bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfection of 2,6 and 2,3‐sialyltransferase genes and GlcNAc‐transferase genes into human glioma cell line U‐373 MG affects glycoconjugate expression and enhances cell death
Authors: Dawson G. , Moskal J. R. , Dawson S. A. 
Published: June 2004

AbstractHuman glioma cell line U‐373 MG expresses CMP‐NeuAc : Galβ1,3GlcNAc α2,3‐sialyltransferase [EC No. 2.4.99.6] (α2,3ST), UDP‐GlcNAc : β‐d‐mannoside β1,6‐N‐acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT‐V) and UDP‐GlcNAc3: β‐d‐mannoside β1,4‐N‐acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT‐III) but not CMP‐NeuAc : Galβ1,4GlcNAc α2,6‐sialyltransferase [EC 2.4.99.1] (α2,6ST) under normal culture conditions. We have previously shown that transfection of the α2,6ST gene into U‐373 cells replaced α2,3‐linked sialic acids with α2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U‐373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U‐373 cell viability showed a three‐fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2‐ceramide or etoposide, when either α2,6ST and GnT‐V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as α2,3ST and GnT‐III, were able to decrease viability two‐fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase‐transfected U‐373 cells to pro‐apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase‐3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U‐373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3‐kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.
Blood‐to‐retina transport of creatine via creatine transporter (CRT) at the rat inner blood–retinal barrier
Authors: Nakashima Toshihisa , Tomi Masatoshi , Katayama Kazunori , Tachikawa Masanori , Watanabe Masahiko , Terasaki Tetsuya , Hosoya Ken‐ichi 
Published: June 2004

AbstractThe purpose of this study was to elucidate the mechanisms of blood‐to‐retina creatine transport across the blood–retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR‐iBRB2 cells) were examined using [14C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT‐PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [14C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [14C]Creatine uptake by TR‐iBRB2 cells was saturable, Na+‐ and Cl–‐dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT‐PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR‐iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood‐to‐retina creatine transport across the inner BRB.
Mitochondria dysfunction of Alzheimer's disease cybrids enhances Aβ toxicity
Authors: Cardoso Sandra M. , Santana Isabel , Swerdlow Russell H. , Oliveira Catarina R. 
Published: June 2004

AbstractAlzheimer's disease (AD) brain reveals high rates of oxygen consumption and oxidative stress, altered antioxidant defences, increased oxidized polyunsaturated fatty acids, and elevated transition metal ions. Mitochondrial dysfunction in AD is perhaps relevant to these observations, as such may contribute to neurodegenerative cell death through the formation of reactive oxygen species (ROS) and the release of molecules that initiate programmed cell death pathways. In this study, we analyzed the effects of beta‐amyloid peptide (Aβ) on human teratocarcinoma (NT2) cells expressing endogenous mitochondrial DNA (mtDNA), mtDNA from AD subjects (AD cybrids), and mtDNA from age‐matched control subjects (control cybrids). In addition to finding reduced cytochrome oxidase activity, elevated ROS, and reduced ATP levels in the AD cybrids, when these cell lines were exposed to Aβ 1–40 we observed excessive mitochondrial membrane potential depolarization, increased cytoplasmic cytochrome c, and elevated caspase‐3 activity. When exposed to Aβ, events associated with programmed cell death are activated in AD NT2 cybrids to a greater extent than they are in control cybrids or the native NT2 cell line, suggesting a role for mtDNA‐derived mitochondrial dysfunction in AD degeneration.
Brain‐specific potential guanine nucleotide exchange factor for Arf, synArfGEF (Po), is localized to postsynaptic density
Authors: Inaba Yuji , Tian Qing Bao , Okano Akira , Zhang Jing‐Ping , Sakagami Hiroyuki , Miyazawa Shoko , Li Weidong , Komiyama Atsushi , Inokuchi Kaoru , Kondo Hisatake , Suzuki Tatsuo 
Published: June 2004

AbstractWe cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585‐bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled‐coil structure in the N‐terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP‐ribosylation factor (Arf)‐binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region‐specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD‐95, SAP97 and Homer/Vesl 1/PSD‐Zip45 via its C‐terminal PDZ‐binding motif and co‐localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.
Reduction of excitotoxicity and associated leukocyte recruitment by a broad‐spectrum matrix metalloproteinase inhibitor
Authors: Campbell Sandra J. , Finlay Malcolm , Clements John M. , Wells Graham , Miller Karen M. , Perry V. Hugh , Anthony Daniel C. 
Published: June 2004

AbstractAn important step in the cascade leading to neuronal cell death is degradation of laminin and other components of the brain extracellular matrix by microglia‐derived proteases. Excitotoxic cell death of murine hippocampal neurones in vivo can be prevented by inhibitors of tissue plasminogen activator (tPA) or by inhibitors of plasmin. Plasmin is a potent activator of the matrix metalloproteinases (MMPs), which are made by resident and recruited leukocytes following CNS injury. In this study, we show, using Taqman RT‐PCR, that MMP mRNAs, but not other calcium‐dependent proteases such as calpain mRNAs, are acutely up‐regulated after an excitotoxic challenge in vivo. α2‐antiplasmin or BB‐3103, a broad‐spectrum inhibitor of the MMPs, co‐injected with kainic acid into the striatum, inhibits excitotoxic cell death in the rat striatum, and reduces both the number of recruited macrophages and the size of the lesion. We also show that leukocyte populations differentially express MMPs, which may account, in part, for the expression profile we observe in the challenged brain. Our results show that inhibition of the MMPs in the rat will prevent kainic acid‐induced cell death in the brain. These studies suggest that MMP inhibitors have therapeutic potential for use in stroke, and support the increasing evidence that microglial activation may contribute to neuronal cell death.
4‐Hydroxy‐trans‐2‐nonenoic acid is a γ‐hydroxybutyrate receptor ligand in the cerebral cortex and hippocampus
Authors: Murphy Tonya C. , Poppe Claire , Porter James E. , Montine Thomas J. , Picklo Sr Matthew J. 
Published: June 2004

AbstractElevated production of 4‐hydroxy‐trans‐2‐nonenal (HNE) occurs in numerous neurological disorders involving oxidative damage. HNE is metabolized to the non‐toxic 4‐hydroxy‐trans‐2‐nonenoic acid (HNEAcid) by aldehyde dehydrogenases in the rat cerebral cortex. Based upon the structural similarity of HNEAcid to ligands of the γ‐hydroxybutyrate (GHB) receptor, we hypothesized that HNEAcid is an endogenous ligand for the GHB receptor. HNEAcid displaced the specific binding of the GHB receptor ligand 3H‐NCS382 (30 nm) in membrane preparations of human frontal cerebral cortex and whole rat cerebral cortex with IC50s of 3.9 ± 1.1 and 5.6 ± 1.2 µm, respectively. Inhibition was attenuated when the carboxyl group of HNEAcid was replaced with an aldehyde or an alcohol. HNEAcid (300 µm) did not displace the binding of β‐adrenergic receptor and GABAB receptor antagonists, demonstrating the selectivity of HNEAcid for the GHB receptor. HNEAcid is formed in homogenates of human frontal cortical gray matter in an NAD+‐dependent (VMax, 0.71 nmol/min/mg) and NADP+‐dependent (VMax, 0.12 nmol/min/mg) manner. Lastly, 3H‐NCS382 binding is elevated 2.7‐fold with age in the cerebral cortex of rats. Our data demonstrate that an HNE metabolite, formed in rat and human brain, is a signaling molecule analogous to other bioactive lipid peroxidation products.
3,4‐Methylenedioxymethamphetamine increases interleukin‐1β levels and activates microglia in rat brain: studies on the relationship with acute hyperthermia and 5‐HT depletion
Authors: Orio Laura , O'Shea Esther , Sanchez Veronica , Pradillo Jesus M. , Escobedo Isabel , Camarero Jorge , Moro Maria A. , Green A. Richard , Colado M. Isabel 
Published: June 2004

Abstract3,4‐Methylenedioxymethamphetamine (MDMA) administration to rats produces acute hyperthermia and 5‐HT release. Interleukin‐1β (IL‐1β) is a pro‐inflammatory pyrogen produced by activated microglia in the brain. We examined the effect of a neurotoxic dose of MDMA on IL‐1β concentration and glial activation and their relationship with acute hyperthermia and 5‐HT depletion. MDMA, given to rats housed at 22°C, increased IL‐1β levels in hypothalamus and cortex from 1 to 6 h and [3H]‐(1‐(2‐chlorophenyl)‐N‐methyl‐N‐(1‐methylpropyl)3‐isoquinolinecarboxamide) binding between 3 and 48 h. Increased immunoreactivity to OX‐42 was also detected. Rats became hyperthermic immediately after MDMA and up to at least 12 h later. The IL‐1 receptor antagonist did not modify MDMA‐induced hyperthermia indicating that IL‐1β release is a consequence, not the cause, of the rise in body temperature. When MDMA was given to rats housed at 4°C, hyperthermia was abolished and the IL‐1β increase significantly reduced. The MDMA‐induced acute 5‐HT depletion was prevented by fluoxetine coadministration but the IL‐1β increase and hyperthermia were unaffected. Therefore, the rise in IL‐1β is not related to the acute 5‐HT release but is linked to the hyperthermia. Contrary to IL‐1β levels, microglial activation is not significantly modified when hyperthermia is prevented, suggesting that it might be a process not dependent on the hyperthermic response induced by MDMA.
Increased expression of the gene for the Y1 receptor of neuropeptide Y in the amygdala and paraventricular nucleus of Y1R/LacZ transgenic mice in response to restraint stress
Authors: Mele Paolo , Oberto Alessandra , Serra Mariangela , Pisu Maria Giuseppina , Floris Ivan , Biggio Giovanni , Eva Carola 
Published: June 2004

AbstractA sustained increase in the brain concentrations of neuroactive steroids was previously shown to induce Y1 receptor gene expression in the amygdala of Y1R/LacZ transgenic mice which harbour a construct comprising the murine Y1 receptor gene promoter and the lacZ reporter gene. We now investigated the effects of restraint stress on both the cerebrocortical concentrations of neuroactive steroids and Y1 receptor gene expression in the amygdala and hypothalamic paraventricular nucleus (PVN) of Y1R/LacZ transgenic mice. The cerebrocortical concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone were significantly increased immediately after a 1‐h exposure to restraint stress and had returned to control values within 30 min. Expression of Y1R/LacZ was increased in the amygdala and PVN 6 h after restraint. The 5α‐reductase inhibitor finasteride, that prevented the increase in neuroactive steroid concentrations, did not block that in transgene expression induced by 1‐h restraint. Daily exposure to restraint for 10 days also increased the cerebrocortical concentrations of neuroactive steroids but failed to affect transgene expression. Acute but not repeated restraint thus increases Y1 receptor gene expression in the amygdala and PVN, suggesting that tolerance develops towards this stressor. The effect of acute restraint is not mediated by the increase in the brain concentrations of neuroactive steroids but may rather reflect a ligand‐induced increase in Y1 receptor gene transcription. Data support a role of Y1 receptors in the behavioural and neuroendocrine responses to stress.
Parkin attenuates manganese‐induced dopaminergic cell death
Authors: Higashi Youichirou , Asanuma Masato , Miyazaki Ikuko , Hattori Nobutaka , Mizuno Yoshikuni , Ogawa Norio 
Published: June 2004

AbstractManganese as environmental factor is considered to cause parkinsonism and induce endoplasmic reticulum stress‐mediated dopaminergic cell death. We examined the effects of manganese on parkin, identified as the gene responsible for familial Parkinson's disease, and the role of parkin in manganese‐induced neuronal cell death. Manganese dose‐dependently induced cell death of dopaminergic SH‐SY5Y and CATH.a cells and cholinergic Neuro‐2a cells, and that the former two cell types were more sensitive to manganese toxicity than Neuro‐2a cells. Moreover, manganese increased the expression of endoplasmic reticulum stress‐associated genes, including parkin, in SH‐SY5Y cells and CATH.a cells, but not in Neuro‐2a cells. Treatment with manganese resulted in accumulation of parkin protein in SH‐SY5Y cells and its redistribution to the perinuclear region, especially aggregated Golgi complex, while in Neuro‐2a cells neither expression nor redistribution of parkin was noted. Manganese showed no changes in proteasome activities in either cell. Transient transfection of parkin gene inhibited manganese‐ or manganese plus dopamine‐induced cell death of SH‐SY5Y cells, but not of Neuro‐2a cells. Our results suggest that the attenuating effects of parkin against manganese‐ or manganese plus dopamine‐induced cell death are dopaminergic cell‐specific compensatory reactions associated with its accumulation and redistribution to perinuclear regions but not with proteasome system.
Dopamine D2 receptor activates extracellular signal‐regulated kinase through the specific region in the third cytoplasmic loop
Authors: Takeuchi Yusuke , Fukunaga Kohji 
Published: June 2004

AbstractTo investigate whether the third cytoplasmic loop and the C‐terminal cytoplasmic tail of dopamine D2 receptor (D2R) are involved in extracellular signal‐regulated kinase (ERK) activation and subsequent regulation of transcription factors, we established NG108‐15 cells stably expressing D2LR and D2SR deleted 40 amino acid residues in the third cytoplasmic loop (NGD2LR‐3rd‐dele and NGD2SR‐3rd‐dele) or the C‐terminal cytoplasmic tail (NGD2LR‐C‐dele and NGD2SR‐C‐dele) and evaluated these receptors' functions using luciferase reporter gene assay. Immunocytochemical studies showed similar intracellular distributions of D2LR‐3rd‐dele and D2SR‐3rd‐dele to D2LR and D2SR, respectively. Quinpirole‐induced inhibition of forskolin‐induced cyclic AMP responsive element (CRE) activation was not affected by the deletion of 40 amino acid residues. However, nuclear factor‐kappa B (NF‐κB) activation by D2R‐3rd‐dele was largely attenuated compared to that by D2R. Similarly, ERK or serum‐responsive element (SRE) activation by quinpirole treatment was totally abolished in NGD2R‐3rd‐dele cells. Moreover, D2R‐C‐dele was diffusely distributed or clustered in the cell bodies and lost the receptor functions. Taken together, the 40 amino acid residues in the third cytoplasmic loop are essential for the ERK activation but not for inhibition of adenylyl cyclase through Gi/o proteins. In addition, the C‐terminal cytoplasmic tail is essential for membrane association of D2Rs to elicit the receptor functions.
Immature nicastrin stabilizes APH‐1 independent of PEN‐2 and presenilin: identification of nicastrin mutants that selectively interact with APH‐1
Authors: Shirotani Keiro , Edbauer Dieter , Kostka Marcus , Steiner Harald , Haass Christian 
Published: June 2004

Abstractγ‐Secretase is a high molecular mass aspartyl protease complex composed of presenilin (PS1 or PS2), nicastrin (Nct), anterior pharynx‐defective‐1 (APH‐1) and presenilin enhancer‐2 (PEN‐2). The complex mediates the intramembraneous proteolysis of β‐secretase cleaved β‐amyloid precursor protein (APP) leading to the secretion of the Alzheimer's disease‐associated amyloid β‐peptide (Aβ). In order to dissect functionally important domains of Nct required for γ‐secretase complex assembly, maturation, and activity we mutated evolutionary conserved amino acids. The mutant Nct variants were expressed in a cellular background with significantly reduced endogenous Nct. Mutant Nct was functionally investigated by its ability to restore PS, APH‐1 and PEN‐2 expression as well as by monitoring the accumulation of the APP C‐terminal fragments, the immediate substrates of γ‐secretase. We identified three independent mutations within the ectodomain of Nct, which rescued expression of APH‐1 but not of PEN‐2 or PS and thus failed to restore γ‐secretase activity. Interestingly, these immature Nct variants selectively bound to APH‐1, suggesting a stable Nct/APH−1 interaction independent of PS and PEN‐2. Consistent with this finding, expression of APH‐1 remained largely unaffected in the PS double knock‐out and immature Nct co‐immunoprecipitated with APH‐1 in the absence of PS and PEN‐2. Taken together, our findings suggest that immature Nct can stably interact with APH‐1 to form a potential scaffold for binding of PS and PEN‐2. Moreover, binding of the latter two complex partners critically depends on the integrity of the Nct ectodomain.
Neuroprotective effects of the mGlu5R antagonist MPEP towards quinolinic acid‐induced striatal toxicity: involvement of pre‐ and post‐synaptic mechanisms and lack of direct NMDA blocking activity
Authors: Popoli Patrizia , Pintor Annita , Tebano Maria Teresa , Frank Claudio , Pepponi Rita , Nazzicone Valeria , Grieco Rosa , Pèzzola Antonella , Reggio Rosaria , Minghetti Luisa , De Berardinis Maria Anna , Martire Alberto , Potenza Rosa Luisa , Domenici Maria Rosaria , Massotti Marino 
Published: June 2004

AbstractThe aim of this work was to investigate the potential neuroprotective effects of the metabotropic glutamate receptor 5 (mGlu5R) antagonist 2‐Methyl‐6‐(phenylethynyl)‐pyridine (MPEP) towards quinolinic acid (QA)–induced striatal excitoxicity. Intrastriatal MPEP (5 nmol/0.5 µL) significantly attenuated the body weight loss, the electroencephalographic alterations, the impairment in spatial memory and the striatal damage induced by bilateral striatal injection of QA (210 nmol/0.7 µL). In a second set of experiments, we aimed to elucidate the mechanisms underlying the neuroprotective effects of MPEP. In microdialysis studies in naive rats MPEP (80–250 µm through the dialysis probe) significantly reduced the increase in glutamate levels induced by 5 mm QA. In primary cultures of striatal neurons MPEP (50 µm) reduced the toxicity induced by direct application of glutamate [measured as release of lactate dehydrogenase [LDH]). Finally, we found that 50 µm MPEP was unable to directly block NMDA‐induced effects (namely field potential reduction in corticostriatal slices, as well as LDH release and intracellular calcium increase in striatal neurons). We conclude that: (i) MPEP has neuroprotective effects towards QA‐induced striatal excitotoxicity; (ii) both pre‐ and post‐synaptic mechanisms are involved; (iii) the neuroprotective effects of MPEP do not appear to involve a direct blockade of NMDA receptors.
A novel lipopolysaccharide‐modulated Jun binding repressor in intron 2 of CYP2E1
Authors: Tindberg Niclas , Bengtsson Inger , Hu Yin 
Published: June 2004

AbstractCytochrome P450 2E1 (CYP2E1) exhibits a pronounced oxidase activity that may mediate apoptotic injury in glial cells as well as hepatocytes. Strict regulation of CYP2E1 and it's activity is therefore thought to be crucial. We have studied CYP2E1 transcriptional regulation in primary cortical glial cells and have identified a novel repressor element at +1452/+1460 in intron 2 of the rat CYP2E1 gene. The element very potently repressed CYP2E1 and SV40 promoters and consisted of the non‐palindromic core sequence 5′‐TTCCACTCA‐3′. Jun proteins were found to interact with the site. The protein complexes were also found to contain an as yet unidentified protein of ≈60 kDa, probably with DNA binding properties similar to G‐box binding factors found in, e.g. Arabidopsis thaliana. Stimulation with lipopolysaccharide, or overexpression of the mitogen‐activated protein kinase kinase kinase, MEKK‐1, further deepened the repression in primary cortical glial cells. It is suggested that this novel Jun binding repressor helps to control basal expression levels of CYP2E1, and modulates the response to inflammatory factors. Future in vivo experiments will, however, be required for a full appreciation of the role of this repressor in the complex regulation of CYP2E1 during inflammatory conditions.
Multi‐modal regulation of endogenous D1 dopamine receptor expression and function in the CAD catecholaminergic cell line
Authors: Pasuit Jennifer B. , Li Zhuting , Kuzhikandathil Eldo V. 
Published: June 2004

AbstractDopamine receptors exhibit tissue‐ and cell type‐specific expression that is modulated during development, aging and in diseases such as Parkinson's. The molecular mechanisms regulating expression of dopamine receptors are not well understood, in part due to the lack of a model cell line that not only expresses endogenous dopamine receptors but also has the requisite regulatory mechanisms. Here, we demonstrate that the CAD catecholaminergic cell line expresses D1, D2, D3 and D5 dopamine receptor subtypes and associated signaling proteins. CAD cell differentiation induced by serum withdrawal increases the levels of D1 receptor mRNA by transcriptional up‐regulation. This increase is also mimicked by the neurotrophin NT3. Interestingly, the increase of D1 receptor mRNA does not result in increased levels of D1 receptor protein in differentiated CAD cells. Furthermore, while the D1 receptor protein is expressed in differentiated CAD cells, it loses its ability to activate adenylyl cyclase. We demonstrate that the post‐transcriptional regulation is not due to decreased D1 receptor mRNA stability or generation of a truncated D1 receptor mRNA, and that the down‐regulation of D1 receptor function in differentiated CAD cells is mediated by post‐translational mechanisms that decrease cell surface receptor expression by altering receptor processing and trafficking.