bluuebunny/crossref_metadata_2025 huggingface dataset:
Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2α‐carbomethoxy‐3α‐(4‐fluorophenyl)tropane
Authors: Chen Nianhang , Sun LingLing , Reith Maarten E. A.
Published: March 2003

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+‐free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+] from 20 to 155 mm reduced Li+‐induced increase in DA Ki, but not CFT Kd. The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+‐independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+‐insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+'s inhibitory effect, though they caused Na+ protection of DA Ki against H+'s inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+‐bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.
Association of protein kinase C with GABAA receptors containing α1 and α4 subunits in the cerebral cortex: selective effects of chronic ethanol consumption
Authors: Kumar Sandeep , Sieghart Werner , Morrow A. Leslie
Published: March 2003

Previous studies have suggested that protein kinase C (PKC) isoforms differentially influence the sensitivity of γ‐aminobutyric acidA (GABAA) receptor responses in brain. Both PKCγ and PKCε knock‐out mice exhibit altered ethanol potentiation of GABAA receptor mediated Cl– flux. Furthermore, chronic ethanol consumption alters GABAA receptor function and receptor subunit peptide expression by mechanisms that are not yet understood. The present study explored the possibility that PKC isoforms are directly associated with GABAA receptors, and this association is influenced by chronic ethanol exposure. GABAA receptors containing α1 or α4 subunits were immunoprecipitated from solubilized protein derived from the membrane fraction of rat cerebral cortex using selective antibodies. Immunoprecipitated receptors were screened by western blot analysis for the presence of PKCδ, γ and ε isoforms. We found pronounced labeling of PKCγ but not PKCδ or PKCε in the α1 and α4 subunit immunoprecipitates. Immunoprecipitation with PKCγ, but not with IgG antibody also yielded GABAA receptor α1 and α4 subunits in the immunoprecipitate. The association of PKCγ with α1‐containing receptors was decreased 44 ± 11% after chronic ethanol consumption. In contrast, PKCγ associated with α4‐containing receptors was increased 32 ± 7% after chronic ethanol consumption. These results suggest that PKCγ may be involved in GABAA receptor adaptations following chronic ethanol consumption.
Effects of intrahippocampal CT105, a carboxyl terminal fragment of β‐amyloid precursor protein, alone/with inflammatory cytokines on working memory in rats
Authors: Matsumoto Yuji , Watanabe Shigenori , Suh Yoo‐Hun , Yamamoto Tsuneyuki
Published: March 2003

In this study, we examined the effects of a 105 amino acid carboxyl terminal fragment of β‐amyloid precursor protein (CT105) and inflammatory cytokines on working memory in rats, by using a three‐panel runway set‐up. CT105 at 10 nmol/side significantly impaired working memory when it was administered bilaterally into the hippocampus. Furthermore, to elucidate the interaction of CT105 with inflammatory cytokines, we co‐administered tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1β (IL‐1β) in combination with CT105. Concurrent injections of CT105 (1.0 nmol/side) and TNF‐α (100 ng/side) produced a synergistic deficit of working memory, whereas IL‐1β (100 ng/side) combined with CT105 (1.0 nmol/side) did not affect the working memory performance. These results indicate that the CT105‐induced impairment of working memory is strongly aggravated by an increase in the level of the inflammatory cytokine TNF‐α, which may occur in the brains of patients with Alzheimer's disease.
Stimulation of 5‐hydroxytryptamine (5‐HT2C) receptors in the ventrotegmental area inhibits stress‐induced but not basal dopamine release in the rat prefrontal cortex
Authors: Pozzi Laura , Acconcia Sabrina , Ceglia Ilaria , Invernizzi Roberto W. , Samanin Rosario
Published: March 2003

The present study investigated whether 5‐HT2C receptors in the ventrotegmental area and prefrontal cortex regulate basal and stimulus‐evoked dopamine release in the prefrontal cortex. Using the in vivo microdialysis technique in conscious rats, we studied the effect of a selective 5‐HT2C receptor agonist, Ro60–0175, on basal and immobilization stress‐induced dopamine release in the prefrontal cortex. Ro60–0175 intraperitoneally (2.5 mg/kg) and into the ventrotegmental area (10 µg/0.5 µL) completely antagonized the effect of stress on extracellular dopamine without altering basal levels. Infusion of 10 µm Ro60–0175 through the cortical probe had no significant effect on basal and stress‐induced dopamine release. SB242084 (10 mg/kg), a selective antagonist of 5‐HT2C receptors, significantly increased basal extracellular dopamine and completely prevented the effect of intraperitoneal and intraventrotegmental Ro60–0175 on the stress‐induced rise of extracellular dopamine, but had no effect itself in stressed rats. The results show that Ro60–0175 suppresses cortical dopamine release induced by immobilization stress through the stimulation of 5‐HT2C receptors in the ventrotegmental area. While confirming that endogenous 5‐HT acting on 5‐HT2C receptors tonically inhibit basal dopamine release in the prefrontal cortex, the present findings suggest that the stimulation of 5‐HT2C receptors with an exogenous agonist preferentially inhibit stimulated release.
Generation of reactive oxygen species and activation of NF‐κB by non‐Aβ component of Alzheimer's disease amyloid
Authors: Tanaka Seigo , Takehashi Masanori , Matoh Naomi , Iida Shinya , Suzuki Tomoki , Futaki Shiroh , Hamada Hirofumi , Masliah Eliezer , Sugiura Yukio , Ueda Kunihiro
Published: March 2003

Non‐amyloid beta (Aβ) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Aβ protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC50 = 5.6 µm) than in astrocytes (IC50≈ 50 µm). The neuron‐specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre‐treatment with the antioxidants, propyl gallate (PG) and N‐t‐butyl‐phenylnitrone (BPN), or overexpression of human Bcl‐2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2′,7′‐dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl‐tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF‐κB) and enhanced its DNA‐binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF‐κB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.
Inhibition of oxidative stress produced by plasma membrane NADH oxidase delays low‐potassium‐induced apoptosis of cerebellar granule cells
Authors: Martín‐Romero Francisco Javier , García‐Martín Elena , Gutiérrez‐Merino Carlos
Published: March 2003

From 1 to 3 h after the onset of cerebellar granule cells (CGC) apoptosis in a low‐K+(5 mm KCl) medium there was a large decay of NADH and a 2.5‐fold increase of the rate of reactive oxygen species (ROS) production (measured using CGC loaded with dichlorodihydrofluorescein). During the same time period, the ascorbate‐dependent NADH oxidase activity, which accounted for more than 90% of both total NADH oxidase activity and NADH‐dependent ·O2– production of CGC lysates, increased 2.5‐ to threefold. The stimulation of the ascorbate‐dependent NADH oxidase activity by oxidized cytochrome c, 2.5‐fold at saturation with a K0.5 of 4–5 µm cytochrome c, can at least partially explain this activation. The plasma membrane ascorbate‐dependent NADH oxidase activity accounted for more than 70% of the total activity (both in terms of NADH oxidase and ·O2– release) of CGC lysates. 4‐Hydroxyquinazoline (4‐HQ), which was found to block this apoptotic process, prevented the increase of ROS production. 4‐HQ protection against cell viability loss and DNA fragmentation correlated with the inhibition by 4‐HQ of the ascorbate‐dependent NADH oxidase activity of CGC lysates, showing the same K0.5‐value (4–5 mm 4‐HQ). The efficient blockade of CGC apoptosis by addition of superoxide dismutase to the medium further supports the neurotoxic role of ·O2– overproduction by the plasma membrane ascorbate‐dependent NADH oxidase.
Matrix metalloproteinase‐1 activates a pertussis toxin‐sensitive signaling pathway that stimulates the release of matrix metalloproteinase‐9
Authors: Conant Katherine , Haughey Norman , Nath Avindra , Hillaire Coryse St. , Gary Devin S. , Pardo Carlos A. , Wahl Larry M. , Bilak Masako , Milward Elizabeth , Mattson Mark P.
Published: March 2003

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non‐ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP‐1 coimmunoprecipitates with the α2β1integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP‐1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Giα containing heterotrimers. As an example, α2β1can form a complex with CD47 that may associate with Giα. In the present study we have therefore investigated the possibility that MMP‐1 may affect intracellular changes that are linked to the activation of a Gi protein‐coupled receptor. We show that treatment of neural cells with MMP‐1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP‐1 potentiates proteinase activated receptor‐1 (PAR‐1) agonist‐linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein‐coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP‐1 stimulates pertussis toxin sensitive release ofMMP‐9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP‐1 signals through a pertussis toxin‐sensitive G protein‐coupled receptor.
Membrane‐bound histamine N‐methyltransferase in mouse brain: possible role in the synaptic inactivation of neuronal histamine
Authors: Barnes William G. , Hough Lindsay B.
Published: March 2003

In the CNS, histamine is a neurotransmitter that is inactivated by histamine N‐methyltransferase (HNMT), a soluble enzyme localized to the cytosol of neurons and endothelial cells. However, it has not been established how extracellular histamine, a charged molecule at physiological pH, reaches intracellular HNMT. Present studies investigated two potential routes of histamine inactivation in mouse brain nerve terminal fractions (synaptosomes): (i) histamine uptake and (ii) histamine metabolism by HNMT. Intact synaptosomes demonstrated a weak temperature‐dependent histamine uptake (0.098 pmol/min‐mg protein), but contained a much greater capacity to metabolize histamine by HNMT (1.4 pmol/min‐mg protein). Determination of the distribution of HNMT within synaptosomes revealed that synaptosomal membranes (devoid of soluble HNMT) contribute HNMT activity equivalent to intact synaptosomes (14.3 ± 2.2 and 18.2 ± 4.3 pmol/min‐tube, respectively) and suggested that histamine‐methylating activity is associated with the membrane fraction. Additional experimental findings that support this hypothesis include: (i) the histamine metabolite tele‐methylhistamine (tMH) was found exclusively in the supernatant fraction following an HNMT assay with intact synaptosomes; (ii) the membrane‐bound HNMT activity was shown to increase 6.5‐fold upon the solubilization of the membranes with 0.1% Triton X‐100; and (iii) HNMT activity from the S2 fraction, ruptured synaptosomes, and synaptosomal membranes displayed different stability profiles when stored over 23 days at − 20°C. Taken together, these studies demonstrate functional evidence for the existence of membrane‐bound HNMT. Although molecular studies have not yet identified the nature of this activity, the present work suggests that levels of biologically active histamine may be controlled by an extracellular process.
Prostaglandin H2 (PGH2) accelerates formation of amyloid β1−42 oligomers
Authors: Boutaud Olivier , Ou Joyce J. , Chaurand Pierre , Caprioli Richard M. , Montine Thomas J. , Oates John A.
Published: March 2003

Epidemiologic evidence implicates cyclooxygenase activity in the pathogenesis of Alzheimer's disease, in which amyloid plaques have been found to contain increased levels of dimers and higher multimers of the amyloid β peptide. The product of the oxygenation of arachidonic acid by the cyclooxygenases, prostaglandin H2 (PGH2), rearranges non‐enzymatically to several prostaglandins, including the highly reactive γ‐keto aldehydes, levuglandins E2 and D2. We demonstrate that PGH2 markedly accelerates the formation of dimers and higher oligomers of amyloid β1−42. This is associated with the formation of levuglandin adducts of the peptide. These findings provide the molecular basis for a hypothesis linking cyclooxygenase activity to the formation of oligomers of amyloid β.
Repeated cocaine administration alters the expression of genes in corticolimbic circuitry after a 3‐week withdrawal: a DNA macroarray study
Authors: Toda Shigenobu , McGinty Jacqueline F. , Kalivas Peter W.
Published: March 2003

Addiction to psychostimulants elicits behavioral and biochemical changes that are assumed to be mediated by alterations of gene expression in the brain. The changes in gene expression after 3 weeks of withdrawal from chronic cocaine treatment were evaluated in the nucleus accumbens core and shell, dorsal prefrontal cortex and caudate using a complementary DNA (cDNA) array. The level of mRNA encoded by several genes was identified as being up‐ or down‐regulated in repeated cocaine versus saline subjects. The results from the cDNA array were subsequently confirmed at the protein level with immunoblotting. Of particular interest, parallel up‐regulation in protein and mRNA was found for the adenosine A1 receptor in the accumbens core, neuroglycan C in the accumbens shell, and the GluR5 glutamate receptor subtype in dorsal prefrontal cortex. However, there was an increase in TrkB protein in the nucleus accumbens core of cocaine‐treated rats without a corresponding alteration in mRNA. These changes of gene expression in corticolimbic circuitry may contribute to the psychostimulant‐induced behavioral changes associated with addiction.
Structural role of zinc ions bound to postsynaptic densities
Authors: Jan Huei‐Hsuan , Chen I‐Tong , Tsai Yu‐Yi , Chang Yen‐Chung
Published: March 2003

Postsynaptic densities (PSDs) isolated from porcine cerebral cortices are large aggregates consisting of more than 30 different proteins. Inductively coupled plasma–mass spectrometric analyses revealed that isolated PSDs contained zinc at a concentration of 4.1 nmol per mg protein. Treatment with 8 murea lead to dissociation of the PSDs into small components and, concomitantly, depletion of most of their bound zinc. After removal of the urea by dialysis, urea‐dissociated PSD proteins did not reassemble into aggregates by themselves. Adding ZnCl2to urea‐treated PSD samples resulted in the assembly of urea‐dissociated proteins into large aggregates with morphology and protein composition closely resembling those of the original PSDs. Mg2+, Ca2+, Co2+, Cd2+, Cu2+, Mn2+, Fe3+, K+and Na+ions at higher concentrations also induced the aggregation of urea‐dissociated PSD protein. The structures of the K+‐, Na+‐, Mg2+‐ and Ca2+‐induced aggregates were distinct from that of the original PSDs. Our results indicate that the structure of the PSD could be disassembled and reassembled underin vitroconditions. They further suggest that Zn2+ions, by binding to certain zinc‐binding proteins, play an important role in the formation and maintenance of the structure of the PSD.
Mapping sites responsible for interactions of agrin with neurons
Authors: Burgess Robert W. , Dickman Dion K. , Nunez Lorna , Glass David J. , Sanes Joshua R.
Published: March 2003

The multidomain proteoglycan agrin is a critical organizer of postsynaptic differentiation at the skeletal neuromuscular junction. Agrin is also abundant in the brain, but its roles there are unknown. As a step toward understanding these roles, we mapped sites responsible for interactions of neurons with agrin. First, we used a series of recombinant agrin fragments to show that at least four sites on agrin interact with chick ciliary neurons. Use of blocking antibodies and peptides indicated that neurons adhere to a site in the second of three G domains by means of αVβ1 integrin, and to a site in the last of four epidermal growth factor (EGF) repeats via a distinct β1 integrin. A third, integrin‐independent adhesion site is near to but distinct from the site that induces postsynaptic differentiation in muscles. These domains are insufficient, however, to account for neurite outgrowth‐inhibiting properties of full‐length agrin, which are mediated by the N‐terminal half of the molecule. We then used a second set of agrin mutants to demonstrate and map a transmembrane domain in the amino‐terminus of the SN‐isoform of agrin. The extracellular matrix‐bound form of agrin, called LN, bears an amino‐terminus required for secretion and binding to laminin. The SN form, which is selectively expressed by neurons, bears a variant amino terminus that converts agrin from a secreted, matrix‐associated protein to a type‐II transmembrane protein, providing a mechanism for presenting agrin in central, as opposed to neuromuscular, synaptic clefts. The SN‐amino terminus can mediate externalization and membrane anchoring of heterologous proteins, but is insufficient to target them to the synapse. Together, these studies define sites that contribute to the subcellular localization of and signaling by neuronal agrin.
SEL‐10 interacts with presenilin 1, facilitates its ubiquitination, and alters A‐beta peptide production
Authors: Li Jinhe , Pauley Adele M. , Myers Rick L. , Shuang Rongqing , Brashler John R. , Yan Riqiang , Buhl Allen E. , Ruble Cara , Gurney Mark E.
Published: March 2003

Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma‐secretase, modulate gamma‐cleavage of the amyloid protein precursor (APP) to the amyloid β‐peptide (Aβ). Mutations in sel‐12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL‐10. SEL‐10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1‐Cdc53/CUL1‐F‐box protein) E2‐E3 ubiquitin ligase family. In this study, we show that human SEL‐10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N‐ and C‐terminal fragments. Co‐transfection of sel‐10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid β‐peptide, the principal component of amyloid plaque in Alzheimer's disease.
Inhibition of lipopolysaccharide‐induced cyclooxygenase‐2, tumor necrosis factor‐α and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195
Authors: Choi Hyan B. , Khoo C. , Ryu Jae K. , Van Breemen Edo , Kim Seung U. , McLarnon James G.
Published: March 2003

The anti‐inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1‐(2‐chloro‐ phenyl)‐N‐methyl‐N‐(1‐methylpropyl)‐3‐isoquinoline‐carboxamide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expressions of the inducible enzyme, cyclooxygenase‐2 (COX‐2) and the pro‐inflammatory cytokine, tumor necrosis factor‐α (TNF‐α). PK11195 (at 50 µm) significantly inhibited the LPS‐induced up‐regulation of both inflammatory factors; at a lower concentration of PK11195 (2 µm) expression of TNF‐α, but not COX‐2, was reduced. Production of both factors, using immunocytochemistry for COX‐2 and ELISA for TNF‐α, was markedly reduced with 50 µm of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca2+[Ca2+]i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca2+]i consisted primarily of a Ca2+ influx component accompanied by a smaller mobilization from intracellular Ca2+ stores. In the presence of PK11195, the amplitude of the [Ca2+]i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS‐induced expression of COX‐2 and TNF‐α or the endotoxin increase in [Ca2+]i. These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS‐induced microglial expressions of COX‐2 and TNF‐α and that modulation of Ca2+‐mediated signaling pathways could be involved in the anti‐inflammatory actions.
Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons
Authors: Counts Scott E. , McGuire Susan O. , Sortwell Caryl E. , Crawley Jacqueline N. , Collier Timothy J. , Mufson Elliott J.
Published: March 2003

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH‐immunoreactive (ir) neurons in VM cultures. However, 1 µm GAL reduced an approximately 100% increase in TH‐ir neurons in 1 mm dibutyryl cAMP (dbcAMP)‐treated cultures by ∼50%. TH‐ir neuron number in dbcAMP‐treated VM cultures was dose‐responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi‐quantitative RT‐PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific ∼200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)‐sensitive opening of G protein‐gated K+ channels (GIRKs). GAL reduction of TH‐ir neuron number in dbcAMP + GAL‐treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1‐ mediated reduction of TH in midbrain dopaminergic neurons.
Analysis of the PC12 cell transcriptome after differentiation with pituitary adenylate cyclase‐activating polypeptide (PACAP)
Authors: Vaudry David , Chen Yun , Ravni Aurélia , Hamelink Carol , Elkahloun Abdel G. , Eiden Lee E.
Published: March 2003

Pituitary adenylate cyclase‐activating polypeptide (PACAP) promotes neurite outgrowth and inhibits proliferation of rat pheochromocytoma (PC12) cells. Characterizing the PACAP‐differentiated PC12 cell transcriptome should provide genetic insight into how these processes occur in these cells, and in neuronal precursors in vivo. For this purpose, RNA samples were collected from PC12 cells before or after a 6‐h treatment with PACAP, from which a labeled cDNA was hybridized to a high‐density cDNA array containing 15 365 genes. The genomic response to PACAP involves at least 73 genes. Among the genes differentially expressed in the presence of PACAP, 71% were up regulated, and 29% down regulated, 2‐fold or more. Sixty‐six percent of the messages affected by PACAP code for functionally categorized proteins, most not previously known to be regulated during PC12 cell differentiation. PACAP has been shown to induce PC12 cell neurite outgrowth through the mitogen‐activated protein kinase kinase (MEK) pathway independently of protein kinase A (PKA). Therefore treatments were conducted in the absence or presence of the PKA inhibitor H89, or the MEK inhibitor U0126 in order to identify subsets of genes involved in specific aspects of PC12 cell differentiation. Co‐treatment of PC12 cells with PACAP plus H89 revealed a cluster of five genes specifically regulated through the PKA pathway and co‐treatment of the cells with PACAP and U0126 revealed a cluster of 13 messages specifically activated through the MEK pathway. Many of the known genes regulated by PACAP have been associated with neuritogenesis (i.e. villin 2 or annexin A2) or cell growth (i.e. growth arrest specific 1 or cyclin B2). Thus, some of the expressed sequence tags (ESTs) that exhibit the same regulation pattern (i.e. AU016391 or AW552690) may also be involved in the neuritogenic and anti‐mitogenic effects of PACAP in PC12 cells. Among the 73 PACAP regulated genes, 10 are disqualified on pharmacological grounds as actors in PACAP‐mediated neurite outgrowth or growth arrest, leaving 63 new PACAP‐regulated genes implicated in neuronal differentiation. Thirteen of these are candidates for mediating ERK‐dependent neurite outgrowth, and 47 are possibly involved in the ERK‐independent growth arrest induced by PACAP.
Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter, GLYT1a
Authors: Pearlman Rhonda Jo , Aubrey Karin R. , Vandenberg Robert J.
Published: March 2003

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo‐oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1‐methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212–2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.
In developing Drosophila neurones the production of γ‐amino butyric acid is tightly regulated downstream of glutamate decarboxylase translation and can be influenced by calcium
Authors: Küppers Barbara , Sánchez‐Soriano Natalia , Letzkus Johannes , Technau Gerhard M. , Prokop Andreas
Published: March 2003

The presented work pioneers the embryonic Drosophila CNS for studies of the developmental regulation and function of γ‐amino butyric acid (GABA). We describe for the first time the developmental pattern of GABA in Drosophila and address underlying regulatory mechanisms. Surprisingly, and in contrast to vertebrates, detectable levels of GABA occur late during Drosophila neurogenesis, after essential neuronal proliferation and growth have taken place and synaptogenesis has been initiated. This timeline is almost unchanged when the GABA synthetase glutamate decarboxylase (GAD) is strongly misexpressed throughout the nervous system suggesting a tight post‐translational regulation of GABA expression. We confirmed such GABA control mechanisms in an independent model system, i.e. primary Drosophila cell cultures raised in elevated [K+]. The data suggest that, in both systems, GABA suppression occurs via control of GAD activity. Using developing embryos and cell cultures as parallel assay systems for pharmacological and genetic studies we show that the negative regulation of GAD can be overridden by drugs known to elevate intracellular free [Ca2+]. Our results provide the basis for investigations of genetic mechanisms underlying the observed phenomenon, and we discuss the potential implications of this work for Drosophila neurogenesis but also for a general understanding of GAD regulation.
The basic helix‐loop‐helix differentiation factor Nex1/MATH‐2 functions as a key activator of the GAP‐43 gene
Authors: Uittenbogaard Martine , Martinka Debra L. , Chiaramello Anne
Published: March 2003

Nex1/MATH‐2 is a neurogenic basic Helix‐Loop‐Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP‐43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP‐43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP‐43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP‐43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP‐43 promoter. The Nex1‐mediated activity is restricted to the conserved E1–E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site‐directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E‐box is a high affinity binding site. We further found that Nex1 released the ME1 E‐protein‐mediated repression in a concentration dependent manner. Thus, the E1–E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N‐terminal and C‐terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP‐43 promoter and cellular environment.
Involvement of c‐Jun N‐terminal kinase in amyloid precursor protein‐mediated neuronal cell death
Authors: Hashimoto Yuichi , Tsuji Osahiko , Niikura Takako , Yamagishi Yohichi , Ishizaka Miho , Kawasumi Masaoki , Chiba Tomohiro , Kanekura Kohsuke , Yamada Marina , Tsukamoto Emi , Kouyama Keisuke , Terashita Kenzo , Aiso Sadakazu , Lin Anning , Nishimoto Ikuo
Published: March 2003

Amyloid precursor protein (APP), the precursor of Aβ, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti‐APP antibody. The c‐Jun N‐terminal kinase (JNK) can mediate various neurotoxic signals, including Aβ neurotoxicity. However, the relationship of APP‐mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Aβ independent. Here we examined whether JNK is involved in APP‐mediated neuronal cell death and found that: (i) neuronal cell death by antibody‐bound APP was inhibited by dominant‐negative JNK, JIP‐1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK‐mediated cell death closely coincided with that of APP‐mediated cell death. Pertussis toxin (PTX) suppressed APP‐mediated cell death but not caJNK‐induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP‐mediated cytotoxicity. In the presence of PTX, the PTX‐resistant mutant of Gαo, but not that of Gαi, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP‐mediated neuronal cell death as a downstream signal transducer of Go.
Altered cAMP‐dependent protein kinase subunit immunolabeling in post‐mortem brain from patients with bipolar affective disorder
Authors: Chang Annisa , Li Peter P. , Warsh Jerry J.
Published: March 2003

Previous findings of reduced [3H]cAMP binding and increased activities of cAMP‐dependent protein kinase (PKA) in discrete post‐mortem brain regions from patients with bipolar affective disorder (BD) suggest that PKA, the major downstream target of cAMP, is also affected in this illness. As prolonged elevation of intracellular cAMP levels can modify PKA regulatory (R) and catalytic (C) subunit levels, we sought to determine whether these PKA abnormalities are related to changes in the abundance of PKA subunits in BD brain. Using immunoblotting techniques along with PKA subunit isoform‐specific polyclonal antisera, levels of PKA RIα, RIβ, RIIα, RIIβ and Cα subunits were measured in cytosolic and particulate fractions of temporal, frontal and parietal cortices of post‐mortem brain from BD patients and matched, non‐neurological, non‐psychiatric controls. Immunoreactive levels of cytosolic Cα in temporal and frontal cortices, as well as that of cytosolic RIIβ in temporal cortex, were significantly higher in the BD compared with the matched control brains. These changes were independent of age, post‐mortem interval or pH and unrelated to ante‐mortem lithium treatment or suicide. These findings strengthen further the notion that the cAMP/PKA signaling system is up‐regulated in discrete cerebral cortical regions in BD.
Do multidisciplinary integrated care pathways improve interprofessional collaboration?
Authors: Atwal Anita , Caldwell Kay
Published: March 2003

This paper reports on the evaluation stage of an action research project on interprofessional collaboration in discharge planning. Findings from interviews with health care professionals working in the acute sector had revealed concerns about discharge planning and multidisciplinary teamwork. In the United Kingdom the National Health Service (NHS) Plan has reinforced the need for an integrated approach to health care. Effective health care integration requires effective communication, teamwork and the commitment to deliver integrated care. Integrated documentation is a key strategy for enhancing interprofessional collaboration and reducing the isolation of professionals, and has been successfully implemented in a range of health care settings. Presented with the concerns about the collaborative process in discharge planning, an action research strategy was chosen to bring about change in an orthopaedic ward in one London teaching hospital. This paper will evaluate the implementation of an integrated care pathway with fractured neck of femurs in one London teaching hospital. Care pathways facilitate the management of defined patient groups using interdisciplinary plans of care. The emphasis will be on understanding whether integrated care pathways enhance and develop interprofessional collaboration and enable effective information access and flow across the professions and the organization. The criteria for evaluation, forming the hypotheses of the study, were that interprofessional nonverbal and verbal communication would be enhanced and that interprofesisonal collaboration would increase. Methods of evaluation used were: (i) stakeholder interviews, (ii) interprofessional audit and (iii) analysis of the variances from the integrated care pathway. The evaluation revealed that although integrated care pathways led to improved outcomes for the health care trust there was little evidence to suggest that interprofessional relationships and communication were enhanced. Furthermore, key factors in discharge delays appeared to be organizational rather than professional.
A critique of avianCHD‐based molecular sexing protocols illustrated by a Z‐chromosome polymorphism detected in auklets
Authors: Dawson Deborah A. , Darby Steven , Hunter Fiona M. , Krupa Andrew P. , Jones Ian L. , Burke Terry
Published: March 2003

The sexes of non‐ratite birds can be determined routinely by PCR amplification of theCHD‐ZandCHD‐Wgenes.CHD‐based molecular sexing of four species of auklets revealed the presence of a polymorphism in the Z chromosome. No deviation from a 1:1 sex ratio was observed among the chicks, though the analyses were of limited power. Polymorphism in theCHD‐Zgene has not been reported previously in any bird, but if undetected it could lead to the incorrect assignment of sex. We discuss the potential difficulties caused by a polymorphism such as that identified in auklets and the merits of alternativeCHD‐based sexing protocols and primers.
Highly polymorphic microsatellite loci for Speyeria idalia (Lepidoptera: Nymphalidae)
Authors: Williams Barry L. , Brawn Jeffrey D. , Paige Ken N.
Published: March 2003

Four polymorphic microsatellite loci were identified in the butterfly Speyeria idalia. We constructed a phagemid library and screened approximately 120 000 inserts. Probing with GT15, we identified 36 positives and designed polymerase chain reaction (PCR) primers for 12 potential loci. Of those loci, only four consistently produced polymorphic, diallelic PCR products in the expected size range. These results are consistent with previous studies concerning the low frequency of microsatellite loci in the lepidoptera, although these four loci are highly polymorphic and therefore likely to provide information on the fine scale genetic structure among populations in this species.
Microsatellite loci for great white herons and great blue herons (Aves, Ardeidae, Ardea herodias)
Authors: McGuire Heather L. , Noor Mohamed A. F.
Published: March 2003

We used an enrichment technique to isolate 60 microsatellite loci in Ardea herodias. We developed primers for 17 loci, screened for variation in A. herodias and attempted to amplify these loci in three closely related species (A. alba, A. cinerea and A. cocoi). Fifteen loci were polymorphic in A. herodias. Observed heterozygosities ranged from 0.10 to 0.81. Two loci appeared to be monomorphic in A. herodias, but exhibited variation in product size among species within the genus. Our ability to amplify polymorphic products in closely related species suggests that these markers may be useful in other herons.
A set of polymorphic microsatellites for Vochysia ferruginea, a promising tree for land reclamation in the Neotropics
Authors: Lowe A. J. , Goodall‐Copestake W. P. , Caron H. , Kremer A. , Decroocq S.
Published: March 2003

Vochysia ferruginea Mart. (Vochysiaceae) is a gap colonist of Neotropical forest. As a result of its high tolerance of low‐nutrient acidic conditions and high aluminium and iron concentrations, and its high potential seed and pollen dispersal, it is a promising timber species for commercial development as regenerated or reclaimed forest on degraded land. We present here the primer sequences for 10 polymorphic simple sequence repeat loci for use with V. ferruginea to assess fine‐scale genetic structure and gene flow dynamics.
Species‐diagnostic microsatellite loci for the fig wasp genus Pegoscapus
Authors: Molbo D. , Krieger M. J. B. , Herre E. A. , Keller L.
Published: March 2003

To obtain tools for the estimation of inbreeding and assignment of offspring to matrilines, we developed 13 microsatellite loci from the fig wasps that pollinate Ficus obtusifolia. Based on morphological studies, it was thought that a single species (Pegoscapus hoffmeyeri) pollinated this fig. However, our data revealed the presence of two coexisting cryptic species. Several diagnostic microsatellite markers may be used to distinguish these two cryptic species. The new microsatellites can be used across a wide range of fig‐pollinating wasp species for both evolutionary and population genetic studies.
Eight polymorphic microsatellite loci for bottlenose dolphin and other cetacean species
Authors: Caldwell Marthajane , Gaines Michael S. , Hughes Colin R.
Published: March 2003

We developed eight polymorphic microsatellite loci that can be used to elucidate population structure and aid management of bottlenose dolphins, Tursiops truncatus, in the northwest Atlantic. These loci averaged nine alleles, with no evidence for null alleles. Their cross‐species utility was examined in Cephalorhynchus commersonii, Delphinus delphis, Delphinapterus leucas, Eschrictius robustus, Globicephala macrorhynchus, Lagenorhynchus obliquidens, Orcinus orca, Steno bredanensis and Stenella clymene. On average, 75% of the loci were polymorphic in these species (range = 50–88%). Our results indicate that these loci will be useful for elucidating population structure of bottlenose dolphin as well as of other cetacean species.
Development and characterization of microsatellite markers for Liporrhopalum tentacularis Grandi, the pollinator fig wasp of Ficus montana Blume
Authors: Zavodna M. , Arens P. , Vosman B. , Van Dijk P. J. , Van Damme J. M. M.
Published: March 2003

Microsatellite markers for the pollinator fig wasp Liporrhopalum tentacularis were developed using genomic libraries enriched for di‐, tri‐ and tetranucleotide repeats. A subset of 31 positive clones was sequenced and primers were designed. Eleven primer pairs produced polymorphic amplification products in L. tentacularis. Eight markers gave unambiguously scorable patterns and were further characterized on 29 individuals collected from different fruits of the dioecious host fig Ficus montana in Indonesia. Three to 19 alleles per locus were detected in this set of samples. The observed heterozygosity ranged between 0.10 and 0.55.
Isolation and characterization of microsatellite loci in Lampsilis abrupta (Bivalvia: Unionidae) and cross‐species amplification within the genus
Authors: Eackles Michael S. , King Tim L.
Published: March 2003

We have documented the first microsatellites isolated from a unionid and demonstrated that these markers can be useful for surveys of neutral genetic variation in several Lampsilis species. We describe the isolation and characterization of 15 polymorphic microsatellite DNA loci for the endangered unionid Lampsilis abrupta. Among individuals from five collections, allelic diversity ranged from six to 17 alleles and averaged 10.4 alleles per locus. Individual heterozygosity was observed to range from 20.0% to 86.7% and averaged 46.9%. Cross‐species amplification was investigated in nine additional Lampsilis species. A high level of flanking sequence similarity was inferred as 13 of 15 loci amplified in at least seven species.
Characterization of microsatellite loci in Humboldt penguin (Spheniscus humboldti) and cross‐amplification in other penguin species
Authors: Schlosser J. A. , Garner T. W. J. , Dubach J. M. , McElligott A. G.
Published: March 2003

The Humboldt penguin (Spheniscus humboldti) was once very common throughout its range along the coast of Peru and Chile. Today, listed as endangered, it is crucial to gain an understanding of gene flow and levels of genetic variation between breeding colonies to protect this species effectively. We developed seven microsatellite primers to investigate gene flow and population structure among four colonies. Locus‐specific allelic diversity ranges from five to 11 alleles and observed heterozygosity ranges from 0.50 to 0.88. These markers cross‐amplify in eight penguin species over five genera.
Microsatellite loci for the eusocialLasioglossum malachurumand other sweat bees (Hymenoptera, Halictidae)
Authors: Paxton R. J. , Arévalo E. , Field J.
Published: March 2003

Sweat bees (family Halictidae) comprise a numerous and diverse group that are arguably among the most socially labile of all insect taxa. Given the lack of highly variable markers for eusocial species of the family, we developed a suite of dinucleotide and trinucleotide markers for one of its members, the EurasianLasioglossum malachurum, and used them to amplify DNA from other halictids. Loci were highly variable inL. malachurumand amplified DNA from many other halictids.
Adult survival and productivity of Hutton's Shearwaters
Authors: Cuthbert Richard , Davis Lloyd S.
Published: March 2003

Accurate estimation of breeding and survival parameters is essential to assess the population viability of the endangered Hutton's ShearwaterPuffinus huttoni. This study investigated the accuracy of using field signs, an infrared ‘burrowscope’ and inspection hatches to monitor burrow occupancy and breeding success. Inspection hatches proved to be the most reliable means of measuring burrow occupancy and breeding success, and Hutton's Shearwaters appear to tolerate disturbance from this method. Data collected from inspection hatches over a 10‐year period indicate that breeding success has averaged 46.5%, and that 70.5% of burrows are occupied by an incubating bird at the start of the breeding season. This occupancy figure suggests that the population of Hutton's Shearwaters may be closer to 106 000 breeding pairs than the 134 000 pairs previously estimated. Annual adult survival is estimated to be 93.1%. These values of breeding success and adult survival are very similar to published data onPuffinusspecies breeding in environments free from introduced predators.
Anti‐predator adaptations in nesting Marsh Tits Parus palustris: the role of nest‐site security
Authors: Wesołowski Tomasz
Published: March 2003

A proposal that nest predation is the most important selective pressure shaping nest‐site use of Marsh Tits Parus palustris was checked in a long‐term study (almost 500 nests) carried out in the Białowieża National Park (eastern Poland) in primeval conditions, where the birds breed in natural holes and their nests are at risk from a variety of predators. It was predicted that predation rates would depend on hole attributes, so that Marsh Tits should use the most secure holes. Predation was responsible for 70% of total nest losses. Predation risk depended on hole attributes. Nests in dead wood were predated significantly more often than those in live wood. Nests in old woodpecker holes were predated more frequently than nests in holes of other origin, and nests situated closer to the entrance were more at risk than ones further from it. The entrance size did not influence overall predation risk, but small entrance size was important in preventing access by larger predators. These differences can account for the very rare use of holes in dead wood or of woodpecker holes, the fact that they nest in holes with small entrances and relatively far from the entrance. It is concluded that the patterns of nest‐site use found in this species are best explained as anti‐predator adaptations, which have evolved and are maintained by the pressure of nest predators. This study also indicates the possible limits of, and constraints on, these adaptations.