Vol. 17 No. 2 June 2012

DOI: 10.2478/s11658-012-0001-z Volume 17 (2012) pp 171-181
Title RED BLOOD CELL SHAPE AND DEFORMABILITY IN THE CONTEXT OF THE FUNCTIONAL EVOLUTION OF ITS MEMBRANE STRUCTURE#
Authors Sasa Svetina*
Abstract It is proposed that it is possible to identify some of the problems that had to be solved in the course of evolution for the red blood cell (RBC) to achieve its present day effectiveness, by studying the behavior of systems featuring different, partial characteristics of its membrane. The appropriateness of the RBC volume to membrane area ratio for its circulation in the blood is interpreted on the basis of an analysis of the shape behavior of phospholipid vesicles. The role of the membrane skeleton is associated with preventing an RBC from transforming into a budded shape, which could form in its absence due to curvature-dependent transmembrane protein-membrane interaction. It is shown that, by causing the formation of echinocytes, the skeleton also acts protectively when, in vesicles with a bilayer membrane, the budded shapes would form due to increasing difference between the areas of their outer and inner layers.
Keywords Red blood cell, Vesicle shapes, Deformability, Membrane skeleton, Functional evolution
Address and Contact Information Institute of Biophysics, Faculty of Medicine, University of Ljubljana and Jožef Stefan Institute, 1000 Ljubljana, Slovenia
# Paper authored by participant 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: sasa.s vetina@mf.uni-lj.si, tel: +386 1 5437602, fax: +386 1 5437601
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DOI: 10.2478/s11658-012-0004-9 Volume 17 (2012) pp 182-195
Title MAINTENANCE OF RAT HEPATOCYTES UNDER INFLAMMATION BY COCULTURE WITH HUMAN ORBITAL FAT-DERIVED STEM CELLS
Authors Xia Chen1,2, Shichang Zhang1, Tao Liu1, Yong Liu1 and Yingjie Wang1,*
Abstract Preservation of hepatocyte functions in vitro will undoubtedly help the management of acute liver failure. The coculture system may be able to prevent functional decline of hepatocytes. It has already been shown that hepatocytes, when cocultured with bone marrow mesenchymal stem cells, could undergo long-term culture in vitro without loss of functions. In this study, human orbital fat-derived stem cells were isolated and cocultured with rat hepatocytes. When treated with serum from an acute liver failure patient, rat hepatocyte monoculture showed reducti on of cell viability and loss of liver-specific functions. However, rat hepatocyt es in the coculture system were still able to secret albumin and synthesize urea. IL-6 was significantly elevated in the coculture of rat hepatocyte with orbital fat-derived stem cells, and it might be the key immunoregulator which protects rat hepatocytes against inflammation. Our data confirmed that orbital fat-derived stem cells, or other adipose tissue-derived stem cells, are an ideal candidate to support rat hepatocyte functions in vitro.
Keywords Acute liver failure, Coculture, Differentiation, Fat tissue, Hepatocytes, Inflammation, Interleukin-6, Mesenchymal stem cells, Orbital fat-derived stem cells, Serum
Address and Contact Information 1Department of Infectious Disease, Southwest Hospital Affiliated to Third Military Medical University, Chongqing 400038, China,
2Department of Pediatrics, the 324th Hospital of PLA, Chongqing 400020, China
* 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/s11658-012-0002-y Volume 17 (2012) pp 196-205
Title THE EFFECT OF HYPEROSMOLAR STIMULI AND CYCLOPHOSPHAMIDE ON THE CULTURE OF NORMAL RAT UROTHELIAL CELLS in vitro
Authors Kajetan Juszczak1,2*, Jolanta Kaszuba-Zwoińska1, Paulina Chorobik3, Agata Ziomber1 and Piotr Jan Thor1
Abstract Highly concentrated urine may induce a harmful effect on the urinary bladder. Therefore, we considered osmolarity of the urine as a basic pathomechanism of mucosal damage. The influence of both cyclophosphamide (CYP) and hyperosmolar stimuli (HS) on the urothelium are not well described. The purpose was to evaluate the effect of CYP and HS on rat urothelial cultured cells (RUCC). 15 Wistar rats were used for RUCC preparation. RUCC were exposed to HS (2080 and 3222 mOsm/l NaCl) for 15 min and CYP (1 mg/ml) for 4 hrs. APC-labelled annexin V was used to quantitatively determine the percentage of apoptotic cells and propidium iodide (PI) as a standard flow cytometric viability probe to distinguish necrotic cells from viable ones. Annexin V-APC (+), annexin V-APC and PI (+), and PI (+) cells were analysed as apoptotic, dead, and necrotic cells, respectively. The results were presented in percentage values. The flow cytometric analysis was done on a FACSCalibur Flow Cytometer using Cell-Quest software. Treatment with 2080 and 3222 mOsm/l HS resulted in 23.7 ± 3.9% and 26.0 ± 1.5% apoptotic cells, respectively, 14.3 ± 1.4% and 19.4 ± 2.7% necrotic cells, respectively and 60.5 ± 1.4% and 48.6 ± 5.3% dead cells, respectively. The effect of CYP on RUCC was similar to the effect of HS. After CYP the apoptotic and necrotic cells were 23.1 ± 0.3% and 17.9 ± 7.4%, respectively. The percentage of dead cells was 57.7 ± 10.8%. CYP and HS induced apoptosis and necrosis in RUCC. 3222 mOsm/l HS had the most harmful effect based on the percentage of necrotic and apoptotic cells.
Keywords Urothelial cell, Culture, Cyclophosphamide, Hyperosmolarity, Bladder, Rat, Apoptosis, Necrosis, Overactive bladder
Address and Contact Information 1Department of Pathophysiology, Jagiellonian University, Medical College, Czysta 18, 31-121 Cracow, Poland,
2Department of Urology, Rydygier Memorial Hospital, Cracow, Poland,
3Department of Immunology, Jagiellonian University, Medical College, Cracow, Poland
* Author for correspondence. e-mail: kajus13@poczta.onet.pl, tel.: +48 12 633 3947, fax: + 48 12 632 9056
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DOI: 10.2478/s11658-012-0003-x Volume 17 (2012) pp 206-216
Title DIFFERENTIAL FUCOSYLTRANSFERASE IV EXPRESSION IN SQUAMOUS CARCINOMA CELLS IS REGULATED BY PROMOTER METHYLATION
Authors Hongyan Li1,2, Shaoming Tong1,2, Jiwei Liu3, Li Han1, Xuesong Yang1, Hesheng Hou2, Qiu Yan1 and Xiao-Qi Wang4,*
Abstract Enhanced fucosyltransferase IV (FUT4) expression correlates with increased tumor malignancy in many carcinomas. However, little is known about the regulation of FUT4 expression, and whether FUT4 expression is influenced by the methylation status of the FUT4 promoter is unclear. In this study, we demonstrated that FUT4 expression is negatively correlated with the methylation degree of a CpG island in the FUT4 promoter, suggesting that the methylation status of FUT4 promoter regulates the expression of FUT4. The results indicate that manipulating the methylation status of the FUT4 promoter to regulate FUT4 expression may be a novel approach in the treatment of malignant tumors.
Keywords Fucosyltransferase, FUT4 promoter, Methylation, A431 cells, SCC12 cells
Address and Contact Information 1Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, Liaoning, People’s Republic of China,
2College of Life Science, Liaoning Normal University, Dalian, Liaoning, People’s Republic of China,
3Department of Oncology, 1st Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, People’s Republic of China,
4Departments of Dermatology and Pediatrics, Northwestern University’s Feinberg School of Medicine, Chicago, Illinois, USA
* Author for correspondence. e-mail: x-wang1@northwestern.edu, tel.: 312-503-0294, fax: 312-503-0296
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DOI: 10.2478/s11658-012-0005-8 Volume 17 (2012) pp 217-227
Title STRESS-FREE STATE OF THE RED BLOOD CELL MEMBRANE AND THE DEFORMATION OF ITS SKELETON#
Authors Tjasa Svelc1* and Sasa Svetina1,2
Abstract The response of a red blood cell (RBC) to deformation depends on its membrane, a composite of a lipid bilayer and a skeleton, which is a closed, two-dimensional network of spectrin tetramers as its bonds. The deformation of the skeleton and its lateral redistribution are studied in terms of the RBC resting state for a fixed geometry of the RBC, partially aspirated into a micropipette. The geometry of the RBC skeleton in its initial state is taken to be either two concentric circles, a references biconcave shape or a sphere. It is assumed that in its initial state the skeleton is distributed laterally in a homogeneous manner with its bonds either unstressed, presenting its stress-free state, or prestressed. The lateral distribution was calculated using a variational calculation. It was assumed that the spectrin tetramer bonds exhibit a linear elasticity. The results showed a significant effect of the initial skeleton geometry on its lateral distribution in the deformed state. The proposed model is used to analyze the measurements of skeleton extension ratios by the method of applying two modes of RBC micropipette aspiration.
Keywords Red blood cell, Stress-free shape, Membrane skeleton, Skeleton deformation, Spectrin, Micropipette aspiration
Address and Contact Information 1Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Slovenia,
2Jožef Stefan Institute, Ljubljana, Slovenia
# Paper authored by participant 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: tjasa.svelc@mf.uni-lj.si, tel.: +386 1 5437600, fax: +386 1 5437601
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DOI: 10.2478/s11658-012-0006-7 Volume 17 (2012) pp 228-239
Title PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A NEW TARGET MOLECULE FOR GENE THERAPY
Authors Anna Banaszewska, Michal Piechota and Robert Plewa*
Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for controlling plasma levels of low-density lipoprotein cholesterol (LDL-C) and decreasing the risk of cardiovascular diseases. At present it is clear that the major classes of commonly prescribed lipid-lowering medications increase serum PCSK9 levels and fail to protect a significant percentage of patients from cardiovascular events. Therefore development of new LDL-C lowering medications that either do not increase circulating PCSK9 levels or work through inhibition of PCSK9 expression and protease activity is a highly desirable approach to overcome hypercholesterolemia. Since there are several agents which are being evaluated in human preclinical and clinical trials, this review summarizes current therapeutic strategies targeting PCSK9, including specific antibodies, antisense o ligonucleotides, small interfering RNAs (siRNAs) and other small-molecule inhibitors.
Keywords PCSK9, LDL cholesterol, LDL receptor degradation, Statins, Fibrates, Ezetimibe, Ani-PCSK9 antibody, Antisense oligonucleotides, RNAi, Hypercholesterolemia
Address and Contact Information Department of Animal Physiology and Development, Adam Mickiewicz University, 89 Umultowska St. 61-614 Poznan, Poland
* Author for correspondence. e-mail: rdplewa@gmail.com, tel.: +48 61 829 5926
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DOI: 10.2478/s11658-012-0007-6 Volume 17 (2012) pp 240-248
Title PROTECTIVE EFFECT OF PLACENTA EXTRACTS AGAINST NITRITE-INDUCED OXIDATIVE STRESS IN HUMAN ERYTHROCYTES #
Authors Svitlana Rozanova*, Yana Cherkashina, Svitlana Repina, Katherina Rozanova and Oleg Nardid
Abstract Aqueous-saline human placenta extract (HPE) is known to possess antioxidant activity due to the high concen tration of bioactive substances. This fact allows its application in clinical practice in order to treat oxidation-induced diseases. Extract antioxidant activity is mainly conditioned by proteins. Freezing of extracts has been shown to lead to their antioxidant activity increasing due to protein conformation changes. Different biological models are widely used in order to evaluate efficacy of novel antioxidants and mechanisms of their action. One such model appears to be erythrocytes under nitrite-induced oxidative stress. Nitrite is known to be able to penetrate erythrocyte membrane and to oxidize hemoglobin. In order to investigate whether HPE is able to decrease nitrite-induced oxidative injuries and to evaluate the protein contribution to this process, spectrophotometric and electron spin resonance (ESR) assays were used. Experimental data have revealed that antioxidant activity of extracts and of some of their fractions correlates with methemoglobin concentration lowering. Preliminary erythrocyte incubation with an extract fraction of 12 kDa allows preservation of the structural-dynamic cytosol state the closest to the control.
Keywords Human placenta extract, Protein, Antioxidant activity, Oxidative stress, Nitrite, Erythrocyte, Methemoglobin, Cytosol, Freezing, Thawing
Address and Contact Information Department of Cryobiophysics, Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine, Kharkiv, Ukraine
# Paper authored by participant 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: sv.rosanova@gmail.com
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DOI: 10.2478/s11658-012-0009-4 Volume 17 (2012) pp 249-257
Title AUTOPHAGY FAVORS Brucella melitensis SURVIVAL IN INFECTED MACROPHAGES
Authors Fei Guo, Hui Zhang, Chuangfu Chen1*, Shengwei Hu2, Yuanzhi Wang3, Jun Qiao1, Yan Ren3, Ke Zhang1, Yong Wang1 and Guoqing Du1
Abstract This study investigated the role of autophagy in the survival of the invasive bacterium Brucella melitensis strain 16M in murine macrophages. Here, Brucella melitensis 16M was found to trigger autophagosome formation, enhance autophagy flux and increase the expression level of the autophagy marker protein LC3-II. When autophagy was pharmacologically inhibited by 3-methyladenine (3-MA), Brucella replication efficiency was significantly decreased (p < 0.05). These results suggest that autophagy favors Brucella melitensis 16M survival in murine macrophages.
Keywords Autophagy, Brucella melitensis 16M, LC3-II, LC3-I, Autophagosome, Autophagy flux, 3-Methyladenine, Intracellular pathogens, Murine macrophage RAW264.7, Autophagic vesicle
Address and Contact Information 1College of Animal Science and Technology, Shihezi University, Shihezi 832003, China,
2Key Laboratory of Xijiang Endemic and Ethnic Disease, Shihezi University, Shihezi 832003, China,
3College of Medicine, Shihezi University, Shihezi, 832003, China
§ Both authors contributed equally to the work
* Author for correspondence. e-mail: chuangfu_chen@163.com, tel.: +86-0993-2058002, fax: +86-0993-2058612
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DOI: 10.2478/s11658-012-0008-5 Volume 17 (2012) pp 258-273
Title Rab3D REGULATES AMYLASE LEVELS, NOT AGONIST-INDUCED AMYLASE RELEASE, IN AR42J CELLS
Authors Saima Limi1, George Ojakian2 and Robert Raffaniello1*
Abstract Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (R ab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.
Keywords Amylase, AR42J cells, Exocrine, Rab3D, Secretion, Zymogen granules, GTP-binding protein, Pancreas, Cholecystokinin, Digestive enzyme
Address and Contact Information 1Hunter College, School of Health Professions, Medical Laboratory Sciences Program, Box 617, 425 East 25th Street, New York, NY 10010, USA,
2SUNY-Downstate Medical Center, Department of Cell Biology, Brooklyn, New York, USA
* Author for correspondence. e-mail: rraffani@hunter.cuny.edu
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DOI: 10.2478/s11658-012-0011-x Volume 17 (2012) pp 274-288
Title ANALYSIS OF THE ROLE OF THE INTEGRIN SIGNALING PATHWAY IN HEPATOCYTES DURING RAT LIVER REGENERATION
Authors Cunshuan Xu1,2,*, Yanjie Yang1,2, Junying Yang2, Xiaoguang Chen2 and Gaiping Wang2
Abstract To explore the role of the integrin signaling pathway in hepatocytes during rat liver regeneration, the integrin signaling pathway–related gene expression profile in hepatocytes of regenerative liver was detected using Rat Genome 230 2.0 array. The chip data showed that 265 genes of the integrin signaling pathway were included by Rat Genome 230 2.0 array and 132 genes showed significant expression changes in hepatocytes of regenerative liver. The numbers of up-, down- and up/down- re gulated genes were 110, 15 and 7 respectively. In addition, bioinformatics and systems biology methods were used to analyze the role of the integrin signaling pathway in hepatocytes. The analysis of gene synergy value indicated that paths 1, 8, 12, and 15 promoted hepatocyte proliferation at the priming phase of liver regeneration; paths 1, 3, 8, and 12-15 enhanced hepatocyte proliferation at the progressing phase; paths 11 and 14 promoted hepatocyte proliferation, while paths 12 and 13 reduced hepatocyte proliferation at the terminal phase. Additionally, the other 8 paths (2, 4, 5-7, 9-10 and 16) were not found to be related to liver regeneration. In conclusion, 132 genes and 8 cascades of the integrin signaling pathway participated in regulating hepatocyte proliferation during rat liver regeneration.
Keywords Integrin signaling pathway, Rat, Liver regeneration, Partial hepatectomy, Immunochemistry, Hepatocyte proliferation, Rat Genome 230 2.0 array, RT-PCR, Gene expression profile, Gene synergy value
Address and Contact Information 1College of Life Science, Henan Normal University, Xinxiang 453007, P.R. China,
2Key Laboratory for Cell Differentiation Regulation, Xinxiang 453007, P.R. China
* Author for correspondence. e-mail: cellkeylab@126.com, tel.:+086 373 3326001, fax: +086 373 3326524
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DOI: 10.2478/s11658-012-0010-y Volume 17 (2012) pp 289-308
Title INTERACTION OF SELECTED ANTHOCYANINS WITH ERYTHROCYTES AND LIPOSOME MEMBRANES#
Authors Dorota Bonarska-Kujawa*, Hanna Pruchnik and Halina Kleszczyńska
Abstract Anthocyanins are one of the main flavonoid groups. They are responsible for, e.g., the color of plants and have antioxidant features and a wide spectrum of medical activity. The subject of the study was the following compounds that belong to the anthocyanins and which can be found, e.g., in strawberries and chokeberries: callistephin chloride (pelargonidin-3-O-glucoside chloride) and ideain chloride (cyanidin-3-O-gal actoside chloride). The aim of the study was to determine the compounds’ antioxidant activity towards the erythrocyte membrane and changes incurred by the tested anthocyanins in the lipid phase of the erythrocyte membrane, in liposomes composed of erythrocyte lipids and in DPPC, DPPC/cholesterol and egg lecithin liposomes. In particular, we studied the effect of the two selected anthocyanins on red blood cell morphology, on packing order in the lipid hydrophilic phase, on fluidity of the hydrophobic phase, as well as on the temperature of phase transition in DPPC and DPPC/cholesterol liposomes. Fluorimetry with the Laurdan and Prodan probes indicated increased packing density in the hydrophilic phase of the membrane in the presence of anthocyanins. Using the fluorescence probes DPH and TMA-DPH, no effect was noted inside the hydrophobic phase of the membrane, as the lipid bilayer fluidity was not modified. The compounds slightly lowered the phase transition temperature of phosphatidylcholine liposomes. The study has shown that both anthocyanins are incorporated into the outer region of the erythrocyte membrane, affecting its shape and lipid packing order, which is reflected in the increasing number of echinocytes. The investigation proved that the compounds penetrate only the outer part of the external lipid layer of liposomes composed of erythrocyte lipids, DPPC, DPPC/cholesterol and egg lecithin lipids, changing its packing order. Fluorimetry studies with DPH-PA proved that the tested anthocyanins are very effective antioxidants. The antioxidant activity of the compounds was comparable with the activity of Trolox®
Keywords Anthocyanins, Fluorescent probes, Liposomes, Erythrocyte membrane, Echinocytes, DSC, Anisotropy, Generalized polarization, Phase transition
Address and Contact Information Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
# Paper authored by participant 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: dorota.bonarska-kujawa@up.wroc.pl, tel.: +48 71 3205275
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DOI: 10.2478/s11658-012-0012-9 Volume 17 (2012) pp 309-322
Title DEFICIENCY IN TR4 NUCLEAR RECEPTOR ABROGATES Gadd45a EXPRESSION AND INCREASES CYTOTOXICITY INDUCED BY IONIZING RADIATION
Authors Shian-Jang Yan1,§, Yi-Fen Lee1,§, Huei-Ju Ting1,§, Ning-Chun Liu1, Su Liu1, Shin-Jen Lin1, Shauh-Der Yeh2, Gonghui Li1 and Chawnshang Chang1,3,*
Abstract The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a , the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient ( TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.
Keywords TR4, GADD45A, Ionizing radiation, Mouse embryonic fibroblast, Genotoxic stress, TR4 response element
Address and Contact Information 1George Whipple Lab for Cancer Research , Departments of Pathology, Urology, and Radiation Oncology, University of Rochester Medical Center, Rochester, NY 14642,
2Department of Urology, Taipei Medical University, Taipei 110, Taiwan,
3Sex Hormone Research Center, China Medical University/Hospital, Taichung 404, Taiwan
§ These authors contributed equally to this paper.
* Author for correspondence. e-mail: chang@urmc.rochester.edu
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