Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 13 (2008) No. 1

DOI: 10.2478/s11658-007-0030-1 Volume 13 (2008) pp 1-10
Title	POLYMORPHISMS OF THE URIDINEDIPHOSPHOGLUCURONOSYLTRANSFERASE 1A1 GENE AND CORONARY ARTERY DISEASE
Authors	Chia-Jung Hsieh¹, Meng-Jung Chen²*, Yung-Liang Liao³ and Tung-Nan Liao¹
Abstract	Bilirubin, an antioxidant in the blood, plays a role in protection from atherosclerosis. The level of bilirubin is highly correlated to the incidence of coronary artery disease (CAD). Unconjugated bilirubin is conjugated with glucuronic acid through the reaction of uridine 5’-diphosphate-glucuronosyl transferase 1A1 (UGT1A1). The interactions of CAD and the variations in the coding regions of the UGT1A1 gene have never been evaluated. The purpose of this study was to analyze the influence of the UGT1A1 variant on the incidence of CAD. There were 135 participants in this study: 61 in the experimental group, who had CAD, and 74 in the control group, who did not have CAD. The blood samples from all 135 participants were collected and assayed to clarify the relationship between bilirubin and CAD. The assay of the polymerase chain reaction and the sequence of the UGT1A1 gene were examined to find the gene’s polymorphisms. The bilirubin levels for the participants in the control group were significantly higher than for the patients in the CAD group. Although the concentration of bilirubin in the UGT1A1 variant was higher than the wild type for the patients in the CAD group, there was no significant difference in the polymorphism of UGT1A1 between the patients in the CAD group and the participants in the control group.
Keywords	Atherosclerosis, Coronary artery disease, UGT1A1, Bilirubin, Antioxidant
Adress and Contact Informations	¹Chung-Hwa University of Medical Technology 89, Wen-Hwa 1st Street, Tainan, Taiwan, ²Chi-Mei Medical Center 901, Chung-Hwa Road, Tainan, Taiwan, ³Department of Pathology, Chi-Mei Medical Center 901, Chung-Hwa Road, Tainan, Taiwan * Author for correspondence; e-mail: ericmjc@yahoo.com.tw, tel: +88 69 5566 7035, fax: +886 6283 3806

DOI: 10.2478/s11658-007-0031-0 Volume 13 (2008) pp 11-19
Title	*THE INHIBITION OF in vivo* TUMORIGENESIS OF OSTEOSARCOMA (OS)-732 CELLS BY ANTISENSE HUMAN OSTEOPONTIN RNA**
Authors	Si-Jin Liu^1,6*, Dao-Qiang Zhang², Xiu-Mei Sui², Lin Zhang³, Zi-Wei Cai⁴, Li-Qiu Sun⁴, Ya-Jun Liu⁵, Yan Xue⁵ and Guo-Fa Hu¹
Abstract	Osteopontin (OPN) is a secreted, non-collagenous, sialic acid-rich protein which functions by mediating cell-matrix interactions and cellular signaling via binding with integrins and CD44 receptors. An increasing number of studies have shown that OPN plays an important role in controlling cancer progression and metastasis. OPN was found to be expressed in many human cancer types, and in some cases, its over-expression was shown to be directly associated with poor patient prognosis. In vitro cancer cell line and animal model studies have clearly indicated that OPN can function in regulating the cell signaling that ultimately controls the oncogenic potential of various cancers. Previous studies in our laboratory demonstrated that OPN is highly expressed in human osteosarcoma (OS) cell line OS-732. In this study, we successfully reduced the tumorigenecity of OS-732 cells xenotransplanted into nude mice, using the antisense human OPN (hOPN) RNA expression vector.
Keywords	Osteopontin, Osteosarcoma, Antisense RNA
Adress and Contact Informations	¹Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China, ²Wendeng Central Hospital, Weifang Medical College, Weihai, Shandong Province 264400, China, ³Research Institute of Sports Medicine, Soochow University, Suzhou, Jiangsu Province 215021, China ⁴Department of Pathophysiology, Medical College of Shantou University, Shantou 515031, China, ⁵Beijing Jishuitan Hospital, Peking University, Beijing 100035, China, ⁶Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02142, USA * Author for correspondence. Present address: Harvard-MIT Division of Health Sciences and Technology, MIT, 45 Carleton Str, E25-537, Cambridge, MA, 02142, USA. E-mail: sjliu@mit.edu, tel: 617-253-1446, fax: 617-253-3459

DOI: 10.2478/s11658-007-0032-z Volume 13 (2008) pp 20-37
Title	Hemidesmus indicus PROTECTS AGAINST ETHANOL-INDUCED LIVER TOXICITY
Authors	Nadana Saravanan¹ and Namasivayam Nalini²*
Abstract	Alcoholic liver disease (ALD) is one of the most common diseases in modern society. A large number of studies are in progress aiming to identify natural substances that would be effective in reducing the severity of ALD. Although there are currently a number of drugs on the market, their long-term use can have numerous side effects. Hemidesmus indicus is an indigenous Ayurvedic medicinal plant used in soft drinks in India. In this study, we examined the effects of its ethanolic root extract on experimental liver damage in order to evaluate its hepatoprotective effects against hepatotoxicity induced in rats by ethanol at a dosage of 5 g/kg body weight for 60 days. The H. indicus root extract was given at a dose of 500 mg/kg body weight for the last 30 days of the experiment. The animals were monitored for food intake and weight gain. The liver was analysed for the degree of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) and antioxidant status using the activities of glutathione-depedendant enzymes. The degree of liver damage was analysed using serum marker enzyme activities, the total protein, albumin, globulin, ceruloplasmin and liver glycogen contents, and the A/G ratio. The Fourier transform infrared spectra (FT-IR) of the liver tissues were recorded in the region of 4000-400 cm-1. The ethanol-fed rats showed significantly elevated liver marker enzyme activities, lipid peroxidation levels and reduced antioxidant levels as compared to the control rats. Oral administration of H. indicus for the latter 30 days resulted in an increased food intake and weight gain, decreased TBARS levels, near normal levels of glutathione-dependent enzymes, increased total protein, albumin, globulin and liver glycogen contents, an increased A/G ratio, and decreased liver marker enzyme activities and ceruloplasmin levels. The relative intensity of the liver FT-IR bands for the experimental groups were found to be altered significantly (p < 0.05) compared to the control samples. For the group that had H. indicus co-administered with ethanol, the intensity of the bands was near normal. Moreover, the results of the FT-IR study correlated with our biochemical results.
Keywords	FT-IR spectroscopy, Lipid peroxidation, Hemidesmus indicus, Rat liver
Adress and Contact Informations	¹Rani Meyammai College of Nursing, Faculty of Medicine, ²Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamilnadu, India * Author for correspondence; e-mail: nalininam@yahoo.com, tel: (91) 4144-238343 ext. 227, fax: (91) 4144-238343

DOI: 10.2478/s11658-007-0033-y Volume 13 (2008) pp 38-48
Title	*THE EFFECT OF CALNEXIN DELETION ON THE EXPRESSION LEVEL OF PDI IN Saccharomyces cerevisiae* UNDER HEAT STRESS CONDITIONS**
Authors	Huili Zhang¹, Jianwei He², Yanyan Ji¹, Akio Kato² and Youtao Song¹*
Abstract	We cultured calnexin-disrupted and wild-type Saccharomyces cerevisiae strains under conditions of heat stress. The growth rate of the calnexin-disrupted yeast was almost the same as that of the wild-type yeast under those conditions. However, the induced mRNA level of the molecular chaperone PDI in the ER was clearly higher in calnexin-disrupted S. cerevisiae relative to the wild type at 37ºC, despite being almost the same in the two strains under normal conditions. The western blotting analysis for PDI protein expression in the ER yielded results that show a parallel in their mRNA levels in the two strains. We suggest that PDI may interact with calnexin under heat stress conditions, and that the induction of PDI in the ER can recover part of the function of calnexin in calnexin-disrupted yeast, and result in the same growth rate as in wild-type yeast.
Keywords	Calnexin, Molecular chaperone, PDI, Heat stress
Adress and Contact Informations	1Department of Life Science, Liaoning University, Shenyang 110036, China, 2Department of Biological Chemistry, Yamaguchi University, Yamaguchi 753-8515, Japan * Author for correspondence; e-mail: ysong@lnu.edu.cn, tel: +86-24-62202280; fax: +86- 24-86864476

DOI: 10.2478/s11658-007-0034-x Volume 13 (2008) pp 49-57
Title	ACTIVATION OF THE INTRINSIC AND EXTRINSIC PATHWAYS IN HIGH PRESSURE-INDUCED APOPTOSIS OF MURINE ERYTHROLEUKEMIA CELLS
Authors	Takeo Yamaguchi*, Kenji Hashiguchi, Satoshi Katsuki, Wakako Iwamoto, Shoichiro Tsuruhara And Shigeyuki Terada
Abstract	We previously demonstrated that caspase-3, an executioner of apoptosis, is activated in the pressure-induced apoptosis of murine erythroleukemia (MEL) cells (at 100 MPa). Here, we examined the pathway of caspase-3 activation using peptide substrates and caspase inhibitors. Using the substrates of caspases-8 and -9, it was found that both are activated in cells under high pressure. The production of nuclei with sub-G1 DNA content in 100 MPa-treated MEL cells was suppressed by inhibitors of caspases-8 and -9, and pan-caspase. In 100 MPa-treated cells, pan-caspase inhibitor partially prevented the cytochrome c release from the mitochondria and the breakdown of mitochondrial membrane potential. These results suggest that the intrinsic and extrinsic pathways are activated in apoptotic signaling during the high pressureinduced death of MEL cells.
Keywords	Apoptosis, Caspases, Cytochrome c, Flow cytometry, Membrane potential, High pressure
Adress and Contact Informations	Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka, 814-0180, Japan *Author for correspondence; e-mail: takeo@fukuoka-u.ac.jp

DOI: 10.2478/s11658-007-0035-9 Volume 13 (2008) pp 58-66
Title	PLATELET-ACTIVATING FACTOR CHANGES IN PHOSPHOLIPID EXTRACTS FROM THE PLASMA, PERIPHERAL BLOOD MONONUCLEAR CELLS AND BONE MARROW MONONUCLEAR CELLS OF PATIENTS WITH ACUTE LEUKEMIA – A ³¹P MRS in vitro STUDY
Authors	Małgorzata Kuliszkiewicz-Janus^1,2*, Mariusz Adam Tuz^1,3, Marek Kiełbiński¹, Stanisław Baczyński⁴, Bożena Jaźwiec¹ and Helena Śladowska⁵
Abstract	The aim of this investigation was to evaluate the changes in PAF concentrations in the plasma, PBMC and BMMC of patients with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). The plasma was from 23 healthy volunteers (HV) and 44 patients with AL (16 ALL, 28 AML). The PBMC were from 15 HV and 55 patients with AL (18 ALL, 37 AML), and the BMMC from 40 patients with AL (11 ALL, 29 AML). Methanol-chloroform phospholipid extraction from 60 x 10⁶ cells (PBMC or BMMC) was performed according to a modified version of Folch’s method. ³¹P MRS data was obtained on an AMX 300 Bruker spectrometer (7.05 T). The PAF concentration in the plasma of the patients with ALL or AML was lower than that for the healthy volunteers. The PAF concentration in the plasma of the patients with ALL did not differ significantly from that of the patients with AML. In the case of both the PBMC and BMMC, the PAF concentration was significantly diminished in patients with ALL relative to the concentration for those with AML and for the healthy volunteers. No differences were observed in the PAF concentrations for the AML patients and the healthy volunteers.
Keywords	PAF, Acute leukemia, ³¹P MRS in vitro
Adress and Contact Informations	¹Department of Haematology and Oncology, Wrocław Medical University, Poland, ²Academic Centre for the Biotechnology of Lipid Aggregates, Poland, ³Institute of Experimental Physics, University of Wrocław, Poland, ⁴Faculty of Chemistry, University of Wrocław, Poland, ⁵Department of Chemistry of Drugs, Wrocław Medical University, Wrocław, Poland *Author for correspondence; e-mail: mkj@hemat.am.wroc.pl

DOI: 10.2478/s11658-007-0037-7 Volume 13 (2008) pp 67-73
Title	THE EFFECT OF TRIBUTYLTIN ON HUMAN EOSINOPHYLIC LEUKEMIA EoL-1 CELLS^#
Authors	Jolanta Sroka*, Przemysław Włosiak, Anna Wilk, Justyna Antonik, Jarosław Czyż and Zbigniew Madeja
Abstract	Organotin compounds are chemicals that are widely used in industry and agriculture as plastic stabilizers, catalysts and biocides. Many of them, including tributyltin (TBT), have been detected in human food and, as a consequence, detectable levels have been found in human blood. As organotin compounds were shown to possess immunotoxic activity, we focused our attention on the effect of TBT on the basic determinants of the function of eosinophils, i.e. cell adhesiveness and motility. We used human eosinophylic leukemia EoL-1 cells, a common in vitro cellular model of human eosinophils. Here, we demonstrate that TBT causes a dose-dependent decrease in the viability of EoL-1 cells. When administered at sub-lethal concentrations, TBT significantly decreases the adhesion of EoL-1 cells to human fibroblasts (HSFs) and inhibits their migration on fibroblast surfaces. Since the basic function of eosinophils is to invade inflamed tissues, our results indicate that TBT, and possibly other organotin compounds, may affect major cellular properties involved in the determination of in vivo eosinophil function.
Keywords	Tributyltin, Cytotoxicity, Cell adhesion, Cell migration, EoL-1 cells
Adress and Contact Informations	Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-378 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: jola@mol.uj.edu.pl

DOI: 10.2478/s11658-007-0038-6 Volume 13 (2008) pp 74-91
Title	*THE CONSTRUCTION AND CHARACTERISTICS OF A BAC LIBRARY FOR Cucumis sativus* L. ‘B10’**
Authors	Wojciech Gutman¹*, Magdalena Pawełkowicz², Rafał Woycicki², Ewa Piszczek¹ and Zbigniew Przybecki²
Abstract	Cloning using bacterial artificial chromosomes (BACs) can yield high quality genomic libraries, which are used for the physical mapping, identification and isolation of genes, and for gene sequencing. A BAC genomic library was constructed from high molecular weight DNA (HMW DNA) obtained from nuclei of the cucumber (Cucumis sativus L. cv. Borszczagowski; B10 line). The DNA was digested with the HindIII restriction enzyme and ligated into the pCC1BAC vector. The library consists of 34,560 BAC clones with an average insert size of 135 kb, and 12.7x genome coverage. Screening the library for chloroplast and mitochondrial DNA content indicated an exceptionally low 0.26% contamination with chloroplast DNA and 0.3% with mitochondrial DNA.
Keywords	Cucumis sativus L., Bacterial artificial chromosome, High molecular weight DNA, Pulsed field gel electrophoresis
Adress and Contact Informations	¹Department of Biochemistry, Warsaw Agricultural University, 02-776 Warsaw ²Department of Genetics, Breeding and Biotechnology of Plant, Warsaw Agricultural University, 02-776 Warsaw *Author for correspondence; e-mail: wojciech_gutman@sggw.pl

DOI: 10.2478/s11658-007-0039-5 Volume 13 (2008) pp 92-102
Title	THE STAGE-SPECIFIC FUNCTION OF GAP JUNCTIONS DURING TUMOURIGENESIS ^#
Authors	Jarosław Czyż*
Abstract	Tumour development is a process resulting from the disturbance of various cellular functions including cell proliferation, adhesion and motility. While the role of these cell parameters in tumour promotion and progression has been widely recognized, the mechanisms that influence gap junctional coupling during tumorigenesis remain elusive. Neoplastic cells usually display decreased levels of connexin expression and/or gap junctional coupling. Thus, impaired intercellular communication via gap junctions may facilitate the release of a potentially neoplastic cell from the controlling regime of the surrounding tissue, leading to tumour promotion. However, recent data indicates that metastatic tumour cell lines are often characterized by relatively high levels of connexin expression and gap junctional coupling. This review outlines current knowledge on the role of connexins in tumorigenesis and the possible mechanisms of the interference of gap junctional coupling with the processes of tumour invasion and metastasis.
Keywords	Gap junctions, Connexin, Tumour, Neoplasia, Metastasis
Adress and Contact Informations	Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-378 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: jaro@mol.uj.edu.pl

DOI: 10.2478/s11658-007-0040-z Volume 13 (2008) pp 103-111
Title	ASCORBIC ACID INHIBITS THE MIGRATION OF WALKER 256 CARCINOSARCOMA CELLS ^#
Authors	Ewa Wybieralska, Monika Koza, Jolanta Sroka, Jarosław Czyż and Zbigniew Madeja*
Abstract	The results of several experimental studies have shown that ascorbic acid inhibits tumor growth and metastasis. Ascorbic acid is an antioxidant that acts as a scavenger for a wide range of reactive oxygen species (ROS). Both tumour metastasis and cell migration have been correlated with the intracellular ROS level, so it was postulated that the inhibitory effect of ascorbic acid derivatives on cell motility may be caused by scavenging of ROS. Time-lapse analyses of Walker 256 carcinosarcoma cell migration showed that both the speed of movement and the cell displacement were inhibited by ascorbic acid applied in concentrations ranging from 10 to 250 μM. This effect correlated with a reduction in the intracellular ROS level in WC 256 cells, suggesting that ROS scavenging may be a mechanism responsible for the inhibition of WC 256 cell migration. However, another potent antioxidant, N-acetyl-L-cysteine, also efficiently decreased the intracellular ROS level in WC 256 cells, but did not affect the migration of the investigated cells. These results demonstrate that intact, unmodified ascorbic acid applied in physiologically relevant and nontoxic concentrations exerts an inhibitory effect on the migration of WC 256 carcinosarcoma cells, and that this may be one of the factors responsible for the anti-metastatic activity of vitamin C. However, our data does not support the hypothesis that the scavenging of intracellular ROS is the main mechanism in the inhibition of cancer cell migration by ascorbic acid.
Keywords	Ascorbic acid, Cell migration, Metastasis, Reactive oxygen species
Adress and Contact Informations	Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-378 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: zibi@mol.uj.edu.pl

DOI: 10.2478/s11658-007-0041-y Volume 13 (2008) pp 112-118
Title	THE TRANSCRIPTION REINITIATION PROPERTIES OF RNA POLYMERASE III IN THE ABSENCE OF TRANSCRIPTION FACTORS
Authors	Roberto Ferrari and Giorgio Dieci*
Abstract	Transcription reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated reinitiation during subsequent cycles is compromised.
Keywords	RNA polymerase III, Transcription reinitiation, TFIIIB, G-less cassette
Adress and Contact Informations	Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Parma, Viale G.P. Usberti 23/A, 43100 Parma, Italy *Author for correspondence; e-mail: giorgio.dieci@unipr.it, tel: +39-0521-905649, fax: +39-0521-905151

DOI: 10.2478/s11658-007-0042-x Volume 13 (2008) pp 119-129
Title	ACIDIFICATION INDUCES BAX TRANSLOCATION TO THE MITOCHONDRIA AND PROMOTES ULTRAVIOLET LIGHT-INDUCED APOPTOSIS
Authors	Lin Yang*¹, Yongyu Mei¹, Qifeng Xie¹, Xiaoyan Han², Fucheng Zhang², Lin Gu¹, Yufeng Zhang¹, Youming Chen¹, Gang Li¹ and Zhiliang Gao¹
Abstract	It has been suggested that Bax translocation to the mitochondria is related to apoptosis, and that cytosol acidification contributes to apoptosis events. However, the mechanisms remain obscure. We investigated the effect of acidification on Bax translocation and on ultraviolet (UV) light-induced apoptosis. The Bax translocation assay in vitro showed that Bax translocated to the mitochondria at pH 6.5, whereas no Bax translocation was observed at pH 7.4. VHDBB cells expressing the GFP-Bax fusion protein were treated for 12 h with a pH 6.5 DMEM medium, nigericin (5 μg/ml) and UV light (50 J/cm²), separately or in combination, and Bax translocation to the mitochondria was determined by SDS-PAGE and Western blot, and apoptotic cell death was detected by flow cytometry. The results showed that some of the Bax translocated to the mitochondria in the cells treated with the normal medium, nigericin and UV in combination, whereas all of the Bax translocated to the mitochondria in the cells treated with the pH 6.5 medium, nigericin and UV in combination. In VHDBB cells treated for 12 h with nigericin, UV alone,and UV and nigericin in combination, the respective rates of apoptotic cell death were 25.08%, 33.25% and 52.88%. In cells treated with pH 6.5 medium and nigericin, pH 6.5 medium and UV, and pH 6.5 medium, nigericin and UV in combination, the respective rates of apoptotic cell death increased to 37.19%, 41.42% and 89.44%. Our results indicated that acidification induces Bax translocation from the cytosol to the mitochondria, and promotes UV lightmediated apoptosis. This suggests that there is a possibility of improving cancer treatment by combining acidification with irradiation or chemotherapeutic drugs.
Keywords	Acidification, Bax, Translocation, Ultraviolet light, Apoptosis, Cancer
Adress and Contact Informations	¹Research Unit of Viral Hepatitis, Department of Infectious Diseases, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, P. R. China, ²Medical Research Center, The Third Affiliated Hospital, Sun Yat-sen University, 600 Tianhe Road, Guangzhou 510630, P. R. China *Author for correspondence; e-mail: linyang1962@hotmail.com

DOI: 10.2478/s11658-007-0047-5 Volume 13 (2008) pp 130-143
Title	SCLEROTIA OF THE ACELLULAR (TRUE) SLIME MOULD Fuligo septica AS A MODEL TO STUDY MELANIZATION AND ANABIOSIS ^#
Authors	Anna Krzywda¹, Elżbieta Petelenz^1,2, Dominika Michalczyk¹ And Przemysław M. Płonka¹*
Abstract	Acellular (true) slime moulds (Myxomycetes) are capable of a transition to the stage of sclerotium – a dormant form of plasmodium produced under unfavourable environmental conditions. In this study, sclerotia of Fuligo septica were analyzed by means of electron paramagnetic resonance (EPR) spectroscopy. The moulds were cultivated in vitro on filter paper, fed with oat flour, and kept until the plasmodia began to produce sclerotia. The obtained sclerotia differed in colour from yellow through orange to dark-brown. The EPR spectra revealed a free radical, melanin-like signal correlated with the depth of the colour; it was strongest in the dark sclerotia. Sclerotization only took place when the plasmodia were starved and very slowly dried. Only the yellow sclerotia were able to regenerate into viable plasmodia. This suggests that myxomycete cytoplasm dehydration is an active process regulated metabolically. Plasmodial sclerotization may therefore serve as a convenient model system to study the regulation of cytoplasmatic water balance, and sclerotia as a convenient material for EPR measurements, combining the quality of plasmodia with the technical simplicity of the measurements characteristic of dry spores. Darkening of the sclerotia is most probably a pathological phenomenon connected with the impairment of water balance during sclerotization.
Keywords	Aquaporins, Dehydration, EPR, Melanin, Myxomycetes, Pigmentation
Adress and Contact Informations	¹Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, ²Department of Cell and Molecular Biology, Microbiology, Göteborg University, Göteborg, Sweden ^# 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; Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland; tel: 4812 664-6350, fax: 4812 66 4-6907, e-mail: mieszko@mol.uj.edu.pl

DOI: 10.2478/s11658-007-0043-9 Volume 13 (2008) pp 144-154
Title	SYNTAXIN 8 HAS TWO FUNCTIONALLY DISTINCT DI-LEUCINEBASED MOTIFS
Authors	Kazuo Kasai^1,2,3, Kei Suga¹, Tetsuro Izumi³ and Kimio Akagawa^1,*
Abstract	Syntaxin 8 has been shown to form the SNARE complex with syntaxin 7, vti1b and endobrevin. These have been shown to function as the machinery for the homotypic fusion of late endosomes. Recently, we showed that syntaxins 7 and 8 cycle through the plasma membrane, and that the dileucine- based motifs in the cytoplasmic domain of syntaxins 7 and 8 respectively function in their endocytic and exocytic processes. However, we could not elucidate the mechanism by which syntaxin 8 cycles through the plasma membrane. In this study, we constructed several different syntaxin 8 molecules by mutating putative di-leucine-based motifs, and analyzed their intracellular localization and trafficking. We found a di-leucine-based motif in the cytoplasmic domain of syntaxin 8. It is similar to that of syntaxin 7, and functions in its endocytosis. These results suggest that in the cytoplasmic domain, syntaxin 8 has two functionally distinct di-leucine-based motifs that act independently in its endocytic and exocytic processes. This is the first report on two di-leucine-based motifs in the same molecule acting independently in distinct transport pathways.
Keywords	Syntaxin, Di-leucine-based motif, Endocytosis, Exocytosis
Adress and Contact Informations	¹Department of Cell Physiology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan, ²Toyama Chemical Co., Ltd., Shinjuku, Tokyo 160- 0023, Japan, and ³Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan *Author for correspondence; e-mail: akagawak@kyorin-u.ac.jp, tel: +81-0422-47-5511, fax: +81-0422-47-4801

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