bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a cysteine proteinase inhibitor induced during neuronal cell differentiation
Authors: Hong Jia , Yoshida Keiko , Rosner Marsha Rich 
Published: March 2003

AbstractA rat homolog of human cystatin E/M was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the family 2 cystatins, rat cystatin E/M is secreted, glycosylated and developmentally regulated. Rat cystatin E/M is expressed in brain, and is induced during differentiation of a conditionally immortalized E17 rat hippocampal cell line (H19‐7) by bFGF or activated Raf via MEK‐dependent and ‐independent signaling pathways. Rat cystatin E/M protein is increased post‐transcriptionally in PC12 cells, and the protein is secreted into the medium of primary embryonal hippocampal cultures. Analysis of the Ki of recombinant His‐tagged rat cystatin E/M toward cathepsins B and H revealed that rat cystatin E/M has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between rat cystatin E/M and human cystatin C, a well‐characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Taken together, these results describe a member of the cystatin family that has a distinct inhibitor profile and may play a role in neuronal development.
Detergent‐insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane
Authors: Taylor Christopher M. , Coetzee Timothy , Pfeiffer Steven E. 
Published: March 2003

AbstractGlycosphingolipids and cholesterol form lateral assemblies, or lipid ‘rafts’, within biological membranes. Lipid rafts are routinely studied biochemically as low‐density, detergent‐insoluble complexes (in non‐ionic detergents at 4°C; DIGs, detergent‐insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re‐evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol‐rich myelin membrane, using four detergents representing different chemical classes (TX‐100, CHAPS, Brij 96 and TX‐102), under four conditions: detergent extraction of myelin either at (i) 4°C or (ii) 37°C, or at 4°C after pre‐extraction with (iii) saponin or (iv) methyl‐β‐cyclodextrin (MβCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre‐extracted with saponin were denser than DIGs pre‐extracted with MβCD. Furthermore, pre‐extraction with MβCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre‐extraction with saponin did not, suggesting that saponin is less effective as a cholesterol‐perturbing agent than is MβCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent‐specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.
PI 3‐kinase regulation of dopamine uptake
Authors: Carvelli Lucia , Morón José A. , Kahlig Kristopher M. , Ferrer Jasmine V. , Sen Namita , Lechleiter James D. , Leeb‐Lundberg L. M. Fredrik , Merrill Gerald , Lafer Eileen M. , Ballou Lisa M. , Shippenberg Toni S. , Javitch Jonathan A. , Lin Richard Z. , Galli Aurelio 
Published: March 2003

AbstractThe magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3‐kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [3H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3‐kinase, increased [3H]DA uptake and blocked the amphetamine‐induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3‐kinase. The LY294002‐induced reduction in [3H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin‐dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.
D3 receptor ligands modulate extracellular dopamine clearance in the nucleus accumbens
Authors: Zapata A. , Shippenberg T. S. 
Published: March 2003

AbstractAn involvement of the D3 dopamine receptor in the regulation of extracellular dopamine has been suggested. However, the mechanisms mediating this effect are unclear. We have used the technique of no net flux microdialysis under transient conditions to examine the influence of the D3‐preferring agonist (+)‐PD128907 upon extracellular dopamine levels in the nucleus accumbens of the mouse. (+)‐PD 128907 (0.1 mg/kg intraperitoneally) significantly decreased extracellular dopamine. This decrease was associated with a marked increase in the extraction fraction, which suggests an increase in dopamine clearance. The ability of D3‐preferring compounds to modulate dopamine uptake was investigated in vitro using rotating disk electrode voltammetry. (+)‐PD 128907 (10 nm) significantly increased the initial clearance rate of 3 μm dopamine in rat nucleus accumbens tissue suspensions. Kinetic analysis revealed no change in the apparent Km of uptake but it showed a 33% increase in Vmax. In contrast, the D3 antagonist GR 103691 (10 nm) significantly decreased dopamine uptake. Consistent with the low levels of D3 receptors in the dorsal striatum, neither compound affected uptake in tissue suspensions from this brain region. These data indicate that D3 receptor activation increases dopamine uptake in the nucleus accumbens and suggest that this receptor subtype can regulate extracellular dopamine by modulating the DA transporter activity.
Heterogeneity of normal prion protein in two‐dimensional immunoblot: presence of various glycosylated and truncated forms
Authors: Pan Tao , Li Ruliang , Wong Boon‐Seng , Liu Tong ,   Pierluigi Gambetti and Man‐Sun Sy
Published: March 2003

AbstractThe common use of one‐dimensional (1‐D) immunoblot with a single monoclonal antibody (Mab) engenders the notion that the normal or cellular prion protein (PrPC) comprises few and simple forms. In this study we used two‐dimensional (2‐D) immunoblot with a panel Mabs to various regions of the prion protein to demonstrate the complexity of the PrPC present in human brain. We distinguished over 50 immunoblot spots, each representing a distinct PrPC species based on combinations of different molecular weights and isoelectric points (pIs). The PrPC heterogeneity is due to the presence of a full‐length and two major truncated forms as well as to the diversity of the glycans linked to most of these forms. The two major truncated forms result from distinct cleavage sites located at the N‐terminus. In addition, enzymatic removal of sialic acid and lectin binding studies indicate that the glycans linked to the full‐length and truncated PrPC forms differ in their structure and ratios of the glycoforms. The truncation of PrPC and the heterogeneity of the linked glycans may play a role in regulating PrPC function. Furthermore, the presence of relatively large quantities of different PrPC species may provide additional mechanisms by which the diversity of prion strains could be generated.
Alternative splicing isoform of tau protein kinase I/glycogen synthase kinase 3β
Authors: Mukai Fumiko , Ishiguro Koichi , Sano Yumiko , Fujita Shinobu C. 
Published: March 2003

AbstractGlycogen synthase kinase 3 (GSK3) plays important roles in Wnt and insulin signaling, cell fate determination, and Alzheimer‐like tau phosphorylation. We discovered an isoform of tau protein kinase I (TPKI)/GSK3β with a 13 amino acid insert in the catalytic domain owing to alternative splicing. The alternative transcripts were found in the brains of the mouse, rat and human, with highly conserved sequences. The variant protein, named TPKI2/GSK3β2, was abundant in the brain. Immunohistochemistry indicated differential distribution of the conventional and the new TPKI/GSK3β isoforms within young neurons. TPKI2/GSK3β2 showed decreased kinase activities towards two phosphorylation sites on tau compared with the conventional isoform. Immunohistochemistry indicated that TPKI2/GSK3β2 occurs predominantly in the neuronal soma, while TPKI1/GSK3β1 is found both in the soma and processes. These results indicate that the new splice isoform has a different function. Because the amino acid insert occurs in the domain implicated in interaction with a protein phosphatase in a homologous kinase cdk‐2, the alternative splicing can regulate multiprotein complex formation and function involving TPKI/GSK3β.
Poly(ADP‐ribose) polymerase inhibition prevents both apoptotic‐like delayed neuronal death and necrosis after H2O2 injury
Authors: Cole Kasie K. , Perez‐Polo J. Regino 
Published: March 2003

AbstractToxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3‐aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP‐ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H2O2 injury. Exposure to 0.5 mm H2O2 resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12–24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H2O2‐induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase‐3 like activity was absent. Moreover, pretreatment with AB restored caspase‐3‐like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.
Effect of ethanol on protein kinase Cζ and p70S6 kinase activation by carbachol: a possible mechanism for ethanol‐induced inhibition of glial cell proliferation
Authors: Guizzetti Marina , Costa Lucio G. 
Published: March 2003

AbstractThe signal transduction pathways that mediate the mitogenic response of muscarinic acetylcholine receptors in astroglial cells have not been fully elucidated. In this study we investigated the activation of p70S6 kinase (p70S6K) by carbachol in 1321 N1 astroctyoma cells. Carbachol induced a dose‐ and time‐dependent activation of p70S6K, as evidenced by increased phosphorylation at Thr‐389, Thr‐421 and Ser‐424, by increased p70S6K activity, and by a shift in its molecular weight. Activation of p70S6K was mediated by M3 muscarinic acetylcholine receptors (mAChRs) and was inhibited by two phosphatidylinositol‐3‐kinase (PI3‐K) inhibitors, by a pseudosubstrate to protein kinase C (PKC) ζ, and by the p70S6K inhibitor rapamycin. Carbachol‐induced DNA synthesis was strongly inhibited by rapamycin, suggesting that p70S6K activation plays an important role in carbachol‐induced cell proliferation. Ethanol (25–100 mm) has been shown to inhibit carbachol‐induced proliferation of astroglial cells. In the same range of concentrations, ethanol also inhibits carbachol‐induced activation of PKCζ and of p70S6K. On the other hand, inhibition of PI3‐kinase was only observed at higher ethanol concentrations. These results indicate that activation of the PKCζ→ p70S6K pathway by M3 mAChRs may play a role in the increased DNA synthesis and may represent a target for ethanol‐induced inhibition of astroglial cell proliferation.
Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures
Authors: Patel Ravi , McIntosh Laura , McLaughlin John , Brooke Sheila , Nimon Vitaliy , Sapolsky Robert 
Published: March 2003

AbstractGlucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non‐energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.
Lithium inhibits aluminum‐induced apoptosis in rabbit hippocampus, by preventing cytochrome c translocation, Bcl‐2 decrease, Bax elevation and caspase‐3 activation
Authors: Ghribi Othman , Herman Mary M. , Spaulding Natalie K. , Savory John 
Published: March 2003

AbstractA variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis‐regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti‐apoptotic proteins Bcl‐2 and Bcl‐XL, increases levels of the pro‐apoptotic Bax, activates caspase‐3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum‐induced translocation of cytochrome c, and up‐regulates Bcl‐2 and Bcl‐XL, down‐regulates Bax, abolishes caspase‐3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis‐controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis‐regulatory proteins present in the subcellular organelles of rabbit brain.
Isolation of various forms of sterol β‐d‐glucoside from the seed of Cycas circinalis: neurotoxicity and implications for ALS‐parkinsonism dementia complex
Authors: Khabazian I. , Bains J. S. , Williams D. E. , Cheung J. , Wilson J. M. B. , Pasqualotto B. A. , Pelech S. L. , Andersen R. J. , Wang Y.‐T. , Liu L. , Nagai A. , Kim S. U. , Craig U‐K. , Shaw C. A. 
Published: March 2003

AbstractThe factors responsible for ALS‐parkinsonism dementia complex (ALS‐PDC), the unique neurological disorder of Guam, remain unresolved, but identification of causal factors could lead to clues for related neurodegenerative disorders elsewhere. Earlier studies focused on the consumption and toxicity of the seed of Cycas circinalis, a traditional staple of the indigenous diet, but found no convincing evidence for toxin‐linked neurodegeneration. We have reassessed the issue in a series of in vitro bioassays designed to isolate non‐water soluble compounds from washed cycad flour and have identified three sterol β‐d‐glucosides as potential neurotoxins. These compounds give depolarizing field potentials in cortical slices, induce alterations in the activity of specific protein kinases, and cause release of glutamate. They are also highly toxic, leading to release of lactate dehydrogenase (LDH). Theaglycone form, however, is non‐toxic. NMDA receptor antagonists block the actions of the sterol glucosides, but do not compete for binding to the NMDA receptor. The most probable mechanism leading to cell death may involve glutamate neuro/excitotoxicity. Mice fed cycad seed flour containing the isolated sterol glucosides show behavioral and neuropathological outcomes, including increased TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) positivity in various CNS regions. Astrocytes in culture showed increased caspase‐3 labeling after exposure to sterol glucosides. The present results support the hypothesis that cycad consumption may be an important factor in the etiology of ALS‐PDC and further suggest that some sterol glucosides may be involved in other neurodegenerative disorders.
Oligodendroglial cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells
Authors: Hirrlinger Johannes , Resch Alexandra , Gutterer Jan Mirko , Dringen Ralf 
Published: March 2003

AbstractTo investigate the antioxidative capacities of oligodendrocytes, rat brain cultures enriched for oligodendroglial cells were prepared and characterized. These cultures contained predominantly oligodendroglial cells as determined by immunocytochemical staining for the markers galactocerebroside and myelin basic protein. If oligodendroglial cultures were exposed to exogenous hydrogen peroxide (100 µm), the peroxide disappeared from the incubation medium following first order kinetics with a half‐time of approximately 18 min. Normalization of the disposal rate to the protein content of the cultures by calculation of the specific hydrogen peroxide detoxification rate constant revealed that the cells in oligodendroglial cultures have a 60% to 120% higher specific capacity to dispose of hydrogen peroxide than cultures enriched for astroglial cells, microglial cells or neurones. Oligodendroglial cultures contained specific activities of 133.5 ± 30.4 nmol × min−1 × mg protein−1 and 27.5 ± 5.4 nmol × min−1 ×  mg protein−1 of glutathione peroxidase and glutathione reductase, respectively. The specific rate constant of catalase in these cultures was 1.61 ± 0.54 min−1 × mg protein−1. Comparison with data obtained by identical methods for cultures of astroglial cells, microglial cells and neurones revealed that all three of the enzymes which are involved in hydrogen peroxide disposal were present in oligodendroglial cultures in the highest specific activities. These results demonstrate that oligodendroglial cells in culture have a prominent machinery for the disposal of hydrogen peroxide, which is likely to support the protection of these cells in brain against peroxides when produced by these or by surrounding brain cells.
Nitric oxide regulates cGMP‐dependent cAMP‐responsive element binding protein phosphorylation and Bcl‐2 expression in cerebellar neurons: implication for a survival role of nitric oxide
Authors: Ciani Elisabetta , Guidi Sandra , Bartesaghi Renata , Contestabile Antonio 
Published: March 2003

AbstractNitric oxide (NO) is a small, diffusible, highly reactive molecule with a dichotomous regulatory role in the brain: an intra‐ and intercellular messenger under physiological conditions and a neurodegenerative agent under pathological conditions. We have recently demonstrated that long‐lasting exposure to an neuronal nitric oxide synthase (nNOS) inhibitor down‐regulated serine/threonine kinase (Akt) survival pathway and caused apoptosis in cerebellar granule cell cultures. The present study further substantiates the role of NO in neuronal survival by demonstrating that blocking its production down‐regulates the activity of cAMP‐responsive element binding protein (CREB), a transcription factor involved in cell survival and synaptic plasticity. Pharmacological dissection of the pathway linking NO to CREB shows that cGMP and its kinase are intermediate effectors. We also identify Bcl‐2 as one of the anti‐apoptotic genes down‐regulated by NO shortage and decreased CREB phosphorylation. These results not only confirm the role of CREB in neuronal survival but also provide circumstantial evidence for a novel link among NO, CREB activation and survival.
Macrophage colony stimulating factor prevents NMDA‐induced neuronal death in hippocampal organotypic cultures
Authors: Vincent Valerie A. M. , Robinson Christopher C. , Simsek Dilek , Murphy Greer M. 
Published: March 2003

AbstractMacrophage colony stimulating factor (M‐CSF) and its receptor are up‐regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M‐CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M‐CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase‐3 activation in organotypic hippocampal cultures, whereas treatment with M‐CSF protected hippocampal neurons from NMDA‐induced apoptosis. Caspase‐3 activation was inhibited by M‐CSF treatment to the same degree as with the caspase inhibitor Z‐VAD‐FMK. These results suggest that M‐CSF has neuroprotective properties through inhibition of caspase‐3 that could promote neuronal survival after excitotoxic insult. The role of M‐CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.