Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 14 (2009) No. 4

DOI: 10.2478/s11658-009-0017-1 Volume 14 (2009) pp 537-547
Title	THE FUNCTIONAL ANTAGONIST MET-RANTES: A MODIFIED AGONIST THAT INDUCES DIFFERENTIAL CCR5 TRAFFICKING
Authors	Debra L. Kiss^§, James Longden^§, Gregory A. Fechner and Vicky M. Avery*
Abstract	CC chemokine receptor 5 (CCR5) is a pro-inflammatory chemokine receptor that is expressed on cells of the immune system, and specializes in cell migration in response to inflammation and tissue damage. Due to its key role in cell communication and migration, this receptor is involved in various inflammatory and autoimmune diseases, in addition to HIV infection. Met- RANTES is a modified CCR5 ligand that has previously been shown to antagonize CCR5 activation and function in response to its natural ligands in vitro. In vivo, Met-RANTES is able to reduce inflammation in models of induced inflammatory and autoimmune diseases. However, due to the fact that Met-RANTES is also capable of partial agonist activity regarding receptor signaling and internalization, it is clear that Met-RANTES does not function as a conventional receptor antagonist. To further elucidate the effect of Met-RANTES on CCR5, receptor trafficking was investigated in a CHO-CCR5- GFP cell line using the Opera confocal plate reader. The internalization response of CCR5 was quantified, and showed that Met-RANTES internalized CCR5 in a slower, less potent manner than the agonists CCL3 and CCL5. Fluorescent organelle labeling and live cell imaging showed CCL3 and CCL5 caused CCR5 to traffic through sorting endosomes, recycling endosomes and the Golgi apparatus. In contrast, Met-RANTES caused CCR5 to traffic through sorting endosomes and the Golgi apparatus in a manner that was independent of recycling endosomes. As receptor trafficking impacts on cell surface expression and the ability of the receptor to respond to more ligand, this information may indicate an alternative regulation of CCR5 by Met-RANTES that allows the modified ligand to reduce inflammation through stimulation of a pro- inflammatory receptor.
Keywords	Met-RANTES, CCR5 trafficking, CCR5 internalization
Adress and Contact Informations	Discovery Biology, Eskitis Institute for Cell and Molecular Therapies, Brisbane Innovation Park, Griffith University, Don Young Road, Nathan, QLD, 4111, Australia ^§Contributed equally *Author for correspondence. e-mail: v.avery@griffith.edu.au, tel.: +061 737 356056, fax: +061 737 356001

DOI: 10.2478/s11658-009-0020-6 Volume 14 (2009) pp 548-574
Title	THE TRANSCRIPTIONAL REGULATION AND CELL-SPECIFIC EXPRESSION OF THE MAPK-ACTIVATED PROTEIN KINASE MK5
Authors	Nancy Gerits, Alexey Shiryaev, Sergiy Kostenko, Helle Klenow, Olga Shiryaeva, Mona Johannessen and Ugo Moens*
Abstract	The mitogen-activated protein kinase (MAPK) cascades regulate important cellular processes, including growth, differentiation, apoptosis, embryogenesis, motility and gene expression. Although MAPKs mostly appear to be constitutively expressed, the transcript levels of some MAPK-encoding genes increase upon treatment with specific stimuli. This applies to the MAPK- activated protein kinases MK2 and MK3. By contrast, the transcriptional regulation of the related MK5 has not yet been studied. The MK5 promoters of mouse, rat and human contain a plethora of putative transcription factor sites, and the spatio-temporal expression of MK5 suggests inducible transcription of the gene. We examined the transcription pattern of MK5 in different tissues, and studied the kinetics of MK5 expression at the transcriptional and/or translation level in PC12 cells exposed to arsenite, forskolin, KCl, lipopolysaccharide, spermine NONOate, retinoic acid, serum, phorbol ester, temperature shock, and vanadate. Cells exposed to forskolin display a transient increase in MK5 mRNA, despite their unaltered MK5 protein levels. The MK5 promoters of human, mouse and rat contain a cAMP-responsive element that binds the cAMP- responsive element-binding protein (CREB) in vitro. Luciferase reporter constructs containing an 850-base pair human MK5 promoter fragment encompassing the CRE showed a basal activity that was 10-fold higher than the corresponding construct in which the CRE motif was deleted. siRNA-mediated depletion of CREB had no effect on the endogenous MK5 protein levels. Several binding motifs for heat shock factor are dispersed in the mouse and rat promoter, and temperature shock transiently enhanced the MK5 transcript levels. None of the other tested stimuli had an effect on the MK5 mRNA or protein levels. Our results indicate an inducible regulation of MK5 transcription in response to specific stimuli. However, the MK5 protein levels remained unaffected by all the stimuli tested. There is still no explanation for the discrepancy between the increased mRNA and unchanged MK5 protein levels.
Keywords	Mitogen-activated protein kinase-activated protein kinase, MK5, Promoter, CREB, Heat-shock, Oxidative stress
Adress and Contact Informations	Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway * Author for correspondence. e-mail: ugom@fagmed.uit.no, tel.: +47-77644622, fax: +47- 77645350

DOI: 10.2478/s11658-009-0021-5 Volume 14 (2009) pp 575-586
Title	THE ANTI-APOPTOTIC ACTIVITY OF ALBUMIN FOR ENDOTHELIUM IS INHIBITED BY ADVANCED GLYCATION END PRODUCTS RESTRICTING INTRAMOLECULAR MOVEMENT
Authors	Hans Zoellner¹*, Salman Siddiqui¹, Elizabeth Kelly¹ and Heather Medbury²
Abstract	Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intra- molecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p < 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.
Keywords	Apoptosis, Cryptic domain, Endothelium, HSA, HSA-AGE
Adress and Contact Informations	¹Cellular and Molecular Pathology Research Unit, Department of Oral Pathology and Oral Medicine, Westmead Centre for Oral Health, The University of Sydney, Westmead, NSW 2145, Australia, ²Department of Surgery, Westmead Hospital, Westmead, NSW, 2145, Australia * Author for correspondence. e-mail: hzoellne@mail.usyd.edu.au, tel.: + 61 2 9845 7892, fax: +61 2 9893 8671

DOI: 10.2478/s11658-009-0023-3 Volume 14 (2009) pp 587-608
Title	TRANSCRIPTIONAL PROFILES DURING THE DIFFERENTIATION AND MATURATION OF MONOCYTE-DERIVED DENDRITIC CELLS, ANALYZED USING FOCUSED MICROARRAYS
Authors	Weixue Zhong^1#, Min Fei^1#, Yibei Zhu^1,2 and Xueguang Zhang^1,2*
Abstract	Dendritic cells (DC) are professional antigen-presenting cells capable of initiating primary immune responses. They have been intensively studied and are used in both basic immunology research and clinical immunotherapy. However, the genetic pathways leading to DC differentiation and maturation remain poorly understood. Using focused microarrays with oligonucletotide probes for 120 genes encoding co-stimulatory molecules, chemokines, chemokine receptors, cytokines, cytokine receptors, TLRs, and several other related molecules, we analyzed the kinetics of gene expression for the overall differentiation process of monocytes into mature DC. In parallel, we compared the transcriptional profiles in DC maturation in the presence of LPS, TNF-α or trimeric CD40L. We found similar transcriptional profiles for early immature DC and immature DC, respectively generated by culturing monocytes with GM- CSF and IL-4 for three or six days. We identified sets of common and stimuli- specific genes, the expression of which changed following stimulation with LPS, TNF-α or CD40L. A dynamic analysis of the entire DC differentiation and maturation process showed that some important inflammatory and constitutive chemokines are transcribed in both immature and mature DC. The correlative expression kinetics of the gene pairs IL1R1/IL1R2, IL15/IL15RA, DC-SIGN/ICAM-2 and DC-SIGN/ICAM-3 imply that they all play crucial roles in mediating DC functions. Thus, our analysis with focused microarrays shed light on the transcriptional kinetics of DC differentiation and maturation, and this method may also prove useful for identifying novel marker genes involved in DC functions.
Keywords	Dendritic cells, Monocytes, Microarray, Transcriptional profile
Adress and Contact Informations	¹Institute of Biotechnology and Clinical Immunology Research Laboratory of Jiangsu Province, Soochow University, Suzhou, P.R. China, ²Stem Cell Research Laboratory of Jiangsu Province, Soochow University, Suzhou, P.R. China ^#These authors contributed equally to this work. * Author for correspondence. e-mail: smbxuegz@public1.sz.js.cn, tel/fax: +86 512 6510 4908

DOI: 10.2478/s11658-009-0022-4 Volume 14 (2009) pp 609-621
Title	*THE INFLUENCE OF JNK AND P38 MAPK INHIBITION ON IL-12P40 AND IL-23 PRODUCTION DEPENDING ON IL12B* PROMOTER POLYMORPHISM**
Authors	Zlatka Georgieva Dobreva*, Spaska Angelova Stanilova and Lyuba Dineva Miteva
Abstract	The interleukin-12p40 gene (IL12B) encodes the p40 polypeptide chain, which, together with p19, composes IL-23. A bi-allelic promoter polymorphism (IL12Bpro) located at -2703 bp of the transcription initiation site has been reported to show associations with IL-12p40 production. To elucidate the dependence of IL-12p40 and IL-23 production on IL12Bpro polymorphism in relation to MAPK signal transduction pathways, we examined the effect of JNK and p38 inhibition on the secretion of these cytokines by stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with 1.1 and 1.2/2.2 IL12Bpro genotypes. Stimulation with LPS and C3bgp resulted in approximately equal IL-12p40 production from PBMC with the 1.1 and 1.2/2.2 genotypes. The inhibition of JNK and p38 before stimulation significantly upregulated IL-12p40 production by PBMC with the 1.1 genotype, but did not influence IL-12p40 production from PBMC with the 1.2/2.2 genotype. Cultures of PBMC with the 1.1 genotype produced significantly more IL-12p40 than PBMC with the 1.2/2.2 genotype after stimulation with PHA. Inhibition of p38 kinase upregulated p40 production only in cultures with the 1.1 genotype. Decreased IL-23 production was observed in C3bgp-stimulated cultures after the inhibition of p38 regardless of the genotype of the tested cells. We concluded that IL-12p40 and IL-23 expression, which is mediated by the p38 and JNK intracellular signaling pathways, is influenced by IL12Bpro polymorphism.
Keywords	IL-12p40, IL-23, IL12B, Promoter polymorphism
Adress and Contact Informations	Department of Molecular Biology, Immunology & Genetics, Faculty of Medicine, Trakia University, Armeiska 11 St., 6000 Stara Zagora, Bulgaria * Author for correspondence. e-mail: zdobreva@mf.uni-sz.bg, tel.: +359-42-600-675, fax: +359-42-600-705

DOI: 10.2478/s11658-009-0025-1 Volume 14 (2009) pp 622-635
Title	THE IMMUNOSUPPRESSIVE ACTIVITIES OF NEWLY SYNTHESIZED AZAPHENOTHIAZINES IN HUMAN AND MOUSE MODELS
Authors	Michał Zimecki¹, Jolanta Artym¹, Maja Kocięba¹, Krystian Pluta²*, Beata Morak-Młodawska² and Małgorzata Jeleń²
Abstract	In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutinin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 µg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.
Keywords	Azaphenothiazines, Immune response, PBMC, Proliferation, Cytokines
Adress and Contact Informations	¹Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Therapy, R. Weigla 12, 53-114 Wrocław, Poland, ²The Medical University of Silesia, Department of Organic Chemistry, Jagiellońska 4, 41-200 Sosnowiec, Poland * Author for correspondence. e-mail: pluta@sum.edu.pl, fax: (+48 32) 266 78 60

DOI: 10.2478/s11658-009-0018-0 Volume 14 (2009) pp 636-656
Title	MECHANISMS FOR THE FORMATION OF MEMBRANOUS NANOSTRUCTURES IN CELL-TO-CELL COMMUNICATION ^#
Authors	Karin Schara^1,2, Vid Jansa¹, Vid Sustar¹, Drago Dolinar^1,2, Janez Ivan Pavlic^3,4, Marusa Lokar⁴, Veronika Kralj-Iglic¹, Peter Veranic⁵ and Ales Iglic⁴*
Abstract	Cells interact by exchanging material and information. Two methods of cell-to-cell communication are by means of microvesicles and by means of nanotubes. Both microvesicles and nanotubes derive from the cell membrane and are able to transport the contents of the inner solution. In this review, we describe two physical mechanisms involved in the formation of microvesicles and nanotubes: curvature-mediated lateral redistribution of membrane components with the formation of membrane nanodomains; and plasma- mediated attractive forces between membranes. These mechanisms are clinically relevant since they can be affected by drugs. In particular, the underlying mechanism of heparin’s role as an anticoagulant and tumor suppressor is the suppression of microvesicluation due to plasma-mediated attractive interaction between membranes.
Keywords	Membrane nanostructures, Cell-to-cell communication, Microvesicles, Nanotubes, Trousseau syndrome, Heparin
Adress and Contact Informations	¹Laboratory of Clinical Biophysics, Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Lipičeva 2, SI-1000 Ljubljana, Slovenia, ²University Medical Centre Ljubljana, Zaloška 9, SI-1000 Ljubljana, Slovenia, ³Faculty of Health Studies, University of Ljubljana, Poljanska 26a, SI-1000 Ljubljana, Slovenia, ⁴Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, SI-1000 Ljubljana, Slovenia, ⁵Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Lipičeva 2, SI-1000 Ljubljana, Slovenia ^#The content of this Review was first presented in a shortened form at the 12th Mejbaum-Katzenellenbogen Seminar “Membrane Skeleton. Recent Advances and Future Research Directions”, June 15-18, 2008, Zakopane, Poland. Publication cost was partially covered by the organizers of this meeting. * Author for correspondence. e-mail: ales.iglic@fe.uni-lj.si, tel.: +386 1 4768 825

DOI: 10.2478/s11658-009-0024-2 Volume 14 (2009) pp 657-669
Title	ARGININE METHYLATION ANALYSIS OF THE SPLICING- ASSOCIATED SR PROTEIN SFRS9/SRP30C
Authors	Gustavo C. Bressan^1,2, Eduardo C. Moraes^1,2, Adriana O. Manfiolli³, Tais M. Kuniyoshi¹, Dario O. Passos^1,2, Marcelo D. Gomes³ and Jorg Kobarg^1,2*
Abstract	The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.
Keywords	Nuclear bodies, Speckles, RGG boxes, Arginine methylation, Protein-protein interaction
Adress and Contact Informations	¹Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brasil, ²Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil, ³Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, Ribeirão Preto, SP, Brasil * Author for correspondence: Jörg Kobarg, Laboratório Nacional de Luz Síncrotron, Centro de Biologia Molecular Estrutural, Rua Giuseppe Máximo Scolfaro 10.000, C.P. 6192, 13084-971 Campinas - SP, Brasil, e-mail: jkobarg@lnls.br, tel.: (+55) 19-3512-1125, fax: (+55) 19-3512-1006

DOI: 10.2478/s11658-009-0027-z Volume 14 (2009) pp 670-678
Title	OVEREXPRESSION OF HUMAN OSTEOPONTIN INCREASES CELL PROLIFERATION AND MIGRATION IN HUMAN EMBRYO KIDNEY-293 CELLS
Authors	Ya-Jun Liu¹, Dao-Qiang Zhang², Xiu-Mei Sui² and Wei Tian¹
Abstract	Malignant tumors are characterized by dysregulated cell growth and the metastasis of secondary tumors. Numerous studies have documented that osteopontin (OPN) plays a key role in regulating tumor progression and metastasis. Here, we show that the overexpression of OPN in human embryo kidney-293 cells significantly increases both the level of cell proliferation, by provoking the G₁/S transition, and the level of cell migration in vitro. These findings suggest that augmented OPN contributes to cell growth and motility. Inhibiting OPN or the pathway it stimulates may therefore represent a novel approach for the treatment of primary tumors and associated metastases.
Keywords	Osteopontin, Overexpression, Proliferation, Metastasis
Adress and Contact Informations	¹Beijing Jishuitan Hospital, Peking University, Beijing 100035, P. R. China ²Wendeng Central Hospital, Weifang Medical College, Weihai, Shandong Province 264400, P. R. China * Author for correspondence. Ya-Jun Liu, e-mail: yajun.liu001@gmail.com and Wei Tian, tianweia@163bj.com

DOI: 10.2478/s11658-009-0026-0 Volume 14 (2009) pp 679-691
Title	THE TRANSCRIPTIONAL REGULATION OF PODOCIN (NPHS2) BY Lmx1b AND A PROMOTER SINGLE NUCLEOTIDE POLYMORPHISM
Authors	Sigrid Harendza*, Rolf A.K. Stahl and Andre Schneider
Abstract	Podocin (NPHS2) is a component of the glomerular slit membrane with major regulatory functions in the renal permeability of proteins. A loss of podocin and a decrease in its resynthesis can influence the outcome of renal diseases with nephrotic syndrome, such as minimal change glomerulonephritis, focal segmental glomerulosclerosis (FSGS) and membranous nephropathy. The transcriptional regulation of podocin may play a major role in these processes. We defined the transcriptional regulation of the human podocin gene and the influence of single nucleotide polymorphisms (SNPs) within its promoter region in the podocytes using reporter gene constructs and gel shift analysis. In addition, we took genomic DNA from healthy Caucasian blood donors and from biopsies of kidneys with defined renal diseases and screened it for podocin promoter SNPs. Our data shows that the transcription of podocin is mainly regulated by the transcription factor Lmx1b, which binds to a FLAT-F element and displays enhancer function. With the SNP variant -116T, there was a significant reduction in luciferase activity, and nuclear protein binding was observed, while the SNP -670C/T did not display functionality. The allelic distribution of -116C/T in patients with kidney diseases leading to nephrotic syndrome was not significantly different from that in the control group. Our data indicates that among other factors, podocin is specifically regulated by the transcription factor Lmx1b and by the functional polymorphism -116C/T. However, there is no association between -116C/T and susceptibility to minimal change glomerulonephritis, focal segmental glomerulosclerosis or membranous nephropathy.
Keywords	Lmx1b, Nephrotic syndrome, NPHS2 gene, Podocin promoter, Proteinuria, SNP, Transcription
Adress and Contact Informations	Universitätsklinikum Hamburg-Eppendorf, III. Medizinische Klinik, Martinistr. 52, D-20246 Hamburg, Germany * Author for correspondence. e-mail: harendza@uke.de, tel.: +4940428033908, fax: +4940428035186

DOI: 10.2478/s11658-009-0028-y Volume 14 (2009) pp 692-702
Title	*MOLECULAR CHARACTERIZATION OF THE niaD* AND pyrG GENES FROM Penicillium camemberti, AND THEIR USE AS TRANSFORMATION MARKERS**
Authors	Katherinne Navarrete¹, Amanda Roa¹, Inmaculada Vaca², Yeison Espinosa¹, Claudio Navarro¹ and Renato Chavez^1,*
Abstract	Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5’-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.
Keywords	Penicillium camemberti, niaD gene, pyrG gene, Transformation, Aspergillus nidulans
Adress and Contact Informations	¹Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile (USACH), Alameda 3363, Estación Central 9170022, Santiago, Chile, ²Departamento de Química, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ńuńoa, Santiago, Chilel * Author for correspondence. e-mail: renato.chavez@usach.cl, tel.: +56-2-7181091, fax: +56-2-6812108

DOI: 10.2478/s11658-009-0029-x Volume 14 (2009) pp 703-714
Title	INCREASED EXPRESSION OF PAD2 AFTER REPEATED INTRACEREBROVENTRICULAR INFUSIONS OF SOLUBLE Aβ_25-35 IN THE ALZHEIMER’S DISEASE MODEL RAT BRAIN: EFFECT OF MEMANTINE
Authors	Mohammad Arif^1,2* and Takeshi Kato¹
Abstract	Peptidylarginine deiminases (PADs) convert the arginine residues in proteins into citrulline residues in a Ca ²⁺ -dependent manner. We previously showed that a bilateral injection of ibotenic acid into the rat nucleus basalis magnocellularis elevated the PAD2 activity in the hippocampus and striatum. In this study, we examined whether repeated intracerebroventricular infusions of soluble Aβ_25-35 would affect the PAD2 expression in any regions of the rat brain. We also assessed the protective effect of memantine on Aβ-induced PAD2 alterations. The infusion of Aβ_25-35 increased the activity and protein level of PAD2 in the hippocampus, and co-treatment with memantine suppressed these changes. An immunohistochemical analysis showed that an increased level of PAD2 was coincident with GFAP-positive astrocytes and CD11b-positive microglia. In addition, immunofluoresecence staining revealed that citrulline- postive immunoreactivity coincided with the occurrence of GFAP-positive astrocytes. Co-treatment with memantine reversed the activation of the astrocytes and microglia, thus attenuating the PAD2 increment. These biochemical and immunohistochemical results suggest that PAD2 might play an important role in the pathology of early Alzheimer’s disease, and may correlate with the changes in glial cells that are recovered by memantine treatment.
Keywords	Peptidylarginine deiminase, Alzheimer’s disease, Astrocytes, Microglia, Memantine
Adress and Contact Informations	¹Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan, ²Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh * Author for correspondence. e-mail: marif@univdhaka.edu; marif567@yahoo.com, tel.: 880-2-9661920-59, ext: 7643, fax: 880-2-8615583

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