bluuebunny/crossref_metadata_2025_split huggingface dataset:
The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly
Authors: Bongiorno‐Borbone Lucilla , Onofri Franco , Giovedì Silvia , Ferrari Rosaria , Girault Jean‐Antoine , Benfenati Fabio 
Published: March 2003

AbstractFocal adhesion kinase (FAK) and the related proline‐rich tyrosine kinase 2 (PYK2) are non‐receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane‐associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane‐associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.
Characterization of [125I]epibatidine binding and nicotinic agonist‐mediated 86Rb+ efflux in interpeduncular nucleus and inferior colliculus of β2 null mutant mice
Authors: Marks Michael J. , Whiteaker Paul , Grady Sharon R. , Picciotto Marina R. , McIntosh J. Michael , Collins Allan C. 
Published: March 2003

AbstractThe β2 nicotinic acetylcholine receptor subunit null mutation eliminated most high affinity [3H]epibatidine binding in mouse brain, but significant binding remained in accessory olfactory nucleus, medial habenula, inferior colliculus and interpeduncular nucleus. Residual [125I]epibatidine binding sites in the inferior colliculus and interpeduncular nucleus were subsequently characterized. Inhibition of [125I]epibatidine binding by 12 agonists and six antagonists was very similar in these regions. Most acetylcholine‐stimulated 86Rb+ efflux is eliminated in thalamus and superior colliculus of β2 null mutants, but significant activity remained in inferior colliculus and interpeduncular nucleus. This residual activity was subsequently characterized. The 12 nicotinic agonists tested elicited concentration‐dependent 86Rb+ efflux. Epibatidine was the most potent agonist. Cytisine was also potent and efficacious. EC50 values for quaternary agonists were relatively high. Cytisine‐stimulated 86Rb+ efflux was inhibited by six classical nicotinic antagonists. Mecamylamine and d‐tubocurarine were most potent, while decamethonium was the least potent. Agonists and antagonists exhibited similar potency in both brain regions. α‐Bungarotoxin (100 nm) did not significantly inhibit cytisine‐stimulated 86Rb+ efflux, while the α3β4 selective antagonist, αConotoxinAuIB, inhibited a significant fraction of the response in both brain regions. Thus, β2 null mutant mice express residual nicotinic activity with properties resembling those of α3β4*‐nAChR.
Alternative non‐coding exons support serotonin transporter mRNA expression in the brain and gut
Authors: Ozsarac N. , Santha E. , Hoffman B. J. 
Published: March 2003

AbstractA number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5′ rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5′ untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue‐specific manner, we used RT–PCR and exon‐specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.
Different pathways for iNOS‐mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression
Authors: Golde Sabine , Chandran Siddharthan , Brown Guy C. , Compston Alastair 
Published: March 2003

AbstractCo‐localization of activated microglia and damaged neurones seen in brain injury suggests microglia‐induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co‐cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)‐derived NO aloneis responsible for neuronal death from interferon γ(IFNγ) +lipopolysaccharide (LPS)‐activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO‐mediated toxicity to immature neurones, MK801 rescues 60–70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO‐donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO‐mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.
Insulin‐like growth factor (IGF)‐I binding to a cell membrane associated IGF binding protein‐3 acid‐labile subunit complex in human anterior pituitary gland
Authors: Wilczak Nadine , Kühl Nicole , Chesik Daniel , Geerts Albert , Luiten Paul , De Keyser Jacques 
Published: March 2003

AbstractThe binding characteristics of [125I]insulin‐like growth factor (IGF)‐I were studied in human brain and pituitary gland. Competition binding studies with DES(1‐3)IGF‐I and R3‐IGF‐I, which display high affinity for the IGF‐I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [125I]IGF‐I binding to IGF‐I receptors and IGFBPs. Specific [125I]IGF‐I binding in brain regions and the posterior pituitary was completely displaced by DES(1‐3)IGF‐I and R3‐IGF‐I, indicating binding to IGF‐I receptors. In contrast, [125I]IGF‐I binding in the anterior pituitary was not displaced by DES(1‐3)IGF‐I and R3‐IGF‐I, suggesting binding to an IGF‐binding site that is different from the IGF‐I receptor. Binding affinity of IGF‐I to this site was about 10‐fold lower than for the IGF‐I receptor. Using western immunoblotting we were also unable to detect IGF‐I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150‐kDa IGFBP‐3‐acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT‐PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP‐3 was easily washed away during pre‐incubation procedures as used in the [125I]IGF‐I binding experiments. In contrast, the IGFBP‐3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane‐bound 150‐kDa IGFBP‐3–ALS complex that is absent in brain and posterior pituitary.
Muscarinic receptor‐mediated phosphorylation of cyclic AMP response element binding protein in human neuroblastoma cells
Authors: Greenwood Jeffrey M. , Dragunow Michael 
Published: March 2003

AbstractThis study describes the effect of signalling through muscarinic acetylcholine receptors on two transcription factors implicated in long‐term synaptic plasticity and memory formation, EGR1 and the cyclic AMP response element binding protein (CREB). In SK‐N‐SH neuroblastoma cells, treatment with the cholinergic agonist carbachol led to maximal induction of EGR1 1 h after stimulation. This was preceded by the phosphorylation of CREB, which peaked as early as 5 minutes after carbachol treatment. The levels of both EGR1 and phosphorylated CREB (pCREB) slowly decayed over 4–8 h. CREB phosphorylation and EGR1 induction showed similar sensitivity to carbachol concentration, with EC50 values in the range of 1–10 µM, and the changes in both transcription factors were blocked by the muscarinic antagonist atropine. As has been described elsewhere, EGR1 induction was dependent on activation of p42/44 MAP kinase, as it was blocked by the MEK inhibitor U0126. However, CREB phosphorylation by carbachol was largely unaffected by MAP kinase blockade. As both CREB phosphorylation and EGR1 induction have been linked to long‐term potentiation and some forms of memory consolidation, these results may implicate CREB and EGR1 in independent or partially independent cholinergic signalling pathways involved in memory processes.
Protective effects of docosahexaenoic acid in staurosporine‐induced apoptosis: involvement of phosphatidylinositol‐3 kinase pathway
Authors: Akbar Mohammed , Kim Hee‐Yong 
Published: March 2003

AbstractDocosahexaenoic acid (22:6n‐3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kimet al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase‐3 activity, cleavage of pro‐caspase‐3 to active caspase‐3, DNA strand‐breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3‐kinase (PI3‐K) inhibitors, wortmanin, and LY‐294002 abolished the protective effect of DHA in ST‐induced apoptosis. Concurrently, ST‐treatment significantly decreased the phosphorylation status of Akt at Ser‐473 and Thr‐308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time‐dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine‐free medium, the PS increase diminished and the DHA effect on caspase‐3 activation as well as Akt phosphorylation in ST‐induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST‐induced apoptosis in a PS‐ and PI3‐K‐dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3‐K/Akt pathway, facilitated by DHA‐induced PS accumulation.
Direct visualization of the gamma secretase‐generated carboxyl‐terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus
Authors: Kinoshita A. , Whelan C. M. , Smith C. J. , Berezovska O. , Hyman B. T. 
Published: March 2003

AbstractAmyloid‐β, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase‐mediated intramembranous cleavage. In addition to amyloid‐β, this cleavage produces a carboxyl‐terminal intracellular fragment which has an unknown function. The carboxyl‐terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl‐terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP‐CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP‐CT‐Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP‐CT.
Erucylphosphocholine‐induced apoptosis in glioma cells: involvement of death receptor signalling and caspase activation
Authors: Kugler Wilfried , Erdlenbruch Bernhard , Jünemann Anja , Heinemann Dagmar , Eibl Hansjörg , Lakomek Max 
Published: March 2003

AbstractErucylphosphocholine (ErPC) is a promising anti‐neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti‐cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC‐mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFα or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant‐negative FADD construct did not abolish ErPC‐induced effects. Western blot analysis indicated that ErPC‐triggered apoptosis resulted in a time‐dependent processing of caspases‐3, ‐7, ‐8 and ‐9 into their respective active subunits. Co‐treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase‐independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.
Numb isoforms containing a short PTB domain promote neurotrophic factor‐induced differentiation and neurotrophic factor withdrawal‐induced death of PC12 Cells
Authors: Pedersen Ward A. , Chan Sic L. , Zhu Haiyan , Abdur‐Rahman Lilanie A. , Verdi Joseph M. , Mattson Mark P. 
Published: March 2003

AbstractThe development of the nervous system is regulated by trophic signals that control cell proliferation, differentiation, and survival. Numb is an evolutionarily conserved protein identified by its ability to control cell fate in the nervous system ofDrosophila. Mammals express four isoforms of Numb that differ in the length of a phosphotyrosine‐binding (PTB) domain and a proline‐rich region (PRR). Using PC12 cells stably expressing each of the human isoforms, we show that Numb regulates sensitivity of the cells to neurotrophic factor‐induced differentiation and neurotrophic factor withdrawal‐induced death in an isoform‐specific manner. Numb isoforms containing a short PTB domain enhance the differentiation response to NGF and enhance apoptosis upon NGF withdrawal; Numb isoforms containing a long PTB domain exhibit the same sensitivity to NGF as vector‐transfected cells. These effects of Numb were found to be independent of the length of the PRR. In undifferentiated conditions, the levels of full‐length TrkA and of phosphorylated p44/p42 mitogen‐activated protein kinase (MAPK) are increased in cells expressing Numb isoforms with a short PTB domain, indicating an up‐regulation of NGF signaling pathways. Furthermore, we provide evidence that the mechanism whereby short PTB domain Numb isoforms sensitize cells to trophic factor deprivation‐induced apoptosis involves elevations in intracellular calcium concentrations. Our results suggest that Numb sensitizes cells to neurotrophin responses in an isoform‐specific manner, an effect that may play an important role in the development and plasticity of the nervous system.
Suppression of the morphine‐induced rewarding effect in the rat with neuropathic pain: implication of the reduction in µ‐opioid receptor functions in the ventral tegmental area
Authors: Ozaki Satoru , Narita Minoru , Narita Michiko , Iino Masahiko , Sugita Junichi , Matsumura Yumiko , Suzuki Tsutomu 
Published: March 2003

AbstractThe present study was designed to investigate the rewarding effect, G‐protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham‐operated and sciatic nerve‐ligated rats in the increases in guanosine‐5′‐o‐(3‐[35S]thio)triphosphate ([35S]GTPγS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N‐propylnoraporphine or the D3 receptor agonist 7‐hydroxy‐2‐dipropylaminotetralin (7‐OH DPAT). In contrast, the increases in [35S]GTPγS binding to membranes of the VTA induced by either morphine or a selective µ‐opioid receptor agonist [d‐Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve‐ligated rats as compared with sham‐ operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine‐induced place preference under neuropathic pain may result from a decrease in the morphine‐induced DA release in the N.Acc with reduction in the µ‐opioid receptor‐mediated G‐protein activation in the VTA.
Kinetics of nitric oxide‐cyclic GMP signalling in CNS cells and itspossible regulation by cyclic GMP
Authors: Wykes Victoria , Bellamy Tomas C. , Garthwaite John 
Published: March 2003

AbstractPhysiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO‐evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics ofcGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP‐stimulated PDE (PDE 2). Analysis of NO‐induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.
Survival‐promoting activity of nimodipine and nifedipine in rat motoneurons: implications of an intrinsic calcium toxicity in motoneurons
Authors: Arakawa Yoshihiro , Nishijima Chiaki , Shimizu Nao , Urushidani Tetsuro 
Published: March 2003

AbstractL‐type calcium channel antagonists, nimodipine and nifedipine, were tested for effects on the survival of purified rat motoneurons in culture. They showed significant activity, with maximum survival at 30 µm after 3 days in culture as high as 75%, which was comparable to the maximum effect obtained with brain‐derived neurotrophic factor, a potent neurotrophic factor for rat motoneurons. It was also found that depolarizing conditions with a high potassium concentration (30 mm) were toxic to motoneurons. This toxicity was blocked by co‐treatment with nimodipine. These results implicate a pre‐existing calcium burden through calcium channels in motoneurons; they may offer further insights into understanding the selective death of motoneurons and have therapeutic implications in amyotrophic lateral screlosis.
Synphilin‐1 degradation by the ubiquitin‐proteasome pathway and effects on cell survival
Authors: Lee Gwang , Junn Eunsung , Tanaka Mikiei , Kim Yong Man , Lee Sang Seop , Mouradian M. Maral 
Published: March 2003

AbstractParkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. α‐Synuclein and its interacting partner synphilin‐1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin‐1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG‐tagged synphilin‐1. Pulse‐chase experiments revealed that this protein is relatively stable with a half‐life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin‐1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri‐nuclear inclusions which were immunoreactive for synphilin‐1, ubiquitin and α‐synuclein. Cell viability studies revealed increased susceptibility of synphilin‐1 over‐expressing cells to proteasomal dysfunction. These observations indicate that synphilin‐1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin‐1 due to impaired clearance results in its aggregation as peri‐nuclear inclusions and in poor cell survival.