Vol.14 No.2 June 2009

DOI: 10.2478/s11658-008-0043-4 Volume 14 (2009) pp 175-189
Title THE CERAMIDE STRUCTURE OF GM1 GANGLIOSIDE DIFFERENTLY AFFECTS ITS RECOVERY IN LOW-DENSITY MEMBRANE FRACTIONS PREPARED FROM HL-60 CELLS WITH OR WITHOUT TRITON-X100
Authors Mirosława Panasiewicz1, Hanna Domek1, Grażyna Hoser2, Natalia Fedoryszak1, Maciej Kawalec 2 and Tadeusz Pacuszka1*
Abstract Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [3H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C14, C18 or C18:1 fatty acids (Fas),respectively in GM1(18:1/14), GM1(18:1/18) or GM1(18:1/18:1). The recovery of the GM1s in the LDMs was unrelated to their preference for the liquidordered phase, and differed from that reported for DRMs in terms of dependence on the length and saturation of the Fas and cholesterol content in the cell membranes. Sonication resulted in the redistribution of GM1s from the LDMs to membranes of higher buoyant density. This process depended on the ceramide structure of the GM1s, with GM1(18:1/14) recovered in the highest proportion; on the intensity of sonication; and on the character of the sample. The greater recovery of GM1(18:1/14) in LDMs may be due to its enrichment in membrane domains different from those containing the other GM1s. Cross-linking all the GM1s with cholera toxin increased their resistance to sonication-induced redistribution to membranes of higher density. Extraction of sonicated samples containing GM1(18:1/18) with Triton X-100 resulted in the recovery of at least 75% of this ganglioside in the low density DRMs. Moreover, sonication strongly reduced the LDM CD59 content, but did not significantly affect that of CD14.
Keywords Ceramide, Gangliosides, GM1, Membrane domains, Myristic acid, Sonication
Address and Contact Information Departments of 1Biochemistry and Molecular Biology and 2Clinical Cytology, Medical Center of Postgraduate Education, Marymoncka 99, 01-813 Warszawa, Poland
* Author for correspondence; e-mail: pacuszka@cmkp.edu.pl, tel.: (48 22) 5693814; fax: (48 22) 5693712
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0042-5 Volume 14 (2009) pp 190-198
Title A CONSENSUS MAP OF CHROMOSOME 6R IN RYE (Secale cereale L.)
Authors Stefan Stojałowski, Beata Myśków*, Paweł Milczarski and Piotr Masojć
Abstract Four F2 mapping populations derived from crosses between rye inbred lines DS2×RXL10, 541×Ot1-3, S120×S76 and 544×Ot0-20 were used to develop a consensus map of chromosome 6R. Thirteen marker loci that were polymorphic in more than one mapping population constituted the basis for the alignment of the four maps using the JoinMap v. 3.0 software package. The consensus map consists of 104 molecular marker loci including RFLPs, RAPDs, AFLPs, SSRs, ISSRs, SCARs, STSs and isozymes. The average distance between the marker loci is 1.3 cM, and the total map length is 135.5 cM. This consensus map may be used as a source of molecular markers for the rapid development of new maps of chromosome 6R in any mapping population.
Keywords Rye, Consensus map, Molecular markers
Address and Contact Information Department of Genetics and Plant Breeding, Agricultural University Słowackiego 17, 71-434 Szczecin, Poland
* Author for correspondence; e-mail: beata.myskow@agro.ar.szczecin.pl
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0044-3 Volume 14 (2009) pp 199-221
Title PERTURBATION OF THE LIPID PHASE OF A MEMBRANE IS NOT INVOLVED IN THE MODULATION OF MRP1 TRANSPORT ACTIVITY BY FLAVONOIDS
Authors Olga Wesołowska1,*, Andrzej B. Hendrich1, Barbara Łania-Pietrzak1, Jerzy Wiśniewski1, Joseph Molnar2, Imre Ocsovszki3 and Krystyna Michalak1
Abstract The expression of transmembrane transporter multidrug resistanceassociated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.
Keywords Flavonoids, Multidrug resistance-associated protein 1 (MRP1), Lipid bilayer, Carbocyanine dyes
Address and Contact Information 1Department of Biophysics, Wrocław Medical University, ul. Chałubińskiego 10, 50-368 Wrocław, Poland, 2Institute of Medical Microbiology, University of Szeged, Dom ter 10, H-6720 Szeged, Hungary, 3Department of Biochemistry, University of Szeged, Dom ter 9, H-6720 Szeged, Hungary
* Author for correspondence; e-mail: olawes@biofiz.am.wroc.pl, tel.: (+48 71) 784 14 15, fax: (+48 71) 784 00 88
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0045-2 Volume 14 (2009) pp 222-247
Title THE SIGNALING PATHWAYS OF EPSTEIN-BARR VIRUS-ENCODED LATENT MEMBRANE PROTEIN 2A (LMP2A) IN LATENCY AND CANCER
Authors Mei-Fong Pang1, Kah-Wai Lin2 and Suat-Cheng Peh3*
Abstract Epstein-Barr virus (EBV) is a ubiquitous virus with infections commonly resulting in a latency carrier state. Although the exact role of EBV in cancer pathogenesis remains not entirely clear, it is highly probable that it causes several lymphoid and epithelial malignancies, such as Hodgkin’s lymphoma, NK-T cell lymphoma, Burkitt’s lymphoma, and nasopharyngeal carcinoma. EBV-associated malignancies are associated with a latent form of infection, and several of these EBV-encoded latent proteins are known to mediate cellular transformation. These include six nuclear antigens and three latent membrane proteins. Studies have shown that EBV displays distinct patterns of viral latent gene expression in these lymphoid and epithelial tumors. The constant expression of latent membrane protein 2A (LMP2A) at the RNA level in both primary and metastatic tumors suggests that this protein might be a driving factor in the tumorigenesis of EBV-associated malignancies. LMP2A may cooperate with the aberrant host genome, and thereby contribute to malignant transformation by intervening in signaling pathways at multiple points, especially in the cell cycle and apoptotic pathway. This review summarizes the role of EBV-encoded LMP2A in EBV-associated viral latency and cancers. We will focus our discussions on the molecular interactions of each of the conserved motifs in LMP2A, and their involvement in various signaling pathways, namely the B-cell receptor blockade mechanism, the ubiquitin-mediated (Notch and Wnt) pathways, and the MAPK, PI3-K/Akt, NK-κB and STAT pathways, which can provide us with important insights into the roles of LMP2A in the EBVassociated latency state and various malignancies.
Keywords Epstein-Barr virus, Latent membrane protein, Cancer, Latency
Address and Contact Information 1Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden,
2Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden,
3Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
* Author for correspondence; e-mail: suatcheng_peh@yahoo.com
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0048-z Volume 14 (2009) pp 248-272
Title TOLL-LIKE RECEPTORS AND THEIR ROLE IN CARCINOGENESIS AND ANTI-TUMOR TREATMENT
Authors Anna Wolska, Ewa Lech-Marańda and Tadeusz Robak*
Abstract Toll-like receptors (TLRs) have been described as major components of the innate immune system, recognizing the conserved molecular structures found in the large groups of pathogens called pathogen-associated molecular patterns (PAMPs). TLR expression is ubiquitous, from epithelial to immunocompetent cells. TLR ligation triggers several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators but also anti-inflammatory and anti-tumor cytokines. The result of this activation goes beyond innate immunity to shape the adaptive responses against pathogens and tumor cells, and maintains host homeostasis via cell debris utilization. TLRs have already become potent targets in infectious disease treatment and vaccine therapy and in neoplastic disease treatment, due to their ability to enhance antigen presentation. However, some studies show the dual effect of TLR stimulation on malignant cells: they can be proapoptotic or promote survival under different conditions. It is therefore crucial to design further studies assessing the biology of these receptors in normal and transformed cells. The established role of TLRs in human disease therapy is based on TLR7 and TLR4 agonists, respectively for the novel treatment of some types of skin cancer and for the anti-hepatitis B virus vaccine. Some clinical trials involving TLR agonists as potent enhancers of the anti-tumor response in solid tumors have begun.
Keywords Toll-like receptors, Innate immunity, Treatment, Carcinogenesis, Tumor, Vaccine, Dendritic cells
Address and Contact Information Department of Hematology, Medical University of Łódź, Ciołkowskiego 2, 93-513 Łódź, Poland
* Author for correspondence; e-mail: robaktad@csk.umed.lodz.pl, tel.: +48 42 689 51 91, fax: +48 42 689 51 92
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0046-1 Volume 14 (2009) pp 273-287
Title ACHERON, AN NOVEL LA ANTIGEN FAMILY MEMBER, BINDS TO CASK AND FORMS A COMPLEX WITH ID TRANSCRIPTION FACTORS
Authors Haifeng Weng1,2, Chul Kim1, Christos Valavanis3,4, Zhaohui Wang1 and Lawrence M. Schwartz1,3,5,*
Abstract Acheron, a Lupus antigen ortholog, was identified as a novel deathassociated transcript from the intersegmental muscles of the moth Manduca sexta. Acheron is phylogenetically-conserved and represents a new sub-family of Lupus antigen proteins. Acheron is expressed predominantly in neurons and muscle in vertebrates, and regulates several developmental events including myogenesis, neurogenesis and possibly metastasis. Using Acheron as bait, we performed a yeast two-hybrid screen with a mouse embryo cDNA library and identified CASK-C, a novel CASK/Lin-2 isoform, as an Acheron binding partner. Acheron and CASK-C bind via the C-terminus of Acheron and the CaMKII-like domain of CASK-C. Co-immunoprecipitation assays verify this interaction and demonstrate that Acheron also forms a complex with all members of the Id (inhibitor of differentiation) proteins. Taken together, these data suggest a mechanism by which Acheron may regulate development and pathology.
Keywords Programmed cell death, Manduca sexta, Muscle, Apoptosis, Lupus antigen protein
Address and Contact Information 1Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA,
2Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA,
3Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA,
4Molecular Pathology and Genetics Division, Department of Pathology, Metaxa Cancer Hospital, Botassi 51, Piraeus 185 37 Greece,
5Pioneer Valley Life Sciences Institute, 3601 Main Street, Springfield, Massachusetts, 01199, USA
* Author for correspondence. Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA, e-mail: LMS@bio.umass.edu, tel.: (413) 545-2435, fax: (413) 545-3243
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0005-5 Volume 14 (2009) pp 288
Title ACHERON, AN NOVEL LA ANTIGEN FAMILY MEMBER, BINDS TO CASK AND FORMS A COMPLEX WITH ID TRANSCRIPTION FACTORS
Authors Haifeng Weng1,2, Chul Kim1, Christos Valavanis3,4, Zhaohui Wang1 and Lawrence M. Schwartz1,3,5,*
Abstract Erratum to CMBL, DOI: 10.2478/s11658-008-0046-1
The original version of the article unfortunately contained mistakes:
1. On page 4, line 44: “3.0-3.5 mg” should be changed to “3.0-3.5 µg”;
2. On page 5, line 1: “500 ml” should be changed to “500 µl”;
3. On page 5, line 9: “60 ml” should be changed to “60 µl”;
4. On page 5, line 14: “60 ml” should be changed to “60 µl”;
5. On page 6, line 29: word “dimmer” should be changed to “dimer”.
Keywords
Address and Contact Information 1Molecular and Cellular Biology Program, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA,
2Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA,
3Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA,
sup>4Molecular Pathology and Genetics Division, Department of Pathology, Metaxa Cancer Hospital, Botassi 51, Piraeus 185 37 Greece,
5Pioneer Valley Life Sciences Institute, 3601 Main Street, Springfield, Massachusetts, 01199, USA
* Author for correspondence. Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, USA, e-mail: LMS@bio.umass.edu, tel.: (413) 545-2435, fax: (413) 545-3243
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-008-0050-5 Volume 14 (2009) pp 289-304
Title A TRANSCRIPTIONALLY ACTIVE copia-LIKE RETROELEMENT IN Citrus limon
Authors Bruna De Felice1*, Robert R. Wilson1, Carolina Argenziano1, Ioanis Kafantaris1 and Clara Conicella2
Abstract The plant nuclear genome is largely composed of mobile DNA, which can rearrange genomes and other individual gene structure and also affect gene regulation through various promoted activities: transposition, insertion, excision, chromosome breakage, and ectopic recombination. Ty1-copia-like retrotransposon is a widespread class of transposable elements in the plant kingdom, representing a large part of the total DNA content. Here, a novel retrotransposon-like sequence was isolated and identified as the Ty1-copia-like reverse transcriptase domain (named here CLCoy1), based on the homology of known elements. Fluorescence in situ hybridization, revealed that CLCoy1 was mainly located in telomeric and sub-telomeric regions along the Citrus chromosomes. CLCoy1 composes 3.6% of the genome and, interestingly, while transposons are mostly specific to a species, this element was identified in other Citrus species such as Citrus aurantium, Fortunella margarita and Citrus paradisi, but undetected in Poncirus trifoliata. We also determined that wounding, salt and cell culture stress produced transcriptional activation of this novel retroelement in Citrus limon. The novel Ty1-copia-like element CLCoy1 may have played a major role in shaping genome structure and size during Citrus species evolution.
Keywords Ty1-copia-like, Citrus, Transcription, Biotic and abiotic stress
Address and Contact Information 1Department of Life Sciences, University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy,
2CNR-IGV, Research Institute of Plant Genetics, Via Universitá 133, 80055 Portici, Italy
* Author for correspondence; e-mail: bruna.defelice@unina2.it, bruna.defelice@yahoo.it, tel: +39-823-274543, fax: +39-823-274571
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0001-9 Volume 14 (2009) pp 305-318
Title THE KNOCKDOWN OF c-myc EXPRESSION BY RNAi INHIBITS CELL PROLIFERATION IN HUMAN COLON CANCER HT-29 CELLS in vitro AND in vivo
Authors Xiao Zhang, Yin-Lin Ge* and Run-Hua Tian
Abstract We investigated the effects of RNA interference-mediated silencing of the c-myc gene on celluar proliferation and apoptosis in human colon cancer HT-29 cells in vitro and in vivo. A small interfering RNA (siRNA) targeting c-myc was designed, the DNA template was synthesized, and the siRNA was obtained by in vitro transcription. After siRNA transfection into HT-29 and human neuroblastoma IMR-32 cells with Lipofectamine 2000TM, the proliferation of the HT-29 and IMR-32 cells was assessed via 3-(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetry, and Hoechst 33258 staining was used to observe cell apoptosis. Following gene transfer to HT-29 cells, the expression of c-myc mRNA was examined via reverse transcription polymerase chain reaction, and the level of the protein via Western blot assay. Growth curves were constructed and in vivo experiments were performed on nude mice to assess the effects of c-myc silencing on tumor growth. The c-myc expression in the tumor tissue was measured by reverse transcription polymerase chain reaction and subsequently by immunohistochemistry. Our paper demonstrates that the delivery of siRNA directed against c-myc not only efficiently down-regulated the expression of c-myc, inhibited the proliferation of HT-29 cells and induced apoptosis in vitro, but also suppressed the growth of colon cancer cells in vivo.
Keywords c-myc, Cell proliferation, HT-29, siRNA
Address and Contact Information Department of Biochemistry and Molecular Biology, Medical College, Qingdao University, Qingdao, 266021, China
* Author for correspondence: e-mail: geyinlin@126.com
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0002-8 Volume 14 (2009) pp 319-335
Title ACTIVATION OF THE HEAT SHOCK RESPONSE IN A PRIMARY CELLULAR MODEL OF MOTONEURON NEURODEGENERATION - EVIDENCE FOR NEUROPROTECTIVE AND NEUROTOXIC EFFECTS
Authors Bernadett Kalmar* and Linda Greensmith
Abstract Pharmacological up-regulation of heat shock proteins (hsps) rescues motoneurons from cell death in a mouse model of amyotrophic lateral sclerosis. However, the relationship between increased hsp expression and neuronal survival is not straightforward. Here we examined the effects of two pharmacological agents that induce the heat shock response via activation of HSF-1, on stressed primary motoneurons in culture. Although both arimoclomol and celastrol induced the expression of Hsp70, their effects on primary motoneurons in culture were significantly different. Whereas arimoclomol had survival-promoting effects, rescuing motoneurons from staurosporin and H2O2 induced apoptosis, celastrol not only failed to protect stressed motoneurons from apoptosis under same experimental conditions, but was neurotoxic and induced neuronal death. Immunostaining of celastrol-treated cultures for hsp70 and activated caspase-3 revealed that celastrol treatment activates both the heat shock response and the apoptotic cell death cascade. These results indicate that not all agents that activate the heat shock response will necessarily be neuroprotective.
Keywords Amyotrophic Lateral Sclerosis, Heat shock protein, SOD1 mice, Neuroprotection, Motoneuron, Arimoclomol, Celastrol
Address and Contact Information Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, United Kingdom
* Author for correspondence; e-mail: b.kalmar@ion.ucl.ac.uk, l.greensmith@ion.ucl.ac.uk, tel.:0207 676 2161, fax: 0207 813 3107
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0006-4 Volume 14 (2009) pp 336-346
Title POTASSIUM CURRENTS IN HUMAN MYOGENIC CELLS FROM HEALTHY AND CONGENITAL MYOTONIC DYSTROPHY FOETUSES
Authors Ewa Nurowska1*, Andrew Constanti2, Beata Dworakowska1, Vincent Mouly3, Denis Furling3, Paola Lorenzon4, Tiziana Pietrangelo4, 5, Krzysztof Dołowy1 and Fabio Ruzzier4
Abstract The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K+ (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K+ channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.
Keywords Potassium channels, Myoblast fusion, Congenital myotonic dystrophy, Patch-clamp
Address and Contact Information 1Department of Biophysics, Warsaw University of Life Sciences SGGW, Warsaw, Poland,
2Department of Pharmacology, The School of Pharmacy, London WC1N 1AX, Great Britain,
3UMR S787, Inserm / UPMC-Paris 6/ Institute of Myology, Paris Cedex 13, France,
4Department of Physiology and Pathology, University of Trieste, Trieste, Italy,
5current address: Interuniversity Institute of Myology (IIM), Center for Research on Ageing, University “G. d’Annunzio”, Chieti, Italy
* Author for correspondence: e-mail: ewa_nurowska@sggw.pl, tel.: +48 (22) 59 386 20, fax: +48 (22) 59 386 19
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-009-0003-7 Volume 14 (2009) pp 347-362
Title TARGETED CATIONIC POLY(D,L-LACTIC-CO-GLYCOLIC ACID) NANOPARTICLES FOR GENE DELIVERY TO CULTURED CELLS
Authors Sonsoles Diez, Itziar Migueliz and Conchita Tros De Ilarduya*
Abstract We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.
Keywords Poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP), Asialofetuin, Targeted gene delivery, Pharmaceutical nanotechnology
Address and Contact Information Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Pamplona, Spain
* Author for correspondence; e-mail: ctros@unav.es
[Rozmiar: 1332 bajtów]