bin , Jung Jun‐Sub , Suh Hong‐Won , Huh Sung‐Oh , Park Sang‐Wook , Wie Myung‐Bok , Kim Yung‐Hi
Published: March 2003

Abstract: Intracerebroventricular injection of lipopolysaccharide (LPS) induces a marked increase in circulating interleukin (IL)‐6 levels and in IL‐6 mRNA expression in brain and peripheral organs. Recently, it was reported that intraperitoneal administration of α‐adrenoceptor antagonists inhibits centrally injected LPS‐induced increases in plasma IL‐6 levels, suggesting the involvement of the norepinephrine (NE) system in the central LPS‐induced IL‐6 response. However, the localization (either central or peripheral) of NE involvement in the central LPS‐induced IL‐6 response has not been characterized. In the present study, mice were pretreated with 6‐hydroxydopamine (6‐OHDA) administered intracerebroventricularly or intraperitoneally to deplete central or peripheral stores of NE, respectively. Intracerebroventricular LPS (50 ng/mouse) markedly increased plasma IL‐6 levels and IL‐6 mRNA expression in choroid plexus, hypothalamus, pituitary, adrenals, heart, liver, spleen, and lymph nodes, but with minimal effect in lung, kidney, and testis, as revealed by RT‐PCR. Pretreatment, with intracerebroventricular 6‐OHDA (50 μg/mouse) decreased the LPS‐induced plasma IL‐6 levels by 39% and the LPS‐induced IL‐6 mRNA expression in liver, spleen, and lymph nodes, but not in choroid plexus, hypothalamus, pituitary, adrenals, and heart. Pretreatment with intraperitoneal 6‐OHDA (100 mg/kg) decreased the LPS‐induced plasma IL‐6 levels by 36% and the LPS‐induced IL‐6 mRNA expression in all the peripheral organs displaying increased IL‐6 mRNA. Central LPS‐induced increase in plasma corticosterone levels was decreased slightly by central but not by peripheral NE depletion. These results suggest that central NE and peripheral NE are differentially involved in the central LPS‐induced IL‐6 mRNA expression in peripheral organs.
Increased Cyclic AMP‐Dependent Protein Kinase Activity in Postmortem Brain from Patients with Bipolar Affective Disorder
Authors: Fields Anat , Li Peter P. , Kish Stephen J. , Warsh Jerry J.
Published: March 2003

Abstract : Previous observations of reduced [3H]cyclic

AMP binding in postmortem brainregions frombipolar affective disorder subjects

imply cyclic AMP‐dependent proteinkinase functionmay be altered in this

illness. To test this hypothesis, basal and stimulated cyclic AMP‐dependent

protein kinase activity was determined in cytosolic and particulate fractions

of postmortem brain from bipolar disorder patients and matched controls.

Maximal enzyme activity was significantly higher (104%) in temporal cortex

cytosolic fractions from bipolar disorder brain compared with matched

controls. In temporal cortex particulate fractions and in the cytosolic and

particulate fractions of other brain regions, smaller but statistically

nonsignificant increments in maximal enzyme activity were detected. Basal

cyclic AMP‐dependent protein kinase activity was also significantly higher

(40%) in temporal cortex cytosolic fractions of bipolar disorder brain

compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP‐dependent proteinkinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.
Calcium‐Dependent Dissociation of Synaptotagmin from Synaptic SNARE Complexes
Authors: Leveque Christian , Boudier Jeanne‐Andree , Takahashi Masami , Seagar Michael
Published: March 2003

Abstract: The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+‐dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)‐resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+‐dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30‐300 μM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v‐SNARE/t‐SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N‐ethylmaleimide‐sensitive fusion protein (NSF) was inhibited with N‐ethylmaleimide or adenosine 5′‐O‐(3‐thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+‐triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.
A Response Element for the Homeodomain Transcription Factor Ptx3 in the Tyrosine Hydroxylase Gene Promoter
Authors: Cazorla Pilar , Smidt Marten P. , O'Malley Karen L. , Burbach J. Peter H.
Published: March 2003

Abstract: Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position ‐50 to ‐45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3‐containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight‐ to 12‐fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60‐80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3‐binding site and the major cyclic AMP‐response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP‐modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type‐dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.
Implication of Glutamate in the Expression of Inducible Nitric Oxide Synthase After Oxygen and Glucose Deprivation in Rat Forebrain Slices
Authors: Cárdenas Antonio , Moro María A. , Hurtado Olivia , Leza Juan C. , Lorenzo Pedro , Castrillo Antonio , Bodelón Oscar G. , Boscá Lisardo , Lizasoain Ignacio
Published: March 2003

Abstract: Nitric oxide synthesis by inducible nitric oxide synthase (iNOS) has been postulated to contribute to ischemia‐reperfusion neurotoxicity. The expression of this enzyme has been demonstrated in cells present in the postischemic brain. The mechanisms of iNOS expression after cerebral ischemia are a subject of current research. We therefore decided to investigate whether glutamate, which is released in ischemia and is implicated in neurotoxicity, might be involved in the mechanisms by which oxygen and glucose deprivation (OGD) leads to the expression of iNOS in rat forebrain slices. In this model, we have shown previously that 20 min of OGD causes the expression of iNOS. We have now found that the NMDA receptor antagonist MK‐801 blocks the expression of iNOS, suggesting that the activation of the NMDA subtype of glutamate receptor is implicated in the mechanisms that lead to the expression of this isoform. Moreover, we have found that glutamate alone could trigger the induction process, as shown by the appearance of a Ca2+‐independent NOS activity and by the detection of iNOS mRNA and protein in slices exposed to glutamate. Glutamate‐dependent iNOS expression was concentration‐dependent and was blocked by EGTA and by the inhibitors of nuclear factor κB (NF‐κB) activation pyrrolidine dithiocarbamate and MG132. In addition, glutamate induced NF‐κB translocation to the nucleus, an effect that was inhibited by MG132. Taken together, our data suggest that activation of NMDA receptors by glutamate released in ischemia is involved in the expression of iNOS in rat forebrain slices via a Ca2+‐dependent activation of the transcription factor NF‐κB. To our knowledge, this is the first report showing an implication of excitatory amino acids in the expression of iNOS caused by ischemia.
Transcriptional Responses to Growth Factor and G Protein‐Coupled Receptors in PC12 Cells
Authors: Minneman Kenneth P. , Lee Deborah , Zhong Hongying , Berts Alf , Abbott Karen L. , Murphy T. J.
Published: March 2003

Abstract: Transcriptional responses to growth factor and G protein‐coupled receptors were compared in PC12 cells using retroviral luciferase reporters. In cells stably expressing α1A‐adrenergic receptors, norepinephrine activated all five reporters [AP1 (activator protein‐1), SRE (serum response element), CRE (cyclic AMP response element), NFκB) (nuclear factor‐κB), and NFAT (nuclear factor of activated T cells)], whereas nerve growth factor (NGF) and epidermal growth factor activated only AP1 and SRE. Activation of P2Y2 receptors by UTP did not activate any reporters. Protein kinase C inhibition blocked NFκB activation by norepinephrine, but potentiated CRE. Mitogen‐activated protein kinase kinase inhibition blocked AP1 activation by norepinephrine, but also potentiated CRE. p38 mitogen‐activated protein kinase inhibition reduced most norepinephrine responses, but not NGF responses. inhibition of Src eliminated SRE responses to norepinephrine and NGF, and reduced all responses except CRE. Phosphatidylinositol 3‐kinase inhibitors markedly potentiated CRE activation by norepinephrine, with only small effects on the other responses. Comparison of the three human subtypes showed that the α1A activated all five reporters, the α1B showed smaller effects, and the α1D was ineffective. Cell differentiation caused by norepinephrine, but not NGF, was reduced by all inhibitors studied. These experiments suggest that α1A‐adrenergic receptors activate a wider array of transcriptional responses than do growth factors in PC12 cells. These responses are not linearly related to second messenger production, and different subtypes show different patterns of activation.
Poly(ADP‐Ribose) Synthase Inhibition Reduces Ischemic Injury and Inflammation in Neonatal Rat Brain
Authors: Ducrocq S. , Benjelloun N. , Plotkine M. , Ben‐Ari Y. , Charriaut‐Marlangue C.
Published: March 2003

Abstract: Poly(ADP‐ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3‐aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 ± 2 (untreated) versus 28 ± 4 mm3 (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP‐ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.
Structure—Activity Relationships Among N‐Arachidonylethanolamine (Anandamide) Head Group Analogues for the Anandamide Transporter
Authors: Jarrahian Abbas , Manna Sukumar , Edgemond William S. , Campbell William B. , Hillard Cecilia J.
Published: March 2003

Abstract: Two putative endocannabinoids, N‐arachidonylethanolamine (AEA) and 2‐arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein‐mediated facilitated diffusion. We have synthesized and studied 22 N‐linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB1 cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4‐hydroxybenzyl results in a high‐affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3‐pyridinyl also results in a high‐affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB1 receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements.
Involvement of βγ Subunits of Gq/11 in Muscarinic M1 Receptor Potentiation of Corticotropin‐Releasing Hormone‐Stimulated Adenylyl Cyclase Activity in Rat Frontal Cortex
Authors: Olianas Maria C. , Onali Pierluigi
Published: March 2003

Abstract : In the present study, we investigated the involvement of

βγ subunits of Gq/11 in the muscarinic M1

receptor‐induced potentiation of corticotropin‐releasing hormone

(CRH)‐stimulated adenylyl cyclase activity in membranes of rat frontal cortex.

Tissue exposure to either one of two βγ scavengers, the QEHA

fragment type II adenylyl cyclase and the GDP‐bound form of the α

subunit of transducin, inhibited the muscarinic M1 facilitatory

effect. Moreover, like acetylcholine (ACh), exogenously added βγ

subunits of transducin potentiated the CRH‐stimulated adenylyl cyclase

activity, and this effect was not additive with that elicited by ACh. Western

blot analysis indicated the expression in frontal cortex of both type II and

type IV adenylyl cyclases, two isoforms stimulated by βγ subunits

in synergism with activated Gs. The M1 receptor‐induced

enhancement of the adenylyl cyclase response to CRH was counteracted by the

Gq/11 antagonist GpAnt‐2A but not by GpAnt‐2, a preferential

Gi/o antagonist. In addition, the muscarinic facilitatory effect

was inhibited by membrane preincubation with antiserum directed against the C

terminus of the α subunit of Gq/11, whereas the same

treatment with antiserum against either Gi1/2 or Go was

without effect. These data indicate that in membranes of rat frontal cortex,

activation of muscarinic M1 receptors potentiates CRH‐stimulated

adenylyl cyclase activity through βγ subunits of

Gq/11.
Immunocytochemical Characterization of the Mitochondrially Encoded ND1 Subunit of Complex I (NADH : Ubiquinone Oxidoreductase) in Rat Brain
Authors: Pettus Edward H. , Betarbet Ranjita , Cottrell Barbara , Wallace Douglas C. , Madyastha Vijay , Greenamyre J. Timothy
Published: March 2003

Abstract : In Parkinson's disease, there is a selective defect in complex I of the electron transfer chain. To better understand complex I and its involvement in neurodegenerative disease, we raised an antibody against a conserved epitope of the human mitochondrially encoded subunit 1 of complex I (ND1). Antibodies were affinity purified and assessed by ELISA, immunoblotting, and immunocytochemistry. Immunoblots of brain homogenates from mouse, rat, and monkey brain showed a single 33‐kDa band consistent with the predicted molecular mass of the protein. Subcellular fractionation showed the protein to be enriched in mitochondria. Immunocytochemistry in rat brain revealed punctate labeling in cell bodies and processes of neurons. Immunoreactively generally co‐localized with subunit IV of complex IV. In striatum, ND1 immunoreactively was greatly enriched in large cholinergic neurons and neurons containing nitric oxide synthase, two cell populations that are resistant to excitotoxic and metabolic insults. In substantia nigra, many dopaminergic neurons had little ND1 immunoreactivity, which may help to explain their sensitivity to complex I inhibitors. In spinal cord, ND1 immunoreactively was enriched in motor neurons. We conclude that complex I is differentially distributed across brain regions, between neurons and glia, and between types of neurons. This antibody should provide a valuable tool for assessing complex I in normal and pathological conditions.