Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 13 (2008) No. 2

DOI: 10.2478/s11658-007-0046-6 Volume 13 (2008) pp 155-181
Title	NATRIURETIC PEPTIDES IN CARDIOVASCULAR DISEASES
Authors	Mariusz Piechota¹, Maciej Banach²*, Anna Jacoń³ and Jacek Rysz⁴
Abstract	The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.
Keywords	Atrial natriuretic peptide, Brain natriuretic peptide, C-type natriuretic peptide, Dendroaspis natriuretic peptide, Urodilatin, Endothelin-1
Adress and Contact Informations	¹Department of Anaesthesiology and Intensive Care Unit, Boleslaw Szarecki University Hospital No. 5 in Łódź, Medical University in Łódź, Poland, ²Department Cardiology, 1st Chair of Cardiology and Cardiac Surgery, University Hospital No. 3 in Łódź, Medical University in Łódź, Poland, ³Department of Health Protection Policy, Medical University of Łódź, Poland ⁴2nd Department of Family Medicine, University Hospital No. 2 in Łódź, Medical University in Łódź, Poland * Author for correspondence: Department of Cardiology, Medical University of Łódź, Sterlinga 1/3, 91-425 Łódź, Poland, tel/fax: +48 42 6364 471, e-mail: m.banach@termedia.pl

DOI: 10.2478/s11658-007-0045-7 Volume 13 (2008) pp 182-194
Title	THE INDUCTION OF APOPTOSIS BY DAUNORUBICIN AND IDARUBICIN IN HUMAN TRISOMIC AND DIABETIC FIBROBLASTS
Authors	Sylwia Dragojew¹, Agnieszka Marczak¹, Janusz Maszewski², Krzysztof Ilnicki³ and Zofia Jóźwiak¹*
Abstract	In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2-48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2-6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.
Keywords	Daunorubicin, Idarubicin, Apoptosis, Fibroblasts, Down’s syndrome, Diabetes
Adress and Contact Informations	¹Department of Thermobiology, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland, ²Department of Cytophysiology, University of Łódź, Pilarskiego 14/16, 90-237 Łódź, Poland, ³Department of Medical Genetics, Institute of the Centrum of Child Health, Al. Dzieci Polskich 20, 04-730 Warszawa, Poland *Author for correspondence; e-mail: zjozwiak@biol.uni.lodz.pl

DOI: 10.2478/s11658-007-0044-8 Volume 13 (2008) pp 195-211
Title	ACCUMULATION OF AQUAPORIN-1 DURING HEMOLYSININDUCED NECROTIC CELL DEATH
Authors	Kelly Schweitzer¹, Erran Li², Venkataramana Sidhaye¹, Virginia Leitch¹, Sergey Kuznetsov¹ and Landon S. King¹*
Abstract	Altered tissue water homeostasis may contribute to edema formation during various stresses including bacterial infection. We observed induction of aquaporin-1 (AQP1) during Staphylococcus aureus infection of cultured cells indicating a potential mechanism underlying altered water homeostasis during infection. To investigate mechanisms of AQP1 induction, we examined the effects of the S. aureus α-hemolysin on AQP1 abundance in Balb/c fibroblasts. Fibroblasts incubated with 30 μg/ml hemolysin exhibited a 5-10 fold increase in AQP1 protein within 4-6 hours of exposure. The use of multiple signaling cascade inhibitors failed to affect hemolysin-mediated accumulation of AQP1. However, immunoprecipitation revealed an initial accumulation of ubiquitinated AQP1 followed by a decrease to baseline levels after 4 hours. Immunofluorescence indicated that following hemolysin exposure, AQP1 was no longer on the plasma membrane, but was found in a population of submembrane vacuoles. AQP1 redistribution was further indicated by surface biotinylation experiments suggesting diminished AQP1 abundance on the plasma membrane as well as redistribution out of lipid raft fractions. Live cell confocal microscopy revealed that the pattern of cell volume change observed following hemolysin exposure was altered in cells in which AQP1 was silenced. We conclude that alpha-toxin alters proteasomal processing and leads to intracellular accumulation of AQP1, which may likely contribute to disrupted cell volume homeostasis in infection.
Keywords	Aquaporin, Toxin, Ubiquitin, Lipid raft
Adress and Contact Informations	¹Department of Medicine, Division of Pulmonary and Critical Care Medicine Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle Baltimore, MD 21224, USA, ²Institute of Respiratory Disease, China Medical University, Shenyang, China *Author for correspondence; e-mail: lsking@jhmi.edu, tel: 443-287-3347, fax: 410-287- 3349

DOI: 10.2478/s11658-007-0048-4 Volume 13 (2008) pp 212-229
Title	REGULATION OF BACTERIAL PROTEASE ACTIVITY ^#
Authors	Benedykt Władyka* and Katarzyna Pustelny
Abstract	Proteases, also referred to as peptidases, are the enzymes that catalyse the hydrolysis of peptide bonds in polipeptides. A variety of biological functions and processes depend on their activity. Regardless of the organism’s complexity, peptidases are essential at every stage of life of every individual cell, since all protein molecules produced must be proteolytically processed and eventually recycled. Protease inhibitors play a crucial role in the required strict and multilevel control of the activity of proteases involved in processes conditioning both the physiological and pathophysiological functioning of an organism, as well as in host-pathogen interactions. This review describes the regulation of activity of bacterial proteases produced by dangerous human pathogens, focusing on the Staphylococcus genus.
Keywords	Protease, Protease inhibitor, Zymogen, Operon, Staphylococcus
Adress and Contact Informations	Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland ^# Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7-11, 2007, "The Cell and Its Environment". Publication cost was partially covered by the organisers of this meeting. * Author for correspondence; e-mail: wladykab@interia.pl, tel: +48 12 664 6506, fax: +48 12 664 6915

DOI: 10.2478/s11658-007-0049-3 Volume 13 (2008) pp 230-239
Title	CARVEDILOL MODIFIES ANTIOXIDANT STATUS OF PATIENTS WITH STABLE ANGINA
Authors	Jan Kowalski¹ , Maciej Banach²*, Marcin Barylski¹, Robert Irzmanski¹ and Lucjan Pawlicki¹
Abstract	The aim of this study was to evaluate the effect of carvedilol on the enzymatic antioxidative defence and plasma antioxidative activity in patients with stable angina. The study comprised 30 patients, aged 37-49 years with stable angina. Patients received carvedilol in escalating doses of 12.5 mg/24 h, 25 mg/24 h, and 50 mg/24 h for 4 weeks each. The control group was matched for age and gender, and consisted of 12 healthy volunteers, aged 39-49 years. Blood samples were collected from the cubital vein before and 4, 8 and 12 weeks after the therapy from the patients and once from the control group. For all the subjects, the superoxide dismutase (SOD-1), glutathione peroxidase (GSH-Px), catalase (CAT) activities in the erythrocytes and the antioxidant activity of the blood plasma were determined. The enzymatic antioxidative defence was significantly decreased in patients with stable angina in comparison to the healthy subjects. During the carvedilol therapy, an increase in the SOD-1, GSH-Px and CAT activities was observed. Moreover, 8 and 12 weeks after carvedilol therapy, the GSH-Px activity did not differ significantly from that observed in the group of healthy subjects. Carvedilol also increased the plasma antioxidative activity in patients with stable angina, but its level remained significantly lower than in the control group. In conclusion, carvedilol enhances antioxidant defense mechanisms in patients with chronic stable angina pectoris.
Keywords	Stable angina, Carvedilol, Superoxide dismutase, Peroxidase, Catalase, Plasma antioxidative activity
Adress and Contact Informations	¹Department of Internal Medicine and Cardiological Rehabilitation, Medical University of Łódź, Poland, ²Department of Cardiology, 1st Chair of Cardiology and Cardiac Surgery, Medical University of Łódź, Poland * Author for correspondence: Department of Cardiology, 1st Chair of Cardiology and Cardiac Surgery, Medical University in Łódź, Poland. Sterlinga 1/3; 91-425 Łódź, Poland, e-mail: m.banach@termedia.pl , tel/fax: +48 42 636 44 71

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DOI: 10.2478/s11658-007-0050-x Volume 13 (2008) pp 240-249
Title	CAN CYP1A1 siRNA BE AN EFFECTIVE TREATMENT FOR LUNG CANCER?
Authors	Kulthum Mohammed and Amal Shervington٭
Abstract	Previously, we identified a novel correlation between the upregulated expression of telomerase (hTERT) and cytochrome P450 1A1 (CYP1A1) in A549 human lung cancer cell line. The expression correlation was confirmed by silencing CYP1A1 expression using siRNA technology and observing a silencing of hTERT transcription. Furthermore, silencing CYP1A1 and subsequently downregulating hTERT resulted in the reduction of cancer cell viability by more than 40%, which appeared as early as 24 hours after the treatment. The concomitant downregulation of CYP1A1 and hTERT resulted in rapid cell death. This finding can be further exploited to develop new molecular targets for the treatment of lung cancer.
Keywords	siRNA, Gene knockdown, CYP1A1, hTERT, Transfection
Adress and Contact Informations	University of Central Lancashire, Department of Biological Sciences, Preston, PR1 2HE, United Kingdom *Author for correspondence; e-mail: aashervington@uclan.ac.uk, tel.: +44(0)1772 893598

DOI: 10.2478/s11658-007-0052-8 Volume 13 (2008) pp 250-259
Title	THE CYTOPLASMIC DOMAIN OF CHONDROLECTIN INTERACTS WITH THE β-SUBUNIT OF RAB GERANYLGERANYL TRANSFERASE
Authors	An Claessens, Christine Weyn and Joseph Merregaert*
Abstract	Mouse chondrolectin (chodl) was isolated out of the tail tip of fourday old 129/SvJ mice as a by-product of a PCR-based subtractive cDNA library screening. The gene is predominantly expressed in adult skeletal muscle, heart, testes and lungs and in embryonic stadia. Chodl is the mouse homologue of human chondrolectin (CHODL), a gene that encodes for a type Ia transmembrane protein and that is expressed in human testis, prostate, heart and skeletal muscle tissue. CHODL-splice variants (CHODL_f, CHODL_fΔE, CHODL_ΔE) are detected in human leukocytes. The proteins of the chondrolectin family belong to the family of C-type lectins. As the members of this protein family are important for a wide array of biological processes, the function of chodl was investigated by searching for its protein interaction partners. The β-subunit of Rab geranylgeranyl transferase (Rabggtb) was isolated 8 times after a complete Sos recruitment system (SRS) screen with the cytoplasmic domain of chodl. The interaction was confirmed with in vitro transcription/translation and co-immunoprecipitation (co-IP) experiments.
Keywords	C-type lectin, Chondrolectin, SRS
Adress and Contact Informations	C-type lectin, Chondrolectin, SRS * Author for correspondence; e-mail: joseph.merregaert@ua.ac.be, tel.: 32-3-820-2311, fax: 32-3-820-2248.

DOI: 10.2478/s11658-007-0054-6 Volume 13 (2008) pp 260-270
Title	INCREASED PRESSURE STIMULATES ABERRANT DENDRITIC CELL MATURATION
Authors	David H. Craig^1,4, Keri L. Schaubert⁴, Hiroe Shiratsuchi¹, June Kan-Mitchell⁴, and Marc D. Basson^1,2,3,4*
Abstract	Patients with malignancy typically exhibit abnormal dendritic cell profiles. Interstitial tumor pressure is increased 20-50mmHg over that in normal tissue. We hypothesized that elevated pressure in the tumor microenvironment may influence dendritic cell (DC) phenotype and function. Monocyte-derived immature and mature DC isolated from healthy human donors were exposed to either ambient or 40 mmHg increased pressure at 37°C for 12 hours, then assessed for expression of CD80, CD86, CD83, CD40, MHC-I and MHC-II. IL-12 production and phagocytosis of CFSE-labeled tumor lysate were assessed in parallel. Elevated pressure significantly increased expression of all co-stimulatory and MHC molecules on mature DC. Immature DC significantly increased expression of CD80, CD86, CD83 and MHC-II, but not MHC-I and CD40, versus ambient pressure controls. Pressure-treated immature DC phenotypically resembled mature DC controls, but produced low IL-12. Phenotypic maturation correlated with decreased phagocytic capacity. These results suggest increased extracellular pressure may cause aberrant DC maturation and impair tumor immunosurveillance.
Keywords	Pressure, Dendritic cell, Maturation, Cancer, Immunosurveillance
Adress and Contact Informations	¹Departments of Surgery, ²Anesthesiology, ³Anatomy and Cell Biology, ⁴Karmanos Cancer Institute, John D. Dingell VA Medical Center and Wayne State University, 4646 John R. Street, Detroit, MI 48201, USA *Author for correspondence; e-mail: marc.basson@va.gov, tel.: 313-576-3598, fax: 313-576-1002

DOI: 10.2478/s11658-007-0055-5 Volume 13 (2008) pp 271-282
Title	THE GENES AND ENZYMES INVOLVED IN THE BIOSYNTHESIS OF THIAMIN AND THIAMIN DIPHOSPHATE IN YEASTS ^#
Authors	Ewa Kowalska* and Andrzej Kozik
Abstract	Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino -5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)- 4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl- 2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.
Keywords	Thiamin biosynthesis, Thiamin diphosphate, Thiazole, Pyrimidine, THI genes, Saccharomyces cerevisiae
Adress and Contact Informations	Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland # Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7-11, 2007, "The Cell and Its Environment". Publication cost was partially covered by the organisers of this meeting.

DOI: 10.2478/s11658-008-0001-1 Volume 13 (2008) pp 283-302
Title	A STUDY ON THE FUNDAMENTAL FACTORS DETERMINING THE EFFICACY OF siRNAs WITH HIGH C/G CONTENTS
Authors	Jie-Ying Liao¹, James Q. Yin¹, Fang Chen¹, Tie-Gang Liu² and Jia-Chang Yue¹
Abstract	Although there are many reports about the efficacy of siRNAs, it is not clear whether those siRNAs with high C/G contents can be used to silence their target mRNAs efficiently. In this study, we investigated the structure and function of a group of siRNAs with high C/G contents. The results showed that single siRNAs against the Calpain, Otoferlin and Her2 mRNAs could induce different silencing effects on their targets, suggesting that the accessibility to target sequences influences the efficacy of siRNA. Unexpectedly, a single siRNA could target its cognate sequence in the 3’UTR of EEF1D or the 5’UTR of hTRF2 or CDC6. Their interaction induced different modes of gene silencing. Furthermore, the introduction of mutations into the 3’ end of the passenger strand showed that the position and number of mutated nucleotides could exert some influence on the efficacy of siRNA. However, these mutations did not completely block the passenger strand from exerting its RNAi effect. Interestingly, our findings also indicated that the target mRNA might play essential roles in maintaining or discarding the guide strand in RISCs. Thus, the conclusion could be drawn that favorable siRNA sequences, accessible target structures and the fast cleavage mode are necessary and sufficient prerequisites for efficient RNAi.
Keywords	siRNA, RNAi, mRNA, Local structure, Gene silencing
Adress and Contact Informations	¹Systems Biology Research Center, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, P.R. China ²Molecular Oncology Research Institute, Tufts-NEMC, Boston, MA 02111, USA *Authors for correspondence. James Q. Yin, e-mail: jqwyin@sun5.ibp.ac.cn, tel.: 86-010-64888572, fax: 86-010-64888572 or Jiachang Yue, e-mail: yuejc@sun5.ibp.ac.cn

DOI: 10.2478/s11658-008-0002-0 Volume 13 (2008) pp 303-311
Title	*hnRNP-R REGULATES THE PMA-INDUCED c-fos* EXPRESSION IN RETINAL CELLS**
Authors	Jia Huang¹, Shu-Jing Li¹, Xian-Hua Chen², Yu Han and Ping Xu²
Abstract	This study focused on the function of hnRNP-R in the regulation of c-fos expression. We demonstrated that hnRNP-R accelerated the rise and decline phases of c-fos mRNAs and Fos proteins, allowing PMA to induce an augmented pulse response of c-fos expression. Then, we examined the role of the c-fos-derived AU-rich element (ARE) in hnRNP-R-regulated mRNA degradation. Studies with the ARE-GFP reporter gene showed that hnRNP-R significantly reduced the expression of GFP with an inserted ARE. Moreover, immunoprecipitation-RT-PCR analysis demonstrated that in R28 cells and rat retinal tissues, the c-fos mRNA was co-immunoprecipitated with hnRNP-R. These findings indicate that hnRNP-R regulates the c-fos expression in retinal cells, and that the ARE of c-fos mRNAs contributes to this regulation.
Keywords	hnRNP-R, Retina, c-fos, mRNA turnover, ARE
Adress and Contact Informations	¹Laboratory of Genomic Physiology and ²State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai, 200032, P. R. China *Authors for correspondence; e-mail: matibuck@yahoo.com or xhchen@fudan.edu.cn, tel: 086-21-54237094, fax: 086-21-54237094

DOI: 10.2478/s11658-008-0004-y Volume 13 (2008) pp 312-326
Title	CELL ELECTROPHORESIS – A METHOD FOR CELL SEPARATION AND RESEARCH INTO CELL SURFACE PROPERTIES ^#
Authors	WŁODZIMIERZ KOROHODA* and ANNA WILK
Abstract	In this paper, we discuss the application of various methods of cell electrophoresis in research into cell surface properties (analytical methods), and the separation of uniform cell subpopulations from cell mixtures (preparative methods). The emphasis is on the prospects of the development of simplified and versatile methodologies, i.e. microcapillary cell electrophoresis and horizontal cell electrophoresis under near-isopycnic conditions. New perspectives are considered on the use of analytical and preparative cell electrophoresis in research on cell differentiation, neoplastic transformation, cell-cell interactions and the biology of stem cells.
Keywords	Cell electrophoresis, Cell separation, Cell surface
Adress and Contact Informations	Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland # Paper authored by participants of the international conference: XXXIV Winter School of the Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University, Zakopane, March 7-11, 2007, "The Cell and Its Environment". Publication cost was covered by the organisers of this meeting. *Author for correspondence; e-mail: korohoda@moj.uj.edu.pl

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