bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of Influenza Vaccine Formulation with a Detailed Analysis of the Cytokine Response
Authors: Szyszko E. , Brokstad K. , Cox R. J. , Hovden A.‐O. , Madhun A. , Haaheim L. R.
Published: October 2006

Vaccination provides the most effective method of limiting the impact of influenza. Inactivated influenza vaccines are available in three formulations and more information needs to be generated on how antigen presented in different vaccine formulations influences the subsequent immune response. In the present study, we have investigated the effect of two different influenza vaccine formulations on the resulting antibody and cytokine responses and compared these responses with influenza infection. Mice were vaccinated intramuscularly with one or two doses of whole or split virus vaccine or alternatively intranasally infected with influenza virus. Lymphocytes were isolated from spleen cells and stimulatedin vitrofor 24 or 72 h for analysis of cytokine profile at the gene expression and at the protein level. Additionally, whole blood was collected and the serum antibody response investigated by haemagglutination inhibition (HI) and enzyme‐linked immunosorbent assay (ELISA). We found that one dose of whole virus vaccine induced higher antibody and cytokine responses and thus was more immunogenic in unprimed mice than split virus vaccine. Whole virus vaccine induced a strong IFN‐γ(type 1) immune response after one dose of vaccine and a more mixed cytokine response after the second dose. Split virus vaccine induced a type 2 response, particularly after two vaccine doses. Our results show that two doses of vaccine (both vaccine formulation) were more effective in induction of Th2 type of cytokines and thus indicate that both the formulation and also the number of vaccine doses substantially influences the magnitude and quality of the immune response.
Modulation of Chemokine Gene Expression in CD133+ Cord Blood‐Derived Human Mast Cells by Cyclosporin A and Dexamethasone
Authors: Holm M. , Kvistgaard H. , Dahl C. , Andersen H. B. , Hansen T. K. , Schiøtz P. O. , Junker S.
Published: October 2006

We have recently developed a protocol for generating huge numbers of mature and functional mast cells from in vitro differentiated umbilical cord blood cells. Using CD133 as a positive selection marker to isolate haematopoietic progenitors we routinely expand the number of recovered cells at least 150‐fold, which vastly exceeds the yields of conventional protocols using CD34+ cells as a source of progenitors. Taking advantage of the large quantities of in vitro differentiated mast cells, here we assess at the levels of transcription and translation the kinetics of chemokine gene induction following receptor mediated mast cell activation or following pharmacological activation of specific signal transduction cascades that become activated upon classical FcɛRI receptor crosslinking. We demonstrate that chemokine genes encoding IL‐8, MCP‐1, MIP‐1α, and MIP‐1β are induced with different kinetics and with different amplitudes in a receptor activation dependent manner, and that these events can be mimicked using pharmacological agents which activate distinct signal transduction pathways. These findings were corroborated by adding immunomodulators such as cyclosporin A and dexamethasone prior to mast cell activation. Finally, we demonstrate that the same modulators added after mast cell activation can differentially quench ongoing chemokine gene induction. Thus, considering the vast yields of mast cells, our protocol is valuable not only for studying regulation of gene expression in mast cells in general, but also as an experimental tool to develop better and more balanced treatments of mast cell related disorders.
Immune Regulatory T Cells in Siblings of Children Suffering from Type 1 Diabetes Mellitus
Authors: Michalek J. , Vrabelova Z. , Hrotekova Z. , Kyr M. , Pejchlova M. , Kolouskova S. , Faresjö M. , Stechova K.
Published: October 2006

Patients with type 1 diabetes are suffering from defects in immune regulatory cells. Their siblings may be at increased risk of type 1 diabetes especially if they are carriers of certain human leucocyte antigen (HLA) alleles. In a prospective non‐randomized study, we intended to evaluate 31 healthy siblings of paediatric patients with type 1 diabetes and explore immune regulatory populations of CD4+CD25+ T cells and natural killer (NK) T cells. Tested siblings of type 1 diabetes patients were stratified according to the HLA‐associated risk of possible diabetes development. Immune regulatory function of CD4+CD25+ T cells was tested in vitro. Significant differences in CD4+CD25+ but not in NK T cells have been identified. Siblings of type 1 diabetes patients carrying high risk HLA alleles (DQA1*05, DQB1*0201, DQB1*0302) had significantly lower number of immune regulatory CD4+CD25+ T cells than the age‐matched healthy controls or siblings carrying low‐risk HLA alleles (DQB1*0301, DQB1*0603, DQB1*0602). Regulatory function of CD4+CD25+ T cells demonstrated a dose‐escalation effect. In siblings of type 1 diabetes patients, the defect in immune regulatory CD4+CD25+ T cells exists in association with genetic HLA‐linked risk for type 1 diabetes.
Benznidazole Treatment during Early‐indeterminate Chagas’ Disease Shifted the Cytokine Expression by Innate and Adaptive Immunity Cells toward a Type 1‐modulated Immune Profile
Authors: Sathler‐Avelar R. , Vitelli‐Avelar D.M. , Massara R.L. , Borges J.D. , Lana M. , Teixeira‐Carvalho A. , Dias J.C.P. , Elói‐Santos S.M. , Martins‐Filho O.A.
Published: October 2006

Trypanosoma cruzi‐infected children was treated with benznidazole (Bz) during the early‐indeterminate disease (E‐IND) and the cytokine pattern of innate and adaptive immune compartments were evaluated prior to the treatment and 1 year after it. At first, we observed that the ex vivo cytokine profile of circulating leukocytes from E‐IND (n = 6) resembled the one observed for healthy schoolchildren (n = 7). Additionally, in vitro stimulation with T. cruzi antigens drove the E‐IND cytokine pattern toward a mixed immune profile with higher levels of IFN‐γ+, TNF‐α+ and IL‐4+ NK cells, increased numbers of IFN‐γ+, TNF‐α+ and IL‐10+ CD4+ T cells in addition to enhanced frequency of TNF‐α+/IL‐4+ CD19+ lymphocytes. Interestingly, upon T. cruzi antigen in vitro stimulation, E‐IND CD8+ lymphocytes displayed a selective enhancement of IFN‐γ expression, accounting for a global type 1‐modulated cytokine microenvironment. A shift toward a type 1‐modulated profile was also the hallmark of Bz‐treated children (E‐INDT). In this context, despite the mixed overall ex vivo cytokine profile observed for NK and CD8+ T cells, increased ability of these leukocytes to produce IFN‐γ in response to T. cruzi antigens was reported. Most noteworthy was the IL‐10 production evidenced at T lymphocytes, mainly CD4+ cells, as well as B lymphocytes, both ex vivo and upon antigen stimulation. Together, these findings gave evidence that NK cells and CD8+ T lymphocytes are the major sources of IFN‐γ, a pivotal cytokine for successful therapeutic response in human Chagas’ disease. Moreover, our data have also brought additional information, pointing out IL‐10 production by CD4+ cells and B lymphocytes, as the putative key element for parasite clearance in the absence of deleterious tissue damage.
Association of the CT gene (CA) polymorphism with BMD in osteoporotic mexican women
Authors: Magaña JJ , Gómez R , Cisneros B , Casas L , Castorena F , Miranda A , Diez P , Castro C , Rubio J , Valdés M
Published: October 2006

Calcitonin (CT) plays a role in the pathogenesis of osteoporosis and genetic variations in or adjacent to the CT gene may be associated with loss of bone mineral density (BMD). The correlation between a dinucleotide (cytosine–adenine) repeat polymorphism at the CT locus and BMD was examined in 70 osteoporotic women, 70 non‐osteoporotic women and 500 subjects from the Mexican population. The allele A and genotype AA frequencies were significantly higher in osteoporotic women than in non‐osteoporotic women (60% vs 32%; p < 0.0001 and 41% vs 14%; p = 0.0007, respectively). Genotype AA was associated with the presence of osteoporosis [odds ratio 2.58; 95% confidence interval (CI); 1.62–4.12]. Likewise, the loss of lumbar BMD and T scores were related to the presence of allele A: subjects with a single A allele displayed lower values for lumbar BMD and T score (84.02% and −1.51, respectively) than those who do not present any A allele (89.61% and −0.88, respectively). Individuals with two alleles A showed the lowest lumbar BMD and T‐score values (73.77% and −2.51, respectively). Analysis of potential confounder demonstrated that aging has a significant effect on osteoporosis development (odds ratio 1.1; 95% CI; 1.1052–1.152).
Early occurrence of hepatocellular carcinoma in biliary atresia treated by liver transplantation
Authors: Brunati Andrea , Feruzi Zéphyrin , Sokal Etienne , Smets Françoise , Fervaille Caroline , Gosseye Serge , Clapuyt Philippe , De Ville de Goyet Jean , Reding Raymond
Published: October 2006

Abstract:  A case of liver transplantation for HCC complicating BA in an eight‐month old infant is reported. HCC in BA is extremely rare. Screening of AFP and ultrasonographic examination should be performed regularly in patients with secondary biliary cirrhosis for early detection of HCC.
MOUSTERIAN MUSICIANSHIP? THE CASE OF THE DIVJE BABE I BONE
Authors: MORLEY IAIN
Published: October 2006

Summary. The reputed Neanderthal ‘flute’ from the Slovenian site of Divje babe I has stimulated much interest and detailed research since the original publication of its discovery in 1997. In spite of nearly ten years' worth of analysis and discussion its status as an artefact has remained ambiguous; nevertheless it is still frequently cited as a ‘flute’. This paper examines the literature and research regarding this object, and finds that much of the ambiguity regarding the object's status derives from the literature itself. It concludes that there is no need to invoke hominin agency in explaining the features of the bone.
Pre‐attack stress‐load, appraisals, and coping in children's responses to the 9/11 terrorist attacks
Authors: Lengua Liliana J. , Long Anna C. , Meltzoff Andrew N.
Published: October 2006

Background:  Appraisal and coping following a disaster are important factors in children's post‐traumatic stress (PTS) symptoms. However, little is known about predictors of disaster coping responses. This study examined stress‐load, appraisals and coping styles measured prior to the September 11 terrorist attacks as predictors of 9/11‐specific appraisals, coping and PTS.

Methods:  A community sample of children and parents (N = 143) participating in an ongoing study were interviewed by phone approximately 1 month following 9/11.

Results:  Pre‐attack stress‐load, appraisal and coping styles predicted children's 9/11‐specific appraisals, coping, and PTS. 9/11‐specific threat appraisals and avoidant coping predicted higher PTS and mediated the effects of pre‐attack stress‐load and threat appraisal.

Conclusions:  Pre‐disaster stress‐load, appraisal and coping styles predict disaster‐specific appraisal and coping, which in turn, contribute to PTS. Coping interventions might mitigate PTS symptoms following a disaster.
Efficacy of Interpersonal Psychotherapy‐Adolescent Skills Training: an indicated preventive intervention for depression
Authors: Young Jami F. , Mufson Laura , Davies Mark
Published: October 2006

Background:  Indicated interventions for adolescents with elevated depressive symptoms may help decrease rates of depression. The current study reports on the efficacy of Interpersonal Psychotherapy‐Adolescent Skills Training (IPT‐AST), a group indicated preventive intervention.

Methods:  Forty‐one adolescents with elevated depression symptoms were randomized to receive either IPT‐AST or school counseling (SC) as delivered by guidance counselors and social workers. Adolescents in the two intervention conditions were compared on depression symptoms, overall functioning, and depression diagnoses post‐intervention and at 3‐month and 6‐month follow‐up.

Results:  Adolescents who received IPT‐AST had significantly fewer depression symptoms and better overall functioning post‐intervention and at follow‐up. Adolescents in IPT‐AST also reported fewer depression diagnoses than adolescents in usual care.

Conclusions:  These results provide preliminary evidence of the efficacy of IPT‐AST as an intervention for adolescents with subthreshold depression. Future research is needed to confirm the efficacy of IPT‐AST in a larger and more diverse sample and to determine its long‐term impact on depression symptoms and depression diagnoses.
Reduced Phosphoinositide Concentrations in Anterior Temporal Cortex of Alzheimer‐Diseased Brains
Authors: Stokes Cathryn E. , Hawthorne John N.
Published: October 2006

Abstract: Samples of brain anterior temporal cortex from 17 patients with Alzheimer's disease and 18 age‐matched controls have been analysed for myo‐inositol and the three phosphoinositides. There was significantly less phosphati‐dylinositol in the Alzheimer samples (1.36 μmol/g wet weight) than in the controls (2.28 μmol/g). The concentrations of phosphatidylinositol 4‐phosphate and phosphati‐dylinositol 4,5‐bisphosphate were also lower in Alzheimer cortex, but differences from the control group were not statistically significant. Free myo‐inositol concentrations were 5.11 μmol/g (Alzheimer) and 4.44 μmol/g (control) and again the difference was not significant. The lack of phosphoinositides in Alzheimer temporal cortex may impair receptor function.
[3H]SCH 23390 Binding to Human Putamen D‐1 Dopamine Receptors: Stereochemical and Structure‐Affinity Relationships Among l‐Phenyl‐1H‐3‐Benzazepine Derivatives as a Guide to D‐l Receptor Topography
Authors: O'Boyle Kathy M. , Waddington John L.
Published: October 2006

Abstract: A series of l‐phenyl‐1H‐3‐benzazepine analogues were assessed for enantiomeric and structure‐affinity relationships at human putamen D‐1 dopamine receptors labelled with [3H]SCH 23390. Substitution at the 7‐position of both 3‐H and 3‐methyl benzazepine molecules critically affected affinity for these receptors over a 500‐fold range. The general rank order of potency of 7‐substituents was Cl = Br ≫ CH3 > OH ≥ H. 3‐Methyl substituents increased the affinity of 7‐H and 7‐OH compounds two‐ to fivefold compared to desmethyl counterparts. The displacement of [3H]SCH 23390 binding showed substantial enantioselec‐tivity; the R‐enantiomer of SKF 83566 was 500‐fold more potent that its S‐antipode. However, the displacement of [3H]spiperone binding from D‐2 sites in the same tissue showed negligible enantioselectivity. Through such structure‐affinity relationships, these studies may help to define the topography of the human brain D‐1 dopamine receptor and guide the design of more selecive agents for functional studies.
The Effect of Antibodies to Gangliosides on Ca2+ Channel‐Linked Release of γ‐Aminobutyric Acid in Rat Brain Slices
Authors: Frieder B. , Rapport M. M.
Published: October 2006

Abstract: Antibodies to Gm1 ganglioside enhance the release of γ‐aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 μM veratrine. Three new observations are now reported, (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization‐induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization, (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 μM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.
Efflux of Putative Transmitters from Superfused Rat Brain Slices Induced by Low Chloride Ion Concentrations
Authors: Turner J. D. , Boakes R. J. , Hardy J. A. , Virmani M. A.
Published: October 2006

Abstract:Slices of rat cerebral cortex, preloaded with [14C]γ‐aminobutyric acid (GABA) and either [3H]5‐hy‐droxytryptamine (5‐HT) or [3H]noradrenaline, were super‐fused with media in which varying concentrations of Cl−had been replaced with other monovalent anions. Rapid reduction of [Cl″], by superfusion with media containing instead the impermeant anions propionate, isethionate, glu‐conate, or methyl sulphate, caused increases in the efflux of tritiated biogenic amines, but the increase in that of [14C]‐GABA was not significant. The increased efflux of [3H]5‐HT evoked by superfusion with low Cl−levels when propionate was the replacement anion, was transient and was linearly related to the log[Cl−]−1. It was not affected by removal of Ca2+or by addition of 10 mMMg2+and was delayed but not abolished by tetrodotoxin. The low C1–‐evoked efflux of [3H]5‐HT was not affected by pretreatment with neuronal reuptake blockers but was inhibited by picrotoxin, strychnine, and 4‐acetamido‐4‐isothiocyanostilbene‐2,2‐disul‐phonic acid and was enhanced by glycine. Muscimol and GABA were without effect. These observations are taken to indicate that the efflux of biogenic amines is brought about by terminal depolarisation due to outward movement of Cl−in low chloride‐containing media. They are of relevance to other physiological and pharmacological studies in which anion concentrations are manipulated and suggest that the anion‐evoked release phenomenon may provide a model for the analysis of Cl−‐dependent mechanisms in nerve terminals.
Position of the Peptide Linkage in Glutamyl‐Taurine from Calf Brain Synaptic Vesicles
Authors: Marnela Kirsi‐Marja , Varga Vince , Dibó Gábor , Lähdesmäki Pekka
Published: October 2006

Abstract: To elucidate the position of the peptide bond in glutamyl‐taurine this dipeptide was extracted from calf brain synaptic vesicles and subjected to paper electrophore‐sis. It was analyzed further in an automatic amino acid analyzer prior and subsequent to acid hydrolysis. Both á‐ and γ‐forms were found to be present in approximately equal amounts.
Vasoactive Intestinal Peptide Gene: Putative Mechanism of Information Storage at the RNA Level
Authors: Gozes Illana , Giladi Eliezer , Shani Yael
Published: October 2006

Abstract: The human gene coding for vasoactive intestinal peptide was recently isolated and shown to contain seven exons. We now demonstrate that an intron‐containing precursor RNA can be the major vasoactive intestinal peptide‐related RNA in the cell, which is in contrast to most known genes. By RNA blot analysis using a variety of genomic and cDNA‐related probes we show that in a human tumor producing vasoactive intestinal peptide, most of the RNA encoding the peptide is of the precursor type. Similar precursor transcripts were found in total rat brain RNA as well. As proof of the identity of the intron‐containing RNA, cDNA clones corresponding to this RNA sequence have been isolated.
Arachidonoyl‐Coenzyme A Synthetase and Nonspecific AcylCoenzyme A Synthetase Activities in Purified Rat Brain Microvessels
Authors: Morand O. , Carré J. B. , Homayoun P , Niel E , Baumann N. , Bourre J. M.
Published: October 2006

Abstract: Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl‐CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both ara‐chidonoyl‐CoA and palmitoyl‐CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl‐CoA synthesis by purified rat brain microvessels were 4.0 μM and 5.8 μM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol min−1 mg protein−1 for palmitic acid and arachidonic acid, respectively. Cross‐competition experiments showed inhibition of radiolabelled arachidonoyl‐CoA formation by 15 μM unlabelled arachidonic acid, with a Ki of 7.1 μM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 μM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl‐CoA synthesis. In comparison, radiolabelled palmitoyl‐CoA formation was inhibited competitively by 15 μM unlabelled palmitic acid, with a Ki of 5.0 μM and to a much lesser extent by arachidonic acid (Ki, 23 μM). The Vmax of palmitoyl‐CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl‐CoA synthesis. Both arachidonoyl‐CoA and palmitoyl‐CoA synthesis activities were thermolabile. Arachidonoyl‐CoA formation was inhibited by 75% after 7 min at 40°C whereas a 3‐min heating treatment was sufficient to produce the same relative inhibition of palmitoyl‐CoA synthesis. These data together strongly suggest that rat brain microvessels have the capacity to catalyze specifically the formation of acyl‐CoA derivatives from several polyunsaturated long‐chain fatty acids, including arachidonic acid in the first place. Besides this particular arachidonoyl‐CoA synthetase, palmitic acid could be activated with the aid of a second acyl‐CoA synthetase.
Properties and Localization of the Sulfate‐Activating System in Rat Brain
Authors: Brion Françoise , Schwartz Jean‐Charles , Vargas Froylan
Published: October 2006

Abstract: The formation of the sulfate donor [35S]3′‐phos‐phoadenosine 5′‐phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to β‐naphthol under the action of phenolsulfo‐transferase activity from rat brain cytosol, with the [35S]β‐naphthyl sulfate formed being isolated by polystyrene bead c'iromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chroma‐tography. [35S]PAPS formation by a high‐speed supernatant of rat cerebral cortex occurred with an optimal pH of ∼7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+‐ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1–5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micro‐r olar concentrations inhibited the reaction. The apparent Km values of Mg2+‐ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+‐ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5′‐phos‐phosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity. Among subcellular fractions from cerebral cortex, [35S]PAPS formation only occurred in the cytosolic fractions, including that derived from hypoosmotically shocked synaptosomes. [35S]PAPS formation was not detectable in the crude cortical homogenate, and this was attributable to a thermolabile inhibitory effect of cerebral membranes toward the cytosolic synthesizing system.
Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes
Authors: Sanchez‐Prieto Jose , Harvey Stephen A. K. , Clark John B.
Published: October 2006

Abstract: Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemilumines‐cent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+‐dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygen‐ation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.
Postmortem Changes in Rat Brain: Studies on Membrane‐Bound Enzymes and Receptors
Authors: Syapin Peter J. , Ritchie Terry , Noble Lorna , Noble Ernest P.
Published: October 2006

Abstract: The relationship between the stability of potential neurochemical markers and autolysis time was studied at 4°C and 25°C using postmortem brain samples from two rat strains. In general, qualitatively similar results were obtained with either N/Nih or Sprague‐Dawley rats; however, quantitative differences were often observed, particularly in regard to benzodiazepine receptor changes. For every enzyme activity or binding property examined, no significant change was found when brains were kept at 4°C for up to 72 h prior to freezing at ‐70°C. Na,K‐ATPase and low‐affinity Ca‐ATPase activities were also stable in brains kept at 25°C for up to 72 h. Mg‐ATPase activity was reduced in brains kept at 25°C for 24 and 48 h. [3H]Guanidinoethylmercapto‐succinic acid ([3H]GEMSA) binding to enkephalin conver‐tase in the cytosol was not significantly changed in brains kept at 25°C; however, a small increase was seen for [3H]GEMSA binding to the membrane fraction at 24, but not 48 and 72 h postmortem. [3H]Quinuclidinyl benzilate ([3H]QNB) binding to muscarinic cholinergic receptors decreased in brains kept at 25°C for 72 h. Opioid receptor binding also decreased in brains kept at 25°C. Using [3H]2‐D‐alanine‐5‐D‐leucine enkephalin to label δ opioid receptors, a statistically significant decrease in binding was observed as early as 6 h postmortem, and was completely abolished after 72 h at 25°C. In contrast, [3H]naloxone binding was unchanged after 24 h at 25°C, but was decreased after 48 and 72 h. A statistically significant increase in [3H]diazepam binding was observed in Sprague‐Dawley rat brains kept at 25°C for 24–72 h; however, the increase was not significant when N/Nih rat brains were used. The increase in [3H]diaz‐epam binding was due to an increase in both receptor affinity and apparent number. The mechanism(s) underlying these changes are unclear. The results of this study demonstrate that some, but not all, membrane‐bound enzymes and receptors are subject to postmortem changes when brains are kept at 25°C for varying periods of time. However, the time course and direction of the changes appear unique for each system examined. Extrapolating to humans, the results indicate that postmortem delay time is a potentially important variable that must be considered when utilizing human brain autopsy samples.
Modulatory Effect of Dopamine on High‐Affinity Glutamate Uptake in the Rat Striatum
Authors: Kerkerian Lydia , Dusticier Nicole , Nieoullon André
Published: October 2006

Abstract: In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium‐dependent high‐affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10–8M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. α‐Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating eifects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6‐hydroxydopamine (6‐OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6‐OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.
Effect of Intranigral Administration of 6‐Hydroxydopamine and 5, 7‐Dihydroxytryptamine on Rat Brain Tryptamine
Authors: Juorio A. V. , Greenshaw A. J. , Nguyen T. V.
Published: October 2006

Abstract: Earlier experiments have shown that unilateral electrolytic lesions of the substantia nigra result in significant reductions in the rate of accumulation of rat striatal tryptamine. For elucidation of the type of neuronal degeneration that is associated with tryptamine depletion, the effects of intranigral injections of 6‐hydroxydopamine or 5, 7‐dihydroxytryptamine, which would affect, respectively, dopamine‐ or indoleamine‐containing neurons, have been assessed. Nigral 6‐hydroxydopamine lesions resulted in an ipsilateral reduction in the rate of accumulation of striatal tryptamine, but no changes were observed after nigral injections of 5, 7‐dihydroxytryptamine. The present results suggest that decreases in the pargyline‐induced accumulation of striatal tryptamine may be associated with lesions of the nigral dopamine‐containing cell bodies. Alternatively, there may exist specific tryptamine‐containing neurons that are damaged by 6‐hydroxytryptamine and unaffected by 5, 7‐dihydroxytryptamine.
Enzymatic Protection Against Peroxidative Damage in Isolated Brain Capillaries
Authors: Tayarani Ivan , Chaudiere Jean , Lefauconnier Jeanne‐Marie , Bourre Jean‐Marie
Published: October 2006

Abstract: The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione per‐oxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 μmol/mg of protein, which corresponds to a concentration of ∼3.5 mM. Our results indicate that the blood‐brain barrier contains large amounts of peroxide‐detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.
Selective Transacylation of 1‐O‐Alkylglycerophosphoethanolamine by Docosahexaenoate and Arachidonate in Rat Brain Microsomes
Authors: Ojima Ayako , Nakagawa Yasuhito , Sugiura Takayuki , Masuzawa Yasuo , Waku Keizo
Published: October 2006

Abstract: The mechanism involved in the enzymic acylation of 1‐[3H]alkylglycero‐3‐phosphoethanolamine (1‐[3H]alkyl‐GPE) in brain microsomes was investigated in comparison with the acylation of 1‐[3H]alkylglycero‐3‐phosphocholine (1‐[3H]alkyl‐GPC). Both the alkyllysophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1‐stearoyl‐2‐[14C]arachidonoyl‐GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1‐[3H]alkyl‐GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1‐[3H]alkyl‐GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1‐[3H]alkyl‐GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1‐[3H]alkyl‐GPC by both docosahexaenoate and arachidonate increased 1.5–2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1‐[3H]alkyl‐GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors. These results indicated that 1‐[3H]alkyl‐GPE was mainly acylated, with a high preference for docosahexaenoate and arachidonate, through CoA‐independent direct transfer. Such transacylation is probably responsible for the high proportions of docosahexaenoate and arachidonate in neural phospholipids.
Effects of Extracellular Choline Concentration and K+ Depolarization on Choline Kinase and Choline Acetyltransferase Activities in Superior Cervical Sympathetic Ganglia Excised from Rats
Authors: Ando Masato , Iwata Mitsuyoshi , Takahama Kazuya , Nagata Yutaka
Published: October 2006

Abstract: The activities of choline kinase (CK) and choline acetyltransferase (ChAT) were examined in vitro in superior cervical sympathetic ganglia (SCG) excised from rats following aerobic incubation for 1 h in a medium containing various choline concentrations, with and without application of a high KCl level (70 mM). Ganglionic CK activity was strongly inhibited (by ∼75%) at low extracellular choline concentrations (1–5 μM) but rose as the choline concentration was raised to 10–50 μM in the incubation medium, then fell and rose again with further increases in choline concentration. A similar but moderate accelerative effect on ganglionic CK activity was also observed after addition of acetylcholine (ACh; 1 mM) without eserine. Whereas specific CK activity did not change significantly in axotomized SCG, in which the ratio of glial cells to neurons is greatly increased for a week after the operation, it was remarkably increased after denervation, in which the preganglionic cholinergic nerve terminals had degenerated. When either a high KCl level or hemicholinium‐3 (HC‐3; 50 μM) was added to the medium in the presence or absence of choline, ganglionic CK activity was markedly inhibited. On the other hand, ChAT activity in the SCG remained at a significantly high level during incubation with low choline concentrations (1–10 μM), but the enhanced enzyme activity became inhibited as the extracellular choline concentration was raised to 50–100 μM in the medium. Addition of HC‐3 to the medium did not alter ganglionic ChAT activity at low choline concentrations. However, application of quinacrine (10 μM) considerably reduced ganglionic CK activity and also suppressed ChAT activity induced by high KCl levels. These reciprocal fluctuating changes of ganglionic CK and ChAT activities as a function of extracellular choline concentration in the incubation medium strongly suggest that cytoplasmic CK might contribute to modulating the supply of choline, after uptake from the extracellular fluid, either for storage in membranous phosphatidylcholine (lecithin) or for synthesis of the neurotransmitter ACh by ChAT in cholinergic nerve terminals.
Regional Selectivity of a γ‐Aminobutyric Acid‐Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ‐Aminobutyric Acid Uptake
Authors: Bonanno Giambattista , Raiteri Maurizio
Published: October 2006

Abstract: The effects of γ‐aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01–0.3mM) the basal release of newly synthesized [3H]ACh was increased in a concentration‐dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3mM whereas, at this concentration, (‐)‐baclofen was totally inactive. The GABA‐induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.
Dopamine and Serotonin Metabolism in Brain Sites Ipsi‐ and Contralateral to Direction of Conditioned Turning in Rats
Authors: Schwarting R. , Huston J. P.
Published: October 2006

Abstract: Concentrations of dopamine, serotonin, and some of their metabolites were analyzed by means of HPLC in brain samples obtained from rats operantly conditioned to turn in circles to obtain water reinforcement. In experiment 1 using Wistar rats, no differences in the levels of transmitters or metabolites were detected between brain samples (frontal cortex, ventral striatum, dorsal striatum, septum, amygdala, substantia nigra) from the hemispheres located ipsi‐ and contralateral to the direction of turning. A higher dopamine metabolism (indicated by higher metabolite/transmitter ratios) in ventral striatum, dorsal striatum, and amygdala was found after 15 min than after 5 min of turning in both hemispheres. A higher dopamine metabolism was found in water‐deprived rats compared to nondeprived rats independently of whether or not deprived rats were trained to turn for water reinforcement. In two additional experiments, no differences in dopamine metabolism were found between the ipsi‐ and contralateral striatum of Wistar rats after 25 min and Sprague‐Dawley rats after 10 min of operantly conditioned turning. The present results confirm that dopamine metabolism can change with different behavioral or physiological states; they do not support the hypothesis that conditioned turning is correlated with asymmetrical changes in the metabolism of dopamine or serotonin in the brain.
Stimulated Release of Phosphatidylinositol Phosphodiesterase in an Isolated Phrenic Nerve‐Diaphragm Preparation
Authors: Smith Margaret E. , Shute Janis K. , Morgan‐Harrison Kay
Published: October 2006

Abstract: Electrical stimulation of the phrenic nerve in an isolated nerve‐diaphragm preparation resulted in the release of phosphatidylinositol phosphodiesterase into the organ bath. The released enzyme was Ca2+‐dependent and exhibited two pH optima. The enzyme was released in response to nerve stimulation even in the presence of d‐tubocurarine in concentrations that block neuromuscular transmission, and was not therefore released from the muscle as a consequence of its contractile activity. Phosphatidylinositol phosphodiesterase activity was determined in the soluble cytosol fractions prepared from different regions of skeletal muscles and from normal peripheral nerves and nerves that were degenerating after transection. The specific activity of the enzyme in the cytosol from the endplate‐rich region of the diaphragm was significantly greater than that in cytosol from either the endplate‐free region of the diaphragm or from the phrenic nerve. In degenerating nerve the activity of the enzyme was greater in the distal stump than in the proximal stump at 36 h after nerve section. Possible roles for released phosphatidylinositol phosphodiesterase at the neuromuscular junction are discussed.
Effect of Free Malonate on the Utilization of Glutamate by Rat Brain Mitochondria
Authors: Koeppen Arnulf H. , Riley Kathy M.
Published: October 2006

Abstract:Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L‐glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mMor higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2—oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and γ‐vinyl‐γ‐aminobutyric acid but again added malate allowed transamination to resume. When arsenite and γ‐vinyl‐γ‐aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparentKmandVmaxvalues for uninhibited malate entry were 0.01 mMand 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki=0.75 mM).
Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis
Authors: Damsma G. , Westerink B. H. C. , de Vries J. B. , Van den Berg C. J. , Horn A. S.
Published: October 2006

Abstract: The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC‐assay system that comprises a cation exchanger, a post‐column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 μM) of neostigmineresultedingraduallyincreasingamounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.
Axonal Transport Characteristics of Gangliosides in Sensory Axons of Rat Sciatic Nerve
Authors: Harry G. Jean , Goodrum Jeffry F. , Toews Arrel D. , Morell Pierre
Published: October 2006

Abstract: The distribution of axonally transported gangliosides and glycoproteins along the sciatic nerve was examined from 3 h to 4 weeks following injection of [3H]glucosamine into the fifth lumbar dorsal root ganglion of adult rats. Incorporation of labeled precursor into these glycoconjugates reached a maximal level in the ganglion within 6 h. Outflow patterns of radioactivity for glycoproteins showed a well‐defined crest with a transport rate of approximately 330 mm/day. In contrast, the crest of transported gangliosides was continuously attenuated, implying a significant deposition along the axon, and an alternative method of calculating velocity was required. Analysis of accumulation of labeled material at double ligatures demonstrated both anterograde and retrograde transport of glycoproteins and gangliosides and allowed for the calculation of an anterograde transport rate of about 270 mm/day for each. Additional evidence of ganglioside transport is provided in that the TLC pattern of transported radioactive gangliosides accumulating at a ligature is significantly different from the pattern seen in the dorsal root ganglion or following intra‐neural administration of the labeled precursor. These data indicate that gangliosides are transported at the same rapid rate as glycoproteins but are subject to a more extensive exchange with stationary material than are glycoproteins.
Angiotensin II Inactivation Process in Cultured Mouse Spinal Cord Cells
Authors: Allard M. , Simonnet G. , Dupouy B. , Vincent J. D.
Published: October 2006

Abstract: The pattern of hydrolysis of [3H]angiotensin II ([3H]AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37°C. In the absence of peptidase inhibitors, 70% of [3H]AII was degraded, and the main labeled metabolite was [3H]tyrosine (40% of total radioactivity). Minor quantities of [3H]AII1–5 and [3H]AII4–8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the All‐hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4,14.4) could play a critical role, as suggested by the formation of [3H]Val3‐Tyr4 and [3H]‐Tyr4‐IIe5 in the presence of bestatin (2 ± 10−5M). This hypothesis was confirmed by the potency of dipeptidyl aminopeptidase III inhibitors to inhibit both [3H]AII hydrolysis and formation of these 3H‐labeled dipeptides. An arylamidase‐like activity could also be participating in [3H]AII hydrolysis, because higher concentrations of bestatin (10−4M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited [3H]tyrosine formation, increased protection of [3H]AII and [3H]AII1–7 formed, and provoked a slight accumulation of [3H]AII2–8. These results suggest that the formation of [3H]AII2–8 is due to the action of a bestatininsensitive acidic aminopeptidase and that the Pro7‐Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase. Because care was taken to eliminate interference by enzymes leaking out into the incubation medium, the AII‐hydrolyzing process described on these cultured mouse spinal cord cells presumably occurs under the action of membrane‐bound peptidases.
Inhibition of Catecholamine Secretion from Bovine Chromaffin Cells by Adenine Nucleotides and Adenosine
Authors: Chern Yi‐Juang , Herrera Michelle , Kao Lung Sen , Westhead Edward W.
Published: October 2006

Abstract: ATP, ADP, and adenosine have been found to inhibit acetylcholine‐stimulated secretion from isolated cells of bovine adrenal medulla (chromaffin cells). Maximal inhibition is −30% under the conditions studied; half‐maxmal inhibition occurs at nucleotide concentration in the micromolar range. Cells must be incubated with ATP for ‐90 s for maximal inhibition, but inhibition by adenosine occurs much faster, an observation suggesting the possibility that ATP and ADP exert their effect after being converted to adenosine. Experiments with cells preloaded with the fluorescent calcium chelator quin 2 indicate that external ATP can diminish the rise in cytosolic Ca2+ concentration that follows stimulation by acetylcholine.