bluuebunny/crossref_metadata_2025_split huggingface dataset:
Schirmer tear test results in normal horses and ponies: effect of age, season, environment, sex, time of day and placement of strips
Authors: Beech J. , Zappala R. A. , Smith G. , Lindborg S. 
Published: August 2003

AbstractTear production was evaluated in 39 horses and 29 ponies using Schirmer tear test strips to determine whether diurnal or weekly fluctuations occur, whether location of strip placement has an effect, if values are the same for both eyes in an animal and whether sex, age, stabling vs. pasture and winter vs. summer had an effect. There was no test in which the raw score was less than 10 mm, although there were many occasions where tear wetting exceeded 35 mm. Analysis of the raw (continuous) scores by linear regression provided no evidence that signalment, housing or season or location of strip placement affected results. The distribution of tear test scores for a ‘population’ of eyes did not differ when the right eye was compared with the left eye or when the same eye was compared at different times on the same day. Individual test wetting values for opposing eyes measured at the same time, and also wetting values for the same eye measured at different times on the same day sometimes differed substantially. In winter maximum tear wetting exceeded 35 mm more frequently in the STT I than in the STT II even in housed horses and ponies, but there was no consistent significant difference. There appears to be wide variability in the STT I in normal horses and ponies.
Effects of low‐intensity pulsed ultrasound on wound healing in corneas of dogs following keratoplasty
Authors: Vicenti F. A. M. , Laus J. L. , Neto J. M. Costa , Talieri I. C. , Campos C. F. , Jorge A. T. , Ferreira A. L. , Fantinatti A. P. 
Published: August 2003

AbstractThe effects of low‐intensity pulsed ultrasound on wound healing were evaluated at the graft‐cornea transition in dogs following lamellar keratoplasty using tunica vaginalis preserved in 98% glycerin. Twenty‐one dogs were subdivided into three groups of seven animals. The first group (W/US) received daily treatment of low‐intensity pulsed ultrasound (20 mW/cm2) for 15 min for the first 10 days post surgery. The second group (N/US) was submitted to the same procedure but with the ultrasound apparatus turned off. The third group, the control (CO), underwent the surgical procedure only. The animals were clinically evaluated during the initial (1–15 days), intermediate (16–30 days) and late (31–120 days) postoperative period. The corneas were evaluated by light microscopy at 1, 3, 7, 15, 30, 60 and 120 days after surgery. Clinically, there were no differences which would promote an advantage to any of the treatments. Light microscopy, however, revealed more extensive vascularization and more advanced wound healing in the W/US group, as well as a tendency towards early graft incorporation. Based on the present results, low‐intensity pulsed ultrasound shows advantages, especially in situations where trophic support is a mandatory condition, facilitating better graft incorporation and rapid recovery of stromal organization.
Molecular mechanism of psychosine‐induced cell death in human oligodendrocyte cell line
Authors: Haq Ehtishamul , Giri Shailendra , Singh Inderjit , Singh Avtar K. 
Published: August 2003

AbstractThis study delineates the molecular mechanism underlying psychosine‐induced oligodendroglial cell death. An immortalized human oligodendroglial cell line, MO3.13, was treated with exogenous psychosine (β‐galactosylsphingosine), a toxic metabolite that accumulates in the tissues of patients with Krabbe's disease. The mode of cell death induced by psychosine was found to be apoptotic, as revealed by different apoptotic markers viz., TUNEL, DNA fragmentation and caspase cleavage/activation. The action of psychosine was redox sensitive, as measured by changes in mitochondrial membrane potential (ψΔ), and this effect of psychosine could be reversed by pre‐treatment with the antioxidant molecules N‐acetyl‐l‐cysteine or pro‐cysteine. Psychosine directly affects the mitochondria as revealed by the activation of caspase 9 but not caspase 8. Up‐regulation of the c‐jun/c‐jun N‐terminal kinase pathway by psychosine leads to the induction of AP‐1 and, at the same time, psychosine also down‐regulates the lipopolysaccharide‐induced NF‐κB transactivation. These observations indicate that the mechanism of action of psychosine is, through the up‐regulation of AP‐1, a pro‐apoptotic pathway as well as, through the down‐regulation of the NF‐κB pathway, an antiapoptotic pathway.
Identification of cis‐regulatory regions necessary for robust Nos2 promoter activity in glial cells: indirect role for NF‐κB
Authors: Sanchez Alma C. , Davis Randall L. , Syapin Peter J. 
Published: August 2003

AbstractPrevious reports suggest the nitric‐oxide synthase 2 (Nos2) promoter contains negative and positive cis‐regulatory regions. This study identified such regions using rat C6 glial cells. Activity of the serially deleted rat Nos2 promoter fused to a luciferase reporter gene was found to vary with construct size independent of stimuli, decreasing markedly from 160 to 130 bp then increasing significantly from 110 to 94 bp. In contrast, time to peak activity was stimulus‐dependent but size‐independent; 4–8 h for a cytokine mixture or lipopolysaccharide + interferon‐γ, and 8–16 h for lipopolysaccharide + phorbol 12‐myristate 13‐acetate. Peak activity with heterologous promoters also varied; 4 h for 3.7 kb of the human Nos2A promoter, and 36 h for 1.8 kb of the murine promoter. Electrophoretic mobility shift assays and in vivo DNA footprinting data confirmed nuclear protein binding to promoter regions suspected of containing important regulatory sites based on reporter gene data. A binding site for NF‐κB was not required for Nos2 promoter activity. These findings provide significant new information on the relative importance of different regions of the rat Nos2 promoter for transcriptional activation and nitric oxide production by glial cells and support the existence of cell‐ and species‐specific mechanisms for transcriptional regulation of Nos2 activation.
Assembly and cell surface expression of KA‐2 subunit‐containing kainate receptors
Authors: Gallyas Ferenc , Ball Simon M. , Molnar Elek 
Published: August 2003

AbstractKainate receptors (KARs) modulate synaptic transmission at both pre‐synaptic and post‐synaptic sites. The overlap in the distribution of KA‐2 and GluR6/7 subunits in several brain regions suggests the co‐assembly of these subunits in native KARs. The molecular mechanisms that control the assembly and surface expression of KARs are unknown. Unlike GluR5–7, the KA‐2 subunit is unable to form functional homomeric KAR channels. We expressed the KA‐2 subunit alone or in combination with other KAR subunits in HEK‐293 cells. The cell surface expression of the KAR subunit homo‐ and heteromers were analysed using biotinylation and agonist‐stimulated cobalt uptake. While GluR6 or GluR7 homomers were expressed on the cell surface, KA‐2 alone was retained within the endoplasmic reticulum. We found that the cell surface expression of KA‐2 was dramatically increased by co‐expression with either of the low‐affinity KAR subunits GluR5–7. However, co‐expression with other related ionotropic glutamate receptor subunits (GluR1 and NR1) does not facilitate the cell surface expression of KA‐2. The analysis of subcellular fractions of neocortex revealed that synaptic KARs have a relatively high KA‐2 content compared to microsomal ones. Thus, KA‐2 is likely to contain an endoplasmic reticulum retention signal that is shielded on assembly with other KAR subunits.
Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor‐induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study
Authors: Serra Pier Andrea , Rocchitta Gaia , Delogu Maria R. , Migheli Rossana , Taras Maria G. , Mura Maria P. , Esposito Giovanni , Miele Egidio , Desole Maria S. , Miele Maddalena 
Published: August 2003

Abstract In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor‐induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO‐donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 1.0 mm) induced a long‐lasting increase in DA and 3‐methoxytyramine (3‐MT) dialysate concentrations. SNAP‐induced increases were inhibited either by pre‐infusion of the soluble guanylate cyclase (sGC) inhibitor 1H‐[1,2,4] oxadiazolo[4,3]quinoxalin‐1‐one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re‐introduction restored SNAP effects. SNAP‐induced increases in DA + 3‐MT were unaffected by co‐infusion of the l‐type Ca2+ channel inhibitor nifedipine. The NO‐donor (+/–)‐(E)‐4‐ethyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐3‐hexenamide (NOR‐3, 1.0 mm) induced a short‐lasting decrease in dialysate DA + 3‐MT. Ascorbic acid (0.2 mm) co‐infusion allowed NOR‐3 to increase dialysate DA + 3‐MT. ODQ pre‐infusion inhibited NOR‐3 + ascorbic acid‐induced DA + 3‐MT increases. Infusion of high K+ (75 mm) induced a 2.5‐fold increase in dialysate DA + 3‐MT. The increase was abolished by NOR‐3 co‐infusion. Conversely, co‐infusion of ascorbic acid (0.2 mm) with NOR‐3 + high K+ restored high K+ effects. Co‐infusion of nifedipine inhibited high K+‐induced DA + 3‐MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+‐dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine‐insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.
Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegeneration
Authors: Butterfield D. Allan , Boyd‐Kimball Debra , Castegna Alessandra 
Published: August 2003

AbstractProteomics involves the identification of unknown proteins following their separation, often using two‐dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post‐translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge.
Lipoproteins produced by ApoE–/– astrocytes infected with adenovirus expressing human ApoE
Authors: Peng Dacheng , Song Ching , Reardon Catherine A. , Liao Shutsung , Getz Godfrey S. 
Published: August 2003

AbstractWe have developed an astrocyte cell culture system that is attractive for the study of apoE structure and its impact on astrocyte lipoproteins and neuronal function. Primary astrocytes from apoE–/– mice were infected with adenovirus expressing apoE3 or apoE4 and the nascent lipoproteins secreted were characterized. The nascent apoE‐containing astrocyte particles were predominantly the size of plasma high density lipoprotein (HDL). ApoE4, in contrast to apoE3, appeared to be distributed in two distinct lipoprotein peaks and the apoE4‐containing lipoproteins contained significantly more radiolabeled triglyceride. On electron micrographs the astrocyte particles were both discoidal and spherical in shape with a prevalence of stacked discs in apoE3 particles, but single discs and larger spheres in apoE4 particles. The apoE4 discs were significantly wider than apoE3 discs. These properties of the astrocyte lipoproteins are similar to those obtained from apoE isoform transgenic mice. Astrocyte lipoproteins containing apoE3, but not apoE4, stimulated neurite outgrowth in Neuro‐2a cells. These studies suggest that the isoform‐specific effects of apoE lipoproteins may involve differences in particle size and composition. Finally we demonstrate the usefulness of this system by expressing a truncated apoE3 (Δ202–299) mutant and show preliminary data indicating that a liver X receptor agonist promotes HDL output by the astrocytes without an increase in apoE in the media. This cell culture system is more flexible and allows for more rapid expression of apoE mutants.
Inhibition of protein kinase C reduces ischemia‐induced tyrosine phosphorylation of the N‐methyl‐d‐aspartate receptor
Authors: Cheung Herman H. , Teves Lucy , Wallace M. Christopher , Gurd James W. 
Published: August 2003

AbstractThe role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.
Estrogens inhibit l‐glutamate uptake activity of astrocytes via membrane estrogen receptor α
Authors: Sato Kaoru , Matsuki Norio , Ohno Yasuo , Nakazawa Ken 
Published: August 2003

AbstractWe investigated the effects of estrogen‐related compounds including xenoestrogens [17β‐estradiol (E2), 17α‐ethynylestradiol (EE), diethylstilbestrol (DES), p‐nonylphenol (PNP), bisphenol A (BPA) and 17α‐estradiol (17α)] on l‐glu uptake by cultured astrocytes via glutamate‐aspartate transporter (GLAST). After 24 h treatment, E2 inhibited the l‐glu uptake at 1 µm and higher concentrations. EE and DES also inhibited the l‐glu uptake at 1 nm and higher concentrations. The other four compounds had no effect. The effects of E2, EE and DES were completely blocked by 10 nm of ICI182 780 (ICI). β‐Estradiol 17‐hemisuccinate : bovine serum albumin (E2‐BSA), a membrane‐impermeable conjugate of E2, also elicited the inhibition of l‐glu uptake at 1 nm and higher concentrations, and the effect was blocked by ICI. 16α‐Iodo‐17β‐estradiol (16αIE2), an estrogen receptor α (ERα) selective ligand, revealed an inhibitory effect at 10 nm, while genistein, an ERβ selective ligand, failed to reveal such an effect at this concentration. Western blot analysis showed that the predominant ER of cultured astrocytes was ERα. The colocalization of ERα with GLAST on plasma membranes was immunohistochemically detected in these cells. From these results, we concluded that estrogens down‐regulate l‐glu uptake activity of astrocytes via membrane ERα.
Cooperative interaction between ascorbate and glutathione during mitochondrial impairment in mesencephalic cultures
Authors: Ehrhart Julie , Zeevalk Gail D. 
Published: August 2003

AbstractA decrease in total glutathione, and aberrant mitochondrial bioenergetics have been implicated in the pathogenesis of Parkinson's disease. Our previous work exemplified the importance of glutathione (GSH) in the protection of mesencephalic neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. Additionally, reactive oxygen species (ROS) generation was an early, contributing event in malonate toxicity. Protection by ascorbate was found to correlate with a stimulated increase in protein–glutathione mixed disulfide (Pr‐SSG) levels. The present study further examined ascorbate–glutathione interactions during mitochondrial impairment. Depletion of GSH in mesencephalic cells with buthionine sulfoximine potentiated both the malonate‐induced toxicity and generation of ROS as monitored by dichlorofluorescein diacetate (DCF) fluorescence. Ascorbate completely ameliorated the increase in DCF fluorescence and toxicity in normal and GSH‐depleted cultures, suggesting that protection by ascorbate was due in part to upstream removal of free radicals. Ascorbate stimulated Pr‐SSG formation during mitochondrial impairment in normal and GSH‐depleted cultures to a similar extent when expressed as a proportion of total GSH incorporated into mixed disulfides. Malonate increased the efflux of GSH and GSSG over time in cultures treated for 4, 6 or 8 h. The addition of ascorbate to malonate‐treated cells prevented the efflux of GSH, attenuated the efflux of GSSG and regulated the intracellular GSSG/GSH ratio. Maintenance of GSSG/GSH with ascorbate plus malonate was accompanied by a stimulation of Pr‐SSG formation. These findings indicate that ascorbate contributes to the maintenance of GSSG/GSH status during oxidative stress through scavenging of radical species, attenuation of GSH efflux and redistribution of GSSG to the formation of mixed disulfides. It is speculated that these events are linked by glutaredoxin, an enzyme shown to contain both dehydroascorbate reductase as well as glutathione thioltransferase activities.
Physiological regulation of Munc18/nSec1 phosphorylation on serine‐313
Authors: Craig Tim J. , Evans Gareth J. O. , Morgan Alan 
Published: August 2003

AbstractIncreased protein phosphorylation enhances exocytosis in most secretory cell types, including neurones. However, the molecular mechanisms by which this occurs and the specific protein targets remain unclear. Munc18‐1/nSec1 is essential for exocytosis in neurones, and is known to be phosphorylated by protein kinase C (PKC) in vitro at Ser‐313. This phosphorylation has been shown to decrease its affinity for syntaxin, and to alter the kinetics of exocytosis in chromaffin cells. However, there are no data on the physiological regulation of Ser‐313 phosphorylation. Using phospho‐Ser‐313‐specific antisera, we demonstrate here that Ser‐313 is phosphorylated in intact and permeabilized chromaffin cells in response to histamine and Ca2+ respectively. Furthermore, Ser‐313 is rapidly and transiently phosphorylated in intact synaptosomes in response to depolarization by KCl treatment or by 4‐aminopyridine, and by the metabotropic glutamate receptor agonist dihydroxyphenylglycine. PKC was identified as the kinase, and PP1 and PP2B as the phosphatases responsible for regulating Ser‐313 phosphorylation. As phosphorylation of nSec1 on Ser‐313 affects the rate of transmitter release in chromaffin cells, the demonstration here that this phosphorylation event occurs in neurones suggests that synaptic neurotransmitter release may be similarly regulated by nSec1 phosphorylation. Furthermore, such changes in release kinetics are associated with long‐term potentiation and depression, thus implicating nSec1 phosphorylation as a potential regulatory mechanism underlying presynaptic plasticity.
High‐resolution real‐time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices
Authors: Frenguelli B. G. , Llaudet E. , Dale N. 
Published: August 2003

AbstractWe have used improved miniaturized adenosine biosensors to measure adenosine release during hypoxia from within the CA1 region of rat hippocampal slices. These microelectrode biosensors record from the extracellular space in the vicinity of active synapses as they detect the synaptic field potentials evoked in area CA1 by stimulation of the afferent Schaffer collateral‐commissural fibre pathway. Our new measurements demonstrate the rapid production of adenosine during hypoxia that precedes and accompanies depression of excitatory transmission within area CA1. Simultaneous measurement of adenosine release and synaptic transmission gives an estimated IC50 for adenosine on transmission in the low micromolar range. However, on reoxygenation, synaptic transmission recovers in the face of elevated extracellular adenosine and despite a post‐hypoxic surge of adenosine release. This may indicate the occurrence of apparent adenosine A1 receptor desensitization during metabolic stress. In addition, adenosine release is unaffected by pharmacological blockade of glutamate receptors and shows depletion on repeated exposure to hypoxia. Our results thus suggest that adenosine release is not a consequence of excitotoxic glutamate release. The potential for adenosine A1 receptor desensitization during metabolic stress implies that its prevention may be beneficial in extending adenosine‐mediated neuroprotection in a variety of clinically relevant conditions.
Cloning and characterization of the 3′‐untranslated region of the human excitatory amino acid transporter 2 transcript
Authors: Kim Seon‐Young , Chao Wei , Choi So‐Young , Volsky David J. 
Published: August 2003

AbstractThe 3′‐untranslated region (UTR) of the human excitatory amino acid transporter 2 (EAAT2) transcript was cloned and characterized. The full‐length EAAT2 cDNA of 11 692 bp was found to contain 283 bp of 5′ UTR, a 1725‐bp open reading frame and an unusually long 3′‐UTR of 9684 bp. The 3′‐UTR of EAAT2 cDNA was well conserved among mammals, and human, macaque, rat and mouse cDNA had nearly identical 3′ ends. The human EAAT2 transcripts were detected in brain, spinal cord, liver, adrenal gland, placenta and pancreas by northern hybridization, and many ESTs homologous to the human EAAT2 cDNA were found in numerous tissues. To investigate the role of human EAAT2 3′‐UTR in gene expression, we constructed luciferase expression vectors containing 3′‐UTR fragments spanning the entire length of the region. The individual fragments varied in their effects on reporter gene expression in human astrocytes by a factor of eight to ten suggesting a complex role of the 3′‐UTR in post‐transcriptional regulation of EAAT2 gene expression.