bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of the Progenitor Cell Yield in a Leukapheresis Product by Previous Measurement of CD34+ Cells in the Peripheral Blood
Authors: Möhle Robert , Murea Simona , Pförsich Margit , Witt Barbara , Haas Rainer
Published: May 2003

To assess whether measurement of CD34+ cells in the peripheral blood allows one to estimate the progenitor cell yields of subsequent leukapheresis procedures, 733 corresponding blood and leukapheresis samples were analyzed. Peripheral blood progenitor cells of cancer patients were mobilized with hematopoietic growth factors alone or postchemotherapy, and harvested processing 10 liters of blood for each leukapheresis product. The CD34+ cell count (CD34+ cells/μl blood) correlated most closely with the progenitor cell yield in the corresponding leukapheresis product (CD34+ cells/kg bodyweight, r = 0.80), while the proportion of circulating CD34+ cells to the white blood and mononuclear cells predicted the yield less reliably (r = 0.74 and r = 0.60). The CD34+ cell yield was independent of the white blood count (r = 0.04), whereas a weak correlation was found between the mononuclear cell count and the number of CD34+ cells/kg collected (r = 0.42). It was unlikely to obtain the threshold quantity of 2.5 × 106 CD34+ cells/kg required for rapid engraftment when counts below 10 CD34+ cells/μl blood were detected. At levels between 10 and 30 CD34+ cells/μl sufficient autografts could be harvested, whereas 30–100 CD34+ cells/μl were required to achieve this by a single leukapheresis. A surplus of CD34+ cells was likely above 100 CD34+ cells/μl which could be useful for progenitor cell enrichment techniques. The correlation between the CD34+ cell count and progenitor cell yield was independent of the mobilizing regimen and whether leukaphereses had been performed previously. In conclusion, the number of CD34+ cells/μl blood allows a reliable prediction of the CD34+ progenitor cell yield in subsequent leukapheresis procedures. However, rare cases of unexpectedly sufficient progenitor cell yields may be observed even at CD34+ cell levels below detection limit.
The Low‐Incidence Blood Group Antigen, Wda, Is Associated with the Substitution Val557→ Met in Human Erythrocyte Band 3 (AE1)
Authors: Bruce Lesley J. , Zelinski Teresa , Ridgwell Kay , Tanner Michael J. A.
Published: May 2003

The Waldner blood group antigen (Wda) was first identified in members of a Hutterite kindred. Evidence that the gene governing the Waldner polymorphism is located on chromosome 17, and the observation that the antigen is inactivated by chymotrypsin prompted the investigation of a possible association between Wda and band 3. Single Stranded Conformational Polymorphism (SSCP) analysis and DNA sequence analysis of the AE1 gene, from subjects of known Waldner phenotypes, showed a heterozygous mutation leading to the substitution Val557→ Met in the presumptive Wd(a+) heterozygotes. Therefore the Wda blood group antigen is associated with the presence of Met557 on band 3. The Waldner antigen has been assigned to the Diego blood group system with the International Society of Blood Transfusion number DI5.
Hepatitis A Transmission by Factor IX Concentrates
Authors: Lawlor E. , Graham S. , Davidson F. , Yap P. L. , Cunningham C. , Daly H. , Temperley I. J.
Published: May 2003

Factor IX concentrates unlike factor VIII concentrates have not to date been associated with the transmission of hepatitis A virus (HAV). A retrospective study by reverse transcriptase polymerase chain reaction on a batch of factor IX concentrate used to treat two haemophilia B patients who developed jaundice and IgM anti‐HAV antibodies within 50 days of factor IX administration in 1985 revealed the presence of HAV RNA. These findings indicate that factor IX concentrates can transmit HAV and that appropriate viral inactivation steps to inactivate non‐enveloped viruses as well as enveloped viruses are necessary to ensure the safety of factor IX concentrates.
Removal of Parvovirus and Hepatitis A Virus by Metal Chelate Affinity Chromatography during the Preparation of Replenine ®: A High‐Purity Factor IX Concentrate
Authors: Roberts Peter L.
Published: May 2003

The widespread use of specific virus inactivation techniques such as solvent/detergent, pasteurisation and severe dry‐heat treatment, has greatly increased the safety of coagulation factor concentrates [1]. All these techniques are highly effective against known blood‐borne enveloped viruses such as hepatitis B, hepatitis C and HIV. In addition, they should also prove effective against the recently described GB group of hepatitis viruses [3], given that these are enveloped viruses related to hepatitis C.
The Use of DNA Typing for Human Platelet‐Specific Antigens in the Daily Routine: A Case Report
Authors: Stein Sophia , Drabbels Jos , van 't Sant Paul , Witvliet Marian D. , Bein Gregor , Claas Frans H. J. , Doxiadis Ilyas
Published: May 2003

Human platelets express a variety of immunogenetically determined alloantigens. Besides the classical HLA class I antigens, which are found on almost all nucleated cells, platelet‐specific antigens can cause alloimmunization in susceptible patients.
The effects of exercise and social support on mothers reporting depressive symptoms: A pilot randomized controlled trial
Authors: Armstrong Kylie , Edwards Helen
Published: May 2003

ABSTRACT: A 12‐week randomized controlled trial was conducted (n = 20) investigating the effects of a multi‐intervention programme of exercise and social support compared to a control group. Both groups had given birth in the past 12 months and were experiencing depressive symptomatology. Pre‐test data of physical fitness and structured questionnaires were compared to post‐test data. The results from the study showed that mothers who were in the multi‐intervention group improved their fitness levels and depressive symptomatology significantly more than the control group. However, there were no significant changes to either group's social support levels. These results are encouraging and suggest that a pram push walking intervention might be an effective treatment for mothers suffering postnatal distress.
The association between schizophrenia and cigarette smoking: A review of the literature and implications for mental health nursing practice
Authors: McCloughen Andrea
Published: May 2003

ABSTRACT: Despite a fall in smoking activity among the general population, individuals with schizophrenia continue to smoke at alarming rates and suffer poor health as a consequence. It would appear that limited and conflicting knowledge, outdated perceptions, and ineffective interventions have hindered efforts to promote healthy behaviours among this group. This paper reviews the literature on the association between nicotine dependence and schizophrenia and explores explanations for the phenomenon. Complex psychopathological, biochemical, and neuropharmacological interactions between smoking and schizophrenia are revealed. The interface of schizophrenia and smoking behaviour, particularly among those hospitalized in mental health facilities, and rationales for the management of this manifestation are examined. In addition, inferences regarding the role of mental health nurses in the care of this population are reached, and implications for nursing practice are discussed.
The challenges of mental health care reform for contemporary mental health nursing practice: Relationships, power and control
Authors: Cleary Michelle
Published: May 2003

ABSTRACT: This ethnographic investigation was concerned with the way mental health nurses’ construct their practice in an acute inpatient psychiatric unit in light of the current challenges, demands and influences brought about by service reforms of the 1990s. The setting for this study was a 22‐bed acute inpatient mental health facility. Over a 5‐month period, data were collected through fieldwork observations, focused interviews and discussion groups. In this article, common cultural practices in relation to the imperatives of relationships, power, restrictions and safety are discussed. The findings demonstrate how the client stabilization role of the unit locates these imperatives as central to the delivery of nursing care. The discussion reveals nurses negotiating relationships that are contradictory and challenging in ordinary and everyday ways within the exigencies of daily practice.
What influences patients’ medication adherence? Mental health nurse perspectives and a need for education and training
Authors: Coombs Tim , Deane Frank P. , Lambert Gordon , Griffiths Rhonda
Published: May 2003

ABSTRACT: This paper explores the role of mental health nurses in medication adherence and their perspective of what influences patients’ medication non‐adherence. Forty‐eight mental health nurses with active patient caseloads completed a comprehensive questionnaire assessing a number of variables related to medications, including whom they felt was primarily responsible for monitoring the side‐effects of medication, their knowledge skills and confidence in dealing with medication adherence and their prior education and training in medication adherence strategies. Lack of patient insight was endorsed as the strongest influence on patient non‐adherence. Over 84% of nurses indicated they did not have any prior education or training in medication adherence strategies. Implications of the findings for education and training and nurses’ roles in supporting medication adherence are discussed.
Guidelines for presentations and publications
Authors: Cleary Michelle , Hunt Glenn , Walter Garry , Horsfall Jan
Published: May 2003

ABSTRACT: The oral and written presentation of work arising from clinical practice, service development, quality improvement projects and research is presently encouraged and supported. Because this work may involve consumers, staff and the organization, it is important that proper consideration is given to how the work is disseminated, including any ethical implications. There is a need to ensure that consumers, staff, facilities, the organization and intellectual property are protected from any potential concerns that may arise. This research note provides protocols and a checklist that may be of interest and may be relevant for clinicians and researchers to review and appraise their work before oral or written presentation.
British Nutrition Foundation annual lecture: Chips with everything? Nutritional genomics and the application of diet in disease prevention
Authors: Williams Christine M.
Published: May 2003

The development of high throughput techniques (‘chip’ technology) for measurement of gene expression and gene polymorphisms (genomics), and techniques for measuring global protein expression (proteomics) and metabolite profile (metabolomics) are revolutionising life science research, including research in human nutrition. In particular, the ability to undertake large‐scale genotyping and to identify gene polymorphisms that determine risk of chronic disease (candidate genes) could enable definition of an individual's risk at an early age. However, the search for candidate genes has proven to be more complex, and their identification more elusive, than previously thought. This is largely due to the fact that much of the variability in risk results from interactions between the genome and environmental exposures. Whilst the former is now very well defined via the Human Genome Project, the latter (e.g. diet, toxins, physical activity) are poorly characterised, resulting in inability to account for their confounding effects in most large‐scale candidate gene studies. The polygenic nature of most chronic diseases offers further complexity, requiring very large studies to disentangle relatively weak impacts of large numbers of potential ‘risk’ genes. The efficacy of diet as a preventative strategy could also be considerably increased by better information concerning gene polymorphisms that determine variability in responsiveness to specific diet and nutrient changes. Much of the limited available data are based on retrospective genotyping using stored samples from previously conducted intervention trials. Prospective studies are now needed to provide data that can be used as the basis for provision of individualised dietary advice and development of food products that optimise disease prevention. Application of the new technologies in nutrition research offers considerable potential for development of new knowledge and could greatly advance the role of diet as a preventative disease strategy in the 21st century. Given the potential economic and social benefits offered, funding for research in this area needs greater recognition, and a stronger strategic focus, than is presently the case. Application of genomics in human health offers considerable ethical and societal as well as scientific challenges. Economic determinants of health care provision are more likely to resolve such issues than scientific developments or altruistic concerns for human health.
Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer's disease
Authors: Matsubara Etsuro , Bryant‐Thomas Tara , Pacheco Quinto Javier , Henry Tracey L. , Poeggeler Burkhard , Herbert Donald , Cruz‐Sanchez Felix , Chyan Yau‐Jan , Smith Mark A. , Perry George , Shoji Mikio , Abe Koji , Leone Anna , Grundke‐Ikbal Inge , Wilson Glen L. , Ghiso Jorge , Williams Christina , Refolo Lorenzo M. , Pappolla Miguel A.
Published: May 2003

Increased levels of a 40–42 amino‐acid peptide called the amyloid β protein (Aβ) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from Aβ‐mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of Aβ, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time‐dependent elevation of β‐amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.
A role for semaphorins and neuropilins in oligodendrocyte guidance
Authors: Cohen Rick I. , Rottkamp Daniele M. , Maric Dragan , Barker Jeffery L. , Hudson Lynn D.
Published: May 2003

Oligodendrocytes develop in defined CNS regions as progenitor cells, which migrate to their final destinations, encountering soluble and membrane‐bound signals that influence their differentiation and potential to myelinate axonal projections. To identify the regulatory genes that may be involved in this process, microarray analysis of developing oligodendroglia was performed. Several neural guidance genes, including members of the neuropilin (NP) and semaphorin families were detected. These findings were verified and expanded upon using RT–PCR with RNA from fluorescent activated cell sorted A2B5+ oligodendrocyte progenitors and O4+ pro‐oligodendrocytes isolated from in vitro and in vivo sources. RT–PCR, western and immunocytochemical analyses revealed that oligodendrocytes expressed NP1, several alternatively spliced isoforms of NP2, and a broad spectrum of both soluble (Class 3), membrane‐spanning (Class 4–6), and membrane‐tethered (Class 7) semaphorin ligands. Class 3 semaphorins, in a modified stripe assay, caused the collapse of oligodendrocyte progenitor growth cones, redirection of processes, and altered progenitor migration. Our data support a role for neuropilins and semaphorins in orchestrating the migration patterns of developing oligodendrocytes in the CNS.
Functional study of rat 5‐HT2A receptors using antisense oligonucleotides
Authors: Van Oekelen Dirk , Megens Anton , Meert Theo , Luyten Walter H. M. L. , Leysen Josée E.
Published: May 2003

We studied the effects in rats of a 6‐day intracerebroventricular (i.c.v) infusion of four different end‐capped phosphorothioate‐modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5‐hydroxytryptamine2A (5‐HT2A) receptor mRNA, on central 5‐HT2A receptor expression and 5‐HT2A receptor‐mediated behaviours. Only one of the AOs (sequence 4), directed against the 5′‐untranslated region (from + 557 to + 577), specifically affected central 5‐HT2A receptor expression and receptor‐mediated behaviour. This AO (sequence 4) reduced binding of the 5‐HT2A agonist 1‐(2,5‐dimethoxy‐4‐[125I]iodophenyl)‐2‐aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5‐day infusion of AO (sequence 4) resulted in profound inhibition of the head‐twitch response (HTR) to 1‐(2,5‐dimethoxy‐4‐methylphenyl)‐2‐aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM‐induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5‐HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood–brain barrier and cause a stress‐induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5‐HT2A receptor‐mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5‐HT2A receptor activation. Labelling of central 5‐HT2C receptors by [3H]mesulergine, and 5‐HT2C receptor‐mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down‐regulation of functional 5‐HT2A receptors may shift the balance between various 5‐HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor‐mediated behaviour can be identified.
Pathogenic Aβ induces the expression and activation of matrix metalloproteinase‐2 in human cerebrovascular smooth muscle cells
Authors: Jung Sonia S. , Zhang Weibing , Van Nostrand William E.
Published: May 2003

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid β protein (Aβ). Previously we have shown that pathogenic forms of Aβ induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of Aβ precursor, and apoptotic cell death. Here we show that pathogenic Aβ stimulates the expression and activation of matrix metalloproteinase‐2 (MMP‐2). Furthermore, we demonstrate that the increase in MMP‐2 activation is largely caused by increased expression of membrane type‐1 (MT1)‐MMP expression, the primary MMP‐2 activator. Finally, treatment with MMP‐2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic Aβ. Our findings suggest that increased expression and activation of MMP‐2 may contribute to HCSM cell death in response to pathogenic Aβ. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.
Ethanol consumption and serotonin‐1A (5‐HT1A) receptor function in heterozygous BDNF (+/–) mice
Authors: Hensler J. G. , Ladenheim E. E. , Lyons W. E.
Published: May 2003

Heterozygous brain‐derived neurotrophic factor (BDNF) (+/–) mice display abnormalities in central serotonergic neurotransmission, develop decrements in serotonergic innervation of the forebrain, and exhibit enhanced intermale aggressiveness. As disturbances of serotonin neurotransmission are implicated in alcohol abuse and aggression, we have examined in BDNF (+/–) mice alcohol drinking behavior, as well as central 5‐hydroxytryptamine (5‐HT)1A receptor function at the level of 5‐HT1A receptor–G protein interaction. BDNF (+/–) mice displayed increased ethanol intake in a two‐bottle choice procedure. There was no difference in the preference ratio for non‐alcoholic tastants (i.e. quinine or saccharin) between genotypes. In the brains of alcohol‐naive mice, we measured [35S]GTPγS binding stimulated by the 5‐HT1A receptor agonist (+/–)‐8‐hydroxy‐2‐dipropyl‐aminotetralin hydrobromide (8‐OH‐DPAT; 1 µm). In BDNF (+/–) versus wild‐type (WT) mice, 5‐HT1A receptor‐stimulated [35S]GTPγS binding was significantly attenuated in the median raphe nucleus. There was a decrease in (+/–)8‐OH‐DPAT‐stimulated [35S]GTPγS binding in the dorsal raphe, which did not reach statistical significance. In the hippocampus, 5‐HT1A receptor–stimulated [35S]GTPγS binding was significantly attenuated in BDNF (+/–) mice. 5‐HT1A receptor–stimulated [35S]GTPγS binding was attenuated in the anterior cingulate cortex and lateral septum, although these reductions did not reach statistical significance. 5‐HT1A receptor number was not different between genotypes in any area of brain examined, suggesting that 5‐HT1A receptor function, specifically the capacity of the 5‐HT1A receptor to activate G proteins, is attenuated in BDNF (+/–) mice.
Subtype‐specific coupling with ADP‐ribosyl cyclase of metabotropic glutamate receptors in retina, cervical superior ganglion and NG108‐15 cells
Authors: Higashida Haruhiro , Zhang Jia‐Sheng , Mochida Sumiko , Chen Xiao‐Liang , Shin Yeonsook , Noda Mami , Hossain Kazi Z. , Hoshi Naoto , Hashii Minako , Shigemoto Ryuichi , Nakanishi Shigetada , Fukuda Yutaka , Yokoyama Shigeru
Published: May 2003

Cyclic ADP‐ribose (cADP‐ribose) is a putative second messenger or modulator. However, the role of cADP‐ribose in the downstream signals of the metabotropic glutamate receptors (mGluRs) is unclear. Here, we show that glutamate stimulates ADP‐ribosyl cyclase activity in rat or mouse crude membranes of retina via group III mGluRs or in superior cervical ganglion via group I mGluRs. The retina of mGluR6‐deficient mice showed no increase in the ADP‐ribosyl cyclase level in response to glutamate. GTP enhanced the initial rate of basal and glutamate‐stimulated cyclase activity. GTP‐γ‐S also stimulated basal activity. To determine whether the coupling mode of mGluRs to ADP‐ribosyl cyclase is a feature common to individual cloned mGluRs, we expressed each mGluR subtype in NG108‐15 neuroblastoma × glioma hybrid cells. The glutamate‐induced stimulation of the cyclase occurs preferentially in NG108‐15 cells over‐expressing mGluRs1, 3, 5, and 6. Cells expressing mGluR2 or mGluRs4 and 7 exhibit inhibition or no coupling, respectively. Glutamate‐induced activation or inhibition of the cyclase activity was eliminated after pre‐treatment with cholera or pertussis toxin, respectively. Thus, the subtype‐specific coupling of mGluRs to ADP‐ribosyl cyclase via G proteins suggests that some glutamate‐evoked neuronal functions are mediated by cADP‐ribose.
Glutamate transport alteration triggers differentiation‐state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents
Authors: Ré Diane B. , Boucraut José , Samuel Denise , Birman Serge , Kerkerian‐Le Goff Lydia , Had‐Aissouni Laurence
Published: May 2003

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of l‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC‐induced death was delayed and apoptotic, and death rate was dose and treatment duration‐dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.
Gene profiling of hippocampal neuronal culture
Authors: Dabrowski Michal , Aerts Stein , Van Hummelen Paul , Craessaerts Katleen , De Moor Bart , Annaert Wim , Moreau Yves , De Strooper Bart
Published: May 2003

We performed mRNA expression profiling of mouse primary hippocampal neurones undergoing differentiation in vitro. We show that 2314 genes significantly changed expression during neuronal differentiation. The temporal resolution of our experiment (six time points) permits us to distinguish between gene expression patterns characteristic for the axonal and for the dendritic stages of neurite outgrowth. Cluster analysis reveals that, in the process of in vitro neuronal differentiation, a high level of expression of genes involved in the synthesis of DNA and proteins precedes the up regulation of genes involved in protein transport, energy generation and synaptic functions. We report in detail changes in gene expression for genes involved in the synaptic vesicle cycle. Data for other genes can be accessed at our website. We directly compare expression of 475 genes in the differentiating neurones and the developing mouse hippocampus. We demonstrate that the program of gene expression is accelerated in vitro as compared to the situation in vivo. When this factor is accounted for, the gene expression profiles in vitro and in vivo become very similar (median gene‐wise correlation 0.787). Apparently once the cells have taken a neuronal fate, the further program of gene expression is largely independent of histological or anatomical context. Our results also demonstrate that a comparison across the two experimental platforms (cDNA microarrays and oligonucleotide chips) and across different biological paradigms is feasible.
Heterologous µ‐opioid receptor adaptation by repeated stimulation of κ‐opioid receptor: up‐regulation of G‐protein activation and antinociception
Authors: Narita Minoru , Khotib Junaidi , Suzuki Masami , Ozaki Satoru , Yajima Yoshinori , Suzuki Tsutomu
Published: May 2003

The present study was designed to investigate the effect of repeated administration of a selective κ‐opioid receptor agonist (1S‐trans)‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)cyclohexyl]‐benzeneacetamide hydrochloride [(–)U‐50,488H] on antinociception and G‐protein activation induced by µ‐opioid receptor agonists in mice. A single s.c. injection of (–)U‐50,488H produced a dose‐dependent antinociception, and this effect was reversed by a selective κ‐opioid receptor antagonist nor‐binaltorphimine (nor‐BNI). Furthermore, a single s.c. pre‐treatment with (–)U‐50,488H had no effect on the µ‐opioid receptor agonist‐induced antinociception. In contrast, repeated s.c. administration of (–)U‐50,488H resulted in the development of tolerance to (–)U‐50,488H‐induced antinociception. Under these conditions, we demonstrated here that repeated s.c. injection of (–)U‐50,488H significantly enhanced the antinociceptive effect of selective µ‐opioid receptor agonists endomorphin‐1, endomorphin‐2 and [d‐Ala2,N‐MePhe4,Gly‐ol5] enkephalin (DAMGO). Using the guanosine‐5′‐o‐(3‐[35S]thio) triphosphate ([35S]GTPγS) binding assay, we found that (–)U‐50,488H was able to produce a nor‐BNI‐reversible increase in [35S]GTPγS binding to membranes of the mouse thalamus, which has a high level of κ‐opioid receptors. Repeated administration of (–)U‐50,488H caused a significant reduction in the (–)U‐50,488H‐stimulated [35S]GTPγS binding in this region, whereas chronic treatment with (–)U‐50,488H exhibited the increase in the endomorphin‐1‐, endomorphin‐2‐ and DAMGO‐stimulated [35S]GTPγS bindings in membranes of the thalamus and periaqueductal gray. These results suggest that repeated stimulation of κ‐opioid receptors leads to the heterologous up‐regulation of µ‐opioid receptor functions in the thalamus and periaqueductal gray regions, which may be associated with the supersensitivity of µ‐opioid receptor‐mediated antinociception.
Hydrogen peroxide induces association between glyceraldehyde 3‐phosphate dehydrogenase and phospholipase D2 to facilitate phospholipase D2 activation in PC12 cells
Authors: Kim Jung Hwan , Lee Sukmook , Park Jong Bae , Lee Sang Do , Kim Jong Hyun , Ha Sang Hoon , Hasumi Keiji , Endo Akira , Suh Pann‐Ghill , Ryu Sung Ho
Published: May 2003

Oxidative stress or signaling is widely implicated in apoptosis, ischemia and mitogenesis. Previously, our group reported that the hydrogen peroxide (H2O2)‐dependent activation of phospholipase D2 (PLD2) in PC12 cells is involved in anti‐apoptotic effect. However, the precise mechanism of PLD2 activation by H2O2 was not revealed. To find H2O2‐dependent PLD2‐regulating proteins, we immunoprecipitated PLD2 from PC12 cells and found that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) coimmunoprecipitated with PLD2 upon H2O2 treatment. This interaction was found to be direct by in vitro reconstitution of purified GAPDH and PLD2. In vitro studies also indicated that PLD2‐associated GAPDH was modified on its reactive cysteine residues. Koningic acid, an alkylator of GAPDH on catalytic cysteine residue, also increased interaction between the two proteins in vitro and enhanced PLD2 activity in PC12 cells. Blocking H2O2‐dependent modification of GAPDH with 3‐aminobenzamide resulted in the inhibition of the GAPDH/PLD2 interaction and attenuated H2O2‐induced PLD2 activation in PC12 cells. From the results, we suggest that H2O2 modifies GAPDH on its catalytic cysteine residue not only to inactivate the dehydrogenase activity of GAPDH but also to endow GAPDH with the ability to bind PLD2 and the resulting association is involved in the regulation of PLD2 activity by H2O2.
Cloning, characterization and expression of two alternatively splicing isoforms of Ca2+/calmodulin‐dependent protein kinase Iγ in the rat brain
Authors: Nishimura Hideki , Sakagami Hiroyuki , Uezu Akiyoshi , Fukunaga Kohji , Watanabe Makoto , Kondo Hisatake
Published: May 2003

Ca2+/calmodulin‐dependent protein kinase I (CaMKI), originally identified as a protein kinase phoshorylating synapsin I, has been shown to constitute a family of closely related isoforms (α, β and γ). Here, we have isolated and determined the complete primary structures of two alternatively splicing isoforms of CaMKI termed CaMKIγ1 and ‐γ2. CaMKIγ1 and ‐γ2 contain an identical N‐terminal catalytic domain with different C‐terminal regions due to the deletion of the 425‐bp nucleotide sequence of CaMKIγ1 in CaMKIγ2. In vitro kinase assay has demonstrated the marked enhancement of the Ca2+/CaM‐dependent activity of CaMKIγ1 by the preincubation with Ca2+/calmodulin‐dependent protein kinase kinase (CaMKK), but no significant activation of CaMKIγ2. Northern blot analysis has demonstrated the predominant expression of CaMKIγ in the brain. RT‐PCR analysis has revealed similar expression patterns between CaMKIγ1 and CaMKIγ2 in various brain regions. In situ hybridization analysis has demonstrated that CaMKIγ mRNA is expressed in a distinct pattern from other isoforms of CaMKI with predominant expression in some restricted brain regions such as the olfactory bulb, hippocampal pyramidal cell layer of CA3, central amygdaloid nuclei, ventromedial hypothalamic nucleus and pineal gland. In the primary hippocampal neurons and NG108‐15 cells, transfected CaMKIγ1 and ‐γ2 are localized primarily in the cytoplasm and neurites but not in the nucleus. These findings suggest that both isoforms of CaMKIγ may be involved in Ca2+ signal transduction in the cytoplasmic compartment of certain neuronal population.
Temperature‐dependent, neurotrophic factor‐elicited, neuronal differentiation in adrenal chromaffin cell line immortalized with temperature‐sensitive SV40 T‐antigen
Authors: Murata Tomiyasu , Hikita Kiyomi , Tsuboi Masaru , Niwa Kentaro , Suzuki Misao , Kaneda Norio
Published: May 2003

We established adrenal medullary cell lines from transgenic mice expressing an oncogene, the temperature‐sensitive simian virus 40 large T‐antigen, under the control of the tyrosine hydroxylase promoter. A clonal cell line, named tsAM5D, conditionally grew at a permissive temperature of 33°C and exhibited the dopaminergic chromaffin cell phenotype as exemplified by the expression pattern of mRNA for catecholamine‐synthesizing enzymes and secretory vesicle‐associated proteins. tsAM5D cells proliferated at the permissive temperature in response to basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). At a non‐permissive temperature of 39°C, bFGF and CNTF acted synergistically to differentiate tsAM5D cells into neuron‐like cells. In addition, tsAM5D cells caused to differentiate by bFGF plus CNTF at 39°C became dependent solely on nerve growth factor for their survival and showed markedly enhanced neurite outgrowth. In the presence of bFGF and CNTF, the morphological change induced by the temperature shift was associated with up‐regulated expression of neuronal marker genes including neuron‐specific enolase, growth‐associated protein‐43, microtubule‐associated protein 2, neurofilament, and p75 neurotrophin receptor, indicating that the cells underwent neuronal differentiation. Thus, we demonstrated that tsAM5D cells could proliferate at permissive 33°C, and also had the capacity to terminally differentiate into neuron‐like cells in response to bFGF and CNTF when the oncogene was inactivated by shifting the temperature to non‐permissive 39°C. These results suggest that tsAM5D cells should be a good tool to allow a detailed study of mechanisms regulating neuronal differentiation.
Gliap − a novel untypical l‐asparaginase localized to rat brain astrocytes
Authors: Dieterich Daniela C. , Landwehr Marco , Reissner Carsten , Smalla Karl‐Heinz , Richter Karin , Wolf Gerald , Böckers Tobias M. , Gundelfinger Eckart D. , Kreutz Michael R.
Published: May 2003

l‐asparaginases catalyse the formation of the neuroactive amino acid l‐aspartate by deamination of asparagine. The major pathophysiological significance of l‐asparaginase activity is in its clinical use for the treatment of acute lymphatic leukaemia and neoplasias that require asparagine and obtain it from circulating pools. Here we report the identification and characterization of Gliap, a cytosolic l‐asparaginase, which is the founding member of a new group of l‐asparaginases in mammalia. Structural modelling suggests that Gliap is an atypical mammalian type‐I asparaginase inasmuch as it harbours the active centre of a type‐I glycosylasparaginase but, like plant‐type asparaginases, lacks their auto‐proteolytic site and, in addition, exhibits significant type‐II l‐asparaginase enzymatic activity. Moreover, in contrast to glycosylasparaginases Gliap is enriched in the cytosolic fraction and not in lysosomes. The protein is particularly abundant in liver, testis and brain. In brain Gliap is exclusively expressed in astrocytes and prominently present in structures reminiscent of glial endfeet. These data suggest that Gliap is involved in astroglial production of the neuroactive amino acid l‐aspartate.
An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid‐β peptide1−40‐induced toxicity in septal‐derived cholinergic SN56 cells
Authors: Marin Raquel , Guerra Borja , Morales Araceli , Díaz Mario , Alonso Rafael
Published: May 2003

Although oestrogen [17β‐estradiol (E2)]‐related neuroprotection has been demonstrated in different models, the involvement of non‐classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen‐dependent inhibition of cell death induced by amyloid‐β peptide (Aβ) toxicity. Similarly to E2 alone, a 15‐min exposure to estradiol‐horseradish peroxidase (E‐HRP) significantly reduced Aβ‐induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC‐20 antibody directed to a region neighbouring the ligand‐binding domain of ERα. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC‐20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC‐20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ERα. Using conjugated forms of the steroid, E‐HRP and E2 conjugated to bovine serum albumin‐FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre‐incubation with E2 and decreased by either ICI 182,780 or MC‐20 antibody in a concentration‐dependent manner. Thus, a membrane‐related ER that shares some structural homologies with ERα may participate in oestrogen‐mediated neuroprotection.
Concerted stimulation and deactivation of pertussis toxin‐sensitive G proteins by chimeric G protein‐coupled receptor‐regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4
Authors: Bahia Daljit S. , Sartania Nana , Ward Richard J. , Cavalli Antonella , Jones Teresa L. Z. , Druey Kirk M. , Milligan Graeme
Published: May 2003

Agonists stimulated high‐affinity GTPase activity in membranes of HEK293 cells following coexpression of the α2A‐adrenoceptor and a pertussis toxin‐resistant mutant of Go1α. Enzyme kinetic analysis of Vmax and Km failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N‐terminus of RGS4 was linked to the C‐terminal tail of the α2A‐adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the α2A‐adrenoceptor‐RGS4 fusion protein stimulated but did not enhance deactivation of a form of Go1α that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against Go1α. Mutation of all three cysteine residues that are sites of post‐translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein‐coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.
Molecular correlates of impaired prefrontal plasticity in response to chronic stress
Authors: Kuipers S. D. , Trentani A. , Den Boer J. A. , Ter Horst G. J.
Published: May 2003

Disturbed adaptations at the molecular and cellular levels following stress could represent compromised neural plasticity that contributes to the pathophysiology of stress‐induced disorders. Evidence illustrates atrophy and cell death of stress‐vulnerable neurones in the prefrontal cortex. Reduced plasticity may be realized through the destabilized function of selective proteins involved in organizing the neuronal skeleton and translating neurotrophic signals. To elucidate the mechanisms underlying these effects, rats were exposed to chronic footshock stress. Patterns of c‐fos, phospho‐extracellular‐regulated protein kinases 1/2 (ERK1/2), calcineurin and phospho‐cyclic‐AMP response‐element binding protein (CREB) expression were subsequently investigated. The results indicate chronic stress‐induced impairments in prefrontal and cingulate signal transduction cascades underlying neuronal plasticity. The medial prefrontal cortex, demonstrated functional hyperactivity and dendritic phospho‐ERK1/2 hyperphosphorylation, while reduced c‐fos and calcineurin immunoreactivity occurred in the cingulate cortex. Significantly reduced phospho‐CREB expression in both cortical regions, considering its implication in brain‐derived neurotrophic factor (BDNF) transcription, suggests reduced synaptic plasticity. This data confirms the damaging effect of stress on cortical activity, on a molecular level. Due to the association of these markers in the regulation of BDNF signalling, these findings suggest a central role for intracellular neurotrophin transduction members in the pathways underlying cellular actions of stress in the brain.
The same cellular signaling pathways mediate survival in sensory neurons that switch their trophic requirements during development
Authors: Salvarezza Susana B. , López Hector S. , Mascó Daniel H.
Published: May 2003

A distinct subpopulation of rat dorsal root sensory (DRG) neurons, termed P‐neurons, switch their trophic requirements for survival during development from nerve growth factor (NGF) at embryonic stages to basic fibroblast growth factor (bFGF) just after birth. We investigated in cultured P‐neurons the intracellular signaling pathways mediating survival before and after this switch. The NGF‐induced survival was completely blocked by either wortmannin (100 nm) or PD98059 (25–50 nm), which selectively inhibit the phosphatidylinositol 3‐kinase‐AKT (PI3 kinase‐AKT) and mitogen‐activated kinase kinase extracellular regulated kinase (MEK‐ERKs) pathways, respectively. NGF activated AKT and ERKs in single embryonic P‐neurons, as assayed by immunofluorescence of phorphorylated proteins. In concordance with the survival assays, wortmannin and PD98059 blocked AKT and ERKs activation, respectively. Following the trophic switch, bFGF used the same signaling pathways to promote survival of post‐natal P‐neurons, as either wortmannin or PD98059 blocked its effect. Also, bFGF activated AKT and ERKs in single P‐neurons, and this activation was blocked by the same inhibitors. These results strongly suggest that both pathways concurrently mediate the action of NGF and bFGF during embryonic and post‐natal periods, respectively. Thus, we report the novel result that the switch in trophic requirements occurs with conservation of the signaling pathways mediating survival.
Chronic nicotine administration does not increase nicotinic receptors labeled by [125I]epibatidine in adrenal gland, superior cervical ganglia, pineal or retina
Authors: Dávila‐García Martha I. , Musachio John L. , Kellar Kenneth J.
Published: May 2003

Neuronal nicotinic acetylcholine receptors (nAChRs) were measured in CNS and peripheral tissues following continuous exposure to saline or nicotine hydrogen tartrate (3.3 or 10 mg/kg/day) for 14 days via osmotic pumps. Initially, binding of [3H](–)nicotine, [3H]cytisine and [3H]epibatidine to nAChRs was compared to determine the suitability of each for these kinds of studies. The predominant nAChR labeled by agonists in the cerebral cortex is an α4β2 subtype, whereas the predominant nicotinic receptors in the adrenal gland, superior cervical ganglia and pineal gland contain an α3 subunit, and they do not bind either [3H](–)nicotine or [3H]cytisine with high affinity. In retina some nAChRs bind all three ligands with high affinity, and others appear to bind only [3H]epibatidine. Thus, only [3H]epibatidine had high enough affinity to be useful for measuring the nAChRs in all of the tissues. The receptors from nicotine‐treated rats were then measured using [125I]epibatidine, which has binding characteristics very similar to [3H]epibatidine. Treatment with the two doses of nicotine hydrogen tartrate increased binding sites in the cerebral cortex by 40% and 70%, respectively. In contrast, no significant changes in the density of receptor binding sites were found in the adrenal gland, superior cervical ganglia, pineal gland or retina. These data indicate that chronic administration of nicotine even at high doses does not increase all nicotinic receptor subtypes, and that receptors containing α3 subunits may be particularly resistant to this nicotine‐induced change.
MCP‐1 (CCL2) protects human neurons and astrocytes from NMDA or HIV‐tat‐induced apoptosis
Authors: Eugenin E. A. , D'Aversa T. G. , Lopez L. , Calderon T. M. , Berman J. W.
Published: May 2003

Acquired immunodeficiency syndrome (AIDS)‐associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV‐1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein‐1 (MCP‐1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA‐induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co‐treatment with MCP‐1 or RANTES but not IP‐10. The protective effect of RANTES was blocked by antibodies to MCP‐1, indicating that RANTES protection is mediated by the induction of MCP‐1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co‐treatment with MCP‐1 inhibited tat and NMDA‐induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP‐1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.