bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of apoptosis in human and rat glioma by agonists of the nuclear receptor PPARγ
Authors: Zander Thomas , Kraus Jürgen A. , Grommes Christian , Schlegel Uwe , Feinstein Douglas , Klockgether Thomas , Landreth Gary , Koenigsknecht Jessica , Heneka Michael T.
Published: March 2003

Malignant astrocytomas are among the most common brain tumours and few therapeutic options exist. It has recently been recognized that the ligand‐activated nuclear receptor PPARγ can regulate cellular proliferation and induce apoptosis in different malignant cells. We report the effect of three structurally different PPARγ agonists inducing apoptosis in human (U87MG and A172) and rat (C6) glioma cells. The PPARγ agonists ciglitazone, LY171 833 and prostaglandin‐J2, but not the PPARα agonist WY14643, inhibited proliferation and induced cell death. PPARγ agonist‐induced cell death was characterized by DNA fragmentation and nuclear condensation, as well as inhibited by the synthetic receptor‐antagonist bisphenol A diglycidyl ether (BADGE). In contrast, primary murine astrocytes were not affected by PPARγ agonist treatment. The apoptotic death in the glioma cell lines treated with PPARγ agonists was correlated with the transient up‐regulation of Bax and Bad protein levels. Furthermore, inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPARγ‐mediated apoptosis, indicating an essential role of Bax in PPARγ‐induced apoptosis. However, PPARγ agonists not only induced apoptosis but also caused redifferentiation as indicated by outgrowth of long processes and expression of the redifferentiation marker N‐cadherin in response to PPARγ agonists. Taken together, treatment of glioma cells with PPARγ agonists may hold therapeutic potential for the treatment of gliomas.
Identification of a novel SNAP25 interacting protein (SIP30)
Authors: Lee Ho‐Ki , Safieddine Saaid , Petralia Ronald S. , Wenthold Robert J.
Published: March 2003

Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs), including synaptosome‐associated proteins of 25 kDa (SNAP25), syntaxins, and vesicle‐associated membrane proteins (VAMP), are essential for regulated exocytosis of synaptic vesicles in neurotransmission. We identified a cDNA coding for a novel protein of 266 amino acids that we have named SIP30 (SNAP25 interacting protein of 30 kDa). SIP30 is expressed abundantly in brain and slightly in testis and kidney. In brain, SIP30 is highly expressed in the inferior and superior colliculi, which contain important relay nuclei of the auditory and visual systems. GST–pull‐down and immunoprecipitation assays showed direct binding of SIP30 to SNAP25. Although SIP30 does not directly interact with syntaxin based on pull‐down assays, syntaxin does co‐immunoprecipitate with SIP30 suggesting that syntaxin is indirectly associated with SIP30, perhaps through SNAP25.
BDNF‐mediated signal transduction is modulated by GSK3β and mood stabilizing agents
Authors: Mai Lian , Jope Richard S. , Li Xiaohua
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) is a major neurotrophin in the brain and abnormal regulation of BDNF may contribute to the pathophysiology of mood disorders. In the present study, we examined if alterations in the activity of glycogen synthase kinase‐3‐beta (GSK3β) or treatment with mood stabilizers modulated BDNF‐mediated signal transduction pathways in differentiated human neuroblastoma SH‐SY5Y cells. BDNF increased the phosphorylation of the forkhead transcription factor FKHRL1 through activation of the phosphatidylinositol 3‐kinase (PI3K)/Akt signaling pathway, and the phosphorylation of the cyclic AMP response element binding protein (CREB) through activation of extracellular signal‐regulated kinase1/2 (ERK1/2). BDNF also increased serine9‐phosphorylation of GSK3β, which inhibits GSK3β activity. Overexpression of GSK3β did not affect BDNF‐induced phosphorylation of Akt, ERK1/2, or FKHRL1, but abolished CREB phosphorylation induced by BDNF. This inhibition of BDNF‐induced CREB phosphorylation in GSK3β‐overexpressing SH‐SY5Y cells was blocked by treatment with lithium. In contrast to lithium, sodium valproate and lamotrigine did not affect BDNF‐mediated signaling, whereas carbamazepine induced a rapid and prolonged phosphorylation of ERK1/2 and CREB in the absence or the presence of BDNF. Therefore, increased GSK3β selectively attenuates BDNF‐induced CREB phosphorylation, and lithium and carbamazepine can facilitate activation of CREB.
Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography
Authors: Perry David C. , Xiao Yingxian , Nguyen Henry N. , Musachio John L. , Dávila‐García Martha I. , Kellar Kenneth J.
Published: March 2003

Comparison of [125I]epibatidine and 5‐[125I]iodo‐3‐(2‐azetidinylmethoxy)pyridine ([125I]A‐85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A‐85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of α4β2, α3β2 and α3β4 receptors. Although the predominant subtype in rat brain was α4β2, non‐α4β2 binding sites were prominent in many regions. In the habenulo‐peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, α3β4‐like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in α3β4‐like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in α3β2‐like binding included the habenulo‐peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selectivecompetitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.
Effects of systemic and central nervous system localized inflammation on the contributions of metabolic precursors to the l‐kynurenine and quinolinic acid pools in brain
Authors: Kita Tomoyuki , Morrison Paul F. , Heyes Melvyn P. , Markey S. P.
Published: March 2003

l‐Kynurenine and quinolinic acid are neuroactive l‐tryptophan‐kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [2H3]‐l‐kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS‐localized immune‐activated, and systemically immune‐activated. The specific activity of l‐kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of l‐kynurenine in brain was derived via local synthesis from l‐tryptophan/formylkynurenine versus 78% from l‐kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue l‐tryptophan/formylkynurenine (10%), blood l‐kynurenine (35%), blood 3‐hydroxykynurenine/3‐hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune‐activation, however, l‐kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood l‐kynurenine (52%), blood 3‐hydroxykynurenine or 3‐hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS‐localized immune activation, > 98% of both l‐kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which l‐kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources.
Estrogen‐mediated neuroprotection against β‐amyloid toxicity requires expression of estrogen receptor α or β and activation of the MAPK pathway
Authors: Fitzpatrick Jennifer L. , Mize Amy L. , Wade Christian B. , Harris Julie A. , Shapiro Robert A. , Dorsa Daniel M.
Published: March 2003

It is well documented that estrogen can activate rapid signaling pathways in a variety of cell types. These non‐classical effects of estrogen have been reported to be important for cell survival after exposure to a variety of neurotoxic insults. Since direct evidence of the ability of the estrogen receptors (ERs) α and/or β to mediate such responses is lacking, the hippocampal‐derived cell line HT22 was stably transfected with either ERα (HTERα) or ERβ (HTERβ). In HTERα and HTERβ cells, but not untransfected cells, an increase in ERK2 phosphorylation was measured within 15 min of 17β‐estradiol treatment. The ER antagonist ICI 182, 780 (1 µm) and the MEK inhibitor, PD98059 (50 µm) blocked this increase in ERK2 phosphorylation. Treatment of HT22, HTERα and HTERβ cells with the β‐amyloid peptide (25–35) (10 µm) resulted in a significant decrease in cell viability. Pre‐treatment for 15 min with 10 nm 17β‐estradiol resulted in a 50% increase in the number of living cells in HTERα and HTERβ cells, but not in HT22 cells. Finally, ICI 182, 780 and PD98059 prevented 17β‐estradiol‐mediated protection. This study demonstrates that both ERα and ERβ can couple to rapid signaling events that mediate estrogen‐elicited neuroprotection.
GABA–opioid interactions in the globus pallidus: [D‐Ala2]‐Met‐enkephalinamide attenuates potassium‐evoked GABA release after nigrostriatal lesion
Authors: Schroeder J. A. , Schneider J. S.
Published: March 2003

The motor signs of Parkinson's disease have been partly attributed to an overinhibition of the external globus pallidus (GP) that results from hyperactivity of striatopallidal GABA/enkephalinergic neurons. The goals of this study were to measure basal levels of extracellular fluid GABA in the GP of normal cats, 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐treated parkinsonian cats and cats spontaneously recovered from MPTP‐induced parkinsonism, and to examine the effects of opioid receptor activation on potassium (K+)‐evoked GABA release in the GP in these animals. Basal GP GABA levels were increased 75% from normal in parkinsonian animals 1 week after MPTP administration and returned to control levels in recovered animals 6 weeks afterMPTP administration. No significant differences were observed in K+‐evoked GABA release across conditions. The opioid receptor agonist [D‐Ala2]‐Met‐Enkephalinamide (DALA) significantly attenuated K+‐evoked GABA release in the GP of MPTP‐treated symptomatic and recovered cats, but had no significant effect on GABA release in normal animals. These data show that basal GP GABA levels are elevated coincident with expression of parkinsonian signs and return to normal in animals that have functionally compensated for a nigrostriatal lesion. DALA‐induced inhibition of pallidal GABA release after a dopamine‐depleting lesion, suggests that enkephalin may attenuate GABA release in the GP specifically after striatal dopamine loss.
Blockade by ferrous iron of Ca2+ influx through N‐methyl‐d‐aspartate receptor channels in immature cultured rat cortical neurons
Authors: Nakamichi Noritaka , Ohno Hiroshi , Nakamura Yoichi , Hirai Takao , Kuramoto Nobuyuki , Yoneda Yukio
Published: March 2003

Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo‐3 for assessment of intracellular free calcium ion (Ca2+) concentrations with the aid of a confocal laser‐scanning microscope. In the absence of added MgCl2, the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration‐dependent manner at a concentration range of 1–100 µm with the increase by KCl being transient. The addition of FeCl2, but not FeCl3, markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10–200 µm, without affecting that by KCl. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCl2 was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCl2 invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain.
Mitochondrial permeability transition mediates apoptosis induced by N‐methyl(R)salsolinol, an endogenous neurotoxin, and is inhibited by Bcl‐2 and rasagiline, N‐propargyl‐1(R)‐aminoindan
Authors: Akao Yukihiro , Maruyama Wakako , Shimizu Shigeomi , Yi Hong , Nakagawa Yoshihito , Shamoto‐Nagai Masayo , Youdim Moussa B. H. , Tsujimoto Yoshihide , Naoi Makoto
Published: March 2003

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N‐methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH‐SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, ΔΨm, in the early phase of apoptosis, which was not suppressed by a pan‐caspase inhibitor, but was antagonized by Bcl‐2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal‐induced loss of ΔΨm. NM(R)Sal‐induced apoptosis was completely inhibited not only by Bcl‐2 and a pan‐caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal‐induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced ΔΨm, which was inhibited by cyclosporin A and Bcl‐2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N‐propargyl‐1(R)‐aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the ΔΨm reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH‐SY5Y cells. Rasagiline also inhibited the NM(R)Sal‐induced loss of ΔΨm and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal‐induced cell death and the neuroprotective effect of rasagiline.
Molecular characterization of MARTA1, a protein interacting with the dendritic targeting element of MAP2 mRNAs
Authors: Rehbein Monika , Wege Konstanze , Buck Friedrich , Schweizer Michaela , Richter Dietmar , Kindler Stefan
Published: March 2003

In neurones, the somatodendritic microtubule‐associated protein 2 regulates the stability of the dendritic cytoskeleton. Its extrasomatic localization appears to be a multicausal mechanism that involves dendritic mRNA trafficking, a process that depends on a dendritic targeting element in the 3′ untranslated region. Two rat MAP2‐RNA trans‐acting proteins, MARTA1 and MARTA2, exhibit specific high‐affinity binding to the dendritic targeting element. We have now affinity‐purified MARTA1 from rat brain. Analysis of proteolytic peptides revealed that rat MARTA1 is the orthologue of the human RNA‐binding protein KSRP. Rat MARTA1 is a 74‐kDa protein that contains four putative RNA‐binding domains and is 98% identical to human KSRP. Both purified rat MARTA1 and human KSRP preferentially bind to the dendritic targeting element, but do not strongly interact with other investigated regions of mRNAs encoding microtubule‐associated protein 2 and α‐tubulin. In rat brain neurones and cultured neurones derived from superior cervical ganglia, MARTA1 is primarily intranuclear, but is also present in the somatodendritic cytoplasm. Thus, MARTA1 may play a role in nucleocytoplasmic mRNA targeting.
A novel prohormone processing site in Aplysia californica: the Leu–Leu rule
Authors: Hummon Amanda B. , Huang He‐Qing , Kelley Wayne P. , Sweedler Jonathan V.
Published: March 2003

Neuropeptides are a complex set of signaling molecules produced through enzymatic cleavages from longer prohormone sequences. The most common cleavage sites in prohormones are basic amino acid residues; however, processing is observed at non‐basic sites. Cleavage at Leu–Leu sequences has been observed in three Aplysia californica prohormones. To further investigate this unusual event, native and non‐native synthetic peptides containing Leu–Leu residues are incubated with homogenates of Aplysia californica ganglia and the resulting products monitored with MALDI MS. Cleavage near and between Leu–Leu residues is observed in the abdominal and buccal ganglia homogenates, confirming the presence of an unidentified peptidase. In addition, fractions from an HPLC separation of buccal ganglia homogenates also produce cleavages at Leu–Leu residues. Products resulting from cleavage at Leu–Leu sites are observed and are produced in larger amounts in acidic and neutral pH ranges, and cleavage is inhibited by the addition of EDTA, suggesting a metal is required for activity.
Nicotinic‐acetylcholine receptors are functionally coupled to the nitric oxide/cGMP‐pathway in insect neurons
Authors: Zayas Ricardo M. , Qazi Sanjive , Morton David B. , Trimmer Barry A.
Published: March 2003

In addition to their ionotropic role, neuronal nicotinic acetylcholine receptors (nAChRs) can influence second messenger levels, transmitter release and gene transcription. In this study, we show that nAChRs in an insect CNS control cGMP levels by coupling to NO production. In conditions that inhibit spiking, nicotine induced cGMP synthesis. This increase in cGMP was blocked by nicotinic antagonists, and by inhibitors of both nitric oxide synthase and soluble guanylyl cyclase. The nicotinic‐evoked increase in cGMP was localized to specific NO‐sensitive neurons in the CNS, several of which are identified motoneurons. Because NO production requires Ca2+, we investigated the effect of nicotinic stimulation on [Ca2+]i in cultured neurons. We found that activation of nAChRs increased [Ca2+]i, which was blocked by nAChR antagonists. Nicotinic stimulation of neurons in the isolated CNS in low‐Na+, also evoked increases in [Ca2+]i independent of fast changes in voltage. In addition, approximately 10% of the nicotinic‐evoked [Ca2+]i increase in cultured neurons persisted when voltage‐gated Ca2+ channels were blocked by Ni2+. Under the same conditions, nicotinic stimulation of cGMP in the CNS was unaffected. These combined results suggest that nicotinic stimulation is coupled to NOS potentially by directly gating Ca2+.
Caspase‐3 is not activated in seizure‐induced neuronal necrosis with internucleosomal DNA cleavage
Authors: Fujikawa Denson G. , Ke Xingrao , Trinidad Rosen B. , Shinmei Steve S. , Wu Aiguo
Published: March 2003

A caspase‐3‐activated DNase produces internucleosomal DNA cleavage (DNA laddering). We determined whether caspase‐3 is activated by lithium–pilocarpine‐induced status epilepticus in six brain regions with necrosis‐induced DNA laddering. The thymuses of adult rats given methamphetamine or normal saline were used as controls for apoptosis. Some 6–8 h after methamphetamine treatment, thymocytes showed apoptosis by electron‐microscopic examination, positive terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL), DNA laddering, cleavage of caspase‐3 into its active p17 subunit, active caspase‐3 immunoreactivity, and a 25‐fold increase in caspase‐3‐like activity. Six hours after SE, necrotic neurons by electron‐microscopic examination in hippocampus, amygdala and piriform, entorhinal and frontal cortices showed no TUNEL and no DNA laddering. Twenty‐four hours after seizures, most necrotic neurons were negative for TUNEL, some were positive, but all regions showed DNA laddering. However, 6 and 24 h after seizures, active caspase‐3 immunoreactivity was negative, caspase‐3‐like activity did not increase, and western blot analysis failed to show the p17 subunit. In addition, 24 h after seizures,microdialytic perfusion of carbobenzoxy‐valyl‐alanyl‐aspartyl (O‐methylester) fluoromethylketone was not neuroprotective. Thus, caspase‐3 is not activated in brain regions with seizure‐induced neuronal necrosis with DNA laddering. Either caspase‐activated DNase is activated by another enzyme, or a caspase‐independent DNase is responsible for the DNA cleavage.
Activation of the rat dopamine D2 receptor promoter by mitogen‐activated protein kinase and Ca2+/calmodulin‐dependent protein kinase II pathways
Authors: Takeuchi Yusuke , Miyamoto Eishichi , Fukunaga Kohji
Published: March 2003

To investigate regulation of D2 receptor (D2R) gene expression by protein kinases, we evaluated effects of constitutively active MAPK kinase kinase (MEKK), Ca2+/calmodulin‐dependent protein kinase (CaMK) II, CaMKIV and cyclic AMP‐dependent protein kinase (PKA) on D2R promoter activity using luciferase reporter gene assays. A 1.5‐kbp fragment containing the rat D2R promoter was cloned upstream of the reporter and transfected into D2R‐expressing NB2A cells or nonexpressing NG108‐15 and C6 glioma cells. MEKK and CaMKII, but not CaMKIV and PKA, increased promoter activity 4.5‐ and 1.5‐fold, respectively, in NB2A cells. Inhibitory effects of a MEK inhibitor and lack of effect by dominant negative (DN)‐JNK1 or DN‐p38MAPK revealed that ERK but not JNK and p38MAPK is involved in MEKK‐induced promoter activation. Deletion and mutation of the promoter revealed that the MEKK‐responsive region was Sp1 site B between nucleotides −56 and −47. Overexpression of Sp1 suppressed promoter activity without affecting MEKK‐induced activation. Interestingly, overexpression of Zif268 increased promoter activity through the region between nucleotides −56 and −36. Increased activity by Zif268 was additive with CaMKII‐induced activation but not with activation induced by MEKK. Co‐transfection with CaMKII stimulated nuclear translocation of Zif268. These results suggest that ERK and CaMKII positively regulate the D2R promoter and that Zif268 is a potential transcription factor for the CaMKII‐dependent pathway.
Expression of interleukin‐1 receptors and their role in interleukin‐1 actions in murine microglial cells
Authors: Pinteaux Emmanuel , Parker Lisa C. , Rothwell Nancy J. , Luheshi Giamal N.
Published: March 2003

Interleukin (IL)‐1 is an important mediator of acute brain injury and inflammation, and has been implicated in chronic neurodegeneration. The main source of IL‐1 in the CNS is microglial cells, which have also been suggested as targets for its action. However, no data exist demonstrating expression of IL‐1 receptors [IL‐1 type‐I receptor (IL‐1RI), IL‐1 type‐II receptor (IL‐1RII) and IL‐1 receptor accessory protein (IL‐1RAcP)] on microglia. In the present study we investigated whether microglia express IL‐1 receptors and whether they present target or modulatory properties for IL‐1 actions. RT–PCR analysis demonstrated lower expression of IL‐1RI and higher expression of IL‐1RII mRNAs in mouse microglial cultures compared with mixed glial or pure astrocyte cultures. Bacterial lipopolysaccharide (LPS) caused increased expression of IL‐1RI, IL‐1RII and IL‐1RAcP mRNAs, induced the release of IL‐1β, IL‐6 and prostaglandin‐E2 (PGE2), and activated nuclear factor κB (NF‐κB) and the mitogen‐activated protein kinases (MAPKs) p38, and extracellular signal‐regulated protein kinase (ERK1/2), but not c‐Jun N‐terminal kinase (JNK) in microglial cultures. In comparison, IL‐1β induced the release of PGE2, IL‐6 and activated NF‐κB, p38, JNK and ERK1/2 in mixed glial cultures, but failed to induce any of these responses in microglial cell cultures. IL‐1β also failed to affect LPS‐primed microglial cells. Interestingly, a neutralizing antibody to IL‐1RII significantly increased the concentration of IL‐1β in the medium of LPS‐treated microglia and exacerbated the IL‐1β‐induced IL‐6 release in mixed glia, providing the first evidence that microglial IL‐1RII regulates IL‐1β actions by binding excess levels of this cytokine during brain inflammation.
Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress
Authors: Scheggi Simona , Leggio Benedetta , Masi Flavio , Grappi Silvia , Gambarana Carla , Nanni Giulio , Rauggi Riccardo , De Montis Maria Graziella
Published: March 2003

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3‐week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate–putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393‐stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.
Control of the Na+/Ca2+exchanger 3 promoter by cyclic adenosine monophosphate and Ca2+in differentiating neurons
Authors: Gabellini Nadia , Bortoluzzi Stefania , Danieli Gian A. , Carafoli Ernesto
Published: March 2003

The human gene for member 3 of solute carrier family 8 (SLC8A3), encoding the Na+/Ca2+exchanger isoform 3 (NCX3), was identified on chromosome 14q24.2. The minimal promoter region was predicted 250 bp upstream of exon 1. This was confirmed by luciferase reporter assays of pGL3‐promoter constructs in transfected SH‐SY5Y cells. The promoter activity was monitored during the differentiation of this cell line elicited by the sequential treatment with retinoic acid and brain‐derived neurotrophic factor (BDNF). The activity was induced by cyclic AMP (cAMP) via the CRE (cAMP response element) and was stimulated by retinoic acid. The increase of intracellular Ca2+induced by the partial depolarization of the plasma membrane with KCl down‐regulated both the basal and the cAMP‐stimulated transcription. The down‐regulation of the latter may be mediated by the phosphorylation of the CRE‐binding protein by a calmodulin‐dependent kinase (CaMKII). The exposure of cells to BDNF after treatment with retinoic acid rapidly induced promoter activity during the initial five hours and phosphorylation of CRE‐binding protein during the first two hours. The promoter activity was further enhanced by cAMP, but became insensitive to Ca2+. In BDNF‐stimulated cells cAMP elevation caused the preferential phosphorylation of ATF1 instead of that of CRE‐binding protein.
Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways
Authors: Noda Mami , Yasuda Satsuki , Okada Mitsuko , Higashida Haruhiro , Shimada Aki , Iwata Nakao , Ozaki Norio , Nishikawa Kaori , Shirasawa Sakiko , Uchida Mayumi , Aoki Shunsuke , Wada Keiji
Published: March 2003

Human serotonin 5A (5‐HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5‐HT5A receptors‐expressing cells, accumulation of cAMP by forskolin was inhibited by 5‐HT as reported previously. Pertussis toxin‐sensitive inhibition of ADP‐ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine‐sensitive Ca2+ mobilization. On the other hand, 5‐HT‐induced outward currents were observed using the patch‐clamp technique in whole‐cell configuration. The 5‐HT‐induced outward current was observed in 84% of the patched 5‐HT5A receptor‐expressing cells and was concentration‐dependent. The 5‐HT‐induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5‐HT‐induced current was significantly attenuated by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5‐HT‐induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5‐HT‐induced current. IP3 showed a transient increase after application of 5‐HT in 5‐HT5A receptor‐expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP‐ribosyl cyclase activity, 5‐HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3‐sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.
Down‐regulation of Bcl‐2 and Bcl‐xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death
Authors: Jiang Zhihong , Zheng Xiao , Rich Keith M.
Published: March 2003

The functions of the antiapoptotic proteins Bcl‐2 and Bcl‐xL were examined in glioblastoma cells. Expression of both Bcl‐2 and Bcl‐xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non‐neoplastic glial cells. Down‐regulation of both bcl‐2 and bcl‐xL expression in glioblastoma cell lines U87 and NS008 with bcl‐2/bcl‐xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase‐dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl‐2/bcl‐xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl‐2 and Bcl‐xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase‐dependent cell death in gliomas. These findings support a potential role of bcl‐2/bcl‐xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase‐dependent cell death in patients with glioblastoma.
Functional analysis of connexin‐26 mutants associated with hereditary recessive deafness
Authors: Wang Hung‐Li , Chang Wen‐Teng , Li Allen H. , Yeh Tu‐Hsueh , Wu Ching‐Yi , Chen Mei‐Shin , Huang Pei‐Chen
Published: March 2003

The physiological importance of connexin‐26 (Cx26) gap junctions in regulating auditory function is indicated by the finding that autosomal recessive DFNB1 deafness is associated with mutations of the Cx26 gene. To investigate the pathogenic role of Cx26 mutation in recessive hearing loss, four putative DFNB1 Cx26 mutants (V84L, V95M, R127H, and R143W) were stably expressed in N2A cells, a communication‐deficient cell line. In N2A cells expressing (R127H) Cx26 gap junctions, macroscopic junctional conductance and ability of transferring neurobiotin between transfected cells were greatly reduced. Despite the formation of defective junctional channels, immunoreactivity of (R127H) Cx26 was mainly localized in the cell membrane and prominent in the region of cell–cell contact. Mutant (V84L), (V95M), or (R143W) Cx26 protein formed gap junctions with a junctional conductance similar to that of wild‐type Cx26 junctional channels. (V84L), (V95M), or (R143W) Cx26 gap junctions also permitted neurobiotin transfer between pairs of transfected N2A cells. The present study suggests that (R127H) mutation associated with hereditary sensorineural deafness results in the formation of defective Cx26 gap junctions, which may lead to the malfunction of cochlear gap junctions and hearing loss. Further studies are required to determine the exact mechanism by which mutant (V84L), (V95M), and (R143W) Cx26 proteins, which are capable of forming functional homotypic junctional channels in N2A cells, cause the cochlear dysfunction and sensorineural deafness.
Meal size and frequency affect neuronal plasticity and vulnerability to disease: cellular and molecular mechanisms
Authors: Mattson Mark P. , Duan Wenzhen , Guo Zhihong
Published: March 2003

Although all cells in the body require energy to survive and function properly, excessive calorie intake over long time periods can compromise cell function and promote disorders such as cardiovascular disease, type‐2 diabetes and cancers. Accordingly, dietary restriction (DR; either caloric restriction or intermittent fasting, with maintained vitamin and mineral intake) can extend lifespan and can increase disease resistance. Recent studies have shown that DR can have profound effects on brain function and vulnerability to injury and disease. DR can protect neurons against degeneration in animal models of Alzheimer's, Parkinson's and Huntington's diseases and stroke. Moreover, DR can stimulate the production of new neurons from stem cells (neurogenesis) and can enhance synaptic plasticity, which may increase the ability of the brain to resist aging and restore function following injury. Interestingly, increasing the time interval between meals can have beneficial effects on the brain and overall health of mice that are independent of cummulative calorie intake. The beneficial effects of DR, particularly those of intermittent fasting, appear to be the result of a cellular stress response that stimulates the production of proteins that enhance neuronal plasticity and resistance to oxidative and metabolic insults; they include neurotrophic factors such as brain‐derived neurotrophic factor (BDNF), protein chaperones such as heat‐shock proteins, and mitochondrial uncoupling proteins. Some beneficial effects of DR can be achieved by administering hormones that suppress appetite (leptin and ciliary neurotrophic factor) or by supplementing the diet with 2‐deoxy‐d‐glucose, which may act as a calorie restriction mimetic. The profound influences of the quantity and timing of food intake on neuronal function and vulnerability to disease have revealed novel molecular and cellular mechanisms whereby diet affects the nervous system, and are leading to novel preventative and therapeutic approaches for neurodegenerative disorders.
Employing a superior BACE1 cleavage sequence to probe cellular APP processing
Authors: Tomasselli Alfredo G. , Qahwash Isam , Emmons Thomas L. , Lu Yifeng , Leone Joseph W. , Lull June M. , Fok Kam F. , Bannow Carol A. , Smith Clark W. , Bienkowski Michael J. , Heinrikson Robert L. , Yan Riqiang
Published: March 2003

The involvement of β‐secretase (BACE1; β‐site APP‐cleaving enzyme) in producing the β‐amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3‐P2′ sequence, …VKM‐↓‐DA…, was replaced by …ISY‐↓‐EV… (‐↓‐ = β site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL‐↓‐DAEFR. The expanded peptide, GLTNIKTEEISEISY‐↓‐EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these β‐site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the β‐site. Insertion of the optimized ISY‐↓‐EV motif at either the β‐site (Asp1) or β′‐site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.
Brain region‐specific alterations of 5‐HT2A and 5‐HT2C receptors in serotonin transporter knockout mice
Authors: Li Qian , Wichems Christine H. , Ma Li , Van de Kar Louis D. , Garcia Francisca , Murphy Dennis L.
Published: March 2003

The aim of the present studies was to determine the effects of reduced or absent serotonin (5‐HT) transporters (5‐HTTs) on 5‐HT2A and 5‐HT2C receptors. The density of 5‐HT2C receptors was significantly increased in the amygdala and choroid plexus of 5‐HTT knockout mice. On the other hand, the density of 5‐HT2A receptors was significantly increased in the hypothalamus and septum, but reduced in the striatum, of 5‐HTT knockout mice. However, 5‐HT2A mRNA was not changed in any brain region measured. 5‐HT2C mRNA was significantly reduced in the choroid plexus and lateral habenula nucleus of these mice. The function of 5‐HT2A receptors was evaluated by hormonal responses to (+/–)‐1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI). Oxytocin, but not adrenocorticotrophic hormone or corticosterone, responses to DOI were significantly greater in 5‐HTT knockout mice. In addition, Gq and G11 proteins were not significantly changed in any brain region measured. The present results suggest that the constitutive alteration in the function of 5‐HTTs changes the density of 5‐HT2A and 5‐HT2C receptors in a brain region‐specific manner. These changes may not be mediated by alterations in their gene expression or in the level of Gq/11 proteins. The alterations in these receptors may be related to the altered behaviors of 5‐HTT knockout mice.
Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells
Authors: Ohsawa Ikuroh , Nishimaki Kiyomi , Yasuda Chie , Kamino Kouzin , Ohta Shigeo
Published: March 2003

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late‐onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2‐deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2‐deficient transfectants were highly vulnerable to exogenous 4‐hydroxy‐2‐nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2‐deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4‐hydroxy‐2‐nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.
Estrogen activates protein kinase C in neurons: role in neuroprotection
Authors: Cordey Myriam , Gundimeda Usha , Gopalakrishna Rayudu , Pike Christian J.
Published: March 2003

It has been previously demonstrated that estrogen can protect neurons from a variety of insults, including β‐amyloid (Aβ). Recent studies have shown that estrogen can rapidly modulate intracellular signaling pathways involved in cell survival. In particular, estrogen activates protein kinase C (PKC) in a variety of cell types. This enzyme plays a key role in many cellular events, including regulation of apoptosis. In this study, we show that 17β‐estradiol (E2) rapidly increases PKC activity in primary cultures of rat cerebrocortical neurons. A 1 h pre‐treatment with E2or phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, protects neurons against Aβ toxicity. Protection afforded by both PMA and E2is blocked by pharmacological inhibitors of PKC. Further, depletion of PKC levels resulting from prolonged PMA exposure prevents subsequent E2or PMA protection. Our results indicate that E2activates PKC in neurons, and that PKC activation is an important step in estrogen protection against Aβ. These data provide new understanding into the mechanism(s) underlying estrogen neuroprotection, an action with therapeutic relevance to Alzheimer's disease and other age‐related neurodegenerative disorders.
Elderly persons’ experiences of living with venous leg ulcer: living in a dialectal relationship between freedom and imprisonment
Authors: Ebbeskog B. , Ekman S.‐L.
Published: March 2003

Elderly persons’ experiences of living with venous leg ulcer: living in a dialectal relationship between freedom and imprisonment

The aim was to illuminate elderly persons’ experiences of living with venous leg ulcer. Fifteen persons 74–89 years of age with active leg ulcer were interviewed. Data were analysed utilizing a phenomenological‐hermeneutic approach inspired by Ricoeur. The analysis includes dialectic movement between understanding and explanation of the text as a whole and its parts. In the structural analysis four themes were identified: emotional consequences of altered body image, living a restricted life, achievement of well‐being in connection with a painful wound and bandage, and struggle between hope and despair with regard to a lengthy healing process. The comprehensive understanding indicated that the meaning of living with venous leg ulcer can be understood as a dialectal relationship between, on the one hand the feeling of being imprisoned in the body, the bandage and the home, and on the other hand, hope of freedom from a burdensome body. The results indicate that the concept, body of image, is vitally relevant to the caring of elderly people with venous leg ulcer. The study shows the importance of recognizing the persons’ perceptions of their leg ulcers and the impact of these perceptions on well‐being. Nurses can thereby anticipate problems and provide more sensitive care.
Pre‐understanding and openness – a relationship without hope?
Authors: Nyström Maria , Dahlberg Karin
Published: March 2003

Pre‐understanding and openness – a relationship without hope?

In all research, quantitative as well as qualitative, we have to deal with our presuppositions in order to remain open throughout the whole process of inquiry. If we fail to do so the findings might be little more than a reflected image of something already existing in our understanding. The question dealt with in this paper is: how can we gain increased openness by being more aware of our presuppositions. The aim was to investigate the intentional structure of pre‐understanding and its effects upon data‐collection as well as data‐analysis and conclusions in research. The question of pre‐understanding is central in Gadamer’s philosophy, even if he is not the first one to discuss this concept. Gadamer further developed the philosophy of understanding. According to him pre‐ understanding is an intentional structure of feelings and thoughts, which is activated when we regard something as something. By studying the structure of pre‐understanding we became more aware of its effect upon understanding. In this paper, theoretical frameworks within cognitive and social psychology as well as psychoanalytic object‐relation‐theory were compared with Gadamer’s philosophy concerning the conditions of human understanding. Finally the possibility of increased openness in the process of research was discussed. (Such openness is related to knowledge about the structure of pre‐understanding.)
`From victim to agent': the clinical and social significance of self‐help group participation for people with life‐threatening diseases
Authors: Adamsen Lis
Published: March 2003

`From victim to agent': the clinical and social significance of self‐help group participation for people with life‐threatening diseases

Aim : The main objective of the paper is to discuss the ways in which self‐help groups can be useful to persons with life‐threatening diseases like cancer or human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). The paper offers an outline of the central characteristics of self‐help groups, highlighted by the participants themselves.

Background : Self‐help groups are a growing phenomenon across national borders. New empirical sociological studies show that nurses have become important agents in self‐help groups, both within and outside the public health‐care system. However, research on self‐help groups is a relatively new area within nursing studies yet it serves an important function in shedding light on a rather unique form of social practice organized and managed partly by the participants themselves.

Methods : Qualitative interviews designed to examine how participants evaluate their experiences with self‐help groups. The study includes 53 self‐help group participants suffering from cancer or HIV/AIDS.

Findings : Contact with other people, forming of friendships, new networks and increased self‐confidence are some of the effects indicated by the participants. Participation in a self‐help group has a positive impact on the patients' ability to cope with the psychological and social consequences of living with a life‐threatening disease.

Conclusions : It is evident that the positive effects of self‐help groups are mainly because of their inherent capacity for universalizing personal problems, which, in itself, legitimizes the scientific relevance of self‐help groups. There is no evidence to indicate that self‐help groups have a positive influence on the life expectancy of participants who suffer from cancer or HIV/AIDS, but it should be noted that this is not the explicit concern when groups are formed. By their very existence self‐help groups indicate the need for a reevaluation of the scope and focus of the individualized care and nursing. The clinical and social significance of self‐help group participation has not been sufficiently accounted for.
`Carer and gatekeeper' – conflicting demands in nurses' experiences of telephone advisory services
Authors: Holmström Inger , Dall'Alba Gloria
Published: March 2003

`Carer and gatekeeper' – conflicting demands in nurses' experiences of telephone advisory services

Millions of calls are made to the telephone advisory services in primary health care in Sweden. The patients seem happy with the advice and counselling they receive, but little has been written about nurses' experiences of performing telephone advisory services. Yet, the nurses are expected to be patient, sensitive and have a broad knowledge of medicine, nursing and pedagogy. The aim of this study was to describe how nurses experience the patient encounter when performing telephone advisory services. A strategic sample of five nurses were interviewed and asked to describe how they experienced the central aspects of the patient encounter by telephone. The transcribed interviews were analysed by the Empirical Phenomenological Psychological method. The nurses' experience of the patient encounter when performing telephone advisory services can be characterized in terms of the conflicting demands of being both carer and gatekeeper. The constituents of these conflicting demands were: reading between the lines while pressed for time; educating patients for self‐care while fearful of misinterpreting the situation; encountering patients' satisfaction and dissatisfaction. The conflicting demands of being both professional carer and gatekeeper caused stress among the nurses. The organization of the telephone advisory services seems to hinder high‐quality care.