bluuebunny/crossref_metadata_2025_split huggingface dataset:
Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus
Authors: Tsukada Toshiyuki , Watanabe Masahiko , Yamashima Tetsumori 
Published: March 2003

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain‐induced leakage of lysosomal cathepsins (calpain‐cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase‐activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post‐ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling (TUNEL) staining on days 3–5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post‐ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase‐3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.
Quantitative dual‐probe microdialysis: mathematical model and analysis
Authors: Chen Kevin C. , Höistad Malin , Kehr Jan , Fuxe Kjell , Nicholson Charles 
Published: March 2003

AbstractSteady‐state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual‐probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual‐probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual‐probe microdialysis.
The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions
Authors: Anderson Michelle F. , Sims Neil R. 
Published: March 2003

AbstractGlutathione is a key cellular antioxidant that is contained in both cytoplasmic and mitochondrial compartments. Previous investigations indicate that depletion of the mitochondrial pool of glutathione can greatly reduce cell viability. In the present investigation, the effect of focal cerebral ischemia on total (reduced plus oxidized) glutathione in mitochondria was assessed using a rat model of middle cerebral artery occlusion. Total glutathione was substantially decreased in mitochondria prepared from severely ischemic focal tissue in both the cerebral cortex and striatum at 2 h of vessel occlusion and persisted for at least the first 3 h of reperfusion. The loss of mitochondrial glutathione was not associated with decreases of the total tissue glutathione content and was not due to the formation of mixed disulfides with mitochondrial proteins. Thus, an imbalance between uptake and release from the mitochondria in the ischemic tissue provides the most likely explanation for the loss. Decreases in glutathione also developed in mitochondria from the moderately ischemic perifocal tissue when the period of arterial occlusion was extended to 3 h. The presence of mitochondrial glutathione depletion during ischemia showed an apparent close association with the subsequent development of tissue infarction. These findings are consistent with a role for the glutathione depletion in determining the susceptibility of brain tissue to focal ischemia.
Lack of regulation of aromatic l‐amino acid decarboxylase in intact bovine chromaffin cells
Authors: Waymire Jack C. , Haycock John W. 
Published: March 2003

AbstractAromatic l‐amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate‐limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32Pi) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.
Species differences in the expression of multiple tyrosine hydroxylase protein isoforms
Authors: Haycock John W. 
Published: March 2003

AbstractIn subprimates, a single form of tyrosine hydroxylase (TH) is expressed, whereas two TH protein isoforms have been identified in monkeys and four isoforms have been demonstrated in humans. In order to establish the evolutionary pattern/emergence of these multiple TH isoforms, adrenal medullae from different mammalian species were analyzed by blot immunolabeling using pan‐specific TH antibodies and antibodies specific to each of the four human TH isoforms. The expression of multiple TH isoforms was primate specific and restricted to anthropoids: only a single TH isoform was detected in adrenal medullae from several subprimate and prosimian species (six species from four families), while two TH isoforms were found in all of the anthropoid species studied. The presence of four TH isoforms could only be demonstrated in human specimens. Contrary to previous suggestions, only one TH protein isoform was found in rats and only four TH protein isoforms were found in humans.
Inhibition of brain mitochondrial respiration by dopamine: involvement of H2O2 and hydroxyl radicals but not glutathione–protein–mixed disulfides
Authors: Gluck Martin , Ehrhart Julie , Jayatilleke Elizabeth , Zeevalk Gail D. 
Published: March 2003

AbstractExamination of the downstream mediators responsible for inhibition of mitochondrial respiration by dopamine (DA) was investigated. Consistent with findings reported by others, exposure of rat brain mitochondria to 0.5 mm DA for 15 min at 30°C inhibited pyruvate/glutamate/malate‐supported state‐3 respiration by 20%. Inhibition was prevented in the presence of pargyline and clorgyline demonstrating that mitochondrial inhibition arose from products formed following MAO metabolism and could include hydrogen peroxide (H2O2), hydroxyl radical, oxidized glutathione (GSSG) or glutathione–protein mixed disulfides (PrSSG). As with DA, direct incubation of intact mitochondria with H2O2 (100 µm) significantly inhibited state‐3 respiration. In contrast, incubation with GSSG (1 mm) had no effect on O2 consumption. Exposure of mitochondria to 1 mm GSSG resulted in a 3.3‐fold increase in PrSSG formation compared with 1.4‐ and 1.5‐fold increases in the presence of 100 µm H2O2 or 0.5 mm DA, respectively, suggesting a dissociation between PrSSG formation and effects on respiration. The lack of inhibition of respiration by GSSG could not be accounted for by inadequate delivery of GSSG into mitochondria as increases in PrSSG levels in both membrane‐bound (2‐fold) and intramatrix (3.5‐fold) protein compartments were observed. Furthermore, GSSG was without effect on electron transport chain activities in freeze–thawed brain mitochondria or in pig heart electron transport particles (ETP). In contrast, H2O2 showed differential effects on inhibition of respiration supported by different substrates with a sensitivity of succinate > pyruvate/malate > glutamate/malate. NADH oxidase and succinate oxidase activities in freeze–thawed mitochondria were inhibited with IC50 approximately 2–3‐fold higher than in intact mitochondria. ETPs, however, were relatively insensitive to H2O2. Co‐administration of desferrioxamine with H2O2 had no effect on complex I‐associated inhibition in intact mitochondria, but attenuated inhibition of rotenone‐sensitive NADH oxidase activity by 70% in freeze–thawed mitochondria. The results show that DA‐associated inhibition of respiration is dependent on MAO and that H2O2 and its downstream hydroxyl radical rather than increased GSSG and subsequent PrSSG formation mediate the effects.
Activation of nuclear Ca2+/calmodulin‐dependent protein kinase II and brain‐derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108‐15 cells
Authors: Takeuchi Yusuke , Fukunaga Kohji , Miyamoto Eishichi 
Published: March 2003

AbstractWe identified the isoforms of Ca2+/calmodulin‐dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II α, β, γ and δ were detected including the isoforms of αB, γA, γA′, γA.B, δ3 and δ7 with nuclear localization signal. We established NG108‐15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo‐3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca2+, which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III‐ and IV‐BDNF mRNA. Overexpression of CaM kinase II δ3 increased exon IV‐ but not exon III‐BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca2+ signaling.
Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins
Authors: Di Stasi A. M. Michela , Mallozzi Cinzia , Macchia Gianfranco , Maura Guido , Petrucci Tamara C. , Minetti Maurizio 
Published: March 2003

AbstractThe reactive species peroxynitrite, formed via the near diffusion‐limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc‐18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1‐43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO3, indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate‐resistant SNARE complex in a dose‐dependent manner (100–1000 µm) and induced the formation of 3‐nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein–protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up‐regulate neuronal signaling.
The internal pH of synaptic‐like microvesicles in rat pinealocytes in culture
Authors: Hayashi Mitsuko , Yamamoto Akitsugu , Moriyama Yoshinori 
Published: March 2003

AbstractSynaptic‐like microvesicles (SLMVs) are morphological and functional equivalents of neuronal synaptic vesicles, and are responsible for the storage and secretion of classical neurotransmitters in various endocrine cells. Vacuolar H+‐ATPase acidifies the internal space of these organelles and provides a driving force for the uptake of neurotransmitters. Thus, the luminal pH is an important determinant of the function of SLMVs, although its value in living cells is unknown. Here, we determined the luminal pH of SLMVs in living rat pinealocytes by means of an immunoelectronmicroscopic procedure basedon the distribution of an amphipathic amine, 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine (DAMP). Use of double‐labeling techniques with antibodies against 2,4‐dinitrophenol for DAMP and synaptophysin for SLMVs, and of frozen ultrathin sections enabled us to determine the number of immunogold particles for DAMP per µm2 of SLMVs. Using the density of gold particles, the luminal pH of SLMVs was calculated to be 5.11 ± 0.01. Treatment with either 1 µm bafilomycin A1, a specific inhibitor of vacuolar H+‐ATPase , or 50 mm ammonium chloride, a dissipater of the transmembrane pH gradient, increased the luminal pH to 6.04 ± 0.07 and 6.05 ± 0.11, respectively. Simultaneously, the lysosomal pH was found to be 5.14 ± 0.07, which increased to 5.77 ± 0.09 and 5.93 ± 0.13 with bafilomycin A1 and ammonium chloride, respectively. It is concluded that the luminal pH of SLMVs is comparable to that of lysosomes in vivo.
Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin‐sensitive step and interaction with the AP‐2 adaptor complex
Authors: Barbosa José , Ferreira Lucimar T. , Martins‐Silva C. , Santos Magda S. , Torres Gonzalo E. , Caron Marc G. , Gomez Marcus V. , Ferguson Stephen S. G. , Prado Marco A. M. , Prado Vania F. 
Published: March 2003

AbstractThe pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)‐tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP‐VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I‐K44A impaired GFP‐VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I‐K44A generated vesicles. A fusion protein containing the VAChT C‐terminal tail precipitated the AP‐2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C‐terminal tail of VAChT interacts with the µ subunit of AP‐2 in a di‐leucine (L485A/L486A) dependent fashion. These observations suggest that the di‐leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.
Gene transfer of the Caenorhabditis elegans n‐3 fatty acid desaturase inhibits neuronal apoptosis
Authors: Ge Yinlin , Wang Xiaoying , Chen Zhihong , Landman Natalie , Lo Eng H. , Kang Jing X. 
Published: March 2003

AbstractPrevious studies have shown that n‐3 polyunsaturated fatty acids (PUFAs) can exert an antiapoptotic effect on neurons. The present study was designed to investigate whether the Caenorhabditis elegans fat‐1 gene encoding an n‐3 fatty acid desaturase (an enzyme that converts n‐6 PUFAs to corresponding n‐3 PUFAs) can be expressed functionally in rat cortical neurons and whether its expression can change the ratio of n‐6 : n‐3 fatty acids in the cell membrane and exert an effect on neuronal apoptosis. Infection of primary rat cortical cultures with Ad‐fat‐1 resulted in high expression of the fat‐1 gene. Lipid analysis indicated a decrease in the ratio of n‐6 : n‐3 PUFAs from 5.9 : 1 in control cells, to 1.45 : 1 in cells expressing the n‐3 fatty acid desaturase. Accordingly, the levels of prostaglandin E2, an eicosanoid derived from n‐6 PUFA, were significantly lower in cells infected with Ad‐fat‐1 when compared with control cells. Finally, there was a significant inhibition of growth factor withdrawal‐induced apoptotic cell death in neurons expressing the fat‐1 gene. These results demonstrate that expression of the fat‐1 gene can inhibit apoptotic cell death in neurons and suggest that the change in the n‐6 : n‐3 fatty acid ratio may play a key role in this protective effect.
Inhibition of tryptophan hydroxylase activity and decreased 5‐HT1A receptor binding in a mouse model of Huntington's disease
Authors: Yohrling IV George J. , Jiang George C.‐T. , DeJohn Molly M. , Robertson Daniel J. , Vrana Kent E. , Cha Jang‐Ho J. 
Published: March 2003

AbstractThe pathogenic mechanisms of the mutant huntingtin protein that cause Huntington's disease (HD) are unknown. Previous studies have reported significant decreases in the levels of serotonin (5‐HT) and its metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) in the brains of the R6/2 transgenic mouse model of HD. In an attempt to elucidate the cause of these neurochemical perturbations in HD, the protein levels and enzymatic activity of tryptophan hydroxylase (TPH), the rate‐limiting enzyme in 5‐HT biosynthesis, were determined. Enzyme activity was measured in brainstem homogenates from 4‐, 8‐, and 12‐week‐old R6/2 mice and compared with aged‐matched wild‐type control mice. We observed a 62% decrease in brainstem TPH activity (p = 0.009) in 4‐week‐old R6/2 mice, well before the onset of behavioral symptoms. In addition, significant decreases in TPH activity were also observed at 8 and 12 weeks of age (61%, p = 0.02 and 86%, p = 0.005, respectively). In the 12‐week‐old‐mice, no change in immunoreactive TPH was observed. In vitro binding showed that TPH does not bind to exon 1 of huntingtin in a polyglutamine‐dependent manner. Specifically, glutathione‐S‐transferase huntingtin exon 1 proteins with 20, 32 or 53 polyglutamines did not interact with radiolabeled tryptophan hydroxylase. Therefore, the inhibition of TPH activity does not appear to result from a direct huntingtin/TPH interaction. Receptor binding analyses for the 5‐HT1A receptor in 12‐week‐old R6/2 mice revealed significant reductions in 8‐OH‐[3H]DPAT binding in several hippocampal and cortical regions. These results demonstrate that the serotonergic system in the R6/2 mice is severely disrupted in both presymptomatic and symptomatic mice. The presymptomatic inhibition of TPH activity in the R6/2 mice may help explain the functional consequences of HD and provide insights into new targets for pharmacotherapy.
Differential regulation of metabotropic glutamate receptor 5‐mediated phosphoinositide hydrolysis and extracellular signal‐regulated kinase responses by protein kinase C in cultured astrocytes
Authors: Peavy Richard D. , Sorensen Scott D. , Conn P. Jeffrey 
Published: March 2003

AbstractThe metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor‐mediated, protein kinase C (PKC)‐dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC‐mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal‐regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5‐mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.