bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury
Authors: Beer Ronny , Franz Gerhard , Krajewski Stanislaw , Pike Brian R. , Hayes Ronald L. , Reed John C. , Wang Kevin K. , Klimmer Christian , Schmutzhard Erich , Poewe Werner , Kampfl Andreas 
Published: March 2003

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact‐induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT‐PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8‐ and caspase 3‐immunopositive cells with apoptotic‐like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2–5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.
Prevalence between different α subunits performing the benzodiazepine binding sites in native heterologous GABAA receptors containing the α2 subunit
Authors: Carlos del Río Juan , Araujo Francisco , Ramos Blanca , Ruano Diego , Vitorica Javier 
Published: March 2003

The presence of two heterologous α subunits and a single benzodiazepine binding site in the GABAA receptor implicates the existence of pharmacologically active and inactive α subunits. This fact raises the question of whether a particular α subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of α subunits with different benzodiazepine binding properties (α1, α2 and α5). Thus, we first demonstrated the existence of α2–α1 (36.3 ± 5.2% of the α2 population) and α2–α5 (20.2 ± 2.1%) heterologous receptors. A similar α2–α1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABAA receptors containing a common (α2) and a different (α1 or α5) α subunit. The α2 subunit pharmacologically prevailed over the α1 subunit in both cortex and hippocampus (there was an absence of high‐affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in α2–α1 double immunopurified receptors (Ki = 295 ± 56 nm and 200 ± 8 nm in hippocampus and cortex, respectively). On the contrary, the α5 subunit pharmacologically prevailed over the α2 subunit (low‐ and high‐affinity binding sites for zolpidem and [3H]L‐655,708, respectively). This prevalence was probed in α2–α5 double immunopurified receptors. Zolpidem displayed a single low‐affinity binding site (Ki = 1.73 ± 0.54 µm). These results demonstrated the existence of a differential dominance between the different α subunits performing the benzodiazepine binding sites in the native GABAA receptors.
Inhibition of Rho‐kinase‐induced myristoylated alanine‐rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H‐1152, a novel and specific Rho‐kinase inhibitor
Authors: Ikenoya Mami , Hidaka Hiroyoshi , Hosoya Takamitsu , Suzuki Masaaki , Yamamoto Naoki , Sasaki Yasuharu 
Published: March 2003

AbstractThe functions of small G protein Rho‐associated kinase (Rho‐kinase) have been determined in muscle and non‐muscle cells, but, particularly in neuronal cells, its effector(s) has not been well known. Recently, we preliminarily reported that Rho‐kinase phosphorylates the Ser159 residue in myristoylated alanine‐rich C kinase substrate (MARCKS)in vitro, but it remains obscurein vivo. To further clarify this point, we developed an isoquinolinesulfonamide derivative, H‐1152, that is a more specific, stronger and membrane‐permeable inhibitor of Rho‐kinase with aKivalue of 1.6 nm, but poor inhibitor of other serine/threonine kinases. H‐1152 dose‐ dependently inhibited the phosphorylation of MARCKS in human neuroteratoma (NT‐2) cells stimulated by Rho‐activator lysophosphatidic acid (LPA), which was determined by phosphorylation site‐specific antibody against phospho‐Ser159 in MARCKS, whereas it hardly inhibited the phosphorylation stimulated by phorbol‐12,13‐dibutyrate (PDBu). In contrast, two other Rho‐kinase inhibitors, HA‐1077 at 30 µmand Y‐27632 at 10–30 µm, inhibited the phosphorylation of MARCKS in the cells stimulated by LPA and PDBu. A PKC inhibitor Ro‐31‐8220 selectively inhibited PDBu‐induced phosphorylation of MARCKS. Taken together with our previous results, the present findings strongly suggest that Rho/Rho‐kinase phosphorylates MARCKS at Ser159 residue in neuronal cells in response to LPA stimulation and that H‐1152 is a useful tool to confirm Rho‐kinase function(s) in cells and tissues.
NS 1231, a novel compound with neurotrophic‐like effects in vitro and in vivo
Authors: Dagø Lone , Bonde Christian , Peters Dan , Møller Arne , Bomholt Signe Farsø , Hartz Barbara P. , Meyer Morten , Drejer Jørgen , Grønborg Mette 
Published: March 2003

AbstractNS 1231 [5‐(4‐chlorophenyl)‐6,7,8,9‐tetrahydro‐1H‐pyrrolo‐[3.2‐h]naphthalene‐2,3‐dione‐3‐oxime] belongs to a chemical series of compounds, which exhibit neurotrophic‐like activities. In vitro, NS 1231 rescued nerve growth factor (NGF)‐ differentiated PC12 cells from death induced by withdrawal of trophic factors. In addition, NS 1231 stimulated NGF‐induced neurite outgrowth of undifferentiated PC12 cells. At the molecular level, NS 1231 enhanced NGF‐induced signalling events, such as TrkA phosphorylation at the Shc‐binding site Tyr490 as well as ERK activation in PC12 cells. Moreover, NS 1231 reduced NMDA‐induced excitotoxicity in organotypic hippocampal slice cultures. In a gerbil model of transient global ischaemia, treatment with NS 1231 reduced the delayed loss of neurons in the hippocampal CA1 layer. Furthermore, NS 1231 treatment resulted in a 43% reduction in total infarct volume in the mouse middle cerebral artery occlusion (MCAO) model. The present data thus implicate a therapeutic potential of NS 1231 or structural analogues in treatment of cerebral ischaemia.
Increased brain histamine levels in Parkinson's disease but not in multiple system atrophy
Authors: Rinne J. O. , Anichtchik O. V. , Eriksson K. S. , Kaslin J. , Tuomisto L. , Kalimo H. , Röyttä M. , Panula P. 
Published: March 2003

AbstractWe investigated histamine concentration in post‐mortem brain samples of patients with Parkinson's disease (PD, n = 24), multiple system atrophy (MSA, n = 8) and age‐matched controls (n = 27). Histamine concentrations were significantly increased in the putamen (to 159% of the control mean), substantia nigra pars compacta (to 201%), internal globus pallidus (to 234%) and external globus pallidus (to 200%), i.e. in areas which play a crucial role in the motor behaviour and which show typical functional alterations in PD. In MSA no significant differences were seen. Tele‐methylhistamine (histamine metabolite) concentrations were unchanged in PD. These results indicate that histamine concentration, but not its metabolism is increased in PD, but not in MSA. This finding may have implications in developing new drug therapies for PD and in differential diagnosis between PD and MSA.
Nordihydroguaiaretic acid potently breaks down pre‐formed Alzheimer's β‐amyloid fibrils in vitro
Authors: Ono Kenjiro , Hasegawa Kazuhiro , Yoshiike Yuji , Takashima Akihiko , Yamada Masahito , Naiki Hironobu 
Published: March 2003

AbstractInhibition of the accumulation of amyloid β‐peptide (Aβ) and the formation of β‐amyloid fibrils (fAβ) from Aβ, as well as the degradation of pre‐formed fAβ in the CNS would be attractive therapeutic objectives for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) inhibited fAβ formation from Aβ(1–40) and Aβ(1–42) dose‐dependently in the range of 10–30 µmin vitro. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that NDGA dose‐dependently breaks down fAβ(1–40) and fAβ(1–42) within a few hours at pH 7.5 at 37°C. At 4 h, the fluorescence of fAβ(1–40) and fAβ(1–42) incubated with 50 µm NDGA was 5% and 10% of the initial fluorescence, respectively. The activity of NDGA to break down these fAβs was observed even at a low concentration of 0.1 µm. At 1 h, many short, sheared fibrils were observed in the mixture incubated with 50 µm NDGA, and at 4 h, the number of fibrils reduced markedly, and small amorphous aggregates were observed. We next compared the activity of NDGA to break down fAβ(1–40) and fAβ(1–42), with other molecules reported to inhibit fAβ formation from Aβ and/or to degrade pre‐formed fAβ both in vivo and in vitro. At a concentration of 50 µm, the overall activity of the molecules examined in this study was in the order of: NDGA >> rifampicin = tetracycline > poly(vinylsulfonic acid, sodium salt) = 1,3‐propanedisulfonic acid, disodium salt > β‐sheet breaker peptide (iAβ5). In cell culture experiments, fAβ disrupted by NDGA were less toxic than intact fAβ, as demonstrated by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Although the mechanisms by which NDGA inhibits fAβ formation from Aβ, as well as breaking down pre‐formed fAβin vitro, are still unclear, NDGA could be a key molecule for the development of therapeutics for AD.
Mitochondrial voltage‐dependent anion channel is involved in dopamine‐induced apoptosis
Authors: Premkumar A. , Simantov R. 
Published: March 2003

AbstractNeuronal NMB cells were used to determine changes in gene expression upon treatment with dopamine. Twelve differentially expressed cDNAs were identified and cloned, one of them having 99.4% sequence homology with isoform 2 of a voltage‐dependent anion channel (VDAC‐2). The known role of VDAC, a mitochondrial outer‐membrane protein, in transport of anions, pore formation, and release of cytochrome C prompted us to investigate the possible role of VDAC gene family in dopamine‐induced apoptosis. Semi‐quantitative PCR analysis indicated that expression of the three VDAC isoforms was reduced by dopamine. Immunoblotting with anti‐VDAC antibodies detected two VDAC protein bands of 33 and 34 kDa. Dopamine decreased differentially the immunoreactivity of the 34 kDa protein. Whether the decrease in VDAC expression influence the mitochondrial membrane potential (ΔΨm) was determined with the dye Rhodamine‐123. Dopamine indeed decreased the mitochondrial ΔΨm, but the maximum effect was observed within 3 h, prior to the decrease in VDAC mRNA or protein levels. Cyclosporin A, a blocker of the mitochondrial pore complex, prevented the decrease in ΔΨm, but did not rescue the cells from dopamine toxicity. To elucidate possible involvement of protease caspases in dopamine‐induced apoptosis, the effect of the caspase inhibitor z‐Val‐Ala‐Asp(Ome)‐FMK (zVAD) was determined. zVAD decreased dopamine toxicity, yet it did not rescue the mitochondrial ΔΨm drop. Dopamine also decreased ATP levels. Finally, transfection of NMB cells with pcDNA–VDAC decreased the cytotoxic effect of dopamine. These findings are in agreement with the notion that the mitochondria, and VDAC, are important participants in dopamine‐induced apoptosis.
Rafts in adult peripheral nerve myelin contain major structural myelin proteins and myelin and lymphocyte protein (MAL) and CD59 as specific markers
Authors: Erne B. , Sansano S. , Frank M. , Schaeren‐Wiemers N. 
Published: March 2003

AbstractThe myelin and lymphocyte protein (MAL) proteolipid is localized in central and peripheral compact myelin membranes, as well as in apical membranes of particular polarized cells. In this study, we addressed the question whether MAL and other peripheral myelin proteins are sorted and targeted to myelin membranes using mechanisms similar to those observed in polarized epithelial cells. To investigate the presence of raft‐mediated sorting pathways in Schwann cells, we have isolated and analysed their composition in myelin membranes. Here, we show that rafts are present in adult human and rat peripheral compact myelin membranes and contain MAL, the GPI‐anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59, and MAL have an almost identical expression pattern within compact myelin. Moreover, immuno‐electron microscopy revealed that MAL, besides its localization in compact myelin, is also localized to Schmidt–Lanterman incisures. Taken together, our results demonstrate the presence of detergent‐insoluble glycolipid‐enriched complexes (DIGs) in different compartments of myelin membranes and indicate an important role for DIG‐mediated transport mechanisms in the maintenance of the adult myelin sheath.
Increase in glutamate‐induced neurotoxicity by activated astrocytes involves stimulation of protein kinase C
Authors: Ahlemeyer Barbara , Kölker Stefan , Zhu Yuan , Hoffmann Georg F. , Krieglstein Josef 
Published: March 2003

AbstractActivation of astrocytes is a common feature of neurological disorders, but the importance of this phenomenon for neuronal outcome is not fully understood. Treatment of mixed hippocampal cultures of neurones and astrocytes from day 2–4 in vitro (DIV 2–4) with 1 µm cytosine arabinofuranoside (AraC) caused an activation of astrocytes as detected by a stellate morphology and a 10‐fold increase in glial fibrillary acidic protein (GFAP) level compared with vehicle‐treated cultures. After DIV 12, we determined 43% and 97% damaged neurones 18 h after the exposure to glutamate (1 mm, 1 h) in cultures treated with vehicle and AraC, respectively. Dose–response curves were different with a higher sensitivity to glutamate in cultures treated with AraC (EC50 = 0.01 mm) than with vehicle (EC50 = 0.12 mm). The susceptibility of neurones to 1 mm glutamate did not correlate with the percentage of astrocytes and was insensitive to an inhibition of glutamate uptake. In cultures treated with vehicle and AraC, glutamate‐induced neurotoxicity was mediated through stimulation of the NR1‐NR2B subtype of NMDA receptors, because it was blocked by the NMDA receptor antagonist MK‐801 and the NR1‐NR2B selective receptor antagonist ifenprodil. Protein levels of the NR2A and NR2B subunits of NMDA receptor were similar in cultures treated with vehicle or AraC. AraC‐induced changes in glutamate‐induced neurotoxicity were mimicked by activation of protein kinase C (PKC), whereas neuronal susceptibility to glutamate was reduced in cultures depleted of PKC and treated with AraC suggesting that the increase in glutamate toxicity by activated astrocytes involves activation of PKC.
Mechanisms of ligand binding and efficacy at the human D2(short) dopamine receptor
Authors: Payne Sarah L. , Johansson Anette M. , Strange Philip G. 
Published: March 2003

AbstractMechanisms of ligand binding and receptor activation for the human D2(short) dopamine receptor have been probed using two homologous series of monohydroxylated and dihydroxylated agonists (phenylethylamines and 2‐dipropylaminotetralins). In ligand binding studies, the majority of compounds exhibited competition curves versus [3H]spiperone that were best fitted using a two site binding model. The compounds had different abilities (potencies and maximal effects) to stimulate [35S]GTPγS binding and to inhibit forskolin‐stimulated cAMP accumulation. From the data it can be concluded that: (i) the ability of an agonist to stabilize receptor/G protein coupling can be used to predict agonist efficacy for some groups of compounds (2‐dipropylaminotetralins) but not for others (phenylethylamines); (ii) the receptor may be activated by unhydroxylated compounds; (iii) single hydroxyl groups or pairs of hydroxyl groups on the agonist may contribute tobinding affinity, potency and efficacy; and (iv) for the 2‐dipropylaminotetralin series two modes of agonist/receptor interaction have been identified associated with different relative efficacy.
Maternal behavior regulates long‐term hippocampal expression of BAX and apoptosis in the offspring
Authors: Weaver Ian C. G. , Grant Rebecca J. , Meaney Michael J. 
Published: March 2003

AbstractNaturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group‐1 tumor necrosis factor family mediated cell death effector) in 90‐day‐old male offspring. BAX‐like immunoreactivity on western blots is significantly increased in the adult offspring of low‐level LG mothers. There is no effect of maternal care on levels of either B‐cell lymphoma‐2 (BCL‐2) (group‐II mitochondria mediated cell death suppressor) or BAD (group‐III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)‐mediated dUTP‐biotin nick‐end labeling (TUNEL) and 4′,6′‐diamidino‐2‐phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL‐positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low‐level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low‐level LG mothers may be more vulnerable to loss through apoptosis.
HNK‐1 sulfotransferase null mice express glucuronyl glycoconjugates and show normal cerebellar granule neuron migration in vivo and in vitro
Authors: Chou Denise K. H. , Schachner Melitta , Jungalwala Firoze B. 
Published: March 2003

AbstractSulfoglucuronyl carbohydrate (SGC), reactive with antibody against human natural killer cell antigen, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system and has been implicated in cell–cell recognition, neurite outgrowth and neuronal migration during development, through its interaction with SGC‐binding protein (SBP) 1. However, sulfotransferase (ST) null mutant mice, which lack SGC, were shown to have normal development with usual gross anatomy of the nervous system and other organs. Failure to observe a severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoproteins of the ST mutant nervous system. In the ST null mice, SGC‐containing molecules were absent; instead the precursor glucuronyl carbohydrate (GC)‐containing molecules accumulated. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites in the nervous system as the SGC molecules in the wild type. In vitro binding studies showed that, similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP‐1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected in the mutant, possibly owing to interaction of SBP‐1 with GC molecules. The results suggested that in vivo SBP‐1–GC interaction was sufficient to allow normal neurite outgrowth and cell migration in the mutant, giving rise to a wild‐type phenotype. However, the role of other compensatory molecules involved in these processes cannot be completely ruled out.
Changes in expression of the neuropeptide Y Y1 receptor gene in the medial amygdala of transgenic mice during pregnancy and after delivery
Authors: Oberto Alessandra , Serra Mariangela , Pisu Maria Giuseppina , Biggio Giovanni , Eva Carola 
Published: March 2003

AbstractLong‐term administration of progesterone or allopregnanolone was previously shown to increase Y1 receptor gene expression in the medial amygdala of Y1R/LacZ transgenic mice, which harbor a construct comprising the murine Y1 receptor gene promoter and a lacZ reporter. We have now investigated the effects of physiological fluctuations in the cerebrocortical concentrations of neuroactive steroids during pregnancy on Y1R/LacZ transgene expression by quantitative histochemical analysis of β‐galactosidase activity. Cerebrocortical concentrations of progesterone and its metabolites allopregnanolone and allotetrahydrodeoxycorticosterone were increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Transgene expression in the medial amygdala was also increased on day 18 of pregnancy and had returned to control values 2 days after delivery. Similar results were obtained after analysis of Y1R mRNA levels in the medial amygdala of pregnant mice by in situ hybridization. Administration of the 5α‐reductase inhibitor finasteride to pregnant mice prevented both the increase in the cerebrocortical concentrations of neuroactive steroids as well as the increase in transgene expression. These data suggest that fluctuations in the brain concentrations of endogenous neuroactive steroids during pregnancy are associated with changes in Y1 receptor gene expression in the medial amygdala, further supporting a functional interaction between the GABAergic and NPY–Y1 receptor systems.
Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices
Authors: Hirai Kiyoshi , Sugawara Taku , Chan Pak H. , Basus Vladimir J. , James Thomas L. , Litt Lawrence 
Published: March 2003

AbstractCellular injury was evaluated in superfused cerebrocortical slices (350 µm) from 7‐day‐old Sprague–Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase‐9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase‐9; (ii) localization of cleaved caspase‐9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase‐9 returned to control values, while whole cell caspase‐9 stayed approximately the same, suggesting translocation of caspase‐9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.