Vol. 18 No. 3 September 2013

DOI: 10.2478/s11658-013-0091-2 Volume 18 (2013) pp 315-327
Title CHARACTERIZATION OF THE 5’-FLANKING REGION OF THE MOUSE ASPARAGINE-LINKED GLYCOSYLATION 12 HOMOLOG GENE
Authors Kentaro Oh-Hashi1,*, Tomomi Tejima1, Yoko Hirata1,2 and Kazutoshi Kiuchi1,2
Abstract Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique headto-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study,we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5’-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reducedALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in thisstudy. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.
Keywords ALG12, CRELD2, ERSE, Ets family, NF-Y, Sp1 family, YY1
Address and Contact Information 1Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,
2United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
* Author for correspondence. e-mail: oohashi@gifu-u.ac.jp, tel: +81-58-293-2659, fax: +81-58-293-2794

DOI: 10.2478/s11658-013-0092-1 Volume 18 (2013) pp 328-339
Title THE PROTECTIVE EFFECT OF CROCIN ON THE AMYLOID FIBRIL FORMATION OF Aβ42 PEPTIDE in vitro
Authors Arezou Ghahghaei1,*, S. Zahra Bathaie2, Hoda Kheirkhah1 and Elmira Bahraminejad1
Abstract Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ40, and the less common but more amyloidogenic form, Aβ42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ42 amyloids. To further investigate the relationship between crocin and Aβ42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβamyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.
Keywords Alzheimer’s disease, Neurotic plaques, Aβ42, Crocin, Amyloid, Neurofibrillary, Aggregation, Oligomerization, Protofibrils, Cytotoxic
Address and Contact Information 1 Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran,
2 Depatmentof Clinical Biochemistry, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran
* Author for correspondence. e-mail: arezou@chem.usb.ac.ir, tel.: 001198 915 190 4052, fax: 001198 541 2446565

DOI: 10.2478/s11658-013-0093-0 Volume 18 (2013) pp 340-354
Title Smad1 STABILIZATION AND DELOCALIZATION IN RESPONSE TO THE BLOCKADE OF BMP ACTIVITY
Authors Lili Li1,2, Jianhe Wang3, Jenny Fung Ling Chau3, Huijuan Liu2, Baojie Li2, Aijun Hao1 and Jing Li4,*
Abstract Signaling at the plasma membrane receptors is generally terminated by some form of feedback regulation, such as endocytosis and/or degradation of the receptors. BMP-Smad1 signaling can also be attenuated by BMP-induced expression of the inhibitory Smads, which are negative regulators of Smad1 transactivation activity and/or BMP antagonists. Here, we report on a novel Smad1 regulation mechanism that occurs in response to the blockade of BMP activity. Lowering the serum levels or antagonizing BMPs with noggin led to upregulation of Smad1 at the protein level in several cell lines, but not to upregulation of Smad5, Smad8 or Smad2/3. The Smad1 upregulation occurs at the level of protein stabilization. Upregulated Smad1 was relocalized to the perinuclear region. These alterations seemto affect the dynamics and amplitude of BMP2-induced Smad1 reactivation. Our findings indicate that depleting or antagonizing BMPs leads to Smad1 stabilization and relocalization, thus revealing an unexpected regulatory mechanism for BMP-Smad1 signaling.
Keywords Smad1, BMPs, Feedback regulation, Degradation, Noggin, BMPRIA, PPM1A, Serum starvation, Intestinal villi, Smad2/3
Address and Contact Information 1 Department of Histology and Embryology, Shandong University School of Medicine 44#, Wenhua Xi Road, Jinan, Shandong, 250012, China,
2 Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China,
3 Institute of Molecular and Cell Biology, Singapore,
4 Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
* Author for correspondence. e-mail: lj.xinhua@gmail.com, tel. and fax: 86-21-25078843

DOI: 10.2478/s11658-013-0095-y Volume 18 (2013) pp 355-367
Title ROLE OF INFLAMMASOMES AND THEIR REGULATORS IN PROSTATE CANCER INITIATION, PROGRESSION AND METASTASIS
Authors Sudhakar Veeranki
Abstract Prostate cancer is one of the main cancers that affect men, especially older men. Though there has been considerable progress in understanding the progression of prostate cancer, the drivers ofits development need to be studied more comprehensively. The emergence of resistant forms has also increased the clinical challenges involved in the treatment of prostate cancer. Recent evidence has suggested that inflammation might playan important role at various stages of cancer development. This review focuses on inflammasome research that is relevant to prostate cancer and indicates future avenues of study into its effective prevention and treatment through inflammasome regulation. With regard to prostate cancer, such research is still in its early stages. Further study is certainly necessary to gain a broader understanding ofprostate cancer development and to create successful therapy solutions.
Keywords Inflammation, Adaptor protein ASC, Caspase-1, Prostate cancer, IFI16, caspase-1, IL-1β, IL-18, NLR
Address and Contact Information University of Louisville, Department of Biochemistry and Molecular Biology, Baxter II, Rm. 117, 580 S. Preston St., Louisville, KY 40202, USA
* Author for correspondence. e-mail: veeransr@gmail.com, tel.: 9736101160

DOI: 10.2478/s11658-013-0094-z Volume 18 (2013) pp 368-397
Title TUBULIN-INTERACTIVE STILBENE DERIVATIVES AS ANTICANCER AGENTS
Authors Renata Mikstacka*, Tomasz Stefański and Jakub Różański
Abstract Microtubules are dynamic polymers that occur in eukaryotic cells and play important roles in cell division, motility, transport and signaling. They form during the process of polymerization of α- and β-tubulin dimers. Tubulin is a significant and heavily researched molecular target for anticancer drugs. Combretastatins are natural cis-stilbenes that exhibit cytotoxic properties in cultured cancer cells in vitro. Combretastatin A-4 (3’-hydroxy-3,4,4’,5-tetramethoxy-cis-stilbene; CA-4) is a potent cytotoxic cis-stilbene that binds to β-tubulin at the colchicine-binding site and inhibits tubulin polymerization. The prodrug CA-4 phosphate is currently in clinical trials as a chemotherapeutic agent for cancer treatment. Numerous series of stilbene analogs have been studied in search of potent cytotoxic agents with the requisite tubulin-interactive properties. Microtubule-interfering agents include numerous CA-4 and trans-resveratrol analogs and other synthetic stilbene derivatives. Importantly, these agents are active in both tumor cells and immature endothelial cells of tumor blood vessels, where they inhibit the process of angiogenesis. Recently, computer-aided virtual screening was usedto select potent tubulin-interactive compounds. This review covers the role of stilbene derivatives as a class of antitumor agents that act by targeting microtubule assembly dynamics. Additionally, we present the results of molecular modeling of their binding to specific sites on the α- and β-tubulin heterodimer. This has enabled the elucidation of the mechanism of stilbene cytotoxicity and isuseful in the design of novel agents with improved anti-mitotic activity. Tubulin-interactive agents are believed to have the potential to play a significant role in the fight against cancer.
Keywords Tubulin polymerization, Tubulin-interactive agents, Stilbenes, Combretastatins
Address and Contact Information Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznań, Poland
* Author for correspondence. e-mail: rmikstac@ump.edu.pl, tel.: +48618546625, fax: +48618542520

DOI: 10.2478/s11658-013-0096-x Volume 18 (2013) pp 398-415
Title TRANSCRIPTIONAL REGULATION OF MOUSE MESENCEPHALIC ASTROCYTE-DERIVED NEUROTROPHIC FACTOR IN Neuro2a CELLS
Authors Kentaro Oh-Hashi1,*, Yoko Hirata1,2 and Kazutoshi Kiuchi1,2
Abstract Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel type of trophic factor. Recent studies indicate that the MANF gene is induced in response to endoplasmic reticulum (ER) stress through ER stress response element II (ERSE-II) in its 5’-flanking region. In this study, we evaluated the roles of six ER stressresponse transcription factors in the regulation of the promoter activities of the mouse MANF gene via ERSE-II using various types of mutant MANF luciferase reporter constructs. Treatment with thapsigargin (Tg) induced MANF mRNA generation in parallel with the elevation of ATF6α, sXBP and Luman mRNA levels in Neuro2a cells. Of the six transcription factors, ATF6α most strongly increased the MANF promoter activity via ERSE-II, while the effects of ATF6β and sXBP1 were moderate. However, overexpression of Luman or OASIS did not enhance ERSE-II-dependent MANF promoter activity inNeuro2a cells. To evaluate the relationships between transcription factors in the regulation of ERSE-II-dependent MANF promoter activity, we transfected two effective transcription factor constructs chosen from ATF6α, ATF6β, uXBP1 and sXBP1 into Neuro2a cells with the MANF reporter construct.The MANF promoter activity induced by co-transfection of ATF6α with ATF6β was significantly lower than that induced by ATF6α alone, while other combinations did not show any effect on the ERSE-II-dependent MANF promoter activityin Neuro2a cells. Our study is the first to show the efficiency of ER stress-related transcription factors for ERSE-II in activating the transcription of the mouse MANF gene in Neuro2a cells.
Keywords ATF6α, ATF6β, ERSE-II, ER stress, Luman, MANF, OASIS, sXBP1, uXBP1
Address and Contact Information 1 Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan,
2 United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
* Author for correspondence. e-mail: oohashi@gifu-u.ac.jp, tel.: +81-58-293-2659, fax: +81-58-293-2794

DOI: 10.2478/s11658-013-0097-9 Volume 18 (2013) pp 416-432
Title IDENTIFICATION OF microRNAsPOTENTIALLY INVOLVED IN MALE STERILITY OF Brassica campestrisssp. chinensis USING microRNA ARRAY AND QUANTITATIVE RT-PCR ASSAYS
Authors Jianxia Jiang, Jingjing Jiang, Yafei Yang and Jiashu Cao*
Abstract microRNAs (miRNAs) are a class of newly identified, noncoding, small RNA molecules that negatively regulate gene expression. Many miRNAs are reportedly involved in plant growth, development and stress response processes. However, their roles in the sexual reproduction mechanisms in flowering plants remain unknown. Pollendevelopment is an important process in the life cycle of a flowering plant, and it is closely related to the yield and quality of crop seeds. This study aimed to identify miRNAs involved in pollen development. A microarray assay was conducted using the known complementary sequences of plant miRNAs as probes on inflorescences of a sterile male line (Bcajh97-01A) and a fertile male line (Bcajh97-01B) of the Brassica campestris ssp. chinensiscv. ‘Aijiaohuang’ genic male sterility sister line system (Bcajh97-01A/B). The results showed that 44 miRNAs were differently expressed in the two lines. Ofthese, 15 had over 1.5-fold changes in their transcript levels, with 9 upregulated and 6 downregulated miRNAs in inflorescences of ‘Bcajh97-01A’ sterile line plants. We then focused on 3 of these 15 miRNAs (miR158, miR168 and miR172). Through computational methods, 13 family members were predicted for these 3 miRNAs and 22 genes were predicted to be their candidate target genes. By using 5’ modified RACE, 2 target genes of miR168 and 5 target genes of miR172 were identified. Then, qRT-PCR was applied to verify the existence and expression patterns of the 3 miRNAs in the flower buds at five developmental stages. The results were generally consistent with those of the microarray. Thus, this study may give a valuable clue for further exploring the miRNA group that may function during pollen development.
Keywords Brassica campestris, Chinese cabbage, Brassica rapa, Microarray, microRNA, Pollen development, Quantitative RT-PCR, 5’ modified RACE, Male sterile line, Male fertile line
Address and Contact Information Laboratory of Cell & Molecular Biology,Institute of Vegetable Science, Zhejiang University, Hangzhou 310058, China
* Author for correspondence. e-mail: jshcao@zju.edu.cn, tel.: +86-571-88982188, fax: +86-571-88982188

DOI: 10.2478/s11658-013-0098-8 Volume 18 (2013) pp 433-446
Title MOLECULAR CLONING AND CHARACTERIZATION OF A NOVEL ANTI-TLR9 INTRABODY
Authors Elisa Reimer1,§, Stefan Somplatzki1,§, Diana Zegenhagen1, Svenja Hänel1, Alina Fels1, Thorsten Bollhorst1, Ludger Grosse Hovest2, Stefan Bauer3, Carsten J. Kirschning4 and Thomas Böldicke1,*
Abstract Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κBdriven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFαproduction was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.
Keywords Recombinant antibodies, Recombinant adenovirus, Protein knockdown, Intracellular toll-like receptors, TLR9, ER intrabodies, Macrophage activation
Address and Contact Information 1 Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation, Inhoffenstraße 7, 38124 Braunschweig, Germany,
2 EberhardKarls-University, Department of Immunology, Auf der Morgenstelle 15, 72076 Tübingen, Germany,
3 Philipps-Universität Marburg, Institut für Immunologie, Hans-Meerwein Straße 2, 35043 Marburg, Germany,
4 Universitätsklinikum Essen, Institut für Medizinische Mikrobiologie, Hufelandstraße 55, 45122 Essen, Germany
§ These authors contributed equally to this work.
* Author for correspondence: e-mail: thomas.boeldicke@helmholtz-hzi.de; tel.: ++49-(0)531-6181-5050; fax: ++49-(0)531-6181-7099

DOI: 10.2478/s11658-013-0099-7 Volume 18 (2013) pp 447-458
Title THE NTPASE/HELICASE DOMAIN OF HEPATITIS C VIRUS NONSTRUCTURAL PROTEIN 3 INHIBITS PROTEIN KINASE C INDEPENDENTLY OF ITS NTPASE ACTIVITY
Authors Philip Hartjen1,2,3,*, Bastian Höchst1,4, Denise Heim2, Henning Von Der Kammer1, Judith Lucke1, Michael Reinholz1, Andrea Baier5, Ralf Smeets3, Henning Wege 2, Peter Borowski† 1,5 and Julian Schulze Zur Wiesch2
Abstract Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3,to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro.PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all ofthe PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCαwas inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50= 0.7 µM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.
Keywords Hepatitis C virus (HCV), NS3 protein, Protein kinase C (PKC), PKC isotypes, Protein kinase inhibitors, Pseudosubstrate inhibition, Hepatocellular carcinoma (HCC)
Address and Contact Information 1 Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany,
2 I Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20251 Hamburg, Germany,
3 Department of Oral and Maxillofacial Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany,
4 Institutes of Molecular Medicine and Experimental Immunology, University Hospital Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany,
5 Department of Molecular Biology, The John Paul II Catholic University of Lublin, ul. Konstantynów 1I, 20-708 Lublin Poland
* Author for correspondence. e-mail: philip.hartjen@gmail.com, Department of Oral and Maxillofacial Surgery, University MedicalCenter Hamburg-Eppendorf, Hamburg, Germany

DOI: 10.2478/s11658-013-0100-5 Volume 18 (2013) pp 459-478
Title VIOLOGEN-PHOSPHORUS DENDRIMERS EXHIBIT MINOR TOXICITY AGAINST A MURINE NEUROBLASTOMA CELL LINE
Authors Joanna Lazniewska1, *, Katarzyna Milowska1, Nadia Katir2, Abdelkim El Kadib3, Maria Bryszewska1, Jean-Pierre Majoral2 and Teresa Gabryelak1
Abstract Dendrimers containing viologen (derivatives of 4,4’-bipyridyl) units in their structure have been demonstrated to exhibit antiviral activity against human immunodeficiency virus (HIV-1). It has also recently been revealed that novel dendrimers with both viologen units and phosphorus groups in their structure show different antimicrobial, cytotoxic and hemotoxic properties, and have the ability to influence the activity of cholinesterases and to inhibit α-synuclein fibrillation. Since the influence of viologen-phosphorus structures on basic cellular processes had not been investigated, we examined the impact of such macromolecules on the murine neuroblastoma cell line (N2a). We selected three water-soluble viologen-phosphorus(VPD) dendrimers, which differ in their core structure, number of viologen units and number and type of surface groups, and analyzed several aspects of the cellular response. These included cell viability, generation of reactive oxygen species (ROS), alterations in mitochondrial activity, morphological modifications, and the induction of apoptosis and necrosis. The MTT assay results suggest that all of the tested dendrimers are only slightly cytotoxic. Although some changes in ROS formation and mitochondrial function were detected, the three compounds did not induce apoptosis or necrosis. In light of these results, we can assume that the tested VPD are relatively safe for mouse neuroblastoma cells. Although more research on their safety is needed, VPD seem to be promising nanoparticles for further biomedical investigation.
Keywords Apoptosis, Cytotoxicity, N2a cell line, ROS, Viologen-phosphorus dendrimers
Address and Contact Information 1 Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pomorska 141/143, 90-236 Łódź, Poland
2 Laboratoire de Chimie de Coordination CNRS, 205 Route de Narbonne, 31077 Toulouse, France,
3 Institute of Nanomaterials and Nanotechnology and Moroccan Foundation for AdvancedScience, Innovation and Research (INANOTECH-MAScIR), ENSET, Avenue de l’Arme ́e Royale, Madinat El Irfane, 10100 Rabat, Morocco
* Author for correspondence: e-mail address: jolazn@biol.uni.lodz.pl, tel.: +48 42 635 43 80, fax: +48 42 635 44 74