Vol. 15 No. 1 March 2010
DOI: 10.2478/s11658-009-0030-4 Volume 15 (2010) pp 1-12 | |
Title | THE AGE-DEPENDENT INDUCTION OF APOPTOSIS-INDUCING FACTOR (AIF) IN THE HUMAN SEMITENDINOSUS SKELETAL MUSCLE |
Authors | Soo Yeon Park1*, Ha Young Kim2, Jung Hwan Lee1, Kyoung Ho Yoon3, Mun Seog Chang2 and Seong Kyu Park2* |
Abstract | To assess the dependence on age of the expression of apoptosis regulatory proteins in the human semitendinosus muscle, we measured the expression levels of several apoptosis-related genes, including apoptosis- inducing factor (AIF), Bax, Bcl-2, caspase-3 and heat shock protein 70 (HSP70), using RT-PCR, immunohistochemistry and TUNEL assays. We found that the DNA fragmentation was proportional to the age of the tissues sample donors. The expression levels of AIF were significantly elevated (by 10 to 25%) in semitendinosus tissue samples from older individuals, but the Bax, Bcl-2, caspase-3 and HSP 70 levels remained almost constant. This data suggests that the morphological and functional changes observed in aged human semitendinosus muscle correlates with the apoptosis of muscle cells through the induction of AIF. |
Keywords | Human skeletal muscle, Semitendinosus, Apoptosis, Aging, Apoptosis-inducing factor |
Address and Contact Information | 1Sports Medicine Center, East-West Neo Medicial Center, 2Department of Prescriptionology, College of Oriental Medicine, Kyung Hee University, Seoul 130-701, Korea, 3Department of Orthopaedic Surgery, School of Medicine,Kyung Hee University, Seoul 130-701, Korea *Author for correspondence: e-mail: comskp@khu.ac.kr, tel.: +8229610536, fax: +8229610536 |
DOI: 10.2478/s11658-009-0033-1 Volume 15 (2010) pp 13-31 | |
Title | TREATMENT WITH TNF-α AND IFN-γ ALTERS THE ACTIVATION OF SER/THR PROTEIN KINASES AND THE METABOLIC RESPONSE TO IGF-I IN MOUSE C2C12 MYOGENIC CELLS |
Authors | Katarzyna Grzelkowska-Kowalczyk* and Wioletta Wieteska-Skrzeczyńska |
Abstract | The aim of this study was to compare the effects of TNF-α, IL-1ß and IFN-γ on the activation of protein kinase B (PKB), p70S6k , mitogen-activated protein kinase (MAPK) and p90rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1ß did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70S6k , p42MAPK, p44MAPK and p90rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42MAPK (a 2.81-fold increase after 50 min), and the activation of p70S6k and p90rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70S6k phosphorylation was significantly diminished, and the increase in p42MAPK and p90rsk phosphorylation was prevented. The basal p42MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pre- treatment of myogenic cells with IL-1ß did not modify the IGF-I-stimulated phosphorylation of PKB, p70S6k , p42MAPK and p90rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1ß, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70S6k . iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle. |
Keywords | Cytokines, Glucose transport, IGF-I resistance, Protein synthesis, Signalling pathways |
Address and Contact Information | Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
University of Life Sciences (SGGW), Nowoursynowska 159, 02-776 Warsaw,
Poland * Author for correspondence. e-mail: k_grzel_kow@poczta.fm, tel/fax. (+48 22) 847 24 52 |
DOI: 10.2478/s11658-009-0032-2 Volume 15 (2010) pp 32-45 | |
Title | THE INTRABODY TARGETING OF hTERT ATTENUATES THE IMMORTALITY OF CANCER CELLS |
Authors | Xiangying Zhu, Nan Yang, Jianguo Cai, Guimei Yang, Shenghua Liang and Daming Ren* |
Abstract | hTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells. |
Keywords | Cancer, Intrabody, Anti-hTERT ScFv, Immortality |
Address and Contact Information | State Key Laboratory of Genetic Engineering, School of Life Science,
Fudan University, P. R. China * Author for correspondence. e-mail: dmren@fudan.edu.cn, tel.: +86 21 65642506, fax: +86 21 65648376 |
DOI: 10.2478/s11658-009-0031-3 Volume 15 (2010) pp 46-54 | |
Title | IMPORTANT RESIDUE (G46) IN ERYTHROID SPECTRIN TETRAMER FORMATION |
Authors | Jianxia Kang, Yuanli Song, Akin Sevinc and Leslie W.-M. Fung* |
Abstract | Spectrin tetramerization is important for the erythrocyte to maintain its unique shape, elasticity and deformability. We used recombinant model proteins to show the importance of one residue (G46) in the erythroid α-spectrin junction region that affects spectrin tetramer formation. The G46 residue in the erythroid spectrin N-terminal junction region is the only residue that differs from that in non-erythroid spectrin. The corresponding residue is R37. We believe that this difference may be, at least in part, responsible for the 15-fold difference in the equilibrium constants of erythroid and non-erythroid tetramer formation. In this study, we replaced the Gly residue with Ala, Arg or Glu residues in an erythroid α-spectrin model protein to give G46A, G46R or G46E, respectively. We found that their association affinities with a ß-spectrin model protein were quite different from each other. G46R exhibited a 10-fold increase and G46E exhibited a 16-fold decrease, whereas G46A showed little difference, when compared with the wild type. The thermal and urea denaturation experiments showed insignificant structural change in G46R. Thus, the differences in affinity were due to differences in local, specific interactions, rather than conformational differences in these variants. An intra-helical salt bridge in G46R may stabilize the partial domain single helix in α-spectrin, Helix C’, to allow a more stable helical bundling in the αß complex in spectrin tetramers. These results not only showed the importance of residue G46 in erythroid α-spectrin, but also provided insights toward the differences in association affinity between erythroid and non-erythroid spectrin to form spectrin tetramers. |
Keywords | Erythroid spectrin, Tetramerization, G46, mutation, ITC |
Address and Contact Information | Department of Chemistry, University of Illinois at Chicago,
845 W. Taylor Street, MC 111, Chicago, IL 60607, USA * Author for correspondence. e-mail: lfung@uic.edu, tel.: (312) 355-5516, fax: (312) 996- 0431 |
DOI: 10.2478/s11658-009-0034-0 Volume 15 (2010) pp 55-69 | |
Title | ESTABLISHING AND FUNCTIONAL CHARACTERIZATION OF AN HEK-293 CELL LINE EXPRESSING AUTOFLUORESCENTLY TAGGED ß-ACTIN (pEYFP-ACTIN) AND THE NEUROKININ TYPE 1 RECEPTOR (NK1-R) |
Authors | Alenka Hrovat1, Apolonija Bedina Zavec2, Azra Pogačnik1, Robert Frangeľ3 and Milka Vrecl1* |
Abstract | This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real- time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total ß-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP- actin:endogenous ß-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine- stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells. |
Keywords | Cytoskeleton, Actin filaments, HEK-293, Neurokinin type 1 receptor, Flow cytometry, Confocal microscopy |
Address and Contact Information | 1Institute of Anatomy, Histology and Embryology, Veterinary Faculty,
Gerbičeva 60, SI-1000 Ljubljana, Slovenia, 2Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia, 3Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, Gerbičeva 60, SI-1000 Ljubljana, Slovenia * Author for correspondence. E- mail: milka.vrecl@vf.uni-lj.si |
DOI: 10.2478/s11658-009-0035-z Volume 15 (2010) pp 70-89 | |
Title | THE IMMUNOGENICITY OF THE LIPOSOME-ASSOCIATED OUTER MEMBRANE PROTEINS (OMPs) OF Moraxella catarrhalis |
Authors | Daria Augustyniak1*, Józef Mleczko1 and Jan Gutowicz2 |
Abstract | The outer membrane proteins (OMPs) are the most immunogenic and attractive of the Moraxella catarrhalis vaccine antigens that may induce the protective immune response. The aim of this study was to determine the effectiveness of two types of OMP-associated phosphatidylcholine (PC) liposomal formulations (OMPs-PC, PC-OMPs) and of Zwittergent-based proteomicelles (OMPs-Z) in potentiating an anti-OMP systemic immune response in mice. The immunogenicities of the above preparations were evaluated by assessing serum anti-OMP IgG and IgA reactivity in the post- immunized mouse antisera using ELISA and Western blotting. Additionally, the cross-reactivity of the most effective anti-OMP response was determined using heterologous sera from both humans and mice. Both the proteoliposomes and the proteomicelles showed high immunogenic properties and did not elicit any distinct quantitative differences in the antibody titer or qualitative differences in the pattern of the mouse antisera. The post-immunized mouse antisera predominantly recognized a ~60-kDa OMP of M. catarrhalis. That protein was also found to be a highly cross-reactive antigen interacting with a panel of pooled mouse antisera produced by immunization either with whole cells or the purified OMPs of heterologous M. catarrhalis strains. Furthermore, normal sera collected from healthy children were found to be preferentially reactive with the 60-kDa OMP. The serum-specific IgG, IgA and IgM were respectively detected via immunoblotting in 90%, 85% and 30% of heterologous human sera. This similar immunogenic effectiveness of both OMP-associated liposomal formulations could contribute to the practical use of such formulations in the future in human vaccination. Moreover, the highly cross-reactive 60-kDa OMP seems to be an important antigenic marker of M. catarrhalis, and, as it is responsible for the induction of an antibody-mediated and long-lasting immune response, studying it may partially aid us in understanding the relatively low degree of pathogenicity of the bacterium in immunocompetent individuals. |
Keywords | Moraxella catarrhalis, Outer membrane proteins, Proteoliposomes, Proteomicelles, Anti-OMP antibodies, Cross-reactivity, Zwittergent |
Address and Contact Information | 1Laboratory of Immunology, 2Department of Physico-Chemistry of Microorganisms, Institute of Genetics and Microbiology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland * Author for correspondence. E-mail: august@microb.uni.wroc.pl, tel: (+48) 71 3756 296 |
DOI: 10.2478/s11658-009-0036-y Volume 15 (2010) pp 90-97 | |
Title | THE ANTIOXIDANT PROPERTIES OF CARNITINE in vitro |
Authors | Katarzyna Solarska1, Anna Lewińska1, Agata Karowicz-Bilińska2 and Grzegorz Bartosz1,3* |
Abstract | Many of the effects of carnitine are ascribed to its antioxidant properties. The aim of this study was to evaluate the antioxidant properties of carnitine in vitro. Carnitine was found to decolorize ABTS●+ , and to protect fluorescein against bleaching induced by AAPH-derived peroxyl radicals and peroxynitrite, thiol groups against oxidation induced by hydrogen peroxide, peroxyl radicals, hypochlorite and peroxynitrite, and erythrocytes against hemolysis induced by peroxyl radicals and hypochlorite. These results show that carnitine has a direct antioxidant action against physiologically relevant oxidants. |
Keywords | Antioxidant, Carnitine, Hydrogen peroxide, Hypochlorite, Peroxyl radical, Peroxynitrite |
Address and Contact Information | 1Department of Biochemistry and Cell Biology, University of Rzeszów, Pigonia 6,
35-959 Rzeszów, Poland, 2Clinic of High-Risk Pregnancy, Medical University of Łódź, Wileńska 37, 94-029 Łódź, Poland, 3Department of Molecular Biophysics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland * Author for correspondence. e-mail: gbartosz@biol.uni.lodz.pl, tel./fax: +48 42 6354476 |
DOI: 10.2478/s11658-009-0037-x Volume 15 (2010) pp 98-117 | |
Title | THE INTERACTION OF PVP COMPLEXES OF GOSSYPOL AND ITS DERIVATIVES WITH AN ARTIFICIAL MEMBRANE LIPID MATRIX |
Authors | Maksim Ionov1,2,*, Ilnora Tukfatullina1, Bakhtiyar Salakhutdinov1, Nina Baram1, Maria Bryszewska2 and Takhir Aripov1 |
Abstract | In this paper, we present the results of a study on the membrane- active properties of gossypol, its derivatives and their polyvinylpyrrolidone complexes as assessed by differential scanning calorimetry and by the fluorescent probe method. The latter revealed the change in polarization of the incident radiation caused by the action of the polyphenol on the artificial membrane lipid matrix. |
Keywords | Gossypol and its derivatives, Lipid membranes, Differential scanning calorimetry, Membrane fluidity |
Address and Contact Information | 1A.S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of
Uzbekistan, 83, st, Kh. Abdullaev, Tashkent, 100125, Uzbekistan, 2Department of General Biophysics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland * Author for correspondence. e-mail: maksion@biol.uni.lodz.pl, tel./fax +48426354474 |
DOI: 10.2478/s11658-009-0038-9 Volume 15 (2010) pp 118-133 | |
Title | THE TELOMERE-SPECIFIC NON-LTR RETROTRANSPOSONS SART1 AND TRAS1 ARE SUPPRESSED BY PIWI SUBFAMILY PROTEINS IN THE SILKWORM, Bombyx mori |
Authors | Tsuneyuki Tatsuke1,2, Kosuke Sakashita1, Yuki Masaki1, Jae Man Lee1, Yutaka Kawaguchi1 and Takahiro Kusakabe1* |
Abstract | The telomere structures in Bombyx mori are thought to be maintained mainly by the transposition of the specialized telomeric retroelements SART and TRAS. The silkworm genome has telomeric TTAGG repeats and telomerase, but this telomerase displays little or no activity. Here, we report that the transcription of the telomeric retroelements SART1 and TRAS1 is suppressed by the silkworm Piwi subfamily proteins BmAgo3 and Siwi. The silkworm Piwi subfamily was found to be expressed predominantly in the gonads and early embryo, as in other model organisms, but in BmN4 cultured cells, these proteins formed granules that were separate from the nuage, which is a different behaviour pattern. The expression of TRAS1 was increased in BmN4 cells when BmAgo3 or Siwi were silenced by RNAi. Our results suggest that B. mori Piwi proteins are involved in regulating the transposition of telomeric retroelements, and that the functional piRNA pathway is conserved in BmN4 cultured cells. |
Keywords | Bombyx mori, SART1, TRAS1, Telomere, Piwi |
Address and Contact Information | 1Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Fukuoka 812-8581, Japan, 2Research Fellow of the Japan Society for the Promotion of Science * Author for correspondence. e-mail: kusakabe@agr.kyushu-u.ac.jp; tel./fax: +81-92-642-2842 |
DOI: 10.2478/s11658-009-0039-8 Volume 15 (2010) pp 134-152 | |
Title | SEVERAL DYSTROPHIN-GLYCOPROTEIN COMPLEX MEMBERS ARE PRESENT IN CRUDE SURFACE MEMBRANES BUT THEY ARE SODIUM DODECYL SULPHATE INVISIBLE IN KCl-WASHED MICROSOMES FROM mdx MOUSE MUSCLE |
Authors | Stéphanie Daval1, Chantal Rocher2, Yan Cherel1 and Elisabeth Le Rumeur2* |
Abstract | The dystrophin-glycoprotein complex (DGC) is a large trans- sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent. |
Keywords | Dystrophin, Muscular dystrophy, Dystrophin-glycoprotein complex, mdx mice, SDS insoluble |
Address and Contact Information | 1INRA, UMR 703, Ecole Nationale Vétérinaire de Nantes, Atlanpole – La Chantrerie, BP 40706, Nantes, 44307, France, 2Faculté de Médecine, UMR CNRS 6026, CS 34317, 35043 Rennes Cedex, France * Author for correspondence. e-mail: elisabeth.lerumeur@univ-rennes1.fr, tel.: +33 2 23 23 44 85, fax: +33 2 23 23 46 06 |
DOI: 10.2478/s11658-009-0040-2 Volume 15 (2010) pp 153-176 | |
Title | MISSENSE MUTATIONS IN IHH IMPAIR INDIAN HEDGEHOG SIGNALING IN C3H10T1/2 CELLS: IMPLICATIONS FOR BRACHYDACTYLY TYPE A1, AND NEW TARGETS FOR HEDGEHOG SIGNALING |
Authors | Shengzhen Guo1,2,3§, Jian Zhou1,2§, Bo Gao1,2,3, Jianxin Hu1,2,3, Hongsheng Wang1,2, Junwei Meng1,2, Xinzhi Zhao1,2, Gang Ma1,2, Chuwen Lin1,2, Yue Xiao1,2, Wei Tang1,2, Xuming Zhu1,2, Kathryn S.E. Cheah3, Guoying Feng4, Danny Chan3* and Lin He1,2,5* |
Abstract | Heterozygous missense mutations in IHH result in Brachydactyly type A1 (BDA1; OMIM 112500), a condition characterized by the shortening of digits due to hypoplasia/aplasia of the middle phalanx. Indian Hedgehog signaling regulates the proliferation and differentiation of chondrocytes and is essential for endochondral bone formation. Analyses of activated IHH signaling in C3H10T1/2 cells showed that three BDA1-associated mutations (p.E95K, p.D100E and p.E131K) severely impaired the induction of targets such as Ptch1 and Gli1. However, this was not a complete loss of function, suggesting that these mutations may affect the interaction with the receptor PTCH1 or its partners, with an impact on the induction potency. From comparative microarray expression analyses and quantitative real-time PCR, we identified three additional targets, Sostdc1, Penk1 and Igfbp5, which were also severely affected. Penk1 and Igfbp5 were confirmed to be regulated by GLI1, while the induction of Sostdc1 by IHH is independent of GLI1. SOSTDC1 is a BMP antagonist, and altered BMP signaling is known to affect digit formation. The role of Penk1 and Igfbp5 in skeletogenesis is not known. However, we have shown that both Penk1 and Igfbp5 are expressed in the interzone region of the developing joint of mouse digits, providing another link for a role for IHH signaling in the formation of the distal digits. |
Keywords | Indian Hedgehog, Brachydactyly type A1, Microarray, EMSA |
Address and Contact Information | 1Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China, 2Bio-X Center, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai 200030, China, 3Department of Biochemistry, The University of Hong Kong, Pokfulam, Hong Kong, China, 4Shanghai Institutes of Mental Health, Shanghai 200030, China, 5Institutes of Biomedical Sciences, Fudan University, Ming De Building, 138 Yi Xue Yuan Road, Shanghai 200032, PR China §These authors contributed equally to this work * Authors for correspondence: Dr. Lin He, Bio-X Center, Shanghai Jiao Tong University, Cental Little White House, 1954 Huashan Road, Shanghai 200030, China, tel./fax: 86-21-62822491, e-mail: helin@bio-x.cn and Dr. Danny Chan, Department of Biochemistry, The University of Hong Kong, 3/F, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China, tel.: 852-28199482, fax: 852-28551254, e-mail: chand@hkusua.hku.hk |