bluuebunny/crossref_metadata_2025 huggingface dataset:
Inter- and intra-variant genetic heterogeneity of human coronavirus OC43 strains in France
Authors: Vabret Astrid , Dina Julia , Mourez Thomas , Gouarin Stéphanie , Petitjean Joëlle , van der Werf Sylvie , Freymuth François
Published: October 2006

Human coronavirus OC43 (HCoV-OC43) causes acute, self-limited respiratory infections. A close relationship between bovine coronaviruses (BCoVs) and HCoV-OC43 has recently been demonstrated. This study includes seven clinical, non-cell culture-adapted, contemporary HCoV-OC43 strains detected in France in 2003. By using RT-PCR and clonal sequencing of the S1 gene of HCoV-OC43, the inter-variant heterogeneity of the HCoV-OC43 circulating strains was studied and the intra-variant diversity was assessed by investigation of a quasispecies cloud. This paper brings to the forefront a high genetic diversity of circulating HCoV-OC43 variants. Genetically different groups are defined among the variants described in this study. One of these variants holds characteristics of an outlier and presents a deletion of 12 nt, also found in BCoV strains. Moreover, the presence of HCoV-OC43 as a quasispecies cloud in vivo during an acute respiratory-tract illness was discovered. It has also been revealed that quasispecies-cloud sizes are similar for the two viral populations tested.
Rise in gamma interferon expression during resolution of duck hepatitis B virus infection
Authors: Narayan Ramamurthy , Buronfosse Thierry , Schultz Ursula , Chevallier-Gueyron Philippe , Guerret Sylviane , Chevallier Michelle , Saade Fadi , Ndeboko Benedicte , Trepo Christian , Zoulim Fabien , Cova Lucyna
Published: October 2006

Gamma interferon (IFN-γ) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-γ (DuIFN-γ) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-γ expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3–14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-γ RNA was detected by real-time RT-PCR at days 6–14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-γ expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-γ in PBMCs (days 12–14), which coincided with the peak in liver DuIFN-γ RNA levels. These data reveal that increased DuIFN-γ expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host–virus interactions that control DHBV infection.
Processing of the AIDA‐I precursor: removal of AIDAC and evidence for the outer membrane anchoring as a β‐barrel structure
Authors: Suhr Martin , Benz Inga , Schmidt M. Alexander
Published: October 2006

The AIDA‐I adhesin known to be responsible for the diffuse adherence (DA) phenotype of the diarrhoea‐genie Escherichia coli (DAEC) strain 2787 has been shown previously to be synthesized as a precursor protein and to undergo additional C‐terminal processing. Here, the C‐terminal processing of the AIDA‐I precursor and the outer membrane topology of the cleaved C‐terminal fragment, AIDAC, were investigated. By isolation of the cleaved AIDAC fragment and N‐terminal sequencing, the C‐terminal cleavage site was identified between Ser‐846 and Ala‐847 thereby indicating a molecular mass of 47.5 kDa for AIDAC. The correct processing to AIDA‐I and AIDAC in OmpT, OmpP and DegP protease‐deficient E. coli strains as well as in avirulent salmonellae and shigellae points to an autocatalytic cleavage mechanism. The cleaved AIDAC was localized in the outer membrane. A leader sequence‐AIDAC fusion was efficiently routed to the outer membrane. Analysis by protease digestion, secondary‐structure prediction and modelling, by comparison with structurally related bacterial proteins like the lgA1 protease from neisseria, the vacuolating toxin from Helicobacter pylori, and the VirG protein of Shigella flexneri, strongly indicates that AIDAC is present in the outer membrane as a β‐barrel structure.
Systemic Antibody Response to Diarrheagenic Escherichia coli and LPS O111, O157 and O55 in Healthy Brazilian Adults
Authors: Zapata‐Quintanilla L. B. , Palmeira P. , Tino‐De‐Franco M. , Amaral J. A. , Carbonare C. B. , Carbonare S. B.
Published: October 2006

Enterohaemorrhagic Escherichia coli (EHEC) can cause a variety of human illnesses ranging from uncomplicated diarrhoea to haemorrhagic colitis and haemolytic uremic syndrome. The serotype O157:H7 has been associated with numerous outbreaks worldwide, but in Brazil the infection is rare. Brazilian adults present antibodies reactive with the principal virulence factors of enteropathogenic E. coli (EPEC) that have many genetic and antigenic similarities with EHEC. Lipopolysaccharides (LPS) are components of outer membranes and important virulence factors of Gram‐negative bacteria. LPS O111 is present in EPEC and EHEC strains. LPS O157 is found only in EHEC strains, but it has some structural similarities with LPS O55 present in EPEC strains. This study investigates the levels of IgG and IgM seric antibodies reactive with EHEC O157:H7, EHEC O111:H–, EPEC O111:H– and the levels of anti‐LPS O111, LPS O157 and LPS O55 antibodies in healthy adults living in São Paulo, Brazil. The antibody levels were determined by an enzyme‐linked immunosorbent assay for 100 individual serum samples, and the presence of anti‐bacterial and anti‐LPS seric antibodies was confirmed. Positive correlations were found among the three kinds of antibodies. The concentrations of IgM anti‐LPS were significantly higher than those of IgG, and surprisingly, the concentrations of anti‐LPS O157 were high in view of the infrequent isolation of O157 bacteria in Brazil. Our results suggest that there is a cross‐reacting immunity to EHEC in the Brazilian population, which may be a result of the immunity to EPEC antigens. Alternatively, Brazilians may be exposed to EHEC more frequently than has previously been thought.
Primary and metastatic perineal adenocarcinoma: aetiology and management
Authors: Leon R. , Ramesh A. , Wilson M. S. , O’Dwyer S. T.
Published: October 2006

Objective:  Adenocarcinoma of the perineum is unusual. This series of nine patients highlights the challenges of management.

Method:  Six male and three female patients aged 29–85 years presented to a single unit, five were T4 and three were T3 tumours.

Results:  Pain and swelling in the perineum and perianal tissues remote from the anal canal occurred in six patients whilst three patients had chronic perineal fistulae, two of whom had longstanding Crohn's disease. In eight patients a primary adenocarcinoma was identified. One patient with fistulating perineal Crohn's disease had multifocal mucinous adenocarcinoma without evidence of a gastrointestinal primary. Radical multimodality treatment including long course chemo‐radiotherapy (CRT) and radical abdominoperineal resection was used in seven patients, four of whom required perineal reconstruction with myocutaneous flaps. Two patients had CRT without surgery (one unresectable and one refused surgery). Negative resection margins were achieved in six of seven patients. All seven patients undergoing resection were well palliated with relief of pain and currently four patients remain free of disease with only one patient developing further perineal disease remote from the surgical resection site.

Conclusion:  Perineal adenocarcinoma may be associated with an occult colorectal primary or chronic fistulae. Lower GI endoscopy and biopsies of non‐healing mucous producing fistulae should be undertaken to establish the diagnosis. Radical surgery often achieves local disease control.
Surgical treatment for urethral recurrence after ileal neobladder reconstruction in patients with bladder cancer
Authors: Yoshida Kenji , Nishiyama Hiroyuki , Kinoshita Hidefumi , Matsuda Tadashi , Ogawa Osamu
Published: October 2006

OBJECTIVE

To report a retrospective study evaluating the management of superficial urethral recurrence after ileal neobladder construction in patients with bladder cancer.

PATIENTS AND METHODS

In 77 consecutive patients with ileal neobladder after radical cystectomy for invasive bladder cancer, urethral recurrence was evaluated and transurethral resection (TUR) used as an initial treatment for superficial urethral recurrence. Urethrectomy with urinary re‐diversion was performed when further recurrence developed.

RESULTS

Four patients (5%) presented with a superficial urethral recurrence and all four were treated by TUR as initial therapy. One patient has had no evidence of recurrence after initial TUR, although the other three patients were later treated with salvage urethrectomy due to repeated urethral recurrence. As a result, the stage of urethral recurrence advanced from pTa to pT1–pT2 in two of the three patients. For urinary re‐diversion, one patient had a conversion from a Studer pouch to an ileal conduit, using the afferent limb, and the other two were converted from a Hautmann pouch to a continent reservoir using the Appe‐Mainz procedure. There was no evidence of metastasis or local recurrence in any of the four patients.

CONCLUSION

Urethral preservation at initial therapy for superficial recurrence might be reasonable, and sequential urethrectomy after attempted urethral preservation might be strategically feasible. Urinary re‐diversion from a neobladder to a catheterizable continent reservoir using the appendix would be a good choice and maintains the quality of life.
Testicular microlithiasis: what does it mean clinically?
Authors: Dagash Haitham , MacKinnon Ewen A.
Published: October 2006

OBJECTIVE

To review our current practice of follow‐up for boys with testicular microlithiasis (TM), an uncommon condition characterized by calcification within the seminiferous tubules, detected by ultrasonography (US); TM has been associated with both benign and malignant conditions of the testes but the natural history of TM in children remains unclear.

PATIENTS AND METHODS

All boys diagnosed with TM over a 14‐year period were included in this study. A search of the radiology database was carried out using the keywords ‘testicular microlithiasis’ and ‘testicular calcification’. A retrospective case‐note review was then used to determine age at diagnosis, presenting symptoms, indication for testicular US, outcome and follow‐up. We also searched Medline/PubMed, using the same keywords for published data on TM from 1970 to 2006.

RESULTS

Over the study period 711 testicular scans were taken in 623 patients; seven cases (1.1%) of TM were identified. The mean (range) age at presentation was 12 (7–15) years. The presenting symptoms were testicular pain (three), undescended testes (two), hydrocele (one) and asymptomatic scrotal swelling (one). In five cases the TM was bilateral and in two a solitary kidney was identified. Only one patient had tumour markers measured (β‐human chorionic gonadotrophin and α‐fetoprotein) and these were within normal limits. On yearly US follow‐up, the TM was less prominent in one patient, unchanged in four and two were lost to follow‐up. Three patients are currently on yearly US follow‐up while two are under the care of adult general surgeons. The analysis of reports published to date indicated that malignancy only develops when TM is associated with other predisposing factors.

CONCLUSION

There is no convincing evidence that TM alone is premalignant. However, when it accompanies other potentially premalignant features we recommend annual US follow‐up.
Government-run Online Discussion Fora: Moderation, Censorship and the Shadow of Control1
Authors: Wright Scott
Published: October 2006

Moderators are widely thought to be crucial to the facilitation of high-quality democratic debate, particularly in government-sponsored participatory exercises. There are, however, persistent fears that moderators censor rather than promote free speech, leading to a ‘shadow of control’. This article analyses the relationship between moderation and censorship on two British central government online discussion fora: Downing Street's Speaker's Corner and Policy Forum, and Citizen Space's E-Democracy Forum. Two models of moderation are developed to help structure the analysis. The main conclusions are that moderation strategies must be clearly linked to the policy goals behind the forum, and that the moderator's roles should be separated to limit the so-called ‘shadow’. The censorial role being conducted by an independent body, with facilitation activities conducted by civil servants linked to the policy being discussed.
Localization of Phospholipid Synthesis to Schwann Cells and Axons
Authors: Gould Robert M. , Holshek John , Silverman Wayne , Spivack Warren D.
Published: October 2006

Abstract: Quantitative electron microscopic autoradiography was used to detect and characterize endoneurial sites of lipid synthesis in mouse sciatic nerve. Six tritiated phospholipid precursors (choline, serine, methionine, inositol, glyc‐erol, and ethanolamine) and a protein precursor (proline) were individually injected into exposed nerves and after 2 h the mice were perfused with buffered aldehyde. The labeled segments of nerve were prepared for autoradiography with procedures that selectively remove nonincorporated precursors and other aqueous metabolites, while preserving nerve lipids (and proteins). At both the light and electron microscope levels, the major site of phospholipid and protein synthesis was the crescent‐shaped perinuclear cytoplasm of my‐elinating Schwann cells. Other internodal Schwann cell cytoplasm, including that in surface channels, Schmidt‐Lanterman incisures, and paranodal regions, was less well labeled than the perinuclear region. Newly formed proteins were selectively located in the Schwann cell nucleus. Lipid and protein formation was also detected in unmyelinated fiber bundles and in endoneurial and perineurial cells. Tritiated inositol was selectively incorporated into phospholip‐ids in both myelinated axons and unmyelinated fibers. Like inositol, glycerol incorporation appeared particularly active in unmyelinated fibers. Quantitative autoradiographic analyses substantiated the following points: (1) myelinating Schwann cells dominate phospholipid and protein synthesis, (2) myelinated axons selectively incorporate tritiated inositol, (3) phospholipid precursors label myelin sheaths and myelinated axons better than proline.
Choline Uptake by Cerebral Capillary Endothelial Cells in Culture
Authors: Estrada Carmen , Bready James , Berliner Judith , Cancilla Pasquale A.
Published: October 2006

Abstract: A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood‐brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier‐mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a Km of 7.59 ± 0.8 μM and a maximum capacity of 142.7 ± 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1–100 μM). When cells were preincubated in the presence of choline, a single saturable component was observed with a Km of 18.5 ± 0.6 μM and a maximum capacity of 452.4 ± 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium‐3, deanol, and AF64A. The presence of ouabain or 2,4‐dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine‐5′‐diphosphocholine and phospholipids; however, under steady‐state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h. Only part of the labelled intracellular free choline was transported back into the incubation medium. Because of their special location, transport properties, and metabolism, cerebral capillary endothelial cells may provide a gating mechanism that can contribute to the physiological control of choline concentration in the brain extracellular fluid.
Phosphate‐Dependent Monoclonal Antibodies to Neurofilaments and Alzheimer Neurofibrillary Tangles Recognize a Synthetic Phosphopeptide
Authors: Coleman Michael P. , Anderton Brian H.
Published: October 2006

Abstract: Five phosphate‐dependent monoclonal antibodies to the neurofilament heavy polypeptide bound strongly to a phosphorylated synthetic peptide, which contains a single Lys‐Ser‐Pro sequence that occurs in human neurofilaments. Three of the antibodies label Alzheimer's disease neurofibrillary tangles and two do not, suggesting that in tangles an epitope similar to the peptide is available to some but not all of the antibodies. In addition, some antibodies were found to be more affected than others by enzymatic dephosphorylation of the antigen, but because all the antibodies bound the same synthetic phosphopeptide they do not bind to mutually exclusive phosphorylation sites. Instead the more phosphate‐dependent antibodies might bind the phosphate group more directly, as suggested by their inhibition by inorganic phosphate and free phosphoserine.
Lipopolyamine‐Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells
Authors: Loeffler J. Ph. , Barthel F. , Feltz P. , Behr J. P. , Sassone‐Corsi P. , Feltz A.
Published: October 2006

Abstract: A simple and efficient transfection technique based on lipopolyamine‐coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans‐acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.
Intra‐ and Interchain Disulfide Bond Generation in S100b Protein
Authors: Mely Yves , Gérard Dominique
Published: October 2006

Abstract: Disulfide‐bridged S100b protein formation, air‐catalyzed and induced by thiol/disulfide exchange, was studied under various ionic conditions. As native, physiological disulfide‐bridged proteins are obtained easily from their reduced counterparts under appropriate redox conditions, this work was performed to determine whether this was the case for disulfide‐bridged S100b proteins, reported to have neurite extension activity. In nondenaturating native medium, no disulfide‐bridged species could be generated from reduced proteins in any of the ion‐induced conformations tested (no ions, Ca2+, Zn2+, or K+) under widely different redox conditions. Only mixed disulfides accumulated, in certain cases.

In contrast, intrasubunit monomeric and intersubunit dimeric disulfide‐bridged species were readily and efficiently generated under denaturating conditions. A brief characterization of these oxidized species suggested that they differed widely in structure from their reduced counterparts and that they probably did not bind Ca2+. Taken together, these data question the physiological relevance of these disulfide‐bridged S100b protein species.
N‐Acetylation of Serotonin Is Correlated with α2‐ but Not with β‐Adrenergic Regulation of Cyclic AMP Levels in Cultured Chick Pineal Cells
Authors: Voisin Pierre , Van Camp Gilles , Collin Jean‐Pierre
Published: October 2006

Abstract: We investigated the role of cyclic AMP (cAMP) in α2‐ and possible β‐adrenergic regulation of arylalkylamine‐N‐acetyltransferase (NAT), the penultimate enzyme in the biosynthesis of melatonin. The study was performed on primary cultures of dispersed chick pineal cells. Electron microscopy indicated that ∼70% of the dispersed cells were modified photoreceptors. A similar proportion of melatoninergic cells was detected by immunocytochemical labeling of hydroxyindole‐O‐methyltransferase, the final enzyme in the biosynthesis of melatonin. Adrenergic agonists caused a sustained 50% inhibition of forskolin‐augmented cAMP levels and NAT activity, with an α2‐adrenergic potency order of UK 14,304 clonidine > norepinephrine > phenylephrine. Noradrenergic inhibition of 3‐isobutyl‐1‐methylxanthine‐augmented cAMP levels and NAT activity was reversed by yohimbine (an α2‐adrenergic antagonist) but not by prazosin (an α1‐adrenergic antagonist). The α2‐adrenergic inhibition of cAMP accumulation and NAT activity was prevented by pertussis toxin. Addition of propranolol (a β‐adrenergic antagonist) was necessary to observe an inhibitory effect of norepinephrine on cAMP levels but not on NAT activity. Similarly, the β‐adrenergic agonist isoproterenol transiently increased cAMP levels but did not affect NAT activity. The data indicate that the α2‐adrenergic inhibition of NAT activity in chick pineal cells is strongly correlated with an inhibition of cAMP accumulation. The lack of β‐adrenergic effect on NAT suggests that β‐adrenoceptors might be on a subset of cells that do not produce melatonin or that the β‐adrenergic‐induced increase in cAMP levels is too transient to affect NAT.
Anatomic and Disease Specificity of NADH CoQ1 Reductase (Complex I) Deficiency in Parkinson's Disease
Authors: Schapira A. H. V. , Mann V. M. , Cooper J. M. , Dexter D. , Daniel S. E. , Jenner P. , Clark J. B. , Marsden C. D.
Published: October 2006

Abstract: 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is thought to produce parkinsonism in humans and other primates through its inhibition of complex I. The recent discovery of mitochondrial complex I deficiency in the substantia nigra of patients with Parkinson's disease has provided a remarkable link between the idiopathic disease and the action of the neurotoxin MPTP. This article shows that complex I deficiency in Parkinson's disease is anatomically specific for the substantia nigra, and is not present in another neurodegenerative disorder involving the substantia nigra. Evidence is also provided to show that there is no correlation between l‐3,4‐dihydroxyphenylalanine therapy and complex I deficiency. These results suggest that complex I deficiency may be the underlying cause of dopaminergic cell death in Parkinson's disease.
[14C]Leucine Incorporation into Brain Proteins in Gerbils After Transient Ischemia: Relationship to Selective Vulnerability of Hippocampus
Authors: Widmann R. , Kuroiwa T. , Bonnekoh P. , Hossmann K.‐A.
Published: October 2006

Abstract: Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to ∼20–40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to <50%. After 1 day, protein synthesis in the CA1 sector returned to ∼70% of control values, followed by a secondary decline to <50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.
Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters
Authors: Blakely Randy D. , Clark Janet A. , Pacholczyk Tadeusz , Amara Susan G.
Published: October 2006

Abstract: The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na+‐dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region‐ and transporter‐specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high‐affinity amino acid uptake systems. Both L‐glutamic acid (Glu) and γ‐aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60–70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L‐glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well‐characterized inhibitors of Glu, GABA, dopamine, and choline transport (D‐aspartate, nipecotic acid, nomifensine, and hemicholinium‐3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region‐specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size‐fractionated on linear (10–31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4–3.0 kb, 4.0–4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0–4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5–3.0 kb with size‐fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.
Enhanced Aspartate Release from Hippocampal Slices of Epileptic (El) Mice
Authors: Flavin Helen J. , Wieraszko Andrzej , Seyfried Thomas N.
Published: October 2006

Abstract: The release of putative neurotransmitters [aspartate, glutamate, and γ‐aminobutyric acid (GABA)] was studied in hippocampal slices from adult normal C57BL/6J (B6) and El (epileptic) mice. The El mice, a genetic model of temporal lobe epilepsy, had an average of 86 seizures. Sets of B6 and El hippocampal slices (400 μm thick) were incubated in a series of normal and high potassium (60 mM) buffers in the presence or absence of calcium. The calcium‐dependent and calcium‐independent potassium‐induced release of amino acids was compared in each mouse strain. Release of endogenous amino acids was measured using liquid chromatog‐raphy with electrochemical detection and was expressed as picomoles of amino acid released per milliliter of incubation buffer per minute of incubation per slice ± SEM. No significant differences were found between the El and B6 mice for the calcium‐dependent potassium‐evoked release of glutamate (18.20 ± 2.62 and 15.41 ± 3.56), or GABA (17.28 ± 2.90 and 12.73 ± 1.37), respectively. Aspartate release, however, was significantly higher in the El mice (6.62 ± 0.69) than in the B6 mice (3.31 ± 0.72). These findings suggest that enhanced aspartate release may be related to seizure expression in El mice.
Characterization of Neuronal and Endothelial Forms of Angiotensin Converting Enzyme in Pig Brain
Authors: Williams Tracy A. , Hooper Nigel M. , Turner Anthony J.
Published: October 2006

Abstract: The molecular forms of angiotensin converting enzyme (ACE; EC 3.4.15.1) in preparations of pig brain cortical microvessels and striatal synaptosomal membranes have been identified by immunoelectrophoretic blot analysis. The cortical microvessels contained only the endothelial form of the enzyme, Mr 180,000, which comigrated with pig kidney ACE on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In contrast, the synaptosomal membranes contained only a smaller form of ACE, Mr 170,000, which represents the neuronal form of the enzyme. No significant differences in inhibitor sensitivity or substrate specificity were detected between the two forms of ACE. In particular, neurokinin A was resistant to hydrolysis by either microvessel or synaptosomal membrane ACE, and the pattern of hydrolysis of substance P by the two preparations was identical.
Creatine Kinase Activity in Postnatal Rat Brain Development and in Cultured Neurons, Astrocytes, and Oligodendrocytes
Authors: Manos Patricia , Bryan Guy K. , Edmond John
Published: October 2006

Abstract: The development and distribution of cytosolic creatine kinase (CK) activity was studied in rat brain and in cell culture. The activity of CK in whole brain increased almost fivefold during the period from birth to day 40 when adult levels of 18–19 U/mg of protein were attained. The distribution of CK activity was examined in dissected regions of the adult brain and was nonuniform; the cerebellum, the striatum, and the pyramidal tracts contained significantly higher CK activity than did whole brain. The cellular compartmentation of CK was investigated using primary cultures of purified neurons, astrocytes, and oligodendrocytes. The CK activity in neurons increased fourfold greater than that measured at the time of isolation to 4 U/mg of protein. The CK activity in astrocytes cultured for 20 days was 3.5 U/mg of protein and was 1.5‐fold greater than that measured at the time of isolation. In contrast, the CK activity in cultured oligodendrocytes (day 20) was three‐ to fourfold higher than that determined in astrocytes and almost sevenfold higher than the activity measured at the time the cells were isolated. The high levels of CK in cultured oligodendrocytes suggest a role for this enzyme in oligodendrocyte function and/or myelinogenesis.
Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ‐Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat
Authors: Globus Mordecai Y.‐T. , Busto Raul , Martinez Elena , Valdés Isabel , Dietrich W. Dalton , Ginsberg Myron D.
Published: October 2006

We evaluated whether regional differences in the magnitude of glutamate, γ‐aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia‐induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10‐ and 20‐min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathoiogical neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathoiogical outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10‐min insult but severely damaged after 20 min of ischemia. Ischemia‐induced increases in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10‐ and 20‐min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20‐min insult Although ischemic neuronal damage was not related to glutamate release, it correlated with the „excitotoxic index,” whose value was derived from the following equation: [glutamate] X [glycine]/[GABA]. No significant changes were observed in the excitotoxic index during ischemia. However, a significant increase in the index was observed in vulnerable brain regions during the early and late recirculation periods. These results suggest that the imbalance between excitation and inhibition, reflected by changes in the excitotoxic index, may account for regional vulnerability to ischemia.
The Susceptibility of Cerebral Endothelial Cells to Astroglial Induction of Blood‐Brain Barrier Enzymes Depends on Their Proliferative State
Authors: Meyer Jörg , Rauh Jürgen , Galla Hans‐Joachim
Published: October 2006

Primary cultures of brain capillary endothelial cells (BCECs) were used to investigate the induction of blood‐brain barrier (BBB) characteristics in vitro. Enzymatic activities of γ‐glutamyltranspeptidase (γ‐GT) and alkaline phos‐phatase (ALP) were taken as indicators for the expression of the BBB phenotype. We were able to show that a coculture system with a direct cell‐cell contact between astroglial cells and BCECs is the necessary precondition for an increase of these enzyme activities that are lost in pure BCEC cultures. Coculture with both astrocytes and C6‐glioma cells reestablishes the BBB phenotype whereas conditioned media as well as an astrocyte‐derived extracellular matrix were ineffective. The susceptibility of the BCECs to an astroglial stimulus depends on the proliferative state of the BCECs. Cells in an early highly proliferate culture phase were stimulated to express an enzymatic activity level similar to the in vivo situation. Confluent BCEC monolayers were not induced at all. With the ALP we observed a spatial induction within a BCEC colony. Astrocyte‐induced ALP activity was first observed at an outer belt of BCEC colonies in direct contact with the astrocyte layer. However, this signal is transferred to the center of the colony with time in culture. We conclude that direct contact of BCECs with astroglial cells is necessary for the induction of the BBB phenotype in cultured BCECs and that this signal may be transferred from induced to noninduced BCECs.
Elevation of Taurine in Hippocampal Extracellular Fluid and Cerebrospinal Fluid of Acutely Hypoosmotic Rats: Contribution by Influx from Blood?
Authors: Lehmann Anders , Carlström Christian , Nagelhus Erlend A. , Ottersen Ole Petter
Published: October 2006

Abstract: Previous work has demonstrated that there is a selective increase in extracellular taurine in the brain during acute water intoxication. One aim of the present study was to investigate whether plasma taurine contributes to this increase. To this end, the concentrations of taurine, other amino acids, and ethanolamine (EA) were measured in plasma and CSF of urethane‐anesthetized rats injected with 150 ml/kg body weight of distilled water. Blood pressure, blood gases, and pH, as well as plasma and CSF osmolality, were also measured. The CSF level of albumin was quantitated to study the function of the blood‐CSF barrier. In separate experiments, hippocampal microdialysis was performed to determine the effects of acute plasma hypoosmolality on extracellular amino acids. Finally, the effect of water injection on hippocampal specific gravity and tissue amino acids was assessed. Blood gases and pH were essentially unchanged after water administration. Mean arterial blood pressure increased to peak levels approximately 50 mm Hg above control. Plasma osmolality decreased rapidly, whereas the depression of CSF osmolality was slower and less pronounced. The average volume of the hippocampus increased by 8%. Water injection was accompanied by a 25‐fold elevation of taurine in plasma, whereas phosphoethanolamine (PEA) and EA increased moderately. A small fraction of the increase in plasma taurine might derive from blood cells because dilution of blood in vitro led to doubled plasma levels of the amino acid. Taurine, PEA, and EA increased consistently in CSF and hippocampal microdialysates. Plasma hypoosmolality transiently opened the blood‐CSF barrier as reflected by augmented CSF concentrations of albumin. The level of taurine in hippocampal tissue (expressed in μmol/g wet weight) was unaffected whereas EA was elevated, and PEA was depressed. The findings suggest that acute plasma hypoosmolality promotes PEA dephosphorylation and/or inhibits EA phosphorylation. Furthermore, they indicate that there is a leakage of plasma taurine into cerebral extracellular fluids in the course of water intoxication. However, in view of earlier observations that taurine is released in response to neural cell swelling in vitro, the augmentation of extracellular taurine in the brain probably reflects a contribution by neurons and/or astrocytes as well as by blood.
Activation by Nitric Oxide of Guanylate Cyclase in Endothelial Cells from Brain Capillaries
Authors: Marsault Robert , Frelin Christian
Published: October 2006

Abstract: Endothelial cells (ECs) from brain microvessels respond to exogenous nitric oxide (NO) donor molecules (N‐ethoxycarbonyl‐3‐morpholinosydnonimine and sodium nitroprusside) with large (> 15‐fold) increases in cyclic GMP (cGMP) levels. Comparable actions of sodium nitroprusside were observed in vascular smooth muscle cells and in neuroblastoma cells. Coculturing brain capillary ECs in the presence of N1E‐115 neuroblastoma cells increased their cGMP levels fourfold. A further increase was observed in the presence of 50 nM neurotensin, although brain capillary ECs lack receptor sites for neurotensin. The neuroblastoma cell‐dependent formation of cGMP was suppressed by 0.1 mM l‐NG‐monomethylarginine, indicating that NO, produced by N1E‐115 cells in response to neurotensin, activated guanylate cyclase in brain capillary ECs. Similarly, culturing brain capillary ECs in the presence of aortic ECs increased their cGMP content in a manner that was amplified by bradykinin and that was inhibited by l‐NG‐monomethylarginine. Bradykinin had no action in pure cultures of brain capillary ECs. It is concluded that brain capillary ECs express high levels of guanylate cyclase activity that could be activated by exogenous NO donor molecules and by NO produced by neuroblastoma cells and by aortic ECs in response to specific agonists. Brain capillary ECs are thus potential target cells for brain‐derived NO.
Terminal Autoreceptor Control of 5‐Hydroxytryptamine Release as Measured by In Vivo Microdialysis in the Conscious Guinea‐Pig
Authors: Lawrence Andrew J. , Marsden Charles A.
Published: October 2006

Abstract: In vivo microdialysis in the frontal cortex of the freely moving guinea‐pig was used to measure extracellular 5‐hydroxytryptamine (5‐HT) and study terminal autoreceptor control of its release. The indoleamine levels were determined by HPLC with electrochemical detection. Release of extracellular 5‐HT and, to a lesser extent, 5‐hydroxyindoleacetic acid was sensitive to tetrodotoxin, confirming the neuronal origin of measured neurotransmitter levels. Both systemic and local administration of the 5‐HT1 agonist 5‐carboxamidotryptamine caused inhibition of extracellular 5‐HT levels, confirming the regulatory role of the terminal, and possibly also the somatodendritic, 5‐HT autoreceptor on neuronal 5‐HT release. Levels of extracellular 5‐hydroxyindoleacetic acid were not affected by 5‐carboxamidotryptamine following either central or peripheral administration.
Stathmin Phosphorylation Is Regulated in Striatal Neurons by Vasoactive Intestinal Peptide and Monoamines via Multiple Intracellular Pathways
Authors: Chneiweiss Hervé , Cordier Jocelyne , Sobel André
Published: October 2006

Abstract: Stathmin is a ubiquitous soluble protein whose phosphorylation is associated with the intracellular mechanisms involved in the regulations of cell proliferation, differentiation, and functions by extracellular effectors. It is present in the various tissues and cell types as at least two distinct isoforms in their unphosphorylated (Mr∼ 19,000; pI ∼ 6.2–6.0) and increasingly phosphorylated forms. Stathmin is particularly abundant in brain, mostly because of its high concentration in neurons, where the protein is a major phosphorylation substrate. In intact striatal neurons grown in primary culture, the cyclic AMP–increasing drug forskolin and the protein kinase. C–activating agent 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) induced a potent phosphorylation of stathmin. Their actions were at least partially additive, appearing actually most likely “sequential” on various phosphorylated states of stathmin. Vasoactive intestinal peptide (VIP) reproduced the forskolin‐like stimulation but stimulated also other, TPA, and/or Ca2+‐like protein phosphorylations. These actions of VIP were already maximal after 5 min and were long lasting, still important after 2 h. In addition, concentrations as low as 1 nM were enough to obtain a significant effect, on both cyclic AMP‐dependent and independent phosphorylations. Dopamine and the β‐adrenergic agonist isoproterenol were also able to stimulate stathmin phosphorylation, but only with a forskolin‐like pattern. Their actions were not additive to those of VIP, confirming previous results on the colocalization of both dopamine D1 and nor‐adrenaline β1 receptors with VIP receptors on striatal neurons. In conclusion, our results show that VIP regulates the functions and differentiation of embryonic striatal neurons through multiple intracellular pathways and further substantiates the role of stathmin as a cytoplasmic relay integrating multiple second messenger signals.
Activity‐Dependent Regulation of Na+,K+‐ATPase α Isoform mRNA Expression In Vivo
Authors: Mata Marina , Hieber Virginia , Beaty Michael , Clevenger Michael , Fink David J.
Published: October 2006

Abstract: To investigate the functional role of the different Na+,K+‐ATPase a (catalytic) subunit isoforms in neuronal cells, we used quantitative in situ hybridization with riboprobes specific for αl, α2, and α3 isoforms to measure the level of a isoform‐specific expression in the neuroendocrine cells of the supraoptic (SON) and paraventricular (PVN) nuclei of rat hypothalamus. A prolonged increase in electrical activity of these cells, achieved by 5 days of salt treatment, increased the amount of α isoform mRNA in the SON and PVN by 50%. Levels of α mRNA in other brain regions and levels of α2 and α3 mRNAs were not affected by salt treatment. We conclude that the α1 isoform Na+,K+‐ATPase may be specifically adapted to pump out Na+, which enters the cells through voltage‐gated channels during neuronal depolarization.
Elevated γ‐Aminobutyric Acid Levels Attenuate the Metabolic Response to Biateral Ischemia
Authors: Abel Marc S. , McCandless David W.
Published: October 2006

Abstract: Bilateral ischemia has been shown to alter the net brain levels of energy metabolites such as ATP, phosphocre‐atine, glucose, and glycogen. The amino acid neurotrans‐mitter γ‐aminobutyric acid (GABA) exerts a tonic inhibitory influence on neural activity. The present studies were designed to evaluate the influence of elevated GABA levels on the metabolic sequelae of ischemia. The GABA transaminase inhibitor γ‐vinyl‐GABA (GVG; vigabatrin) was administered to Mongolian gerbils before the production of a bilateral ischemic incident. GABA levels were elevated in all regions assayed. Levels of energy metabolites were also increased, an indication of reduced energy utilization. In control animals, in the absence of GVG, 1 min of bilateral ischemia produced decreases in the levels of all metabolites. In animals pretreated with GVG, the effects of 1 min of bilateral ischemia were attenuated. These data suggest that the level of ongoing activity may affect the response to an ischemic insult. Furthermore, GVG may have a clinical indication in reducing the effect of minor ischemic incidents.
Chronic Lithium Treatment Impairs Phosphatidylinositol Hydrolysis in Membranes from Rat Brain Regions
Authors: Song Ling , Jope Richard S.
Published: October 2006

Abstract: Membranes prepared from rat brain regions were used to measure the receptor‐coupled and/or guanine nucleotide‐binding protein (G protein)‐mediated hydrolysis of exogenous [3H]phosphatidylinositol ([3H]PI). Guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and NaF (in the presence of AlCl3) caused concentration‐dependent stimulations of [3H]PI hydrolysis, supporting the conclusion that G proteins mediating [3H]PI hydrolysis can be activated in this preparation. Neither of these responses was altered by in vitro incubation with 8 mM LiCl, but both were reduced in hippocampal, striatal, and cortical membranes from rats that had been treated with lithium for 4 weeks compared with controls. Two cholinergic agonists, carbachol and pilocarpine, induced no hydrolysis of [3H]PI GTPγS was also present, in which case each equally stimulated [3H]PI hydrolysis above that obtained with GTPγS alone. In the presence of GTPγS several excitatory amino acid agonists stimulated [3H]PI hydrolysis to an extent similar to that of carbachol. After chronic lithium treatment, [3H]PI hydrolysis stimulated by carbachol was significantly attenuated, but the response to quisqualate was unaffected. Therefore, lithium added in vitro does not have an effect on cholinergic receptor‐ or G protein‐mediated [3H]PI hydrolysis, but each of these is reduced by chronic lithium treatment. Because exogenous [3H]PI was provided as the substrate, it is evident that the inhibitory effect of chronic lithium treatment cannot be due to substrate depletion. Impaired function of G proteins appears to be the most likely mechanism accounting for attenuated [3H]PI hydrolysis after chronic administration of lithium.