Vol. 17 No. 1 March 2012

DOI: 10.2478/s11658-011-0031-y Volume 17 (2012) pp 1-10
Authors Stina K. Carlsson* and J. Peter Gierow
Abstract The lacrimal gland (LG) is an exocrine gland important for secretion of the tear film. The kinase p38 has important signal transduction functions, e.g. in gene transcription, but has previously not been known to modulate exocrine secretion. The aim of the current study was to investigate the role of p38 in carbachol (Cch)-induced LG secretion in LG acinar cells in vitro. Western blotting was used to determine the phosphorylation status of p38 and p42/44 and determine expression of p38 isoforms. To determine the effect of p38 inhibition on LG secretion, PD 16931 6, a general p38 inhibitor, and SB 239063, an inhibitor of p38 α and ß, were added to the cells prior to secretion measurements. The results revealed activation of p38 mediated by Cch stimulation and inhibition of Cch-induced secretion as a result of p38 inhibition. The inhibition was observed with PD 16931 6 isoforms, but not with SB 239063. The p38δ isoform was shown to have robust expression both by Western blotting of acinar cells and immunofluorescence of the whole gland. In conclusion, p38 activation mediates secretion in cholinergic stimulation of rabbit LG cells.
Keywords p38 mitogen-activated protein kinase, Lacrimal gland, Exocrine glands, Signal transduction, Cholinergic receptors
Address and Contact Information School of Natural Sciences, Linnaeus University, SE-39182 Kalmar, Sweden
* Author for correspondence. e-mail: stina.carlsson@lnu.se, phone: +46480-446184, fax: +46480446262
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DOI: 10.2478/s11658-011-0032-x Volume 17 (2012) pp 11-20
Authors Mehrdad Ghashghaeinia1,3, Mahmoud Toulany2, Mohammad Saki2, H. Peter Rodemann2, Ulrich Mrowietz 3, Florian Lang1* and Thomas Wieder4
Abstract Anucleated erythrocytes were long considered as oxygen-transporting cells with limited regulatory functions. Components of different nuclear signaling pathways have not been investigated in those cells, yet. Surprisingly, we repeatedly found significant amounts of transcription factors in purified erythrocyte preparations, i.e. nuclear factor κB (NFκB), and major components of the canonical NFκB signaling pathway. To investigate the functional role of NFκB signaling, the effects of the preclinical compounds Bay 11-7082 and parthenolide on the survival of highly purified erythrocytes were investigated. Interestingly, both inhibitors of the NFκB pathway triggered erythrocyte programmed cell death as demonstrated by enhanced phospholipid scrambling  (phosphatidylserine exposure) and cell shrinkage. Anucleated erythrocytes are an ideal cellular model allowing the study of nongenomic mechanisms contributing to suicidal cell death. As NFκB inhibitors might also interfere with the anti-oxidative defense systems of the cell, we measured the levels of reduced glutathione (GSH) after challenge with the inhibitors. Indeed, incubation of erythrocytes with Bay 11-7082 clearly decreased erythrocyte GSH levels. In conclusion, the pharmacological inhibitors of the NFκB pathway Bay 11-7082 and parthenolide interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression. Besides affecting erythrocyte survival, NFκB inhibition and induction of erythrocyte phosphatidylserine exposure may influence blood clotting. Future studies will be aimed at discriminating between NFκB-dependent and NFκ B-independent GSH-mediated effects of Bay 11-7082 and parthenolide on erythrocyte death.
Keywords Eryptosis, Nuclear factor kappa B, NF κB, IKK-α, I κB- α, Bay 11-7082, Parthenolide
Address and Contact Information 1Department of Physiology, Eberhard Karls University Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany,
2Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University Medical Center Tübingen, Röntgenweg 11, D-72076 Tübingen, Germany,
3Department of Dermatology, University Medical Center Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany,
4Department of Dermatology, University Medical Center Tübingen, Röntgenweg 13/1, D-72076 Tübingen, Germany
# Paper authored by participants of the international conference: 18 th Meeting, European Association for Red Cell Research, Wrocław – Piechowice, Poland, May 12-15 th, 2011. Publication cost was covered by the organizers of this meeting.
* Author for correspondence. e-mail: florian.lang@uni-tuebingen.de, phone: +49 7071 29 72194, fax: +49 7071 29 5618
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DOI: 10.2478/s11658-011-0033-9 Volume 17 (2012) pp 21-35
Authors Barbara Ziemba*, Gabriela Matuszko, Maria Bryszewska and Barbara Klajnert
Abstract Dendrimers, highly branched macromolecules with a specific size and shape, provide many exciting opportunities for biomedical applications. However, most dendrimers demonstrate toxic and haemolytic activity because of their positively charged surface. Masking the peripheral cationic groups by coating them with biocompatible molecules is a method to reduce it. It was proven that modified dendrimers can even diminish haemolytic activity of encapsulated drugs. Experiments confirmed that anionic dendrimers are less haemotoxic than cationic ones. Due to the high affinity of dendrimers for serum proteins, presence of these components in an incubation buffer might also influence red blood cell (RBC)-dendrimer interactions and decrease the haemolysis level. Generally, haemotoxicity of dendrimers is concentration-, generation-, and time-dependent. Various changes in the RBCs’ shape in response to interactions with dendrimers have been observed, from echinocytic transformations through cell aggregation to cluster formation, depending on the dendrimer’s type and concentration. Understanding the physical and chemical origins of dendrimers’ influences on RBCs might advance scientists’ ability to construct dendrimers more suita ble for medical applications.
Keywords Dendrimer, Erythrocytes, Haemolysis, Red blood cell, Red blood cell morphology, Toxicity
Address and Contact Information Department of General Biophysics, University of ŁódĄ, 141/143 Pomorska St., 90-236 ŁódĄ, Poland
# Paper authored by participants of the international conference: 18th Meeting, European Association for Red Cell Research, Wrocław – Piechowice, Poland, May 12-15th, 2011. Publication cost was covered by the organizers of this meeting.
* Author for correspondence. e-mail: barzie@biol.uni.lodz.pl, tel.: (+48 42) 635 41 44, fax: (+48 42) 635 44 74
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DOI: 10.2478/s11658-011-0034-8 Volume 17 (2012) pp 36-48
Authors Hiroko Toyota, Xiao-Zhou Jiang, Hideki Asakura and Junichiro Mizuguchi*
Abstract Thy28 protein is conserved among plants, bacteria, and mammalian cells. Nuclear Thy28 protein is substantially expressed in testis, liver, and immune cells such as lymphocytes. Lymphocyte apoptosis plays a crucial role in homeostasis and formation of a diverse lymphocyte repertoire. In this study, we examined whether Thy28 affects in duction of apoptosis in WEHI-231 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Once they were established, the Thy28-overexpressing WEHI-231 cells showed similar expression levels of IgM and class I major histocompatibility complex (MHC) molecule compared with controls. The Thy28-overexpressing cells were considerably resistant to loss of mitochondrial membrane potential ( ΔΨm), caspase-3 activation, and increase in annexin-positive cells upon mIg engagement. These changes were concomitant with an increase in G1 phase associated with upregulation of p27Kip1. The anti-IgM-induced sustained activation of c-Jun N-terminal kinase (JNK), which was associated with late-phase hydrogen peroxide(H2O2) production, was partially reduced in the Thy28-expressing cells relative to controls. Taken together, the data suggest that in WEHI-231 B lymphoma cells, Thy28 regulates mIg-mediated apoptotic events through the JNK-H2O2 activation pathway, concomitant with an accumulation of cells in G1 phase associated with upregulation of p27Kip1 in WEHI-231 B lymphoma cells.
Keywords Thy28, B lymphoma cells, Apoptosis, Membrane immunoglobulin
Address and Contact Information Department of Immunology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan
* Author for correspondence. e-mail: mizu@tokyo-med.ac.jp
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DOI: 10.2478/s11658-011-0036-6 Volume 17 (2012) pp 49-61
Authors Basak Varol, Muhammet Bektas*, Rustem Nurten and Engin Bermek
Abstract Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as Km=2.2 nM; Vmax=0.25 pmol.min-1. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60-65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo .
Keywords ADP-ribosylation, Diphtheria toxin, Eukaryotic elongation factor-2, F-actin, Fragment A
Address and Contact Information Istanbul University, Istanbul Faculty of Medicine, Department of Biophysics, 34390 Çapa-Istanbul-Türkiye
* Author for correspondence. E-mail: muhbektas@hotmail.com, fax: 00 90 212 4142187
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DOI: 10.2478/s11658-011-0035-7 Volume 17 (2012) pp 62-76
Authors Jihye Lee1, Gunsup Lee2, Jin Hee Park2, Sukchan Lee2, Chang-Hwan Yeom3, Byungjo Na4 and Seyeon Park1*
Abstract Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression and protein modification. Proteome changes of tumor tissue were investigated after intraperitoneal administration of a high concentration of ascorbic acid in BALB/C mice implanted with CT-26 cancer cells using two-dimensional gel electrophoresis and mass spectrometry. Eighteen protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, eukaryotic translation initiation factor 3 subunit 1, nucleophosmin, latexin, actin-related protein 2/3 complex subunit 5, M2-type pyruvate kinase, vimentin, tumor protein translationally-controlled 1, RAS oncogene family Ran, plastin 3 precursor, ATPase, Rho GDT dissociation inhibitor ß, and proteasome activator subunit 2 expression were quantitatively up-regulated. The increase in the level of these proteins was accompanied by an increase in mRNA level. The cytoskeleton protein actin, vimentin, and tumor protein translationally-controlled 1 showed quantitative expression profile differences. A change in actin cytoskelet on distribution, functionally relevant to the proteome result, was observed after treatment with ascorbic acid. These results suggest a previously undefined role of ascorbic acid in the regulation of cytoskeleton remodeling in tumor tissues.
Keywords Cytoskeleton remodelling, Ascorbic acid, Proteomics, Tumor tissue, mRNA
Address and Contact Information 1Department of Applied Chemistry, Dongduk Women’s University, 23-1 Wolgok-dong, Sungbuk-ku, Seoul 136-714, Korea,
2Department of Genetic Engineering, Sungkyunkwan University, Suwon, Korea,
3Yeom's Clinic of Palliative Medicine, Seoul, Korea,
4Weedam Korean Hospital, Seoul, Korea
* Author for correspondence. e-mail: sypark21@dongduk.ac.kr, phone: +82-2-940-4514, fax: +82-2-940-4193
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DOI: 10.2478/s11658-011-0038-4 Volume 17 (2012) pp 77-88
Authors Sylwia Cyboran1*, Jan Oszmiański2 and Halina Kleszczyńska1
Abstract The purpose of these studies was to determine the effect of polyphenols contained in extracts from apple, strawberry and blackcurrant on the properties of the erythrocyte membrane, treated as a model of the biological membrane. To this end, the effect of the substances used on hemolysis, osmotic resistance and shape of erythrocytes, and on packing order in the hydrophilic region of the erythrocyte membrane was studied. The investigation was performed with spectrophotometric and fluorimetric methods, and using the optical microscope. The hemolytic studies have shown that the extracts do not induce hemolysis at the concentrations used. The results obtained from the spectrophotometric measurements of osmotic resistance of erythrocytes showed that the polyphenols contained in the extracts cause an increase in the resistance, rendering them less prone to hemolysis in hypotonic solutions of sodium chloride. The fluorimetric studies indicate that the used substances cause a decrease of packing order in the hydrophilic area of membrane lipids. The observations of erythrocyte shapes in a biological optical microscope have shown that, as a result of the substances’ action, the erythrocytes become mostly echinocytes, which means that the polyphenols of the extracts localize in the outer lipid monolayer of the erythrocyte membrane. The results obtained indicate that, in the concentration range used, the plant extracts are incorporated into the hydrophilic area of the membrane, modifying its properties.
Keywords Erythrocyte membrane, Plant polyphenols, Hemolysis, Osmotic resistance, Echinocytes, Generalized polarization, Lipid packing order
Address and Contact Information 1Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland,
2Department of Fruit, Vegetable and Grain Technology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
# Paper authored by participants of the international conference: 18th Meeting, European Association for Red Cell Research, Wrocław – Piechowice, Poland, May 12-15th, 2011. Publication cost was covered by the organizers of this meeting.
*Author for correspondence. e-mail: sylwia.cyboran@up.wroc.pl; phone: +48 71 320 5418; fax: + 48 71 320 5167
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DOI: 10.2478/s11658-011-0040-x Volume 17 (2012) pp 89-106
Authors Mahboobe Ghaedi1,2,3, Masoud Soleimani4, Iman Shabani5, Yuyou Duan*2 and Abbas S. Lotfi*1,3
Abstract The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a he patocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multi potency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.
Keywords Poly L-lactic acid, Nanofiber, Hepatic differentiation, Mesenchymal stem cells, Electrospinning
Address and Contact Information 1Department of Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, I.R. Iran,
2Transplant Research Program, Department of Internal Medicine, University of California Davis Medical Center, Sacramento, 4635 2nd Ave, Suite 1001, California, CA 95817, USA,
3National Institute for Genetic Engineering and Biotechnology, Pajuhesh Blvd, Tehran, I.R. Iran,
4Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, I.R. Iran,
5Department of Stem Cells and Tissue Engineering, Stem Cell Technology Co. Ltd., Tehran, I.R. Iran
* Authors for correspondence: Abbas S. Lotfi, e-mail: lotfi-ab@nigeb.ac.ir, tel.: +9821 4458 0376; fax: +9821 4458 0395, Yuyou Duan, e-mail: yduan@ucdavis.edu, tel.: (916) 734 7901, fax: (916) 734 8097
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DOI: 10.2478/s11658-011-0041-9 Volume 17 (2012) pp 107-123
Authors Adam Kawalek, Marta Dmochowska-Boguta, Anna Nadolska-Orczyk and Waclaw Orczyk*
Abstract Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:ß molecule was engineered with a unique BamHI site between the open reading frame of ßc (ORF ßc) and poly(A). The mixture of RNA particles α, ßBamHI and γ MCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to ßBamHI and γ MCS. Delivery of the target gene fragment in γ MCS induced stronger silencing, while delivery in ßBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of ß enables simultaneous silencing of two genes. Quantifying the ß and γ particles in virus-inoculated plants revealed a 2.5-fold higher level of γ than ß, while the stability of the insert was higher in ß compared with γ . The possible influence of the relative quantity of ß and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.
Keywords BSMV, Cereals, Functional analysis, Phospholipase D, Phytoene desaturase, Silencing, Wheat, Vector stability, VIGS
Address and Contact Information Plant Breeding and Acclimatization Institute – National Research Institute, 05-870 Błonie, Poland
* Author for correspondence. e-mail: w.orczyk@ihar.edu.pl, tel.: +4822 733 4621, fax: +4822 725 4714
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DOI: 10.2478/s11658-011-0043-7 Volume 17 (2012) pp 124-135
Authors Krishna P. Subedi§, Thoudam Debraj Singh§, Joon-Chul Kim and Sun-Hee Woo*
Abstract Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP3R1 in the rat brain has been reported, the complete sequence of an IP3R1 clone from atrial myocytes has not been reported. We isolated an IP3R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP3R1 that was different from a previously reported IP3R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP3R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693-1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP3R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440-2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further inves tigation is needed on the physiological significance of the new splice variant in atrial cell function.
Keywords Type 1 inositol 1,4,5-trisphosphate receptor type, Splice variant, Rat atrial myocytes, Cloning, Expression
Address and Contact Information College of Pharmacy, IDRD, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, South Korea
§ K. P. Subedi and T. D. Singh contributed equally to this work.
* Author for correspondence. e-mail: shwoo@cnu.ac.kr, tel.: +82-42-821-5924, fax: +82-42-823-6566
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DOI: 10.2478/s11658-011-0042-8 Volume 17 (2012) pp 136-152
Authors Archita Rajasekharan and Sathyanarayana N. Gummadi*
Abstract Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.
Keywords Biogenic membrane flippase, Cholesterol, Endoplasmic reticulum, Flip flop
Address and Contact Information Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India
* Author for correspondence. e-mail: gummadi@iitm.ac.in, tel.: 91-44-22574114, fax: 91-44-22574102
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DOI: 10.2478/s11658-011-0039-3 Volume 17 (2012) pp 153-170
Authors Lisa S. Weingarten1, Hardi Dave2, Hongyan Li2 and Dorota A. Crawford1,2,3*
Abstract P5 ATPases (ATP13A1 through ATP13A5) are found in all eukaryotes. They are currently poorly characterized and have unknown substrate specificity. Recent evidence has linked two P5 ATPases to diseases of the nervous system, suggesting possible importance of these proteins within the nervous system. In this study we determined the relative expression of mouse P5 ATPases in development using quantitative real time PCR. We have shown that ATP13A1 and ATP13A2 were both expressed similarly during development, with the highest expression levels at the peak of neurogenesis. ATP13A3 was expressed highly during organogenesis with one of its isoforms playing a more predominant role during the period of neuronal development. ATP13A5 was expressed most highly in the adult mouse brain. We also assessed the expression of these genes in various regions of the adult mouse brain. ATP13A1 to ATP13A4 were expressed differentially in the cerebral cortex, hippocampus, brainstem and cerebellum while levels of ATP13A5 were fairly constant between these brain regions. Moreover, we demonstrated expression of the ATP13A4 protein in the corresponding brain regions using immunohistochemistry. In summary, this study furthers our knowledge of P5-type ATPases and their potentially important role in the nervous system.
Keywords P5-type ATPases, mRNA expression, Neurogenesis, Parkinson’s disease, Autism spectrum disorders, Real-time PCR, Immunohistochemistry
Address and Contact Information 1Department of Biology, York University, Faculty of Science and Engineering, 4700 Keele Street, Bethune College, Toronto, Ontario M3J-1P3, Canada,
2School of Kinesiology and Health Science, Faculty of Health, York University, Toronto, Canada,
3Neuroscience Graduate Diploma Program, Faculty of Health, York University, Toronto, Canada
* Author for correspondence. e-mail : dakc@yorku.ca, phone: 416-736-2100 ext. 23324, fax: 416-736-5774
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