bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of bovine lactoferrin on in vitro replication of feline herpesvirus
Authors: Beaumont Stephanie L. , Maggs David J. , Clarke Heather E.
Published: August 2003

Objective  To investigate the effects of bovine lactoferrin on in vitro replication of feline herpes virus (FHV‐1) and to determine at what points during viral replication these effects occur.

Sample population  Cultured Crandell‐Reese feline kidney (CRFK) cells and FHV‐1 strain 727.

Procedure  Five concentrations of bovine lactoferrin (0.5, 1, 2, 5, and 10 mg/mL) were added at one or more of three time points during conventional plaque reduction assays: (a) uninfected CRFK cells were incubated in lactoferrin‐containing medium for 30 min prior to viral adsorption; (b) virus was suspended in lactoferrin‐containing medium prior to and during adsorption, or (c) CRFK cells were incubated with lactoferrin‐containing medium for 48 h following viral adsorption. Plaques were counted and antiviral effect expressed as percent inhibition relative to control medium that contained no lactoferrin.

Results  Exposure of CRFK cells to lactoferrin prior to or during viral adsorption inhibited FHV‐1 replication by 87–96% (mean: 91%). Application of lactoferrin following viral adsorption had no appreciable effect on FHV‐1 replication. No additive or synergistic effects were noted when lactoferrin was added at multiple steps. These effects were similar at all concentrations of lactoferrin tested. Cytotoxic effects of lactoferrin on CRFK cells were not observed at any concentration tested.

Conclusions and clinical relevance  Bovine lactoferrin has a notable inhibitory effect on the in vitro replication of FHV‐1 prior to and during, but not following viral adsorption. These findings strongly suggest that lactoferrin inhibits FHV‐1 adsorption to the cell surface and/or penetration of the virus into the cell. Clinical effects of topical lactoferrin in acute or recrudescent herpetic episodes in cats warrant investigation.
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

Tear 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

The 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

This 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

Previous 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

Kainate 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

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

Proteomics 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

We 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

The 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

We 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

A 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

Increased 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

We 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

The 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.
Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system
Authors: Xiao Jingnan , Liu Yuechueng
Published: August 2003

The rat pheochromocytoma PC12 cell line has been an invaluable model system for studying neuritogenesis. Nerve growth factor (NGF) elicits multiple aspects of neurite outgrowth in PC12 cells. It is therefore difficult to dissect and assign an individual signaling pathway to each stage of neuritogenesis. We have recently reported the isolation of a variant PC12 cell line, PC12‐N1 (N1), which spontaneously extends neuritic processes and exhibits an increased sensitivity to NGF. Here, we show that, under different culture conditions, the cells display three distinct phases of neuritogenesis consisting of neurite initiation, rapid neurite elongation, and a maturation process characterized by the thickening of neurites and increase in cell soma sizes. We demonstrate that signaling through ERK, but not p38 or JNK, is required for the spontaneous neurite initiation and extension. Treatment with low concentrations of NGF induces rapid neurite elongation without affecting neurite branching and cell soma sizes. Such a rapid neurite outgrowth can be blocked by the inhibition of ERK, but not JNK, activities. In the presence of higher concentrations of NGF, the N1 cells undergo further differentiation with many characteristics of mature neurons in culture, e.g. larger cell soma and numerous branches/connections. This process can be completely blocked by inhibiting ERK or JNK activities using specific inhibitors. These results suggest that ERK and JNK signals play different roles in neuritogenesis, and that JNK activity is essential in the late stages of neuritogenesis. Furthermore, our results demonstrate that signaling dosage is important in the activation of a specific pathway, leading to distinctive biological outcomes.
Participation of NMDA‐mediated phosphorylation and oxidation of neurogranin in the regulation of Ca2+‐ and Ca2+/calmodulin‐dependent neuronal signaling in the hippocampus
Authors: Wu Junfang , Huang Kuo‐Ping , Huang Freesia L.
Published: August 2003

Neurogranin/RC3 (Ng) is a postsynaptic protein kinase C (PKC) substrate and calmodulin (CaM)‐binding protein whose CaM‐binding affinity is modulated by Ca2+, phosphorylation and oxidation. Ng has been implicated in the modulation of postsynaptic signal transduction pathways and synaptic plasticity. Previously, we showed a severe deficit of spatial memory in Ng knockout (KO) mice. Activation of the NMDA receptor and its downstream signaling molecules are known to be involved in long‐term memory formation. In the present study, using mouse hippocampal slices, we demonstrated that NMDA induced a rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of PKC as evidenced by their phosphorylations, whereas, such activations were greatly reduced in the KO mice. A higher degree of phosphorylation of Ca2+/CaM‐dependent kinase II and activation of cyclic AMP‐dependent protein kinase were also evident in the WT compared to those of the KO mice. Phosphorylation of downstream targets, including mitogen‐activated protein kinases and cAMP response element‐binding protein, were significantly attenuated in the KO mice. These results suggest that by its Ca2+‐sensitive CaM‐binding feature, and through its phosphorylation and oxidation, Ng regulates the Ca2+‐ and Ca2+/CaM‐dependent signaling pathways subsequent to the stimulation of NMDA receptor. These findings support the hypothesis that the derangement of hippocampal signal transduction cascades in Ng KO mice causes the deficits in synaptic plasticity, learning and memory that occur in these mice.
Noradrenaline enhances monocarboxylate transporter 2 expression in cultured mouse cortical neurons via a translational regulation
Authors: Pierre Karin , Debernardi Ruth , Magistretti Pierre J. , Pellerin Luc
Published: August 2003

Regulation of the expression of MCT1 and MCT2, two isoforms of the monocarboxylate transporter (MCT) family, was investigated in primary cultures of mouse cortical neurons. Under basal conditions, both MCT immunoreactivities (IR) were found in the cell soma and dendrites, although IR for MCT1 appeared less bright than for MCT2. Treatment of cultured cortical neurons with 100 μm noradrenaline (NA) led, after a few hours, to a striking enhancement in fluorescence intensity associated with MCT2 IR in the cell soma as well as in dendrites. In contrast, MCT1 IR was not altered by NA treatment. Western blot experiments performed on cultured neurons treated with NA confirmed that MCT2 protein expression was increased. Forskolin and dBcAMP also enhanced MCT2 expression, suggesting the implication of a cAMP‐mediated pathway in the effect of NA. Surprisingly, neither NA, dBcAMP nor forskolin affected MCT2 mRNA expression. Application of cycloheximide, a protein synthesis inhibitor, prevented the enhancement of MCT2 IR, while the mRNA synthesis inhibitor actinomycin D also blocked the effect of NA on MCT2 IR levels. These results suggest that regulation of MCT2 expression in neurons by NA occurs at the translational level despite the requirement for an as yet unknown transcriptional step.
Depolarization‐induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells
Authors: Mendoza Isabel E. , Schmachtenberg Oliver , Tonk Ernesto , Fuentealba Jorge , Díaz‐Raya Pamela , Lagos Verónica L. , García Antonio G. , Cárdenas Ana M.
Published: August 2003

The contribution of Ca2+ entry through different voltage‐activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal‐medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen‐activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization‐induced ERK phosphorylation completely depended on protein kinase C‐α (PKC‐α), but not on Ca2+/calmodulin‐dependent protein kinase nor cyclic AMP‐dependent protein kinase. Blockade of L‐type Ca2+ channels by 3 μm furnidipine, or blockade of N channels by 1 μmω‐conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 μmω‐agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty‐eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.
Activation of p38 mitogen‐activated protein kinase in spinal microglia is a critical link in inflammation‐induced spinal pain processing
Authors: Svensson Camilla I. , Marsala Martin , Westerlund Anna , Calcutt Nigel A. , Campana Wendy M. , Freshwater Jason D. , Catalano Rosanne , Feng Ying , Protter Andrew A. , Scott Brian , Yaksh Tony L.
Published: August 2003

We examined the effect of p38 mitogen‐activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immuncytochemisty revealed that phosphorylated p38 MAPK‐immunoreactive cells were predominantly present in laminae I–IV of the dorsal horn. Double‐staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co‐localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD‐282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo‐oxygenase‐2 and inflammation‐induced appearance of Fos‐positive neurons, was blocked by pretreatment, but not post‐treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation‐induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.
Increased susceptibility to intermittent hypoxia in aging rats: changes in proteasomal activity, neuronal apoptosis and spatial function
Authors: Gozal David , Row Barry W. , Kheirandish Leila , Liu Rugao , Guo Shang Z. , Qiang Fan , Brittian Kenneth R.
Published: August 2003

Obstructive sleep apnea (OSA) is a frequent medical condition characterized by intermittent hypoxia (IH) during sleep, and is associated with neurodegenerative changes in several brain regions along with learning deficits. We hypothesized that aging rats exposed to IH during sleep would be particularly susceptible. Young (3–4 months) and aging (20–22 months) Sprague–Dawley rats were therefore exposed to either room air or IH for 14 days. Learning and memory was assessed with a standard place‐training version of the Morris water maze. Aging rats exposed to room air (RA) or IH displayed significant spatial learning impairments compared with similarly exposed young rats; furthermore, the decrements in performance between RA and IH were markedly greater in aging compared with young rats (p < 0.01), and coincided with the magnitude of IH‐induced decreases in cyclic AMP response element binding (CREB) phosphorylation. Furthermore, decreases in proteasomal activity occurred in both young and aging rats exposed to IH, but were substantially greater in the latter (p < 0.001). Neuronal apoptosis, as shown by cleaved caspase 3 expression, was particularly increased in aging rats exposed to IH (p < 0.01 versus young rats exposed to IH). Collectively, these findings indicate unique vulnerability of the aging rodent brain to IH, which is reflected at least in part, by the more prominent decreases in CREB phosphorylation and a marked inability of the ubiquitin‐proteasomal pathway to adequately clear degraded proteins.
Nerve growth factor induces anti‐apoptotic heme oxygenase‐1 in rat pheochromocytoma PC12 cells
Authors: Liu Heling , Nowak Rebecca , Chao Wei , Bloch Kenneth D.
Published: August 2003

Nerve growth factor (NGF) and heme oxygenases (HOs) both exert neuroprotective effects. To characterize the role of HOs in the prevention of apoptosis by NGF, we investigated the effect of NGF on the expression of HOs in serum‐deprived PC12 cells. Serum deprivation (SD) led to a rapid decrease in HO‐1 gene expression followed by induction of apoptosis. Incubation of serum‐deprived PC12 cells with NGF prevented apoptosis and increased HO‐1 mRNA and protein levels, as well as HO enzyme activity. HO‐2 gene expression was unaffected by SD or NGF. Incubation of cells with mitogen‐activated protein kinase kinase (MEK) inhibitors (PD98059 or U0126) attenuated the ability of NGF to increase HO‐1 expression and to protect PC12 cells against SD‐induced apoptosis. NGF augmented HO‐1 gene transcription but did not alter HO‐1 mRNA stability. HO inhibitors or antisense HO‐1 RNA decreased the ability of NGF to prevent cell apoptosis. Inhibition of HO activity enhanced intracellular reactive oxygen species (ROS) production and attenuated NGF‐induced reduction of ROS in serum‐deprived PC12 cells. These results demonstrate that NGF enhances HO‐1 gene transcription via MEK activation and that the induction of HO‐1 plays an important role in the antioxidative and antiapoptotic effects of NGF in serum‐deprived PC12 cells.
Evidence for an active transport of morphine‐6‐β‐d‐glucuronide but not P‐glycoprotein‐mediated at the blood–brain barrier
Authors: Bourasset Fanchon , Cisternino Salvatore , Temsamani Jamal , Scherrmann Jean‐Michel
Published: August 2003

Morphine‐6‐β‐d‐glucuronide (M6G) is an active metabolite of morphine with high analgesic potency despite a low blood–brain barrier (BBB) permeability. The aim of the study was to elucidate its transport mechanism across the BBB. We first checked if M6G was effluxed by the P‐glycoprotein (P‐gp), as previously reported by others. Second, we investigated the role of anionic transporters like the multidrug resistance‐associated protein mrp1 and the glucose transporter GLUT‐1. The brain uptake of [14C]M6G was measured by the in situ brain perfusion technique in wild‐type and deficient mice [mdr1a(–/–) and mrp1(–/–)], with and without probenecid, digoxin, PSC833 or d‐glucose. No difference was found between P‐gp and mrp1 competent and deficient mice. The brain uptake of [14C]M6G co‐perfused with probenecid in wild‐type mice was not significantly different from that found in group perfused with [14C]M6G alone. The co‐perfusion of [14C]M6G with digoxin or PSC833 was responsible of a threefold decrease of its uptake in mdr1a competent and deficient mice, suggesting that another transporter than P‐gp and sensitive to digoxin and PSC833, may be involved. The co‐perfusion of [14C]M6G with d‐glucose revealed a threefold decrease in M6G uptake. In conclusion, P‐gp and mrp1 are not involved in the transport of M6G at the BBB level in contrast to GLUT‐1 and a digoxin‐sensitive transporter (probably oatp2), which can actively transport M6G but with a weak capacity.
Beta diversity of geometrid moths (Lepidoptera: Geometridae) in an Andean montane rainforest
Authors: Brehm Gunnar , Homeier Jürgen , Fiedler Konrad
Published: August 2003

Abstract. Turnover in species composition of the extremely species‐rich family Geometridae (Lepidoptera) was investigated along an elevational gradient ranging from 1040 m to 2677 m above sea level. Moths were sampled using weak light traps (30 W) in three field periods in 1999 and 2000 in an Andean montane rainforest in the province of Zamora‐Chinchipe in southern Ecuador. A total of 13 938 specimens representing 1010 species were analysed. Similarities of ensembles of all geometrid moths and of the subfamilies Ennominae and Larentiinae were calculated using the NESS index (with mmax). Ordinations performed using nonmetric multidimensional scaling (NMDS) and correspondence analysis depicted a gradual change of the ensembles along the altitudinal gradient. Extracted ordination scores significantly correlate with altitude (−0.97 ≤ r ≤ −0.95, P < 0.001) and with ambient air temperature (0.93 ≤ r ≤ 0.97, P < 0.001). Temperature is therefore assumed to be the most important abiotic determinant responsible for the species turnover among the moths. Matrix correlation tests were performed in order to compare faunal matrices with matrices derived from available environmental factors. Both tree diversity and vegetation structure significantly correlate with faunal data, but tree diversity explains considerably more of the data variability (range: Mantel r = 0.81–0.83, P < 0.001) than vegetation structure (range: Mantel r = 0.35, P < 0.005 to r = 0.43, P < 0.001). Tree diversity also changes gradually and scores of the first NMDS dimension are highly significantly correlated with altitude (r = 0.98, P < 0.001). A common underlying factor such as ambient temperature might also be responsible for such vegetation changes. Additionally, simulated model data was developed that assumed a constant turnover of moth species and equal elevational ranges of all species involved. Despite the simplicity of the models, they fit empirical data very well (Mantel r > 0.80 and P < 0.001 in all models).
Endozoochory by ducks: influence of developmental stage of Bythotrephes diapause eggs on dispersal probability
Authors: Charalambidou Iris , Ketelaars Henk A. M. , Santamaría Luis
Published: August 2003

Abstract. This paper evaluates whether the recent expansion of Bythotrephes longimanus Leydig (Crustacea: Onychopoda) may be a result of endozoochorous avian dispersal. In three experiments, we investigated the survival of diapause eggs in newly produced and end‐of‐winter developmental stages after their passage through the digestive tract of four species of ducks. Resistance of eggs to digestion was low for both developmental stages (< 12%). Most eggs (67–95%) were recovered by four hours after ingestion, with a longer maximum retention time for newly produced (22 h) than end‐of‐winter eggs (4–6 h). Ingestion had a detrimental effect on viability: only eggs retrieved one hour after ingestion hatched and the proportion of those ingested that produced viable neonates was low (0.5% for newly produced and 0.3–3% for end‐of‐winter eggs). We conclude that the probability of endozoochorous dispersal by individual ducks is low but becomes significant when considering the thousands of ducks moving among wetlands. Endozoochorous dispersal of B. longimanus would most likely involve the transport of newly produced diapause eggs from north to south, during the autumn migration of ducks. Based on flight speed estimates of ducks we estimate dispersal probability to drop sharply at distances over 60–80 km.
Regional differences in land use affect population performance of the threatened insectivorous plant Drosophyllum lusitanicum (Droseraceae)
Authors: Garrido B. , Hampe A. , Marañón T. , Arroyo J.
Published: August 2003

Abstract. We analyse populations of the insectivorous plant Drosophyllum lusitanicum (L.) Link (Droseraceae) and their habitats throughout the species’ distribution range (Portugal, Spain and Morocco), and examine their relations. We have surveyed 32 populations and we analyse their demographic structures, accompanying plant communities (by twinspan analyses), and relationships between population features and several edaphic and nonedaphic environmental variables (by multiple regression analyses). Plant communities, their floristic richness and degree of endemism change across the geographical range of Drosophyllum. Populations vary strongly in size, density and age structure. Their recruitment seems largely determined by competition with the surrounding vegetation, while only weak relationships were detected between population performance and other environmental factors. The demographic structure of populations exhibits a marked geographical differentiation, with populations becoming older and sparser towards the north‐western part of the range. The largest and presumably most stable populations are mostly found in southern Spanish heathlands, which are nowadays scarcely affected by man. Moroccan populations suffer from browsing by cattle and experience an accelerated generation turnover, while most Portuguese populations occur in afforested areas and experience strongly reduced recruitment. The historical distribution range of Drosophyllum within its peculiar habitat has probably been relatively stable, but recent regional differences in human land use have resulted in present‐day differences in population performance, as well as in types and degrees of threat across the three countries involved. Conservation strategies should take into account this range‐wide variation and combine approaches on different spatial scales.
Inselbergs in a changing world — global trends
Authors: Burke Antje
Published: August 2003

Abstract. Based on a review of recent literature, this paper puts forward hypotheses for global trends of inselbergs (isolated mountains) with regard to: (a) their bioclimatic position in relation to the surroundings; (b) their potential for providing habitat niches; and (c) human impacts that may influence ecological processes. This review takes a landscape‐level perspective and highlights the challenges ahead in view of changing environmental conditions. Recognizing that inselbergs per se are composed of different microhabitats relative to their surroundings, inselbergs are hypothesized to be bioclimatic ‘islands’ of xeric conditions in a humid matrix in tropical and temperate regions, and ‘islands’ of mesic conditions in arid regions. The bioclimatic status of intermediate positions along this global axis (e.g. semiarid and subtropical savanna regions) is less clear. Here, other environmental variables may be of greater importance (microhabitat composition, size of inselberg, distance to other mountain habitats and biogeographical influences). Whether or not biotic communities match these hypothesized physical and bioclimatic trends warrants investigation and could contribute to explaining global species diversity patterns. Invasion of alien species in tropical and Mediterranean‐climate regions, and altered fire regimes and resource use pose threats to inselberg communities. Their role as sources of native species to recolonize disturbed surroundings is important in degraded semiarid and arid regions. A generalized model is proposed hypothesizing possible processes between inselberg habitats and disturbed areas in their surroundings in different climatic regions. This model may help to direct further research towards substantiating these perceived trends.
Habitat partitioning in European and North American pond‐breeding frogs and toads
Authors: Buskirk Josh Van
Published: August 2003

Abstract. Distributions of species along a freshwater habitat gradient, ranging from ephemeral pools with few predators to permanent lakes with fish, have been used to infer how predation establishes trade‐offs that promote ecological specialization. Larval anurans are said to support the trade‐off model, but there are few comparable and quantitative habitat data available to assess this claim. I performed a survey of field biologists to evaluate the habitats of similar sets of species in the northern parts of Europe (40 respondents and 12 species) and eastern North America (30 respondents and 8 species), using a standard set of criteria. For six European species I also had quantitative field sampling data, and found close agreement between survey results and predator densities experienced by tadpoles in the field. Distributions of most species were restricted to only part of the habitat gradient, as expected under the trade‐off model. The survey confirmed reports that North Ameri‐can Rana species replace one another along the gradient, but this was not true in Europe. European Rana were no different from the North American species in their seasonal and geographical overlap, so the absence of habitat partitioning in European Rana may result from interactions with other species or the special impact of glaciation in Europe. Habitats were unrelated to evolutionary relationships among species, suggesting that changes in habitat evolve quickly. The survey approach was useful for comparing distributions of species, and for generating hypotheses about evolutionary responses to habitat gradients.