Vol. 18 No. 1 March 2013

DOI: 10.2478/s11658-012-0035-2 Volume 18 (2013) pp 1-10
Authors Jacob Grange§, James D. Moody§, Marc P. A. Ascione and Marc D. H. Hansen*
Abstract Cell-cell and cell-substrate adhesions are sites of dramatic actin rearrangements and where actin-membrane connections are tightly regulated. Zyxin-VASP complexes localize to sites of cell-cell and cell-substrate adhesion and function to regulate actin dynamics and actin-membrane connections at these sites. To accomplish these functions, zyxin recruits VASP to cellular sites via proline-rich binding sites near zyxin’s amino terminus. While the prevailing thought has been that zyxin simply acts as a scaffold protein for VASP binding, the identification of a LIM domain-VASP interaction could complicate this view. Here we assess how zyxin-VASP binding through both the proline rich motifs and the LIM domains alters specific VASP functions. We find that neither individual interaction alters VASP’s actin regulatory activities. In contrast, however, we find that full-length zyxin dramatically reduces VASP-mediated actin bundling and actin assembly. Taken together, these results suggest a model where zyxin-VASP complexes occur in complex organizations with suppressed actin regulatory activity.
Keywords Actin, VASP, Zyxin, Adhesion, Migration, Cytoskeleton
Address and Contact Information Physiology and Developmental Biology, Brigham Young University, 574 WIDB Provo, UT 84602, USA
§ These authors contributed equally to this study
*Author for correspondence. e-mail: marchansen@byu.edu, tel: (801) 422-4998, fax: (801) 422-0700

DOI: 10.2478/s11658-012-0036-1 Volume 18 (2013) pp 11-33
Authors Viviana M. Rodriguez-Pardo1 and Jean Paul Vernot2,*
Abstract The purpose of this study was to evaluate the influence of bone marrow-mesenchymal stem cells (BM-MSC) and exogenously added cytokines on the proliferation, primitive cell subpopulation maintenance (including the c-kit+ marker) and clonogenic capacity of hematopoietic stem cells (HSC). BM-MSC were collected from volunteer donors, isolated and characterized. Umbilical cord blood (UCB) samples were collected from healthy full-term deliveries. UCB-CD34+ cells were cultured in the presence or absence of BM-MSC and/or cytokines for 3 and 7 days. CD34+ cell proliferation was evaluated using the CSFE method and cell phenotype was determined by CD34, c-kit, CD33, CD38, HLA-DR, cyCD22 and cyCD3 detection. Cell clonogenic ability was also assessed. Exogenously added SCF, TPO and FLT3L increased CD34+ cell proliferation in the presence or absence of BM-MSC, but with concomitant cell differentiation. Without any added cytokines, BM-MSC are able to increase the percentage of primitive progenitors as evaluated by c-kit expression and CFU-GEMM increase. Interestingly, this latter effect was dependent on both cell-cell interactions and secreted factors. A 7-day co-culture period will be optimal for obtaining an increased primitive HSC level. Including c-kit as a marker for primitive phenotype evaluation has shown the relevance of BM-MSC and their secreted factors on UCB-HSC stemness function. This effect could be dissociated from that of the addition of exogenous cytokines, which induced cellular differentiation instead.
Keywords Hematopoietic stem cell, Mesenchymal stem cell, Bone marrow stroma, CD34+c-kit+ subpopulation, Cytokines, Progenitor cell, Umbilical cord blood
Address and Contact Information 1Grupo de Inmunobiología y Biología Celular, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, Colombia,
2Grupo de Fisiología Celular y Molecular, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá, Colombia
* Author for correspondence. Jean Paul Vernot, Ph.D., Departamento de Ciencias Fisiológicas, Facultad de Medicina, Universidad Nacional de Colombia, Carrera 30 No. 45-03 - Edificio 471, Bogotá D.C., Colombia, tel: +571 3165000 ext. 15077, fax: +571 5300936, e-mail: jpvernoth@unal.edu.co

DOI: 10.2478/s11658-012-0038-z Volume 18 (2013) pp 34-46
Authors Marcin A. Listowski1,§, Elżbieta Heger1,§, Dżamila M. Bogusławska1, Beata Machnicka1, Kazimierz Kuliczkowski2, Jacek Leluk1 and Aleksander F. Sikorski1,3,*
Abstract Cell proliferation and differentiation is a complex process involving many cellular mechanisms. One of the best-studied phenomena in cell differentiation is erythrocyte development during hematopoiesis in vertebrates. In recent years, a new class of small, endogenous, non-coding RNAs called microRNAs (miRNAs) emerged as important regulators of gene expression at the post-transcriptional level. Thousands of miRNAs have been identified in various organisms, including protozoa, fungi, bacteria and viruses, proving that the regulatory miRNA pathway is conserved in evolution. There are many examples of miRNA-mediated regulation of gene expression in the processes of cell proliferation, differentiation and apoptosis, and in cancer genesis. Many of the collected data clearly show the dependence of the proteome of a cell on the qualitative and quantitative composition of endogenous miRNAs. Numerous specific miRNAs are present in the hematopoietic erythroid line. This review attempts to summarize the state of knowledge on the role of miRNAs in the regulation of different stages of erythropoiesis. Original experimental data and results obtained with bioinformatics tools were combined to elucidate the currently known regulatory network of miRNAs that guide the process of differentiation of red blood cells.
Keywords Hematopoiesis, Erythrocyte, Erythroid differentiation, Erythropoiesis, microRNA (miRNA), microRNA expression
Address and Contact Information 1Department of Molecular Biology, University of Zielona Góra, Poland,
2 Department of Hematology, Medical University of Wrocław, Poland,
3 Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wrocław, Poland
§ These authors contributed equally to this work
* Author for correspondence: : e-mail: afsbc@ibmb.uni.wroc.pl, tel.: +48 71 3756 233; fax: +48 71 3756 208

DOI: 10.2478/s11658-012-0037-0 Volume 18 (2013) pp 47-57
Authors Piotr Madanecki1, Niren Kapoor2, Zsuzsa Bebok3, Renata Ochocka1, James F. Collawn3 and Rafal Bartoszewski1,*
Abstract Understanding the cellular pathways that regulate angiogenesis during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. Although the pathways of angiogenesis have been extensively studied, there is limited information on the role of miRNAs in this process. miRNAs or their antagomirs could be used in future therapeutic approaches to regulate hypoxia-induced angiogenesis, so it is critical to understand their role in governing angiogenesis during hypoxic conditions. Although hypoxia and ischemia change the expression profile of many miRNAs, a functional role for a limited number of so-called hypoxamiRs has been demonstrated in angiogenesis. Here, we discuss the best examples that illustrate the role of hypoxamiRs in angiogenesis.
Keywords Angiogenesis, Hypoxia, microRNA, miRNA, HypoxamiR, HIF, VEGF
Address and Contact Information 1 Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Poland,
2 Department of Neurology, University of Alabama at Birmingham, USA,
3 Department of Cell Biology, Developmental, and Integrative, University of Alabama at Birmingham, USA
* Author for correspondence: Department of Biology and Pharmaceutical Botany, Medical University of Gdansk, Hallera 107, 80-416 Gdansk, Poland. e-mail: rafalbar@gumed.edu.pl, tel: +48 58 349 32 14; fax: +48 58 349 32 11

DOI: 10.2478/s11658-012-0039-y Volume 18 (2013) pp 58-74
Authors Patchara Ngok-Ngam1, Piyajit Watcharasit1,2,*, Apinya Thiantanawat1,2 and Jutamaad Satayavivad1,2
Abstract Glycogen synthase kinase-3 (GSK3) and p53 play crucial roles in the mitochondrial apoptotic pathway and are known to interact in the nucleus. However, it is not known if GSK3 has a regulatory role in the mitochondrial translocation of p53 that participates in apoptotic signaling following DNA damage. In this study, we demonstrated that lithium and SB216763, which are pharmacological inhibitors of GSK3, attenuated p53 accumulation and caspase-3 activation, as shown by PARP cleavage induced by the DNA-damaging agents doxorubicin, etoposide and camptothecin. Furthermore, each of these agents induced translocation of p53 to the mitochondria and activated the mitochondrial pathway of apoptosis, as evidenced by the release of cytochrome C from the mitochondria. Both mitochondrial translocation of p53 and mitochondrial release of cytochrome C were attenuated by inhibition of GSK3, indicating that GSK3 promotes the DNA damage-induced mitochondrial translocation of p53 and the mitochondrial apoptosis pathway. Interestingly, the regulation of p53 mitochondrial translocation by GSK3 was only evident with wild-type p53, not with mutated p53. GSK3 inhibition also reduced the phosphorylation of wild-type p53 at serine 33, which is induced by doxorubicin, etoposide and camptothecin in the mitochondria. Moreover, inhibition of GSK3 reduced etoposide-induced association of p53 with Bcl2 and Bax oligomerization. These findings show that GSK3 promotes the mitochondrial translocation of p53, enabling its interaction with Bcl2 to allow Bax oligomerization and the subsequent release of cytochrome C. This leads to caspase activation in the mitochondrial pathway of intrinsic apoptotic signaling.
Keywords GSK3, p53, Mitochondrial translocation, Apoptosis, Bax oligomerization, Cytochrome C, Phosphorylation, Etoposide, Doxorubicin, Camptothecin
Address and Contact Information 1 Chulabhorn Graduate Institute, 54 Kamphaeng Phet 6 Road, Lak Si, Bangkok, 10210, Thailand,
2 Laboratory of Pharmacology, Chulabhorn Research Institute, 54 Kamphaeng Phet 6 Road, Lak Si, Bangkok, 10210 Thailand
* Author for correspondence. e-mail: piyajit@cri.or.th, tel: 662-574-0622 ext. 3402, fax: 662-574-2027

DOI: 10.2478/s11658-012-0040-5 Volume 18 (2013) pp 75-88
Authors Dhanasekaran Marappagounder1,*, Indumathi Somasundaram2, Sudarsanam Dorairaj2 and Rajkumar Janavikula Sankaran1
Abstract Although stem cells are present invarious adult tissues and body fluids, bone marrow has been the most popularsource of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetictherapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.
Keywords Diabetes, Islet-like clusters, Bone marrow, Subcutaneous fat, Mesenchymal stem cells, Transdifferentiation, Flow cytometry, Intracellular staining, Dithizone staining, Glucose challenge assay
Address and Contact Information 1 Lifeline Multispeciality Hospitals, Perungudi, Chennai, India,
2 Loyola College, Nungambakkam, Chennai, India
* Author for correspondence. e-mail: dhanasekar_m@lifelinehospitals.com, tel: 044-42454545

DOI: 10.2478/s11658-012-0041-4 Volume 18 (2013) pp 89-101
Authors Jacek Łuczyński1,*, Renata Frąckowiak1, Aleksandra Włoch2, Halina Kleszczyńska2 and Stanisław Witek1
Abstract Cationic gemini surfactants are an important class of surface-active compounds that exhibit much higher surface activity than their monomeric counterparts. This type of compound architecture lends itself to the compound being easily adsorbed at interfaces and interacting with the cellular membranes of microorganisms. Conventional cationic surfactants have high chemical stability but poor chemical and biological degradability. One of the main approaches to the design of readily biodegradable and environmentally friendly surfactants involves inserting a bond withlimited stability into the surfactant molecule to give a cleavable surfactant. The best-known example of such a compound is the family of ester quats, which are cationic surfactants with a labile ester bond inserted into the molecule. As part of this study, a series of gemini ester quat surfactants were synthesized and assayed for their biological activity. Their hemolytic activity and changes in the fluidity and packing order of the lipid polar heads were used as the measures of their biological activity. A clear correlation between the hemolyticactivity of the tested compounds and their alkyl chain length was established. It was found that the compounds with a long hydrocarbon chain showed higher activity. Moreover, the compounds with greater spacing between their alkyl chains were more active. This proves that they incorporate more easily intothe lipid bilayer of the erythrocyte membrane and affect its properties to a greater extent. A better understanding of the process of cell lysis by surfactants and of their biological activity may assist in developing surfactants with enhancedselectivity and in widening their range of application.
Keywords Gemini surfactants, Cationic amphiphiles, Hemolytic activity, Membrane fluidity, Generalized polarization, Anisotropy, Erythrocyte membrane, Lipid packing order, Hemolysis, Fluorescent probes
Address and Contact Information 1 Faculty of Chemistry, Wrocław University of Technology, Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland,
2 Department of Physics and Biophysics, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
* Author for correspondence. e-mail: jacek.luczynski@pwr.wroc.pl; tel.: +48 71 320 33 94

DOI: 10.2478/s11658-012-0042-3 Volume 18 (2013) pp 102-119
Authors Włodzimierz Korohoda*, Maciej Grys and Zbigniew Madeja*
Abstract Experiments on reversible and irreversible cell electroporation were carried out with an experimental setup based on a standard apparatus for horizontal electrophoresis, a syringe pump with regulated cell suspension flow velocity and a dcEF power supply. Cells in suspension flowing through an orifice in a barrier inserted into the electrophoresis apparatus were exposed to defined localized dcEFs in the range of 0-1000 V/cm for a selected duration in the range 10-1000 ms. This method permitted the determination of the viability of irreversibly electroperforated cells. It also showed that the uptake by reversibly electroperforated cells of fluorescent dyes (calcein, carboxyfluorescein, Alexa Fluor 488 Phalloidin), which otherwise do not penetrate cell membranes, was dependent upon the dcEF strength and duration in any given single electrical field exposure. The method yields reproducible results, makes it easy to load large volumes of cell suspensions with membrane non-penetrating substances, and permitsthe elimination of irreversibly electroporated cells of diameter greater than desired. The results concur with and elaborate on those in earlier reports oncell electroporation in commercially available electroporators. They proved once more that the observed cell perforation does not depend upon the thermal effects of the electric current upon cells. In addition, the method eliminates many of the limitations of commercial electroporators and disposable electroporation chambers. It permits the optimization of conditions in which reversible and irreversible electroporation are separated. Over 90% of reversibly electroporated cells remain viable after one short (less than 400 ms) exposure to the localized dcEF. Experiments were conducted with the AT-2 cancer prostate cell line, human skin fibroblasts and human red blood cells, but they could be run with suspensions of any cell type. It is postulated that the described method could be useful for many purposes in biotechnology and biomedicine and could help optimize conditions for in vivo use of both reversible and irreversible electroporation.
Keywords Irreversible electroporation, Reversible electroporation, Flow through electric field, Fluorescent dye loading, Cell viability, Direct current electric field, Focused electric field, Electrophoresis apparatus, Cell suspension electroporation system
Address and Contact Information Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, JagiellonianUniversity, Gronostajowa 7, 30-387, Cracow, Poland
* Authors for correspondence: e-mail: w.korohoda@uj.edu.pl, tel.: + 48 12 664 61 25, fax: +48 12 664 69 02; e-mail: z.madeja@uj.edu.pl, tel.: + 48 12 664 61 42

DOI: 10.2478/s11658-012-0043-2 Volume 18 (2013) pp 120-136
Authors Hitesh Jagani1, Josyula Venkata Rao1,*, Vasanth Raj Palanimuthu1,2, Raghu Chandrashekar Hariharapura1 and Sagar Gang1
Abstract Overexpression of anti-apoptotic Bcl-2 is often observed in a wide variety of human cancers. It prevents the induction of apoptosis in neoplastic cells and contributes to resistance to chemotherapy. RNA interference has emerged as an efficient and selective technique for gene silencing. The potential to use small interfering RNA (siRNA) as a therapeutic agent for the treatment of cancer has elicited a great deal of interest. However, insufficient cellular uptake and poor stability have limited its therapeutic applications. The purpose of this study was to prepare chitosan nanoparticles via ionic gelation of chitosan by tripolyphosphate for effective delivery ofsiRNA to silence the anti-apoptotic Bcl-2 gene in neoplastic cells. Chitosan nanoparticles loaded with siRNA were in the size range 190 to 340 nm with a polydispersive index ranging from 0.04 to 0.2. They were able to completely bind with siRNA, provide protection against nuclease degradation, and enhance the transfection. Cell culture studies revealed that nanoparticles with entrapped siRNA could efficiently silence the anti-apoptotic Bcl-2 gene. Studies on Swiss albino mice showed that siRNA could be effectively delivered through nanoparticles. There was significant decrease in the tumor volume. Blocking the expression of anti-apoptotic Bcl-2 can enhance the sensitivity of cancerous cells to anti-cancer drugs and the apoptosis rate. Therefore, nanoformulations with siRNA can be promoted as an adjuvant therapy in combination with anti-cancer drugs.
Keywords Chitosan, RNA interference, siRNA, Bcl-2, Apoptosis, Cancer, Nanoformulation
Address and Contact Information 1 Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India-576104,
2 Faculty of Pharmacy, AIMST University, Jalan Bedong Semeling, 08100, Bedong, Kedah Darul Aman, Malaysia
* Author for correspondence. e-mail: rao.josyula@gmail.com, tel.: +91-820-2922482 ext. 771, fax: +91-820-2571998

DOI: 10.2478/s11658-012-0044-1 Volume 18 (2013) pp 137-148
Authors Manuela Molzan1 and Christian Ottmann1,2,*
Abstract Myeloid leukemia factor 1 (MLF1) is associated with the development of leukemic diseases such as acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, information on the physiological function of MLF1 is limited and mostly derived from studies identifying MLF1 interaction partners like CSN3, MLF1IP, MADM, Manp and the 14-3-3 proteins. The 14-3-3-binding site surrounding S34 is one of the only known functional features of the MLF1 sequence, along with one nuclear export sequence (NES) and two nuclear localization sequences (NLS). It was recently shown that the subcellular localization of mouse MLF1 is dependent on 14-3-3 proteins. Based on these findings, we investigated whether the subcellular localization of human MLF1 was also directly 14-3-3-dependent. Live cell imaging with GFP-fused human MLF1 was used to study the effects of mutations and deletions on its subcellular localization. Surprisingly, we found that the subcellular localization offull-length human MLF1 is 14-3-3-independent, and is probably regulatedby other as-yet-unknown proteins.
Keywords MLF1, 14-3-3, Binding site, Protein-protein interactions, Subcellular localization, Acute myeloid leukemia, Myelodysplastic syndrome, Site-directed mutagenesis, Confocal microscopy, Live cell imaging
Address and Contact Information 1 Chemical Genomics Centre of the Max-Planck-Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany,
2 Laboratory of Chemical Biology, Department of Biomedical Engineering, Technische Universiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands
* Author for correspondence. e-mail: christian.ottmann@cgc.mpg.de, c.ottmann@tue.nl, tel.: +49 (0) 231 9742 6481, fax: +49 (0) 231 9742 6479