Vol. 18 No. 2 June 2013

DOI: 10.2478/s11658-013-0081-4 Volume 18 (2013) pp 149-162
Authors Yong Du§, Zhao Du§, Hongping Zheng, Dan Wang, Shifeng Li, Yuanchang Yan and Yiping Li*
Abstract γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABAA receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABAAR-specific agonist muscimol reduced the proliferation ofSSCs. This GABA regulation of SSC proliferation was shown tobe independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.
Keywords GABA, GABAA receptor, Spermatogonial stem cells, Proliferation, Muscimol, Testes
Address and Contact Information State Key Laboratory of Cell Biology, Shanghai Key Laboratory for Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China
§ These authors contributed equally to this study
*Author for correspondence. e-mail: yipingli@sibs.ac.cn, tel.: 86-21-54921413, fax: 86-21-54921415

DOI: 10.2478/s11658-013-0083-2 Volume 18 (2013) pp 163-186
Authors Dana Foudah, Juliana Redondo, Cristina Caldara, Fabrizio Carini, Giovanni Tredici and Mariarosaria Miloso*
Abstract Mesenchymal stem cells (MSCs) are multipotent cells that are able to differentiate into mesodermal lineages (osteogenic, adipogenic, chondrogenic), but also towards non-mesodermal derivatives (e.g. neural cells). Recent in vitro studies revealed that, in the absence of any kind of differentiation stimuli, undifferentiated MSCs express neural differentiation markers, but the literature data do not all concur. Considering their promising therapeutic potential for neurodegenerative diseases, it is very important to expand our knowledge about this particular biological property of MSCs. In this study, we confirmed the spontaneous expression of neural markers (neuronal, glial and progenitor markers) by undifferentiated human MSCs(hMSCs) and in particular, we demonstrated that the neuronal markers βIII-tubulin and NeuN are expressed by a very high percentage of hMSCs, regardless of the number of culture passages and the culture conditions. Moreover, the neuronal markers βIII-tubulin and NeuN are still expressed by hMSCs after in vitroosteogenic and adipogenic differentiation. On the other hand, chondrogenically differentiated hMSCs are negative for these markers. Our findings suggest that the expression of neuronal markers could be common to a wide range of cellular types and not exclusive for neuronal lineages. Therefore, the expression of neuronal markers alone is not sufficient to demonstrate the differentiation of MSCs towards the neuronal phenotype. Functional properties analysis is also required.
Keywords Mesenchymal stem cells, Neural markers, βIII-tubulin, NeuN, Osteogenic differentiation, Adipogenic differentiation, Chondrogenic differentiation, Neuronal differentiation
Address and Contact Information Department of Neurosciences and Biomedical Technologies, University of Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy
* Author for correspondence: Dipartimento di Neuroscienze e Tecnologie Biomediche, Facoltà di Medicina e Chirurgia, Università degli Studi di Milano-Bicocca, Via Cadore 48, 20900 Monza, Italy. e-mail: mariarosaria.miloso@unimib.it,tel: +39-02-64488123, fax: +39-02-64488250.

DOI: 10.2478/s11658-013-0082-3 Volume 18 (2013) pp 187-199
Authors Przemysław Borys*
Abstract Passive calcium influx is one of the theories to explain the cathodal galvanotaxis of cells that utilize the electric field to guide their motion. When exposed to an electric field, the intracellular fluid becomes polarized, leading to positive charge accumulation on the cathodal side and negative charge accumulation on the anodal side. The negative charge on the anodal side attracts extracellular calcium ions, increasing the anodal calcium concentration, which is supposed to decrease the mobile properties of this side. Unfortunately, this model does not capture the Ca2+ dynamics after its presentation to the intracellular fluid. The ions cannot permanently accumulate on the anodal side because that would build a potential drop across the cytoplasm leading to an ionic current, which would carry positive ions (not only Ca2+) from the anodal to the cathodal part through the cytoplasm. If the cytoplasmic conductance for Ca2+ is low enough compared to the membrane conductance, the theory could correctly predict the actual behavior. If the ions move through the cytoplasm at a faster rate, compensating for the passiveinflux, this theory may fail. This paper contains a discussion of the regimes of validity for this theory.
Keywords Galvanotaxis, Electrotaxis, Passive influx, Leak current, PNP equation, Electrodiffusion, Motility
Address and Contact Information Department of Chemistry, Silesian University of Technology, ks. M. Strzody 9, 44-100 Gliwice, Poland
* Author for correspondence. e-mail: Przemyslaw.Borys@polsl.pl; tel/fax: +48 32 237-17-22

DOI: 10.2478/s11658-013-0084-1 Volume 18 (2013) pp 200-208
Authors Jinfeng Huang, Ruifei Wang, Ximing Liu, Xianlu Zeng* and Min Wei*
Abstract Excessive trafficking of leukocytes can lead to serious tissue injury. Here, four regioselectively sulfated chitosans were assessed as inhibitors of HL-60 leukocyte binding to P-selectin, by investigating their effect on leukocyte adhesion to CHO cells expressing human P-selectin under static and flow conditions. The results show that the sulfochitosans exhibit inhibitory activity in this general order: heparin > N-sulfated/6-O-sulfated chitosan ≥3-O,6-O-sulfated chitosan > 6-O-sulfated chitosan >> N-sulfated chitosan. This suggests that the sulfation of the double site in chitosan is essential for efficient inhibition of P-selectin-mediated HL-60 leukocyte adhesion and that such sulfochitosans may have potential as therapeutic agents against inflammatory disease.
Keywords Chitosan, Sulfated chitosan, P-selectin, Inhibition, Cell adhesion, Flow condition, Heparin
Address and Contact Information Institute of Genetics and Cytology, School of Life Sciences, Northeast Normal University, Changchun 130024, P. R. China
* Authors for correspondence: e-mail: weim750@nenu.edu.cn; zengx779@nenu.edu.cn

DOI: 10.2478/s11658-013-0085-0 Volume 18 (2013) pp 209-230
Authors Alessandro Didonna*
Abstract Prion diseases are a class of fatal neurodegenerative disorders that can be sporadic, genetic oriatrogenic. They are characterized by the unique nature of their etiologic agent: prions (PrPSc). A prion is an infectious protein with the ability to convert the host-encoded cellular prion protein (PrPC) into new prion molecules by acting as a template. Since Stanley B. Prusiner proposed the “protein-only” hypothesis for the first time,considerable effort has been put into defining the role played by PrPC in neurons. However, its physiological function remains unclear. This review summarizes the major findings that support the involvement of PrPC in signal transduction.
Keywords Prion, PrPC, PrPSc, Src, MAP kinases, PKA, PKC, AKT, Calcium, Signaling
Address and Contact Information Davee Department of Neurology, Feinberg School of Medicine Northwestern University, E 303 Chicago Ave, Chicago, IL 60611, U.S.A.
* Author for correspondence. e-mail: a-didonna@northwestern.eduor alessandrodidonna@libero.it

DOI: 10.2478/s11658-013-0086-z Volume 18 (2013) pp 231-248
Authors Drosa Anna Cifarelli1, Olimpia D’Onofrio1, Rosalba Grillo1, Teresa Mango1, Francesco Cellini1, Luciana Piarulli2, Rosanna Simeone2, Angelica Giancaspro2, Pasqualina Colasuonno2, Antonio Blanco2 and Agata Gadaleta2*
Abstract Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized themto two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.
Keywords 5-Azacytidine, DNA methylation, Powdery mildew, Microarray, Durum wheat, Near-isogenic line, Candidate gene, Quantitative real-time PCR, Physical mapping, Pm36 gene, Expressed sequence tag
Address and Contact Information 1MetapontumAgrobios, S.S. Jonica 106 - 75010 Metaponto di Bernalda, Province of Matera, Italy,
2 Department of Soil, Plant and Food Sciences, University of Bari “Aldo Moro”, Via G. Amendola 165/A - 70126 Bari, Italy
* Author for correspondence. e-mail: agata.gadaleta@agr.uniba.it, tel.: +39-080-5442995, fax: +39-080-5442200

DOI: 10.2478/s11658-013-0087-y Volume 18 (2013) pp 249-262
Authors Grzegorz Stasiłojć1, Sandra Pinto3, Roksana Wyszkowska1, Magda Wejda1,4, Ewa M. Słomińska2, Martyna Filipska1, Patrycja Koszałka1, Julian Świerczyński2, Jose Enrique O’Connor3 and Jacek Jerzy Bigda1*
Abstract The variant cell line U937V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937V cells was the lack of cellular disintegration, which contrasts to the formation of apoptoticbodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937V cells, the basic molecular events of apoptosis, such asinternucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.
Keywords Apoptosis, U937 cells, Apoptotic bodies, Cell disintegration
Address and Contact Information 1 Department of Cell Biology, Intercollegiate Faculty of Biotechnology UG-MUG, Medical University of Gdansk, Poland,
2 Department of Biochemistry, Faculty of Medicine, Medical University of Gdansk, Poland,
3 Cytomics Laboratory, Mixed Unit University of Valencia - Prince Felipe Research Center, Valencia, Spain,
4 Department for Molecular BiomedicalResearch, Flanders Institute for Biotechnology and the Department ofBiomedical Molecular Biology, Ghent University, Ghent, Belgium (current address)
* Author for correspondence: Department of Cell Biology, Medical University of Gdańsk, ul. Dębinki 1, 80-211 Gdańsk, Poland. Tel. +48 58 3491434; fax. +48 58 3491445; jjbigd@gumed.edu.pl

DOI: 10.2478/s11658-013-0089-9 Volume 18 (2013) pp 263-283
Authors Kamonnaree Chotinantakul1, 2, Chavaboon Dechsukhum3, 4, Duangnapa Dejjuy1, 2 and Wilairat Leeanansaksiri1, 2,*
Abstract Diabetes can impair wound closure, which can give rise to major clinical problems. Most treatments for wound repair in diabetes remain ineffective. This study aimed to investigate the influence on wound closure of treatments using expanded human cord blood CD34+ cells (CB-CD34+ cells), freshly isolated CB-CD34+ cells and a cytokine cocktail. The test subjects were mice with streptozotocin-induced diabetes. Wounds treated with fresh CB-CD34+ cells showed more rapid repair than mice given the PBS control. Injection of expanded CB-CD34 + cells improved wound closure significantly, whereas the injection of the cytokine cocktail alone did not improve wound repair. The results also demonstrated a significant decrease in epithelial gaps and advanced re-epithelialization over the wound bed area after treatment with either expanded CB-CD34+ cells or freshly isolated cells compared with the control. In addition, treatments with both CB-CD34+ cells and the cytokine cocktail were shown to promote recruitment of CD31+-endothelial cells in the wounds. Both the CB-CD34+ cell population and the cytokine treatments also enhanced the recruitment of CD68-positive cells in the early stages (day 3) of treatment compared with PBS control, although the degree of this enhancement was found to decline in the later stages (day 9). These results demonstrated that expanded CB-CD34+ cells or freshly isolated CB-CD34+ cells could accelerate wound repair by increasing the recruitment of macrophages and capillaries and the re-epithelialization over the wound bed area. Our data suggest an effective role in wound closure for both ex vivo expanded CB-CD34+ cells and freshly isolated cells, and these may serve as therapeutic options for wound treatment for diabetic patients. Wound closure acceleration by expanded CB-CD34+ cells also breaks the insufficient quantity obstacle of stem cells per unit of cord blood and other stem cell sources, which indicates a broader potential for autologous transplantation
Keywords CD31+ cells, CD34+ cells, CD68+ cells, Cord blood, Diabetic mice, Ex vivoexpansion, Hematopoietic stem cells, Macrophages, Stem cell therapy, Wound closure
Address and Contact Information 1 Stem Cell Therapy and Transplantation Research Group, Suranaree University of Technology, Thailand,
2 School of Microbiology, Institute of Science, Suranaree University of Technology, Thailand,
3 Gene Therapy and Clinical Application Research Group, Suranaree University of Technology, Thailand,
4 School of Pathology, Institute of Medicine, Suranaree University of Technology, Thailand
* Author for correspondence. e-mail: wilairat@g.sut.ac.th, tel.: +66 44 224628; fax +66 44 224633

DOI: 10.2478/s11658-013-0088-x Volume 18 (2013) pp 284-296
Authors Aruna Korlimarla1,§,*, Lekhana Bhandary1,§, Jyothi S. Prabhu1, Hema Shankar1, Hari Sankaranarayanan1, Pravin Kumar2, Jose Remacle1,#, Dipa Natarajan2 and T.S. Sridhar1
Abstract The breast cancer type 1 susceptibility gene (BRCA1) is a tumor suppressor gene, mutations or loss of which lead to genomic instability and breast cancer. BRCA1 protein is part of a large multi-protein complex involved in a variety of DNA repair and transcription regulatory functions. At least four splice variants have been described and these differ in their function and tissue and spatio-temporal expression patterns. Structural analysis has revealed the presence of two nuclear localization signals (NLS) located in exon 11 of BRCA1. Interestingly, a splice variant of the protein that lacks both of the known NLS still manages to gain entry to the nucleus. While there is experimental proof for the translocation of these proteins bybinding to other established nuclear proteins, we examined the possibility of a hitherto unidentified NLS in this particular variant. In this paper, we present evidence for the existence of a previously unreported non-canonical NLS contained within the first 39 amino acids of exon 11. A fusion protein with this 39mer and a reporter green fluorescent protein translocated into the nucleus when it was expressed in breast epithelial cells. We demonstrate the presence of a hitherto unreported non-canonical NLS in exon 11a of BRCA1. This NLS might aid proteins that were encoded by splice variants and lack the canonical NLS to localize to the nucleus.
Keywords Breast cancer, BRCA1, Splice variants, Nuclear localization, Importin alpha, Green fluorescent protein
Address and Contact Information 1 Division of Molecular Medicine, St. John’s Research Institute (SJRI) Bangalore-560034, India,
2 Prescient Biosciences Pvt.Ltd, Bangalore, India
§ These authors contributed equally to this manuscript
* Author for correspondence. e-mail:aruna@sjri.res.in, tel.: +91 80-22065059, fax: +91 80-25532037
# Visiting Scientist, SJRI

DOI: 10.2478/s11658-013-0090-3 Volume 18 (2013) pp 297-314
Authors Juan Zhou1, Bingbing Wan1,2, Jingxuan Shan1, Huili Shi1, Yanhong Li1,* and Keke Huo1,*
Abstract PTPN4 is a widely expressed non-receptor protein tyrosine phosphatase. Although its overexpression inhibits cell growth, the proteins with which it interacts to regulate cell growth are unknown. In this study, we identified CrkI as a PTPN4-interacting protein using a yeast two-hybrid, and confirmed this interaction using in vitro GST pull-down and co-immunoprecipitation and co-localization assays. We further determined the interactional regions as the SH3 domain of CrkI and the proline-rich region between amino acids 462 and 468 of PTPN4. Notably, overexpression of PTPN4 inhibits CrkI-mediated proliferation and wound healing of HEK293T cells, while knockdown of PTPN4 by siRNA in Hep3B cells enhances CrkI-mediated cell growth and motility. Moreover, our data show that ectopic expression of PTPN4 reduces the phosphorylation level ofCrkI in HEK293T cells. These findings suggest that PTPN4 negatively regulates cell proliferation and motility through dephosphorylation of CrkI.
Keywords PTPN4, PTPMEG, CrkI, CRK, Cell proliferation, Protein tyrosine phosphatase, Wound-healing assay, Subcellular localization, Protein-protein interactions, Yeast two-hybrid
Address and Contact Information 1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai 200433, China,
2 Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor,MI 48109, USA (current address
* Authors for correspondence: e-mail: kkhuo@fudan.edu.cn, tel: +86-21-55664526, fax: +86-21-55664526 (Keke Huo) or lyhong@fudan.edu.cn, tel: +86-21-65642339 (Yanhong Li)