Vol. 11 No. 2 June 2006

DOI: 10.2478/s11658-006-0013-7 Volume 11 (2006) pp 155 – 160
Title THE ASSESSMENT OF DNA FROM MARINE ORGANISMS VIA A MODIFIED SALTING-OUT PROTOCOL
Authors Giovanni Battista Ferrara1, Barbara Murgia1, Anna Maria Parodi1, Laura Valisano2, Carlo Cerrano2*, Giulio Palmisano1, Giorgio Bavestrello3 and Michele Sara2
Abstract We developed a rapid, practical and non-toxic salting-out method for the extraction of DNA from marine organisms, and tested it on two representative species of Porifera and Cnidaria, both living in association with symbiotic zooxanthellae. We tested the efficiency of the protocol by comparing the output of the method for fresh tissue, frozen tissue and tissue stored in ethanol. It proved to be effective for extracting DNA in the case of the methods of preservation considered here, and for obtaining quantities of DNA comparable to those obtained via the traditional approach. The DNA from both species was of good quality. The DNA obtained was amplified by PCR using specific primers for the large ribosomal subunit, allowing the identification of the presence of both the host and symbiont genomes.
Keywords Symbiodinium, Porifera, Cnidaria, DNA, PCR
Address and Contact Information 1Dipartimento di Biologia, Universita di Genova, Viale Benedetto XV, 16132 Genova, Italy,
2Dipartimento per lo studio del Territorio e delle sue Risorse, Universita di Genova, Corso Europa 26, 16132 Genova, Italy,
3Dipartimento di Scienze del Mare, Universita Politecnica delle Marche, Via Brecce Bianche, 60131, Ancona, Italy
* Corresponding author: e-mail: cerrano@dipteris.unige.it; tel: +390103538563; fax: +390103538220

DOI: 10.2478/s11658-006-0014-6 Volume 11 (2006) pp 161 – 170
Title A NOVEL RECENTLY EVOLVED GENE C19orf24 ENCODES A NON-CLASSICAL SECRETED PROTEIN
Authors Xin-Rong Wang1,2, Yu-Bo Zhou2, Feng Liu2, Ke-Sheng Wang2, Yan Shen2, Jian-Hua Liu1 and Ze-Guang Han2*
Abstract Secreted proteins play important roles in many crucial biological processes, and can be new agents or targets for drug therapies. Here, we report on the isolation and characterization of a novel human non-classical secreted protein which is encoded by the hypothetical gene C19orf24 (chromosome 19 open reading frame 24). It has no signal peptide, but can still secrete extracellularly despite the presence of the inhibitor brefeldin A (BFA), proving its non-classical secreted protein status. Via subcellular localization using C19orf24 in vivo and transfected pEYFP-Golgi plasmid in Hela cells, C19orf24 was shown not to co-localize in the Golgi apparatus, which suggested that it secretes via a new and unknown pathway. Deglycosylation analysis with PNGase F verified that it has no N-glycosylation modification sites. Via the reverse transcription-PCR method, it was found to be expressed only in the human liver, and preferentially in normal tissue. In addition, C19orf24 was shown to be a recently evolved gene, found only in Homo sapiens and Pan troglodytes. By calculating its synonymous and non-synonymous substitution rate (dS/dN), we found that it experienced a purifying selection, which suggests that C19orf24 may have a special, irreplaceable biological function in the human organism.
Keywords C19orf24, Late evolution, Non-classical secreted protein, BFA
Address and Contact Information 1School of Life Science and Technology, Shanghai Jiao Tong University, No.1954 Hua-Shan Rd., Shanghai 200030, China,
2Chinese National Human Genome Center at Shanghai, No.351 Guo Shou-Jing Rd., Shanghai 201203, China
* Corresponding author: e-mail: hanzg@chgc.sh.cn; fax: (86-21) 50800402; tel: (86-21) 50800529-111

DOI: 10.2478/s11658-006-0015-5 Volume 11 (2006) pp 171 – 190
Title SERUM DECREASES THE SIZE OF METAFECTANE- and GENEJAMMER-DNA COMPLEXES BUT DOES NOT AFFECT SIGNIFICANTLY THEIR TRANSFECTION ACTIVITY IN SCCVII MURINE SQUAMOUS CELL CARCINOMA CELLS
Authors Krystyna Konopka*, Nathan Overlid, Anitha C. Nagaraj and Nejat Düzgüneş
Abstract Cationic liposome-DNA (lipoplexes) or polymer-DNA (polyplexes) complexes have been used to deliver therapeutic genes, both in vitro and in vivo. However, gene transfer by these non-viral vectors is usually inhibited by biological milieu. A relatively high efficiency of transfection could be achieved in human oral cancer cells transfected with the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in the presence of 60% fetal bovine serum (FBS) (Konopka et al., Cell. Mol. Biol. Lett. 10 (2005) 455-470). Here, we examined the efficacy of these vectors to deliver β-galactosidase (β-gal), luciferase and Herpes Simplex Virus thymidine kinase (HSV-tk) genes to SCCVII murine squamous cell carcinoma cells, which are used to generate an orthotopic murine model of oral cancer. We also evaluated the hydrodynamic size and zeta potential of the vectors and the effect of FBS and mouse serum (up to 60%) on the size of Metafectene and GeneJammer complexes with the pCMV.Luc plasmid. Our results indicate that Metafectene and GeneJammer are highly effective in transfecting SCCVII cells. Approximately 60-70% of SCCVII cells transfected with pCMV.lacZ were positive for β-gal staining. The expression of β-galactosidase was essentially not affected by serum. Mouse serum (20-60%) reduced both Metafectene- and GeneJammer-mediated luciferase expression by ~30-45%, while FBS did not affect transfection efficiency. The delivery of the HSV-tk gene by Metafectene or GeneJammer in the presence of 0% or 60% FBS, followed by GCV treatment for 6 days, resulted in over 90% cytotoxicity. The mean diameters of the DNA complexes of Metafectene and GeneJammer decreased significantly as a function of the serum concentration. The reduction in the size of the lipoplexes and polyplexes by serum was essentially not inhibitory to transfection of SCCVII cells. This is in contrast to previous hypotheses that serum-induced decrease in the size of lipoplexes is the primary cause of serum inhibition of transfection
Keywords Transfection, Metafectene, GeneJammer, Serum inhibition, HSV-tk gene, SCCVII murine squamous cell carcinoma cells
Address and Contact Information Department of Microbiology, University of the Pacific, Arthur A. Dugoni School of Dentistry, 2155 Webster Street, San Francisco, CA 94115, USA
* Corresponding author, e-mail: kkonopka@pacific.edu

DOI: 10.2478/s11658-006-0016-4 Volume 11 (2006) pp 191 – 213
Title In-silico PREDICTION AND OBSERVATIONS OF NUCLEAR MATRIX ATTACHMENT #
Authors Adrian E. Platts1, Amelia K. Quayle2 and Stephen A. Krawetz1,2,3*
Abstract The nuclear matrix is a functionally adaptive structural framework interior to the nuclear envelope. The nature and function of this nuclear organizer remains the subject of widespread discussion in the epigenetic literature. To draw this discussion together with a view to suggest a way forward we summarize the biochemical evidence for the modalities of DNA-matrix binding alongside the in-silico predictions. Concordance is exhibited at various, but not all levels. On the one hand, both the reiteration and sequence similarity of some elements of Matrix Attachment Regions suggest conservation. On the other hand, in-silico predictions suggest additional unique components. In bringing together biological and sequence evidence we conclude that binding may be hierarchical in nature, reflective of a biological role in replicating, transcribing and potentiating chromatin. Nuclear matrix binding may well be more complex than the widely accepted simple loop model.
Keywords Nuclear matrix, Matrix attachment regions, In-silico, Prediction, MARSCAN, MarFinder, MARWIZ, ChrClass, SMARTest, SIDD
Address and Contact Information 1Department of Obstetrics and Gynecology and 2The Center for Molecular Medicine and Genetics,
3Institute for Scientific Computing Wayne State University School of Medicine, 253 C.S. Mott Center, 275 E Hancock, Detroit, MI 48201, USA
# Invited paper
* Corresponding author, e-mail: steve@compbio.med.wayne.edu; tel: (313)-577-6770, fax: (313)-577-8554

DOI: 10.2478/s11658-006-0017-3 Volume 11 (2006) pp 214 – 229
Title En/Spm-LIKE TRANSPOSONS IN POACEAE SPECIES: TRANSPOSASE SEQUENCE VARIABILITY AND CHROMOSOMAL DISTRIBUTION
Authors Ahu Altinkut1,2*, Olga Raskina1, Eviatar Nevo1 and Alexander Belyayev1
Abstract Belonging to Class II of transposable elements, En/Spm transposons are widespread in a variety of distantly related plant species. Here, we report on the sequence conservation of the transposase region from sequence analyses of En/Spm-like transposons from Poaceae species, namely Zingeria biebersteiniana, Zingeria trichopoda, Triticum monococcum, Triticum urartu, Hordeum spontaneum, and Aegilops speltoides. The transposase region of En/Spm-like transposons was cloned, sequenced, and compared with equivalent regions of Oryza and Arabidopsis from the gene bank database. Southern blot analysis indicated that the En/Spm transposon was present in low (Hordeum spontaneum, Triticum monococcum, Triticum urartu) through medium (Zingeria bieberstiana, Zingeria trichopoda) to relatively high (Aegilops speltoides) copy numbers in Poaceae species. A cytogenetic analysis of the chromosomal distribution of En/Spm transposons revealed the concurence of the chromosomal localization of the En/Spm clusters with mobile clusters of rDNA. An analysis of En/Spm-like transposase amino acid sequences was carried out to investigate sequence divergence between 5 genera – Triticum, Aegilops, Zingeria, Oryza and Arabidopsis. A distance matrix was generated; apparently, En/Spm-like transposase sequences shared the highest sequence homology intra-generically and, as expected, these sequences were significantly diverged from those of O. sativa and A. thaliana. A sequence comparison of En/Spm-like transposase coding regions defined that the intra-genomic complex of En/Spm-like transposons could be viewed as relatively independent, vertically transmitted, and permanently active systems inside higher plant genomes. The sequence data from this article was deposited in the EMBL/GenBank Data Libraries under the accession nos. AY707995 - AY707996 - AY707997 - AY707998 - AY707999 - AY708000 - AY708001 - AY708002 - AY708003 - AY708004 - AY708005 - AY708005 - AY265312.
Keywords Transposase, En/Spm, Poaceae, Sequence, Evolution, In situ hybridization
Address and Contact Information 1Institute of Evolution, University of Haifa, Mt. Carmel, Haifa, 31905, Israel,
2Tubitak, Research Institute for Genetic Engineering and Biotechnology, P.O Box 21, 41470, Gebze, Kocaeli, Turkey
* Corresponding author: e-mail: ahu@gmbae.tubitak.gov.tr; ahualtinkut@yahoo.com; tel: +90 262 677 33 32, fax: + 90 262 646 39 29

DOI: 10.2478/s11658-006-0022-6 Volume 11 (2006) pp 230 – 241
Title DECREASED EXPRESSION OF THE HUMAN CARBONYL REDUCTASE 2 GENE HCR2 IN HEPATOCELLULAR CARCINOMA
Authors Shan Liu1, Lijie Ma1, Weixue Huang1, Yin Shai1, Xiaona Ji1, Liya Ding1, Yinkun Liu2, Long Yu1* and Shouyuan Zhao1
Abstract Altered gene expression was associated with the induction and maintenance of hepatocellular carcinoma (HCC). To determine the significance of HCR2 in HCC, here we compare the expression levels of HCR2 in carcinoma and in paired non-carcinoma tissues using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical staining. The expression ratio (ER) of HCR2 between the tumor and paired tumor-free tissues was calculated for each case and the data was clinicopathologically analyzed. The expression of HCR2 mRNA was found to be significantly decreased in HCC tissues compared with paired normal tissues (P < 0.001). HCR2 was downregulated in 58% (n = 22) of 38 HCC patients. The ER of HCR2 was higher in Edmondson’s grade I/II carcinomas than that in Edmondson’s grade III/IV carcinomas (P < 0.05). Western blot analysis showed HCR2 to be notably depressed in carcinoma tissues in 3 out of 4 HCC patients. Immunohistochemical staining indicated most HCR2 protein accumulated in non-carcinoma cells. These results suggested that altered HCR2 expression might play roles in the carcinogenesis and progression of HCC, and it could be a clinical marker for prognosis, and a molecular target for screening potential anti-HCC drugs.
Keywords HCR2, Hepatocellular carcinoma, Gene expression
Address and Contact Information 1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433,
2Institute of Liver Cancer, Shanghai Zhongshan Hospital, 136 Yixueyuan Road, Shanghai 200032, People’s Republic of China
*Corresponding author: tel: +86-21-65643954, fax: +86-21-65643250, e-mail: longyu@fudan.edu.cn

DOI: 10.2478/s11658-006-0018-2 Volume 11 (2006) pp 242 – 248
Title THE BREAKDOWN OF BILAYER LIPID MEMBRANES BY DEDRIMERS
Authors Dzmitry Shcharbin1, Alexander Drapeza2*, Valeri Loban2, Alexej Lisichenok2 and Maria Bryszewska1**
Abstract The BLM-system for studying the electrophysical properties of bilayer lipid membranes (BLM) was applied to investigate interactions between polyamidoamine (PAMAM) dendrimers and lipid bilayers. The cationic PAMAM G5 dendrimer effectively disrupted planar phosphatidylcholine membranes, while the hydroxyl PAMAM-OH G5 and carboxyl PAMAM G4.5 dendrimers had no significant effect on them.
Keywords PAMAM dendrimer, Planar bilayer lipid membrane, Conductivity, Voltammetry
Address and Contact Information 1Department of General Biophysics, University of Łódź ul. Banacha 12/16, 90-237 Łódź Poland,
2Department of Biophysics, Belarussian State University, 4 Skorina Avenue, 220050, Minsk, Belarus
*e-mail: drapeza@bsu.by, **Corresponding author: e-mail: marbrys@biol.uni.lodz.pl

DOI: 10.2478/s11658-006-0019-1 Volume 11 (2006) pp 249 – 255
Title CO-INVOLVEMENT OF THE MITOCHONDRIA AND ENDOPLASMIC RETICULUM IN CELL DEATH INDUCED BY THE NOVEL ER-TARGETED PROTEIN HAP
Authors Hua Xu1, Qing Zhou1 Xin Liu and Yi-Peng Qi*
Abstract HAP (a homologue of the ASY/Nogo-B protein), a novel human apoptosis-inducing protein, was found to be identical to RTN3. In an earlier study, we demonstrated that HAP localized exclusively to the endoplasmic reticulum (ER) and that its overexpression could induce cell apoptosis via a depletion of endoplasmic reticulum (ER) Ca2+ stores. In this study, we show that overexpression of HAP causes the activation of caspase-12 and caspase-3. We still detected the collapse of mitochondrial membrane potential (ΔΨm) and the release of cytochrome c in HAP-overexpressing HeLa cells. All the results indicate that both the mitochondria and the ER are involved in apoptosis caused by HAP overexpression, and suggest that HAP overexpression may initiate an ER overload response (EOR) and bring about the downstream apoptotic events.
Keywords Apoptosis-inducing protein HAP, Mitochondria, Endoplasmic reticulum (ER), Caspase-12, Caspase-3, Mitochondrial membrane potential (ΔΨm), Cytochrome c
Address and Contact Information State Key Laboratory of Virology, Section of Molecular Virology, College of Life Sciences, Wuhan University, Wuhan 430072, P. R. China
1Conributed equally, *Corresponding author; e-mail: yipengqi@whu.edu.cn

DOI: 10.2478/s11658-006-0020-8 Volume 11 (2006) pp 256 – 263
Title BREFELDIN A DECREASES THE ACTIVITY OF THE GENERAL AMINO ACID PERMEASE (GAP1) AND THE MORE SPECIFIC SYSTEMS FOR L-LEUCINE UPTAKE IN Saccharomyces cerevisiae
Authors Manuel Alonso1, Hilda I. Burgos1, Vanesa Pannunzio1, Andrea Monti Hughes1, James R. Mattoon2 and Carlos A. Stella1*
Abstract Brefeldin A is a commonly used antifungal agent that reversibly blocks protein transport from the endoplasmic reticulum to the Golgi complex. In this study, we aimed to characterize L-leucine uptake in Saccharomyces cerevisiae in the presence of brefeldin A. For this purpose, we used a synthetic medium, containing L-proline and the detergent SDS, which allows the agent to permeate into the yeast cell. The results obtained with a wild type strain and a gap1 mutant indicate that BFA causes either direct or indirect modification of the transport and/or processing of L-leucine permeases. The presence of BFA affects the kinetic parameter values for L-leucine uptake and decreases not only the uptake mediated by the general system (GAP1), but also that through the specific BAP2 (S1) and/or S2 systems.
Keywords Brefeldin A, Leucine permeases, Saccharomyces cerevisiae
Address and Contact Information 1Department of Biochemistry, School of Medicine, University of Buenos Aires, Paraguay 2155, 5o Piso, Argentina,
2Biotechnology Center, University of Colorado, Colorado Springs, CO, USA
* Corresponding author; e-mail: cstella@fmed.uba.ar

DOI: 10.2478/s11658-006-0021-7 Volume 11 (2006) pp 264 – 278
Title A PROTEOMICS STUDY OF THE MUNG BEAN EPICOTYL REGULATED BY BRASSINOSTEROIDS UNDER CONDITIONS OF CHILLING STRESS
Authors Bin Huang1, Chien-Hua Chu1, Shu-Ling Chen2, Hsueh-Fen Juan3,4* and Yih-Ming Chen1,3*
Abstract Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77% identity with the Arabidopsis CPD gene. It was strongly suppressed by chilling stress. This implies that exogenous treatment with BR could allow the plant to recover from the inhibited growth caused by chilling. In this study, we used proteomics to investigate whether the mung bean epicotyl can be regulated by brassinosteroids under conditions of chilling stress. Mung bean epicotyls whose growth was initially suppressed by chilling partly recovered their ability to elongate after treatment with 24-epibrassinolde; 17 proteins down-regulated by this chilling were re-up-regulated. These up-regulated proteins are involved in methionine assimilation, ATP synthesis, cell wall construction and the stress response. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment with DL-methionine. This is the first proteome established for the mung bean species. The regulatory relationship between brassinosteroids and chilling conditions was investigated, and possible mechanisms are discussed herein.
Keywords Proteomics, Chilling, Brassinosteroid, Mung bean, Epicotyl
Address and Contact Information 1Institute of Plant Biology, National Taiwan University, Taipei, 106, Taiwan,
2Department of Biotechnology, Ming Chuan University, Taoyuan, 333, Taiwan,
3Department of Life Science, National Taiwan University, Taipei, 106, Taiwan,
4Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, 106, Taiwan
* Corresponding authors: Hsueh-Fen Juan, e-mail: yukijuan@ntu.edu.tw, fax: +886-2-23673374, Yih-Ming Chen; e-mail: yihmingc@ntu.edu.tw, fax: +886-2-83695080

DOI: 10.2478/s11658-006-0024-4 Volume 11 (2006) pp 279 – 290
Title THE DefH9-iaaM-CONTAINING CONSTRUCT EFFICIENTLY INDUCES PARTHENOCARPY IN CUCUMBER #
Authors Zhimin Yin1, Robert Malinowski2, Agnieszka Ziółwska2, Hans Sommer3, Wojciech Plcader2 and Stefan Malepszy2*
Abstract Parthenocarpy (seedless fruits) is a desirable trait that has been achieved in many plant cultivars. We generated parthenocarpic cucumber fruits by introducing the chimeric DefH9-iaaM construct into the cucumber genome using an Agrobacterium tumefaciens-mediated protocol. The construct consists of the DefH9 promoter from Antirrhinum majus and the iaaM coding sequence from Pseudomonas syringae. Transgenic plants were obtained from nine independent transformation events: half of these were tetraploid and did not produce seeds following self-pollination, while the remaining half were capable of displaying parthenocarpy in the subsequent reproductive generation. Of the fruits produced by the transgenic lines, 70-90% were parthenocarpic. The segregation of the marker gene in the transgenic T1 progeny indicated single gene inheritance. The seed set in the transgenic lines and their F1 hybrids was lower than in the non-transgenic control plants. Some of the methodological details and the practical significance of the results are discussed.
Keywords Fruit set, Cucumber, Ovary-specific promoter, Transgenic parthenocarpy
Address and Contact Information 1Institute of Plant Genetics, Polish Academy of Science, Strzeszyńska 34, 60-479 Poznań, Poland,
2Department of Plant Genetics, Breeding and Biotechnology, Faculty of Horticulture and Landscape Architecture, Warsaw Agriculture University, Nowoursynowska 166, 02-787 Warsaw, Poland
3Department of Molecular Plant Genetics, Max Planck Institut für Zuechtungsforschung, 50829 Cologne, Germany
# This paper is dedicated to Prof. Juergen Grunewaldt from Hannover on the occasion of his retirement. *Corresponding author: tel/fax: +48-22-8430982, e-mail: malepszy@alpha.sggw.waw.pl

DOI: 10.2478/s11658-006-0023-5 Volume 11 (2006) pp 291 – 298
Title IDENTIFICATION OF MICROSATELLITE MARKERS IN THE RYE GENOME
Authors Hanna Bolibok1, Monika Rakoczy-Trojanowska1*, Małorzata Wyrzykowska1, Magdalena Radecka2 and Wacław Orczyk2
Abstract The rye genomic library, which consists of DNA fragments in the range of 0.5-1.1 kb, was screened for the presence of tri- and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.
Keywords Rye, Genomic library, SSR, Microsatellite, Wheat-rye addition lines
Address and Contact Information 1Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University, Nowoursynowska 166, 02-787 Warszawa, Poland
2Plant Breeding and Acclimatization Institute, Radzików 05-870 Błonie, Poland
* Corresponding author: e-mail: Monika_Rakoczy_Trojanowska@sggw.pl