bluuebunny/crossref_metadata_2025 huggingface dataset:
(1R,3S)‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (RS‐ACPD) reduces intracellular glutamate levels in astrocytes
Authors: Ye Zu‐Cheng , Ransom Bruce R. , Sontheimer Harald
Published: March 2003

(+/–)‐1‐Aminocyclopentane‐trans‐1,3‐dicarboxylic acid (t‐ACPD) is an equimolar mixture of two enantiomers: (1S,3R)‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (SR‐ACPD) and 1R,3S‐1‐Aminocyclopentane‐1,3‐dicarboxylic acid (RS‐ACPD). t‐ACPD and SR‐ACPD have been commonly used as agonists for metabotropic glutamate receptors (mGluR). Here we demonstrated that RS‐ACPD, but not SR‐ACPD, is transported into astrocytes with a Km of 6.51 ± 2.38 mm and Vmax of 22.8 ± 3.4 nmol/mg/min. This low‐affinity transport is Na+‐dependent and is competitively blocked by glutamate or other substrates for the glutamate transporter. RS‐ACPD therefore is probably taken up by the glutamate transporter. Prolonged incubation with high levels of RS‐ACPD (> 500 µm) induced significant swelling of astrocytes. At lower concentrations (100 µm), RS‐ACPD reduced intracellular glutamate content ([Glu]i) by > 50% without obvious morphological changes. The reduction in [Glu]i was accompanied by an increase in [glutamine]i. The RS‐ACPD's effect on [Glu]i required glutamine and high levels of phosphate, suggesting that RS‐ACPD inhibited phosphate‐activated glutaminase (PAG). These data suggest that astrocytic PAG is actively involved in determining the equilibrium between intracellular glutamate and glutamine. By reducing [Glu]i, RS‐ACPD reduces the amount of glutamate available for release.
Reduction of glial glutamate transporters in the parietal cortex and hippocampus of the EL mouse
Authors: Ingram Esther M. , Wiseman John W. , Tessler Shoshi , Emson Piers C.
Published: March 2003

There is extensive experimental evidence indicating a crucial role for glutamate in epileptogenesis and epileptic activity. The glial glutamate transporters GLT1 and GLAST are proposed to account for the majority of extracellular glutamate re‐uptake. In the present study, polyclonal antibodies specific to GLT1 and GLAST were generated and characterized, revealing distribution patterns for the two transporters confirming those previously reported. In situ hybridization and immunoblotting were then used to compare levels of these two transporters in the parietal cortex and hippocampus of unstimulated and stimulated EL mice with DDY control mice. Additionally, HPLC determined tissue glutamate concentrations in the same regions of these animals. These experiments revealed reductions in GLT1 mRNA and protein in the parietal cortex of unstimulated and stimulated EL mice compared with DDY controls, accompanied by an increase in tissue glutamate concentration in the stimulated EL mice group. GLT1 mRNA was also reduced in the CA3 hippocampal subfield of both unstimulated and stimulated EL mice. GLAST protein was reduced in the hippocampus of the stimulated EL mice group, while no changes in GLAST mRNA or protein were detected in the parietal cortex of EL mice when compared with DDY controls. The glial glutamate transporter down‐regulation reported here may play a role in seizure initiation, spread and maintenance in the EL mouse.
Generation of a constitutively active fragment of PKN in microglia/macrophages after middle cerebral artery occlusion in rats
Authors: Ueyama Takehiko , Ren Yi , Sakai Norio , Takahashi Mikiko , Ono Yoshitaka , Kondoh Takeshi , Tamaki Norihiko , Saito Naoaki
Published: March 2003

PKN is a fatty acid‐ and Rho‐activated serine/threonine kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC). Recent studies have demonstrated that PKN is proteolytically cleaved after apoptotic stimulation and then a constitutively active 55‐kDa fragment is generated. However, the role of the 55‐kDa fragment are poorly understood. Adult Sprague–Dawley (SD) rats underwent middle cerebral artery occlusion (MCAO), and the temporal and spatial changes in the fragmentation of PKN and of PKC δ were examined by immunoblotting. No proteolytic fragment of PKC δ (about 40 kDa) was detected. The 55‐kDa fragment of PKN appeared transiently from 3 days after MCAO at the ipsilateral normal cortex. At the boundary zone of infarction, the 55‐kDa fragment was markedly induced from day 5 then peaked on day 21 and persisted until day 28. Analysis of anti‐phosphoserine immunoprecipitates with an anti‐PKN antibody revealed phosphorylation of the 55‐kDa band. Double staining for PKN and Ox42 was used to examine the source of the 55‐kDa fragment. PKN immunoreactivity was significantly increased in Ox42‐positive cells (microglia/hematogenous macrophages). No DNA laddering and only a few terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL)‐positive cells were observed on day 14 in despite of the high level appearance of the 55‐kDa band. These results suggest that the constitutively active 55‐kDa fragment of PKN does not contribute to apoptosis, but may contribute to a function of microglia/macrophages.
Selective localization of G protein γ5 subunit in the subventricular zone of the lateral ventricle and rostral migratory stream of the adult rat brain
Authors: Asano Tomiko , Shinohara Haruo , Morishita Rika , Ueda Hiroshi , Kawamura Noriko , Katoh‐Semba Ritsuko , Kishikawa Masao , Kato Kanefusa
Published: March 2003

G proteins play important roles in transmembrane signal transduction, and various isoforms of each subunit, α, β and γ, are highly expressed in the brain. The Gγ5 subunit is a minor isoform in the adult brain, but we have previously shown it to be highly expressed in the proliferative region of the ventricular zone in the rat embryonic brain. We show here that Gγ5 is also selectively localized in a proliferative region in the adult rat brain, including the subventricular zone of the lateral ventricle and rostral migratory stream. The Gαi2 subunit colocalized with Gγ5 in these regions, the two subunits being present in neuronal precursors and ependymal cells but not in proliferating astrocytes. In addition, intense staining of Gγ5 was seen in axons of the olfactory neurons, which are known to regenerate. These results suggest specific roles for Gγ5 in precursor cells during neurogenesis so that this isoform might be a useful biological marker.
Insulin‐like growth factor‐1 effects on bovine retinal endothelial cell glucose transport: role of MAP kinase
Authors: DeBosch Brian J. , Deo Baljit K. , Kumagai Arno K.
Published: March 2003

In order to maintain normal metabolism, the neuroretina is completely dependent on the constant delivery of glucose across the retinal microvascular endothelial cells comprising the inner blood–retinal barrier. Glucose uptake into these cells is influenced by various stimuli, including hypoxia and growth factors. Recently, insulin‐like growth factor‐1 (IGF‐1) was shown to enhance retinal endothelial glucose transport in a process that is dependent on protein kinase C (PKC) and phosphatidylinositol‐3 kinase (PI3 kinase). In the current study, the role of mitogen‐activated protein kinase (MAP kinase) in regulating IGF‐1 effects on retinal endothelial cell glucose transport was investigated in a bovine retinal endothelial cell (BREC) culture model. IGF‐1 (25 ng/mL) caused a rapid increase in MAP‐kinase activity and ERK phosphorylation. Inhibition of MAP kinase with PD98059 (100 µm) blocked IGF‐1 enhancement of 2‐deoxyglucose uptake. In order to clarify the relationship between PKC, PI3 kinase and MAP kinase in IGF‐1 signaling in retinal endothelial cells, the effects of selective inhibitors of MAP kinase (PD98059), PKC (GF109203X), and PI3 kinase (wortmannin, LY294002) on signal transduction by IGF‐1 were studied. Inhibition of MAP kinase abolished IGF‐1 stimulation of PKC but had no effect on PI3 kinase activity, whereas inhibition of either PKC and PI3 kinase had no effect on MAP kinase phosphorylation or activity in IGF‐1‐treated cells. Taken together, these data demonstrate that IGF‐1 stimulation of BREC glucose transport requires activation of MAP kinase and that MAP kinase is upstream from PKC but is independent of PI3 kinase in mediating the actions of IGF‐1 on retinal endothelial cells.
Spermine modulation of the glutamatenmda receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex
Authors: Ragnarsson Lotten , Mortensen Martin , Dodd Peter R. , Lewis Richard J.
Published: March 2003

The pharmacology of the N‐methyl‐d‐aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [3H]MK‐801 binding site were delineated along with the enhancement of [3H]MK‐801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate‐mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine‐mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin‐G (con‐G) peptides showed that Ala(7)–con‐G had higher affinity than Lys(7)–con‐G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)–con‐G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)–con‐G. Over‐excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.
Activation of Ca2+/calmodulin‐dependent protein kinase I in cultured rat hippocampal neurons
Authors: Uezu Akiyoshi , Fukunaga Kohji , Kasahara Jiro , Miyamoto Eishichi
Published: March 2003

We have focused on activation mechanisms of calcium/calmodulin‐dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization‐induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P‐labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate‐ and depolarization‐induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated bythe NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.
Lack of up‐regulation of ferritin is associated with sustained iron regulatory protein‐1 binding activity in the substantia nigra of patients with Parkinson's disease
Authors: Faucheux Baptiste A. , Martin Marie‐Elise , Beaumont Carole , Hunot Stéphane , Hauw Jean‐Jacques , Agid Yves , Hirsch Etienne C.
Published: March 2003

Dopaminergic neurones degenerate during Parkinson's disease and cell loss is most extensive in the subpopulation of melanized neurones located in the substantia nigra pars compacta. Iron accumulation, together with a lack of up‐regulation of the iron‐storing protein, ferritin, has been reported and may contribute to increased oxidative stress in this region. We investigated the binding activity of iron regulatory protein‐1 (IRP1) to the iron‐responsive element that precludes ferritin mRNA translation, in the substantia nigra of a group of parkinsonian patients who presented a statistically significant reduction in the number of nigral melanized‐neurones and an increased iron content, together with unchanged H‐ferritin and L‐ferritin subunit levels as compared to matched controls. The levels of ferritin mRNAs and the binding activity of IRP1 to the iron‐responsive element of ferritin mRNA did not differ significantly between the two groups. Moreover, there was no detectable contribution of the iron regulatory protein‐2 (IRP2) binding activity. No change in IRP1 control of ferritin mRNA translation explains the lack of up‐regulation of ferritin expression in cytoplasmic extracts of SNpc that would be normally expected with cytosolic iron accumulation. The data of this study do not favor changes in transcription and post‐transcriptional regulation of ferritin expression in Parkinson's disease and suggest a ‘compartmentalized’ iron accumulation.
Presence of functionally active protease‐activated receptors 1 and 2 in myenteric glia
Authors: Garrido Rosario , Segura Bradley , Zhang Weizhen , Mulholland Michael
Published: March 2003

Protease‐activated receptors (PARs) belong to the family of membrane receptors coupled to G‐proteins; their presence is reported in a wide variety of cells. The object of this study was to demonstrate the presence of PAR‐1 and PAR‐2 in myenteric glia of the guinea pig, and to elucidate the cellular mechanisms that are triggered upon receptor activation. Thrombin and PAR‐1 agonist peptide (PARP‐1) activate PAR‐1 with a maximum mean ± SEM change in intracellular calcium concentration with respect to basal level (Δ[Ca2+]i) of 183 ± 18 nm and 169 ± 6 nm, respectively. Trypsin and PAR‐2 agonist peptide (PARP‐2) activate PAR‐2 with a maximum Δ[Ca2+]i of 364 ± 28 nm and 239 ± 19 nm, respectively. Inhibition of phospholipase C by U73312 (1 µm) decreased the Δ[Ca2+]i due to PAR‐1 activation from 167 ± 10 nm to 87 ± 6 nm. The PAR‐2‐mediated Δ[Ca2+]i decreased from 193 ± 10 nm to 124 ± 8 nm when phospholipase C activity was inhibited. Blockade of sphingosine kinase with dimethylsphingosine (1 µm) decreased the Δ[Ca2+]i due to PAR‐2 activation from 149 ± 19 nm to 67 ± 1 nm, but did not influence the PAR‐1‐mediated Δ[Ca2+]i. PAR‐1 and PAR‐2 were localized in myenteric glia by immunolabeling. Our results indicate that PAR‐1 and PAR‐2 are present in myenteric glia of the guinea pig, and their activation leads to increases in intracellular calcium via different signal transduction mechanisms that involve activation of phospholipase C and sphingosine kinase.
Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain
Authors: Li Shenyang , Moore Christopher L. , Dobretsova Anna , Wight Patricia A.
Published: March 2003

The myelin proteolipid protein (Plp) gene encodes the most abundant protein found in mature CNS myelin. Expression of the gene is regulated spatiotemporally, with maximal expression occurring in oligodendrocytes during the myelination period of CNS development. Plp gene expression is tightly controlled. Misregulation of the gene in humans can result in the dysmyelinating disorder Pelizaeus‐Merzbacher disease, and in transgenic mice carrying a null mutation or extra copies of the gene can result in a variety of conditions, from late onset demyelination and axonopathy, to severe early onset dysmyelination. In this study we have examined the effects of Plp intron 1 DNA in mediating proper developmental expression of Plp‐lacZ fusion genes in transgenic mice. Our results reveal the importance of Plp intron 1 sequences in instigating the expected surge in Plp‐lacZ gene activity during (and following) the active myelination period of brain development. Transgene expression was also detected in the testis (Leydig cells), however, the presence or absence of Plp intron 1 sequences had no effect on the temporal profile in the testis. Surprisingly, expression of the transgene missing Plp intron 1 DNA was always higher in the testis, ascompared to the brain, in all of the transgenic lines generated.
CNS neurons express two distinct plasma membrane electron transport systems implicated in neuronal viability
Authors: Wright M. V. , Kuhn T. B.
Published: March 2003

Trans‐plasma membrane electron transport is critical for maintaining cellular redox balance and viability, yet few, if any, investigations have studied it in intact primary neurons. In this investigation, extracellular reduction of 2,6‐dichloroindophenol (DCIP) and ferricyanide (FeCN) were measured as indicators of trans‐plasma membrane electron transport by chick forebrain neurons. Neurons readily reduced DCIP, but not FeCN unless CoQ1, an exogenous ubiquinone analog, was added to the assays. CoQ1 stimulated FeCN reduction in a dose‐dependent manner but had no effect on DCIP reduction. Reduction of both substrates was totally inhibited by ε‐maleimidocaproic acid (MCA), a membrane‐impermeant thiol reagent, and slightly inhibited by superoxide dismutase. Diphenylene iodonium, a flavoenzyme inhibitor, completely inhibited FeCN reduction but had no affect on DCIP reduction, suggesting that these substrates are reduced by distinct redox pathways. The relationship between plasma membrane electron transport and neuronal viability was tested using the inhibitors MCA and capsaicin. MCA caused a dose‐dependent decline in neuronal viability that closely paralleled its inhibition of both reductase activities. Similarly capsaicin, a NADH oxidase inhibitor, induced a rapid decline in neuronal viability. These results suggest that trans‐plasma membrane electron transport helps maintain a stable redox environment required for neuronal viability.
Constitutive activation of the opioid receptor‐like (ORL1) receptor by mutation of Asn133 to tryptophan in the third transmembrane region
Authors: Kam Kenneth W. L. , New David C. , Wong Yung H.
Published: March 2003

We have introduced a series of point mutations into the human opioid receptor‐like (ORL1) receptor and characterized them for their ability to constitutively activate G protein‐coupled receptor signalling pathways. Among the 12 mutants generated, mutation at Asn133 (N133W) gave increased basal signalling through three separate pathways. N133W increased the basal activity of G14‐ and G16‐dependent pathways by two‐ to three‐fold. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. In HEK‐293 cells stably expressing N133W, signalling through Gi/o‐dependent pathways was also observed. Radioligand binding studies revealed that the affinity for nociceptin of the wild‐type ORL1 receptor and the N133W mutant do not differ significantly, suggesting that the ligand binding and signalling functions of constitutively active mutants of G protein‐coupled receptors are not necessarily intrinsically linked. In conclusion, our results demonstrate that a mutation in the third transmembrane domain is able to increase the basal signalling activity of the human ORL1 receptor.
Spreading depression‐induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors
Authors: Chazot P. L. , Godukhin O. V. , McDonald A. , Obrenovitch T. P.
Published: March 2003

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham‐ and preconditioned cortex, 24 h after treatment. A 30% reduction in α‐amino‐3‐hydroxy‐5‐methyl‐4‐iso‐ xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12–15‐fold increase in α7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the α4 nAChR subunit was not altered. These data point to the α7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating α7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).
Region‐specific and epileptogenic‐dependent expression of six subtypes of α2,3‐sialyltransferase in the adult mouse brain
Authors: Matsuhashi Hitomi , Horii Yoichiro , Kato Keiko
Published: March 2003

Sialylated glycoconjugates play important roles in various biological functions. The structures are also observed in brains and it has been proposed that sialylation may affect neural plasticity. To clarify the effects of sialylation in the brain, particular neurons that exhibit sialylation should first be determined. Using in situ hybridization, we performed systematic surveys of the localization of mRNAs encoding the six α2,3‐sialyltransferases (ST3Gal I–VI) in the adult mouse brain with or without physiological stimulation. First, striking region‐specific patterns of expression were observed: While ST3Gal II, III, and V mRNAs were in neuronal cells throughout the brain, ST3Gal I, IV, and VI mRNAs were in restricted brain regions. Next, to assess whether the expression of the six mRNAs can be regulated, we examined the effect of kindling epileptogenesis on the six mRNA levels. Of the six subtypes, upregulation in the ST3Gal IV level in the thalamus was most pronounced; the number of ST3Gal IV‐expressing neurons in the anterior thalamic nuclei increased from 2% to 21% in a time‐dependent manner during epileptogenesis. Western blot analysis evaluated the increase of the end‐products in the thalamus. These findings provide a molecular basis to clarify when and where sialylated glycoconjugates function accompanied by neural plasticity.
Humanin rescues human cerebrovascular smooth muscle cells from Aβ‐induced toxicity
Authors: Jung Sonia S. , Van Nostrand William E.
Published: March 2003

Cerebral amyloid β‐protein (Aβ) angiopathy (CAA) is a key pathological feature of Alzheimer's disease (AD) and related disorders. We have used human cerebrovascular smooth muscle (HCSM) cells as an in vitro model system to investigate the pathogenic mechanisms of the pathology of CAA. It was previously demonstrated that certain pathogenic forms of Aβ induce several pathologic responses in these cells, including fibril assembly at the cell surface, increased levels of Aβ precursor, degradation of HCSM cell α‐actin and cell death. The recently discovered novel rescue factor humanin (HN) was shown to protect neuronal cells in culture from various AD‐relevant insults including treatment with Aβ. In this report we investigated whether the HN peptide could rescue HCSM cells from Aβ‐induced toxicity. We found that treatment of HCSM cells with 10 μm HN prevented pathogenic Aβ‐induced HCSM cell death using a fluorescent cell viability assay, and degradation of HCSM α‐actin was diminished shown by quantitative immunoblotting. However, Aβ deposition and fibril formation at the cell surface and increased levels of cell‐associated AβPP were not affected by treatment with HN as demonstrated by a thioflavin T fluorescence assay and immunochemical methods, respectively. These results suggest that the protective effects of HN occur downstream of these cell surface molecular events. This is the first demonstration of a rescue factor for HCSM cells from Aβ‐mediated cell death as well as being the first report to show that neuronal cells and HCSM cells may share a common downstream mechanism in the Aβ‐induced cell death pathway.
Accumulation of 7S SNARE complexes in hippocampal synaptosomes from chronically kindled rats
Authors: Matveeva E. A. , Whiteheart S. W. , Slevin J. T.
Published: March 2003

Kindling is a model of complex partial epilepsy wherein periodic application of an initially subconvulsive stimulus leads to first limbic and then generalized tonic‐clonic seizures. Several laboratories have reported that augmented neurotransmitter release of l‐glutamate is associated with the chronically kindled state. Neurotransmitter release requires membrane proteins called SNAREs, which form transmembrane complexes that participate in vesicle docking and are required for membrane fusion. We show here that kindling by entorhinal stimulation is associated with an accumulation of 7S SNARE complexes in the ipsilateral hippocampus. This increase of 7S SNARE complexes appears to begin early in the kindling process, achieves a peak with full kindling, and remains at this level for at least a month following cessation of further kindling stimuli. The increase is focal and permanently limited to the ipsilateral hippocampus despite progression to generalized electrographic and behavioral seizures. It is not seen in animals that receive electroconvulsive seizures, suggesting it is related to the kindling process itself. The duration and focality of increased 7S SNARE complexes with entorhinal kindling suggest that this is an altered molecular process associated with epileptogenesis.
Chloroquine induces activation of nuclear factor‐κB and subsequent expression of pro‐inflammatory cytokines by human astroglial cells
Authors: Park Jinseu , Kwon Daeho , Choi Chulhee , Oh Jae‐Wook , Benveniste Etty N.
Published: March 2003

Chloroquine, an antimalarial lysosomotropic base, is known for its anti‐inflammatory effects and therefore used for treatment of autoimmune diseases. Given its anti‐inflammatory effects, it has been under clinical trials to modify neurodegenerative processes. In this study, we examined whether chloroquine has an anti‐inflammatory effect in the CNS by determining the in vitro effects of chloroquine on LPS‐induced expression of cytokines by glial cells. We observed that (i) chloroquine augmented LPS‐induced expression of pro‐inflammatory cytokines such as lymphotoxin (LT)‐β, tumor necrosis factor (TNF)‐α, interleukin (IL)‐1α, IL‐1β and IL‐6 in human astroglial cells, while the same treatment suppressed LPS‐induced expression of cytokines in monocytic and microglial cells; (ii) chloroquine alone induced expression of pro‐inflammatory cytokines in a dose‐ and time‐dependent manner in astroglial cells; (iii) other lysosomotropic agents such as ammonium chloride and bafilomycin A1 had minimal effects on cytokine expression; and (iv) chloroquine induced the activation of nuclear factor‐kappa B in astroglial cells, which is a required component of chloroquine induction of cytokines. These results suggest that chloroquine may evoke either anti‐ or pro‐inflammatory responses in the CNS depending on the cellular context.
An age‐related increase in resistance to DNA damage‐induced apoptotic cell death is associated with development of DNA repair mechanisms
Authors: Romero Alejandro A. , Gross Stephane R. , Cheng Ke‐Yi , Goldsmith Noriko K. , Geller Herbert M.
Published: March 2003

Neurons in the developing brain die via apoptosis after DNA damage, while neurons in the adult brain are generally resistant to these insults. The basis for this resistance is a matter of conjecture. We report here that cerebellar granule neurons (CGNs) in culture lose their competence to die in response to DNA damage as a function of time in culture. CGNs at either 1 day in vitro (DIV) or 7 DIV were treated with the DNA damaging agents camptothecin, UV or γ‐irradiation and neuronal survival measured. The younger neurons were effectively killed by these agents, while the older neurons displayed a significant resistance to killing. Neuronal survival did not change with time in culture when cells were treated with C2‐ceramide or staurosporine, agents which do not target DNA. The resistance to UV irradiation developed over time in culture and was not due to changes in mitotic rate. Increases in DNA strand breakage, up‐regulation of the levels of both p53 and its phosphorylated form and nuclear translocation of p53 were equivalent in both older and younger neurons, indicating a comparable p53 stress response. In addition, we show that treatment of older neurons with pharmacological inhibitors of distinct components of the DNA repair machinery promotes the accumulation of DNA damage and sensitizes these cells to the toxic effects of UV exposure. These data demonstrate that older neurons appear to be more proficient in DNA repair in comparison to their younger counterparts, and that this leads to increased survival after DNA damage.
Burden of responsibility experienced by family caregivers of elderly dementia sufferers
Authors: Samuelsson Ann Margret , Annerstedt Lena , Elmståhl Sölve , Samuelsson Sven‐Mårten , Grafström Margareta
Published: March 2003

Eight family caregivers of elderly dementia sufferers participated in in‐depth interviews regarding their experiences of giving care. They were selected according to strain, isolation, disappointment and emotional involvement, measured on a ‘caregiver burden’ scale. Structural analyses of the interviews identified six categories reflecting the feelings and experiences of the caregivers. The first symptom of dementia noted by caregiving husbands was a change in personality, whereas other relatives first observed impaired memory. The dementia sufferers were deemed to be helpless, vulnerable and anxious. The quality of the relationship preceding the onset of dementia had a bearing on the carer's situation. All caregivers felt a heavy burden, especially early in the dementia process. Husbands sustained the heaviest burden; they expressed anger, worry, weariness, guilt, distress and isolation. The caregivers used different problem‐ and emotion‐focused strategies to cope with their situation. Visits to the homesof the elderly, for instance by the district nurse or home help, which should include interviews with close relatives, are recommended in order to disclose early signs of dementia and to prepare further individual support for the family caregivers and their relatives suffering from dementia.
Ethical issues in ethnographic nursing research with children and elderly people
Authors: Jokinen Pirkko , Lappalainen Marja , Meriläinen Pirkko , Pelkonen Marjaana
Published: March 2003

Ethical issues in ethnographic nursing research with children and elderly people

The purpose of this article is to stimulate discussion on ethical issues in qualitative nursing research with children and elderly people. Both groups as informants of a qualitative study may need extra protection owing to their vulnerability. There are some references to these issues in articles dealing with nursing research ethics, but this subject is worthy of more debate. This article is based on the authors' experiences of working with informants in two ethnographic nursing studies. Especially difficult and important were those issues related to informed consent and the role of the researcher. The researcher has to respect the informants' humanity and ensure their autonomy, and be sensitive to their expressions and gestures in a reciprocal interaction throughout the research process.
Maternal serum screening for Down syndrome – opinions on acceptance from Swedish women
Authors: Crang‐Svalenius Elizabeth , Dykes Anna‐Karin , Jörgensen Connie
Published: March 2003

Background:  Different screening procedures are becoming an important part of health care. Information about screening and its consequences can be difficult to both impart and understand. This study examined women's theoretical acceptance of a new screening procedure, before its introduction.

Methods:  A group of women (n = 823), who had made an informed choice about the form of foetal diagnosis they wished for, were also asked, in a questionnaire, about their opinion on the acceptability of serum screening for Down syndrome. The main purpose of the this was to ascertain their feelings when making a choice.

Results:  Replies were received from 80.4%. The answers indicated that serum screening would be acceptable to 86% of the women who had chosen a second trimester ultrasound examination. Of the women who had chosen amniocentesis, 51% would consider the test acceptable as a first alternative.

Conclusions:  The ease with which the women were able to make their choice of prenatal diagnosis had bearing on their degree of acceptance of serum screening for Down syndrome.
Universal RAGE–PCR primers to detect mitochondrial polymorphisms among Magnoliophyta
Authors: Wijnants L. , Vasseur J. , Rambaud C.
Published: March 2003

A rapid amplification of genomic ends–polymerase chain reaction (RAGE–PCR) based technique was developed to detect polymorphism in mitochondrial intergenic sequences to compare cytoplasmic male sterile and revertant chicory. Here we describe 84 new mitochondrial primers that were successfully tested with two Liliopsida and four Magnoliopsida species and were useful for exploration of the mitochondrial genome in analyses such as population genetics or phylogenetical studies among Magnoliophyta.
Identification of polymorphic microsatellite loci in the queenless, ponerine ant Diacamma ceylonense
Authors: Gopinath Ashok , Gadagkar Raghavendra , Rao M. R. S.
Published: March 2003

Diacamma ceylonense is a queenless, ponerine ant whose colonies are headed by a single, mated, egg‐laying worker referred to as the gamergate. Thus, new colonies are a result of dispersal by wingless gamergates. This is expected to influence patterns of colony dispersal and spatial distribution of genetic variablity. In order to facilitate the study of population genetic structure we have identified six unique, polymorphic, microsatellite loci. We have used fluorescence tagged primers to detect polymorphism at these loci.
Microsatellite loci in the human blood fluke Schistosoma mansoni and their utility for other schistosome species
Authors: Curtis Jason , Sorensen Robert E. , Page L. Kristen , Minchella Dennis J.
Published: March 2003

Blood flukes in the genus Schistosoma are important human parasites in tropical regions. Genetic heterogeneity of the parasite contributes to the observed phenotypic variation in this host–parasite interaction and may play a role in disease epidemiology. In this paper, we describe the characterization of five polymorphic microsatellite loci from the human blood fluke Schistosoma mansoni, which can now be applied in assessments of schistosome genetic diversity. The five loci revealed extensive polymorphism, as 5–8 alleles per locus were detected among five isolates (from both human patients and snail intermediate hosts) from two Brazilian villages.
Polymorphic microsatellite loci for assigning parentage in least flycatchers (Empidonax minimus)
Authors: Tarof S. A. , Ratcliffe L. M. , Boag P. T.
Published: March 2003

Least flycatchers (Empidonax minimus) are socially monogamous birds that form tight territorial aggregations on the breeding grounds. We designed five polymorphic microsatellite loci for assigning parentage to offspring within least flycatcher clusters. The number of alleles per locus ranged from 7 to 18. Mean polymorphic information content was 83.8%; the probability of exclusion with known maternal genotype was 0.999. These microsatellites are powerful DNA markers for identifying extra‐pair paternity in this species. Preliminary data also suggest that these loci may be useful for other members of this genus.
Isolation and characterization of microsatellites in Aphidius ervi (Hymenoptera: Braconidae) and their applicability to related species
Authors: Hufbauer Ruth A. , Bogdanowicz Steven M. , Perez Luisa , Harrison Richard G.
Published: March 2003

Aphidius ervi (Hymenoptera: Braconidae) is an important biological control agent of aphids, and a model organism for the study of host–parasitoid ecology and evolution. We isolated microsatellite loci from A. ervi to examine the genetic effects of its introduction from Europe to North America. We present primers for 11 microsatellites from A. ervi. We have assayed six of these loci for variability and found from two to 37 alleles. These six primer pairs were tested on 17 related species and appear broadly applicable. These microsatellite loci provide a new tool for research on A. ervi and its relatives.
Development and cross‐species amplification of 18 microsatellite markers in the Sumatran tiger (Panthera tigris sumatrae)
Authors: Williamson J. E. , Huebinger R. M. , Sommer J. A. , Louis E. E. , Barber R. C.
Published: March 2003

Eighteen polymorphic microsatellite loci from the highly endangered Sumatran tiger (Panthera tigris sumatrae) were isolated and characterized. Upon polymerase chain reaction amplification, 16 of these markers produced a single, sharp band in all three tiger and 10 non‐tiger felid species examined. Of the two remaining loci, 6HDZ057 and 6HDZ635 failed to amplify genomic DNA from puma (Felis concolor) and cheetah (Acinonyx jubatus), respectively. The amplification of these markers across four genera is an indication of their usefulness for population genetics studies and conservation work in a wide range of felid species.
papa (package for the analysis of parental allocation): a computer program for simulated and real parental allocation
Authors: Duchesne Pierre , Godbout Marie‐Hélène , Bernatchez Louis
Published: March 2003

papa is a parental pair allocation and simulator program. The allocation method is based on the likelihood of a parental pair producing the multilocus genotype found in the offspring being tested, which will be referred to as the breeding likelihood. Estimated level and structure of allele transmission errors in offspring are parameters fed into the allocation procedure. The embodied Monte‐Carlo simulator also allows modelling of many allocation conditions, including transmission error and the estimated proportion of missing parents. Simulations may be run prior to the collection of real parents in order to define the minimal set of loci that is necessary to reach a desired level of allocation success. Post‐collection simulations aim at statistically assessing the reliability of nonsimulated allocations. Simulations output values for several random variables.
Characterization of dinucleotide microsatellite loci in big brown bats (Eptesicus fuscus), and their use in other North American vespertilionid bats
Authors: Vonhof M. J. , Davis C. S. , Fenton M. B. , Strobeck C.
Published: March 2003

We describe the development of six microsatellite loci for big brown bats, Eptesicus fuscus. Microsatellite markers were isolated from a small insert genomic library, and tested on a population of 44 animals from the Pend d’Oreille Valley, in southeastern British Columbia, Canada. These six loci were highly variable, with 12–23 alleles per locus, and observed and expected heterozygosities of greater than 79.5%. The six primer sets, and three others that were not variable in E. fuscus, were tested on 11 other species in the families Vespertilionidae and Antrozoidae. All the tested loci amplified highly variable products in at least several other species.
Development of novel polymerase chain reaction (PCR) based microsatellite markers in Armillaria gallica by cross‐species amplification and species‐specific cloning
Authors: Lefrancois J.‐B. , Lung‐Escarmant B. , Langrell S. R. H.
Published: March 2003

Cross‐species PCR amplification of Armillaria mellea group taxa with previously reported A. ostoyae microsatellite markers, indicative of flanking sequence conservation, was exploited for the species‐specific isolation of simple sequence repeat (SSR) motifs from A. gallica. Six SSR motifs were sequence characterized from cloned PCR fragments generated with primers previously developed from A. ostoyae. Five novel primer pairs, designed from motif flanking regions, allowed for improved, efficient amplification in this species. One original A. ostoyae primer pair was used directly. Polymorphims were observed at wide geographical levels only. Relative cross‐species amplification intensities generally supported the currently accepted molecular phylogeny of this group.
Development and characterization of SSR markers in Ribes species
Authors: Brennan R. , Jorgensen L. , Woodhead M. , Russell J.
Published: March 2003

Eleven microsatellite loci were identified and characterized in blackcurrant (Ribes nigrum L.) and related species. An enriched library was constructed and screened with simple sequence repeat (SSR) oligonucleotides. Positive clones were sequenced and primers were designed flanking the repeat motifs. These 11 microsatellites produce amplification products polymorphic across a range of Ribes germplasm, predominantly from the Eucoreosma section of the genus. The number of alleles varied from 2 to 18 with levels of diversity ranging from 0.18 to 0.91.
Isolation and characterization of microsatellite loci in the mosquito Anopheles stephensi Liston (Diptera: Culicidae)
Authors: Verardi A. , Donnelly M. J. , Rowland M. , Townson H.
Published: March 2003

The mosquito Anopheles stephensi is an important malaria vector in India, Pakistan, Iran and Afghanistan. Differences in egg morphology and chromosomal characters have been described between urban and rural forms of this mosquito but the population genetic structure remains unclear. In India this species is mainly urban, rural populations are largely zoophilic and not thought to transmit malaria. In eastern Afghanistan and the Punjab and Northwest Frontier Province, Pakistan, it is the major malaria vector. We have developed primers for 16 microsatellite loci to assist in defining the population structure and epidemiological importance of this mosquito.
Microsatellite markers for diverse Salix species
Authors: Barker J. H. A. , Pahlich A. , Trybush S. , Edwards K. J. , Karp A.
Published: March 2003

Forty‐six microsatellites were isolated from an enriched library of Salix burjatica and tested on 20 individuals (of nine species/hybrids) from the National Willows Collection (IACR‐Long Ashton Research Station, UK). Twenty‐nine were monomorphic, gave multilocus or unscorable patterns, or were duplicates. The remaining 17 microsatellites gave 2–22 alleles/locus. Three microsatellites successfully cross‐amplified in 31 additional Salix species. A further six were tested on panels comprising 6–25 individuals from the 31 species. Cross‐amplification was successful in all cases. These results suggest that the microsatellites isolated here should prove useful for population studies in a wide range of Salix species.
A set of primers for the amplification of chloroplast microsatellites in Quercus
Authors: Deguilloux M.‐F. , Dumolin‐Lapègue S. , Gielly L. , Grivet D. , Petit R. J.
Published: March 2003

The increase in demand for the certification of oak seed lots, as well as control of the geographical origin of oak wood, has led us to develop powerful genetic markers permitting us to discriminate among provenance regions. With the aim of detecting new chloroplast variants, we have identified 17 potential cpSSRs motifs from available oak sequences and tested their variability among French oak populations. Six loci were polymorphic at the intraspecific level in Quercus petraea and Q. robur. Moreover, conservation of the primer pairs was checked on a set of 21 forest tree species and they were all shown to work well on several Quercus species, and even within Fagacaea.
microsatellite analyser (MSA): a platform independent analysis tool for large microsatellite data sets
Authors: Dieringer Daniel , Schlötterer Christian
Published: March 2003

In molecular ecology the analysis of large microsatellite data sets is becoming increasingly popular. Here we introduce a new software tool, which is specifically designed to facilitate the analysis of large microsatellite data sets. All common microsatellite summary statistics and distances can be calculated. Furthermore, the microsatellite analyser (msa) software offers an improved method to deal with inbred samples (such as Drosophila isofemale lines). Executables are available for Windows and Macintosh computers.
Isolation of dinucleotide microsatellite loci from red‐backed salamander (Plethodon cinereus)
Authors: Connors Lisa M. , Cabe Paul R.
Published: March 2003

We isolated 13 variable dinucleotide microsatellites from red‐backed salamanders (Plethodon cinereus). After generating fragments using degenerate oligonucleotide primer‐polymerase chain reaction (DOP‐PCR), AC repeats were captured using biotinylated probes and streptavidin‐coated magnetic particles. Captured fragments were cloned into plasmids, screened for microsatellites with a simple PCR reaction, and select plasmids then sequenced. PCR primers were designed and optimized for robust amplification, and nine primers have been further optimized for multiplex reactions with fluorescent primers. These nine loci are variable with an average of 6.11 alleles per locus and an average heterozygosity of 0.61.
Isolation and characterization of polymorphic microsatellite loci in Lobesia botrana Den. & Schiff. (Lepidoptera: Tortricidae)
Authors: Amsellem L. , Risterucci A. M. , Benrey B.
Published: March 2003

The tortricid moth Lobesia botrana is considered to be an important pest of the cultivated grapevine (Vitis vinifera, Vitaceae). It feeds on young flower buds and young fruits, and is the primary vector of the ‘noble mould’ fungus (Botrytis cinerea). In order to study the population dynamics and genetic structure of this species in space and time, we developed seven polymorphic microsatellite markers (three to 12 alleles). Although the presence of null alleles is suspected, these polymorphic loci are likely to provide information on the population genetics of L. botrana, and could help in the development of an efficient control strategy against this pest.
Polymorphic microsatellite loci in Plantago lanceolata
Authors: Hale Marie L. , Wolff Kirsten
Published: March 2003

The genus Plantago is particularly interesting for evolutionary studies because of its wide range of mating systems. We have developed primers for five highly polymorphic microsatellite loci isolated from P. lanceolata. All five loci amplified and were polymorphic in the two populations examined, Lowsteads Beach in the United Kingdom and Duke in the United States. These new markers will allow a comparison of population structure between the outcrossing species P. lanceolata, and the highly selfing species P. major.
Microsatellite markers for the Mallorcan midwife toad Alytes muletensis
Authors: Kraaijeveld‐Smit Femmie J. L. , Rowe Graham , Beebee Trevor J. C. , Griffiths Richard A.
Published: March 2003

The highly endangered midwife toad Alytes muletensis occurs only on the Mediterranean island of Mallorca. Little is known about genetic variability and dispersal among the 19 breeding sites (six of which stem from reintroductions) of this amphibian. The value of such information should be high for successful management of this species. We report here the characterization of eight polymorphic microsatellite loci in A. muletensis using individuals from a single population.
Realizing the cosmetic potential of topical photodynamic therapy
Authors: Morton C A
Published: March 2003

Summary Several open studies report the efficacy of topical photodynamic therapy (PDT) for non‐melanoma skin cancer (NMSC) and precursor lesions. PDT may also be effective in various inflammatory and infectious dermatoses. As a tissue sparing modality, PDT may be preferable for large and/or multiple lesions and those in sites where disfigurement or poor healing from conventional therapies is a particular risk. Limited comparison data confirms that topical PDT achieves superior cosmesis to conventional therapies in actinic keratoses, squamous cell carcinoma in‐situ and basal cell carcinoma. This review assesses the current data and proposes potential future applications for topical PDT including the treatment of photo‐damaged skin.