Vol. 16 No. 1 March 2011

DOI: 10.2478/s11658-010-0024-2 Volume 16 (2011) pp 1-24
Authors Sylwester Głowacki§, Violetta K. Macioszek§ and Andrzej K. Kononowicz*
Abstract Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader's molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity
Keywords Plant innate immunity, Pathogen recognition, Plant resistance protein
Address and Contact Information Department of Genetics, Plant Molecular Biology and Biotechnology, University of Łódź, S. Banacha 12/16, 90-237 Łódź, Poland
§ These authors contributed equally to this work
* Author for correspondence. e-mail: akononow@biol.uni.lodz.pl, tel.: (+48 42) 635 4425, fax: (+48 42) 635 4423
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DOI: 10.2478/s11658-010-0023-3 Volume 16 (2011) pp 25-39
Authors Teresa Janas1,2,* and Tadeusz Janas2
Abstract A growing number of RNA aptamers have been selected experimentally using the SELEX combinatorial approach, and these aptamers have several advantages over monoclonal protein antibodies or peptides with respect to their applications in medicine and nanobiotechnology. Relatively few successful selections have been reported for membrane molecular targets, in contrast to the situation with non-membrane molecular targets. This review compares the procedures and techniques used in selections against membrane proteins and membrane lipids. In the case of membrane proteins, the selections were performed against soluble protein fragments, detergent-membrane protein mixed micelles, whole cells, vesicles derived from cellular membranes, and enveloped viruses. Liposomes were used as an experimental system for the selection of aptamers against membrane lipids. RNA structure-dependent aptamer binding for rafts in lipid vesicles was reported. Based on the selected aptamers against DOPC and the amino acid tryptophan, a specific passive membrane transporter composed of RNA was constructed. The determination of the selectivity of aptamers appears to be a crucial step in a selection, but has rarely been fully investigated. The selections, which use whole cells or vesicles derived from membranes, can yield aptamers not only against proteins but also against membrane lipids.
Keywords RNA, SELEX, Aptamers, Membranes, Membrane proteins, Lipids, Liposomes, Rafts, Membrane transporters
Address and Contact Information 1Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, USA,
2Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland
* Author for correspondence. e-mail: teresa.janas@colorado.edu, tel.: 303-492-8377, fax: 303-492-7744
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DOI: 10.2478/s11658-010-0036-y Volume 16 (2011) pp 40-54
Authors Rob C. A. Keller*
Abstract Protein translocation is an important cellular process. SecA is an essential protein component in the Sec system, as it contains the molecular motor that facilitates protein translocation. In this study, a bioinformatics approach was applied in the search for possible lipid-binding helix regions in protein translocation motor proteins. Novel lipid-binding regions in Escherichia coli SecA were identified. Remarkably, multiple lipid-binding sites were also identified in other motor proteins such as BiP, which is involved in ER protein translocation. The prokaryotic signal recognition particle receptor FtsY, though not a motor protein, is in many ways related to SecA, and was therefore included in this study. The results demonstrate a possible general feature for motor proteins involved in protein translocation.
Keywords Lipid-binding regions, Motor proteins, Protein-lipid interactions, Protein translocation, SecA
Address and Contact Information Section Chemistry, Charlemagne College, Wilhelminastraat 13-15, Nijmegen 3913 NH, The Netherlands
* Author for correspondence. e-mail: r.keller@kgcnijmegen.nl
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DOI: 10.2478/s11658-010-0037-x Volume 16 (2011) pp 55-68
Authors Asa J. Robert McGray, Timothy Gingerich, James J. Petrik and Jonathan LaMarre*
Abstract Thrombospondin-1 (TSP-1) is a matricellular protein that participates in numerous normal and pathological tissue processes and is rapidly modulated by different stimuli. The presence of 8 highly-conserved AU rich elements (AREs) within the 3'-untranslated region (3'UTR) of the TSP-1 mRNA suggests that post-transcriptional regulation is likely to represent one mechanism by which TSP-1 gene expression is regulated. We investigated the roles of these AREs, and proteins which bind to them, in the control of TSP-1 mRNA stability. The endogenous TSP-1 mRNA half-life is approximately 2.0 hours in HEK293 cells. Luciferase reporter mRNAs containing the TSP-1 3'UTR show a similar rate of decay, suggesting that the 3'UTR influences the decay rate. Site-directed mutagenesis of individual and adjacent AREs prolonged reporter mRNA half- life to between 2.2 and 4.4 hours. Mutation of all AREs increased mRNA half life to 8.8 hours, suggesting that all AREs have some effect, but that specific AREs may have key roles in stability regulation. A labeled RNA oligonucleotide derived from the most influential ARE was utilized to purify TSP-1 ARE- binding proteins. The AU-binding protein AUF1 was shown to associate with this motif. These studies reveal that AREs in the 3'UTR control TSP-1 mRNA stability and that the RNA binding protein AUF1 participates in this control. These studies suggest that ARE-dependent control of TSP-1 mRNA stability may represent an important component in the control of TSP-1 gene expression.
Keywords Thrombospondin-1, mRNA stability, Angiogenesis, AU-rich element, Post-transcriptional, Gene expression
Address and Contact Information Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
* Author for correspondence. Fax: 1-519-767-1450, e-mail:jlamarre@uoguelph.ca
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DOI: 10.2478/s11658-010-0040-2 Volume 16 (2011) pp 69-78
Authors Małgorzata Sekuła1, Greta Janawa1, ElĹĄbieta Stankiewicz2 and Ewa StÄ™pieł1,2,*
Abstract Microparticles (MPs) are small membrane vesicles released by stimulated or apoptotic cells, including the endothelium. Hyperhomocysteinemia (HHcy) is a blood disorder characterized by an increase in the plasma concentrations of total homocysteine (Hcy). The plasma Hcy level is determined by environmental factors (dietary habits, i.e. the intake of folic acid, FA) and genetic factors (N5,N10-methylenetetrahydro-folate reductase, MTHFR, polymorphism 677C>T). To evaluate whether moderate Hcy concentrations induce endothelial MP formation, the role of FA supplementation and the influence of MTHFR polymorphism were analysed. Human umbilical vein endothelial cells (HUVEC) were treated in vitro with 50 µM of Hcy and methionine (Met). The MP number and apoptotic phenotype were analyzed using flow cytometry. Increasing doses of FA (5, 15 and 50 µM) were used to reduce the HHcy effect. The MTHFR 677C>T polymorphism was determined. HUVEC stimulated by Hcy produced significantly more MPs than HUVEC under the control conditions: 3,551 ± 620 vs 2,270 ± 657 kMP (p = 0.02). Supplementation with FA at concentrations of 5, 15 and 50 µM reduced the MP count in the cell culture supernatant to 345 ± 332, 873 ± 329, and 688 ± 453 kMP, respectively (p = 0.03). MTHFR 677C>T heterozygosity was associated with a significant increase in MP formation after stimulation with Hcy compared to the control conditions: 3,617 ± 152 vs 1,518 ± 343 kMP (p = 0.02). Furthermore, the MTHFR genotype altered MP formation after Met loading. On average, 24% of the entire MP population was apoptotic (annexin V-positive). Endothelial function impairment due to HHcy is related to MP shedding, which may involve platelets and other blood and vascular cells. MP shedding is a physiological response to moderate HHcy.
Keywords Flow cytometry, Homocysteine, Hyperhomocysteinemia, Microparticles, Human umbilical vein endothelial cells, Methylenetetrahydrofolate reductase
Address and Contact Information 1Institute of Cardiology, Jagiellonian University Medical College, KrakĂłw, Poland,
2Laboratory for Molecular Biology and Research, John Paul II Hospital, PrÄ…dnicka 80, 31-202 KrakĂłw, Poland
* Author for correspondence. e-mail: estepien@szpitaljp2.krakow.pl, tel. +48 12 6143145, fax: +48 12 6143145
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DOI: 10.2478/s11658-010-0039-8 Volume 16 (2011) pp 79-88
Authors Sergey V. Anisimov1,2,3,§, Nicolaj S. Christophersen1,§, Ana S. Correia1,4, Vanessa J. Hall1,5, Ingrid Sandelin1,6, Jia-Yi Li1 and Patrik Brundin1,*
Abstract The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates
Keywords Human embryonic stem cells, Feeder cells, DNA microarray
Address and Contact Information 1Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, BMC A10, SĂślvegatan 17, 221 84 Lund, Sweden,
2Research Department of Cell and Gene Engineering, V. A. Almazov Federal Center for Heart, Blood & Endocrinology, Saint-Petersburg, 197341, Russia,
3Department of Intracellular Signalling and Transport, Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, 194064, Russia,
4Faculty of Medicine, Centre de Recherche du CHUL, Neuroscience Axis, Université Laval, Québec, G1V4G2, QC, Canada,
5Department of Basic Animal and Veterinary Sciences, Faculty of Life Sciences, University of Copenhagen, Copenhagen, DK-1870, Denmark,
6IVF Kliniken Cura, 200 74 Malmo, Sweden
§ These authors contributed equally to this study
* Author for correspondence. e-mail: patrik.brundin@med.lu.se, tel.: +46-46-2220525; fax: +46-46-2220531
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DOI: 10.2478/s11658-010-0041-1 Volume 16 (2011) pp 89-100
Authors Keiko Doi1,2, Takahiro Fujimoto1,2, Midori Koyanagi1,2, Toshiyuki Tsunoda1,2, Yoko Tanaka1,2, Yasuhiro Yoshida1, Yasuo Takashima2, Masahide Kuroki2, Takehiko Sasazuki3 and Senji Shirasawa1,2*
Abstract ZFAT was originally identified as an immune-related transcriptional regulator containing 18 C2H2-type zinc-finger domains and one AT-hook. ZFAT is highly conserved among species and functions as an anti-apoptotic molecule in the lymphoblastic leukemia cell line, MOLT-4. We recently demonstrated that ZFAT is an essential molecule for hematopoietic differentiation in blood islands through the direct regulation of particular transcriptional factors, including Tal1, for endothelial cell assembly, and for the branch point formation of capillary-like structures. However, the molecular mechanisms underlying the anti-apoptotic function of ZFAT remain unknown. Here, we report that ZFAT knockdown by small interfering RNA induced apoptosis in mouse embryonic fibroblasts (MEFs). This response had been similarly observed for MOLT-4 cells. To explore the molecular mechanisms for ZFAT in anti-apoptotic function in both MEFs and MOLT-4 cells, microarray expression analysis and quantitative RT-PCR were done. Of interest was that Bcl-2 and Il6st were identified as commonly down-regulated genes by the depletion of ZFAT for both MEFs and MOLT-4 cells. These results suggest that ZFAT is a critical molecule for cell survival in MEFs and MOLT-4 cells at least in part through the regulation of the apoptosis involved in the BCL-2- and IL6st-mediated pathways. Further elucidation of the molecular functions for ZFAT might shed light on the cellular programs in the mesoderm-derived cells.
Keywords ZFAT, Transcription factor, MEFs, Apoptosis, Gene expression
Address and Contact Information 1Department of Cell Biology, Faculty of Medicine, Fukuoka University,7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan,
2Center for Advanced Molecular Medicine, Fukuoka University, Fukuoka 814-0180, Japan,
3Kyushu University, Fukuoka 812-8581, Japan
* Author for correspondence. e-mail: sshirasa@fukuoka-u.ac.jp; phone: +81-92-801-1011; fax: +81-92-864-3865
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DOI: 10.2478/s11658-010-0043-z Volume 16 (2011) pp 101-113
Authors Amit Khanna1,3, Kulandaivelu Mahalingam3, Debarshi Chakrabarti2 and Giridharan Periyasamy1,*
Abstract Gemcitabine, a novel pyrimidine nucleoside analog, has become the standard chemotherapeutic agent for pancreatic cancer patients. The clinical impact of gemcitabine remains modest owing to the high degree of inherent and acquired resistance. There are various lines of evidence that confirm the role of Ets-1, a proto-oncoprotein, in tumor invasion, progression, and chemoresistance. This study examines a hypothesis that implicates Ets-1 in the development of gemcitabine-resistance in pancreatic cancer cells. Ets-1 protein expression was assessed in parental pancreatic cancer cells and their gemcitabine-resistant clones. Western blot analysis revealed elevated levels of Ets-1 protein expression in gemcitabine-resistant PANC1GemRes (4.8-fold increase; P < 0.05), MIA PaCa2GemRes (3.2-fold increase; P < 0.05), and Capan2GemRes (2.1-fold increase; P < 0.05) cells as compared to their parental counterparts. A time course analysis was conducted to determine the change in Ets-1 expression in the parental cells after incubation with gemcitabine. Reverse transcriptase quantitative real-time PCR (RT-qPCR) and Western blot analysis revealed a significant increase in Ets-1 expression. All the three parental cells incubated with gemcitabine showed elevated Ets-1 protein expression at 6 h. By 24 h, the expression level had decreased. Using small interfering RNA (siRNA) against Ets-1 in gemcitabine-resistant cells, we demonstrated a reversal in gemcitabine chemosensitivity and also detected a marked reduction in the expression of the Ets-1 target genes MMP1 and uPA. Our novel finding demonstrates the significance of Ets-1 in the development of gemcitabine chemoresistance in pancreatic cancer cells. Based on these results, a new siRNA-based therapeutic strategy targeting the Ets-1 genes can be designed to overcome chemoresistance.
Keywords Gemcitabine, Ets-1, MMP1, uPA, PANC1, MIA PaCa2, Capan2
Address and Contact Information 1Department of High Throughput Screening and Biotechnology,
2Department of Biomarker Discovery, Piramal Life Sciences Ltd., 1A, Nirlon Complex, Goregaon-E, Mumbai 400063, Maharashtra, India,
3School of Biotechnology, Chemical and Bio-Medical Engineering, VIT University, Vellore, Tamil Nadu, India
* Author for correspondence. e-mail: periyasamy.giridharan@piramal.com, phone: +91-22-30818807, fax: +91-22-30813000
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DOI: 10.2478/s11658-010-0038-9 Volume 16 (2011) pp 114-148
Authors Magdalena Zaremba-Czogalla, Magda Dubińska-Magiera and Ryszard Rzepecki*
Abstract Laminopathies are rare human degenerative disorders with a wide spectrum of clinical phenotypes, associated with defects in the main protein components of the nuclear envelope, mostly in the lamins. They include systemic disorders and tissue-restricted diseases. Scientists have been trying to explain the pathogenesis of laminopathies and find an efficient method for treatment for many years. In this review, we discuss the current state of knowledge about laminopathies, the molecular mechanisms behind the development of particular phenotypes, and the prospects for stem cell and/or gene therapy treatments.
Keywords Laminopathies, Nuclear lamina, Lamin, Emerin, Gene therapy
Address and Contact Information Laboratory of Nuclear Proteins, Faculty of Biotechnology, University of Wrocław, ul. Przybyszewskiego 63/77, 51-148 Wrocław, Poland
* Author for correspondence. e-mail: rzepecki@ibmb.uni.wroc.pl, phone: +48 71 375 63 08
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DOI: 10.2478/s11658-010-0042-0 Volume 16 (2011) pp 149-161
Authors Barry R. Imhoff and Jason M. Hansen*
Abstract Development is an orderly process that requires the timely activation and/or deactivation of specific regulatory elements that control cellular proliferation, differentiation and apoptosis. While many studies have defined factors that control developmental signaling, the role of intracellular reduction/oxidation (redox) status as a means to control differentiation has not been fully studied. Redox states of intracellular couples may play a very important role in regulating redox-sensitive elements that are involved in differentiation signaling into specific phenotypes. In human mesenchymal stem cells (hMSCs), which are capable of differentiating into many different types of phenotypes, including osteoblasts and adipocytes, glutathione (GSH), cysteine (Cys) and thioredoxin-1 (Trx1) redox potentials were measured during adipogenesis and osteogenesis. GSH redox potentials (Eh) during both osteogenesis and adipogenesis became increasingly oxidized as differentiation ensued, but the rate at which this oxidation occurred was unique for each process. During adipogenesis, Cys Eh became oxidized as adipogenesis progressed but during osteogenesis, it became reduced. Interestingly, intracellular Trx1 concentrations appeared to increase in both adipogenesis and osteogenesis, but the Eh was unchanged when compared to undifferentiated hMSCs. These data show that hMSC differentiation into either adipocytes of osteoblasts corresponds to a unique redox state profile, suggesting that differentiation into specific phenotypes are likely regulated by redox states that are permissive to a specific developmental process.
Keywords Mesenchymal stem cells, Adipogenesis, Osteogenesis, Glutathione, Cysteine, Thioredoxin-1, Redox status
Address and Contact Information Division of Pulmonary, Allergy/Immunology, Cystic Fibrosis and Sleep, Department of Pediatrics, Emory School of Medicine, Emory University Atlanta, 2015 Uppergate Drive Suite 350, Altanta, GA 30322, USA
* Author for correspondence. e-mail: jhansen@emory.edu, phone: 404-727-3145, fax: 404-712-0920
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DOI: 10.2478/s11658-010-0044-y Volume 16 (2011) pp 162-200
Authors Saima Salim and Sharique A. Ali*
Abstract Drug discovery in skin pharmacotherapy is an enormous, continually expanding field. Researchers are developing novel and sensitive pharmaceutical products and drugs that target specific receptors to elicit concerted and appropriate responses. The pigment-bearing cells called melanophores have a significant contribution to make in this field. Melanophores, which contain the dark brown or black pigment melanin, constitute an important class of chromatophores. They are highly specialized in the bidirectional and coordinated translocation of pigment granules when given an appropriate stimulus. The pigment granules can be stimulated to undergo rapid dispersion throughout the melanophores, making the cell appear dark, or to aggregate at the center, making the cell appear light. The major signals involved in pigment transport within the melanophores are dependent on a special class of cell surface receptors called G-protein-coupled receptors (GPCRs). Many of these receptors of adrenaline, acetylcholine, histamine, serotonin, endothelin and melatonin have been found on melanophores. They are believed to have clinical relevance to skin-related ailments and therefore have become targets for high throughput screening projects. The selective screening of these receptors requires the recognition of particular ligands, agonists and antagonists and the characterization of their effects on pigment motility within the cells. The mechanism of skin pigmentation is incredibly intricate, but it would be a considerable step forward to unravel its underlying physiological mechanism. This would provide an experimental basis for new pharmacotherapies for dermatological anomalies. The discernible stimuli that can trigger a variety of intracellular signals affecting pigment granule movement primarily include neurotransmitters and hormones. This review focuses on the role of the hormone and neurotransmitter signals involved in pigment movement in terms of the pharmacology of the specific receptors.
Keywords G-protein-coupled receptors, Melanocytes, Skin pigmentation, Neurotransmitter, Pigment cells, Melanocyte-stimulating hormone, MSH, Drug discovery
Address and Contact Information Postgraduate Department of Biotechnology, Saifia College of Science Bhopal, 462001 India
* Author for correspondence. e-mail: suddin@australia.edu, phone: +91 9893015818
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