Vol. 15 No. 3 September 2010

DOI: 10.2478/s11658-010-0013-5 Volume 15 (2010) pp 365-376
Title A SMALL SEQUENCE IN DOMAIN V OF THE MITOCHONDRIAL LARGE RIBOSOMAL RNA RESTORES Drosophila melanogaster POLE CELL DETERMINATION IN UV-IRRADIATED EMBRYOS
Authors Rossana Psaila1†, Donatella Ponti2,3*, Marta Ponzi1, Franca Gigliani2 and Piero Augusto Battaglia1
Abstract The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.
Keywords mtlrRNA, Drosophila embryo, Drosophila pole cell determination
Address and Contact Information 1Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanita Viale Regina Elena 299, 00161 Roma Italy,
2Dipartimento di Biotecnologie Cellulari ed Ematologia, Universita degliStudi di Roma “Sapienza”, Viale Regina Elena 324, 00161 Roma Italy,
3Dipartimento di Patologia Molecolare, Universita’ degli Studi di Roma “Sapienza”, Corso della Repubblica 79, 04100 Latina, Italy
Present address: Istituto di Neurobiologia e Medicina Molecolare CNR, Roma Italy
* Author for correspondence. e-mail: donatella.ponti@uniroma1.it, tel.: +39 0773 471046, fax: +39 0773 418400
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0014-4 Volume 15 (2010) pp 377-394
Title 12-O-TETRADECANOYLPHORBOL-1,3-ACETATE INDUCES THE NEGATIVE REGULATION OF PROTEIN KINASE B BY PROTEIN KINASE Ca DURING GASTRIC CANCER CELL APOPTOSIS
Authors Bing Zhang1 and Chun Xia2*
Abstract The PKB signaling pathway is essential for cell survival and the inhibition of apoptosis, but its functional mechanisms have not been fully explored. Previously, we reported that TPA effectively inhibited PKB activity and caused PKB degradation, which was correlated with the repression of PKB phosphorylation at Ser473. In this study, we focus on how PKB is regulated by TPA in gastric cancer cells. One of the TPA targets, PKCa, was found to mediate the inhibition of PKB phosphorylation and degredation caused by TPA. Furthermore, TPA induced the import of PKCa into the nucleus, where PKCa exerted an inhibitory effect on PKB expression and phosphorylation. As a result, cancer cell proliferation was arrested. Our study characterizes a novel function of PKCa in mediating the negative regulation of PKB by TPA, and suggests a potential application in the clinical treatment of gastric cancer.
Keywords Protein kinase B (PKB), Protein kinase Ca (PKCa), Translocation, Growth inhibition, TPA
Address and Contact Information 1Medical School, Xiamen University, Xiamen 361005, Fujian Province, China
2Zhongshan Hospital, Xiamen University, Xiamen 361004, Fujian Province, China
* Author for correspondence. e-mail: chunxia99@yahoo.com.cn, chunxia@xmu.edu.cn, tel.: +86 5925921461, fax: +86 5922188680
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0015-3 Volume 15 (2010) pp 395-405
Title INHIBITION OF CALPAIN BUT NOT CASPASE ACTIVITY BY SPECTRIN FRAGMENTS
Authors Ramunas Rolius, Chloe Antoniou#, Lidia A. Nazarova, Stephen H. Kim, Garrett Cobb, Pooja Gala, Priyanka Rajaram, Qufei Li# and Leslie W.-M. Fung*
Abstract Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) a- and b-spectrin and erythroid b-spectrin. The cleavage of erythroid a-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of a-spectrin, and C-terminal region of b-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.
Keywords Spectrin, Calpain, Caspase, Calpain inhibition, Cysteine protease
Address and Contact Information Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, M/C 111, Chicago, IL, 60607, USA
*Author for correspondence. e-mail: lfung@uic.edu, tel.: (312) 355-5516, fax: (312) 996-0431
#Present address: Department of Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, Chicago, IL, 60637, USA
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0012-6 Volume 15 (2010) pp 406-423
Title THE EFFECT OF THE LIPID-BINDING SITE OF THE ANKYRIN- BINDING DOMAIN OF ERYTHROID b-SPECTRIN ON THE PROPERTIES OF NATURAL MEMBRANES AND SKELETAL STRUCTURES
Authors Anna Chorzalska, Agnieszka Łach, Tomasz Borowik2, Marcin Wolny1, Anita Hryniewicz-Jankowska1, Adam Kolondra1, Marek Langner2,3 and Aleksander F. Sikorski1,3*
Abstract It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of b-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482-94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the b-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.
Keywords Spectrin-lipid interactions, Ankyrin-binding domain, Resealed ghosts, Membrane skeleton properties, Transmembrane protein aggregation
Address and Contact Information 1Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wroclaw, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
2Wroclaw University of Technology, Wyb. Wyspiańskiego 27, 50-370 Wrocław, Poland,
3Academic Centre for Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, 51-148 Wrocław, Poland
§These authors equally contributed to this work
* Author for correspondence. e-mail: afsbc@ibmb.uni.wroc.pl, tel.: +4871 3756 233, fax: +4871 3756 208.
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0017-1 Volume 15 (2010) pp 424-439
Title THE HETEROGENEITY OF CELL SUBTYPES FROM A PRIMARY CULTURE OF HUMAN AMNIOTIC FLUID
Authors Shengli Zhang1,2, Hongquan Geng3, Hua Xie1, Qiquan Wu1,2, Xiaorong Ma1,2, Junmei Zhou1,3* and Fang Chen1,3*
Abstract Heterogeneous human amniotic fluid contains various cell types. Herein, we report on the possibility of simultaneously isolating three subtypes of cells from one primary culture. Using a stainless steel instrument named a colony poculum, two of the three cell subtypes could be efficiently cultured, and these were further characterized. The results indicated that these two cell subtypes had different morphologies and were characterized by different cell marker expression profiles, including the differential expression of CD105, CD117 and EBAF. Furthermore, their gene expression array data revealed their different gene expression profiles. Although both cell types expressed several embryonic stem cell-specific markers, they were non-tumorigenic in vivo. This paper not only provides new insight into the heterogeneity of human amniotic fluid, it also presents a simple yet efficient cell isolation method. These results will contribute to the thorough investigation of the properties and potential future applications of human amniotic fluid-derived cells.
Keywords Human amniotic fluid, Cell subtypes, Isolation, Colony poculum
Address and Contact Information 1Department of Urology, Shanghai Children’s Hospital, Shanghai Jiao Tong University School of Medicine, 1400 Beijing Road West, Shanghai, 200040, China,
2Department of Urology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China,
3Tissue Engineering Laboratory, Xinhua Hospital, Shanghai Institute for Pediatric Research, Shanghai Jiao Tong University School of Medicine, Shanghai, China
* Authors for correspondence. e-mail addresses: Fang Chen: cdbxh@yahoo.cn, Junmei Zhou: jemyzh@vip.sina.com, tel.: 86-21-62470059, fax: 86-21-62790494
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0016-2 Volume 15 (2010) pp 440-450
Title CELECOXIB INCREASES RETINOID SENSITIVITY IN HUMAN COLON CANCER CELL LINES
Authors Jian-Pei Liu, Hong-Bo Wei*, Zong-Heng Zheng, Wei-Ping Guo and Jia-Feng Fang
Abstract Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE2 production was measured with the ELISA assay. The expression of RARß was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE2 did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARß in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE2 did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARß and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.
Keywords Colonic neoplasm, Retinoic acid, Celecoxib, Sensitivity
Address and Contact Information Department of Gastrointestinopancreatic Surgery, The Third Affiliated Hospital of Sun Yat-Sen University, Tian He Road 600, Guangzhou 510630, P.R. China
* Author for correspondence. e-mail: drwhb@21cn.com, tel: +86-13060620467
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0018-0 Volume 15 (2010) pp 451-472
Title HYBRID CELLS DIFFERENTIATE TO HEPATIC LINEAGE CELLS AND REPAIR OXIDATIVE DAMAGE
Authors Dan Xu1, Feng Wang1,*, Hongyan Gu2, Jia Wang2, Qinglong Guo2, Yanli Zhang1 and Ziyu Wang1
Abstract Hybrid cells derived from stem cells play an important role in organogenesis, tissue regeneration and cancer formation. However, the fate of hybrid cells and their range of function are poorly understood. Fusing stem cells and somatic cells induces somatic cell reprogramming, and the resulting hybrid cells are embryonic stem cell-like cells. Therefore, we hypothesize that fusion- induced hybrid cells may behave like ES cells in certain microenvironments. In this study, human hepatic cells were induced to apoptosis with H2O2, and then co-cultured with hybrid cells that had been derived from mouse ES cells and human hepatic cells using a transwell. After co-culturing, the degree of apoptosis was evaluated using Annexin-V/PI double-staining analysis, flow cytometry and Western-blot. We observed that H2O2-induced cell apoptosis was inhibited by co-culture. In addition, the activity of injury-related enzymes (GSH-Px, LDH and SOD) and the level of albumin release in the co-culture system trended toward the level of normal undamaged hepatic cells. The stably increased levels of secretion of ALB in the co-culture system also confirmed that co-culture with hybrid cells helped in recovery from injury. The fate of the hybrid cells was studied by analyzing their gene expression and protein expression profiles. The results of RT-PCR indicated that during co-culturing, like ES cells, hybrid cells differentiated into hepatic lineage cells. Hybrid cells transcripted genes from both parental cell genomes. Via immunocytochemical analysis, hepatic directional differentiation of the hybrid cells was also confirmed. After injecting the hybrid cells into the mouse liver, the GFP-labeled transplanted cells were distributed in the hepatic lobules and engrafted into the liver structure. This research expands the knowledge of fusion-related events and the possible function of hybrid cells. Moreover, it could indicate a new route of differentiation from pluripotent cells to tissue-specific cells via conditional co-culture.
Keywords Hybrid cells, Regeneration, Embryonic stem cell, Differentiation, Oxidative damage, Apoptosis
Address and Contact Information 1Center of Embryo Engineering and Technology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China,
2Jiangsu Key Laboratory of Carcinogenesis and intervention (China Pharmaceutical University), Nanjing 210009, China
* Author for correspondence. e-mail: caeet@njau.edu.cn
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0021-5 Volume 15 (2010) pp 473-484
Title SWITCHING p53-DEPENDENT GROWTH ARREST TO APOPTOSIS VIA THE INHIBITION OF DNA DAMAGE-ACTIVATED KINASES
Authors Pavla Hublarova1, Kristina Greplova2, Jitka Holcakova1, Borivoj Vojtesek1 and Roman Hrstka1*
Abstract Cisplatin and doxorubicin are widely used anticancer drugs that cause DNA damage, which activates the ATM-Chk2-p53 pathway in cancer cells. This activation leads to cell cycle block or apoptosis, depending on the nature of the DNA damage. In an attempt to enhance the effects of these agents, we inhibited ATM/ATR and Chk2, which are known upstream regulators of p53. The cancer cell lines A2780 and ARN8, bearing the wild-type p53 protein, were used to study changes in p53 activation and trans-activation. Our results suggest that the G1-checkpoint, normally activated by DNA damage, is functionally overcome by the action of kinase inhibitors that sensitize cells to apoptosis. Both inhibitors show these effects, albeit with variable intensity in different cell lines, which is promising for other studies and theoretically for use in clinical practice.
Keywords Protein p53, ATM/ATR kinases, Chk2, Inhibitors of DNA damage- activated kinases, Doxorubicin, Cisplatin
Address and Contact Information 1Department of Oncological and Experimental Pathology,
2Department of Laboratory Medicine, Masaryk Memorial Cancer Institute, Zluty kopec 7,656 53, Brno, Czech Republic
* Author for correspondence. e-mail: hrstka@mou.cz, tel.: +420543133306, fax: +420543211169
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0022-4 Volume 15 (2010) pp 485-495
Title A STUDY OF THE INTERACTION OF THE C-REACTIVE PROTEIN MONOMER WITH THE U937 MONOCYTE
Authors Jing Zhao1 and Xin-He Shi2,*
Abstract C-reactive protein (CRP) has two structurally distinct isoforms, the CRP pentamer and the CRP monomer. A role for the CRP monomer in atherosclerosis is emerging, but the underlying mechanisms are only beginning to be understood. Monocytes are an important contributor to atherosclerosis, and foam cell formation is the hallmark of atherogenesis. However, whether the CRP monomer can directly interact with the monocytes and modulate their responses remains unknown. Furthermore, although FcgRIII (CD16) has been identified as the receptor for the CRP monomer on neutrophils, its role in mediating the CRP monomer’s biological effects in other cell types has been questioned. In this study, we investigated the interaction of the CRP monomer with the monocytes using the U937 monocytic cell line. The CRP monomer specifically binds to U937 cells. This binding is unique in that it is independent of FcgRs and insensitive to protease digestion of the cell surface proteins. Further assays revealed that the CRP monomer directly incorporates into the plasma membrane. Interestingly, the presence of the CRP monomer efficiently retards oxidized low- density lipoprotein-induced foam cell formation of PMA-differentiated U937 macrophages and peripheral blood monocytic cell-derived macrophages. These findings provide additional evidence for the notion that the CRP monomer is an active CRP isoform that plays a role in atherogenesis via the direct modulation of the behavior of the monocytes.
Keywords C-reactive protein, Monocyte, Low-density lipoprotein, Foam cell
Address and Contact Information 1The School of Life Sciences and 2The Second Hospital of Lanzhou University, Lanzhou 730000, P. R. China
* Author for correspondence. e-mail: xinheshi.lzu@gmail.com, tel./fax: 86-931 -8942893
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0019-z Volume 15 (2010) pp 496-506
Title INTEGRIN RECEPTORS PLAY A ROLE IN THE INTERNALIN B- DEPENDENT ENTRY OF Listeria monocytogenes INTO HOST CELLS
Authors Clementina Auriemma1, Maurizio Viscardi1, Simona Tafuri2, Luigi Michele Pavone2, Federico Capuano1, Laura Rinaldi3, Rossella Della Morte2, Giuseppe Iovane1,3 and Norma Staiano2,*
Abstract Listeria monocytogenes enters non-phagocytic cells by binding its surface proteins inlA (internalin) and inlB to the host’s E-cadherin and Met, respectively. The two internalins play either separate or cooperative roles in the colonization of infected tissues. Here, we studied bacterial uptake into HeLa cells using an L. monocytogenes mutant strain (DinlA) carrying a deletion in the gene coding for inlA. The DinlA mutant strain showed the capability to invade HeLa cells. The monoclonal anti-ß3- and anti-ß1-integrin subunit antibodies prevented bacterial uptake into the cells, while the anti-ß2- and anti-ß4-integrin subunit antibodies failed to affect L. monocytogenes entry into HeLa cells. Three structurally distinct disintegrins (kistrin, echistatin and flavoridin) also inhibited bacterial uptake, showing different potencies correlated to their selective affinity for the ß3- and ß1-integrin subunits. In addition to inducing Met phosphorylation, infection of cells by the L. monocytogenes DinlA mutant strain promoted the tyrosine phosphorylation of the focal adhesion-associated proteins FAK and paxillin. Our findings provide the first evidence that ß3- and ß1-integrin receptors play a role in the inlB-dependent internalization of L. monocytogenes into host cells
Keywords Listeria monocytogenes, Internalin B, Integrins, FAK, Paxillin
Address and Contact Information 1Istituto Zooprofilattico Sperimentale del Mezzogiorno, via Della Salute 2, 80055, Portici (Na), Italy,
2Dipartimento di Strutture, Funzioni e Tecnologie Biologiche,
3Dipartimento di Patologia e Sanita Animale, Universita degli Studi di Napoli Federico II, via F. Delpino 1, 80137 Napoli, Italy
* Author for correspondence. e-mail address: staianor@unina.it, tel.: +39-0812536108, fax: +39-0812536097
[Rozmiar: 1332 bajtów]

DOI: 10.2478/s11658-010-0020-6 Volume 15 (2010) pp 507-516
Title GENDER DIMORPHISM IN THE EXERCISE-NAIVE MURINE SKELETAL MUSCLE PROTEOME
Authors Lauren Ann Metskas1, Mohini Kulp2 and Stylianos P. Scordilis1,2,3*
Abstract Skeletal muscle is a plastic tissue with known gender dimorphism, especially at the metabolic level. A proteomic comparison of male and female murine biceps brachii was undertaken, resolving an average of 600 protein spots of MW 15-150 kDa and pI 5-8. Twenty-six unique full-length proteins spanning 11 KOG groups demonstrated statistically significant (p<0.05) abundance differences between genders; the majority of these proteins have metabolic functions. Identified glycolytic enzymes demonstrated decreased abundance in females, while abundance differences in identified oxidative phosphorylation enzymes were specific to the proteins rather than to the functional group as a whole. Certain cytoskeletal and stress proteins showed specific expression differences, and all three phosphorylation states of creatine kinase showed significant decreased abundance in females. Expression differences were significant but many were subtle ( 2-fold), and known hormonally-regulated proteins were not identified. We conclude that while gender dimorphism is present in non-exercised murine skeletal muscle, the proteome comparison of male and female biceps brachii in exercise-naive mice indicates subtle differences rather than a large or obviously hormonal dimorphism.
Keywords Gender, Muscle proteomics, Glycolysis, Creatine kinase
Address and Contact Information 1Biological Sciences, 2Center for Proteomics, 3Biochemistry, Smith College, Northampton, MA, United States
* Author for correspondence. e-mail: sscordil@smith.edu
[Rozmiar: 1332 bajtów]