Vol. 10 No. 1 March 2005

Volume 10 (2005) pp 3-13
Authors Stanisław Kondracki*, Dorota Banaszewska and Cecylia Mielnicka
Abstract The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 µm2 (P≤0.01) and a flagellum longer by 0.67 μm (P≤0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P≤0.05) and head width (r = 0.36, P≤0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P≤0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P≤0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable.
Address and Contact Information Department of Bioengineering and Animal Breeding, University of Podlasie, B. Prusa 14, 08-110 Siedlce, Poland
*Corresponding author; e-mail: sk@ap.siedlce.pl
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Volume 10 (2005) pp 15-21
Authors Alina Błaszczyk1* and Janusz Skolimowski2
Abstract In our study, we analyzed the cytotoxicity of ethoxyquin (EQ) and its two salts, ethoxyquin hydrochloride (EQ-HCL) and ethoxyquin phosphate (EQP). It was shown that EQ was the most cytotoxic compound (IC50 = 0.09 mM), while the lowest cytotoxic effect was observed for EQ-P (IC50 = 0.8 mM). The properties of ethoxyquin and its salts were also analyzed with the TUNEL method, which evaluates their ability to induce apoptosis. It was shown that EQ induced apoptosis in cultured human lymphocytes, especially at concentrations of 0.25 and 0.5 mM.
Address and Contact Information 1Department of Cytogenetics and Plant Molecular Biology, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland,
2Department of Organic Chemistry, University of Łódź, Narutowicza 68, 90-136 Łódź, Poland
*Corresponding author; e-mail: ablasz@biol.uni.lodz.pl
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Volume 10 (2005) pp 23-35
Authors Gintautas Saulis*
Abstract The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight < 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius < 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4oC, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37oC, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances.
Address and Contact Information Biophysical Research Group, Department of Biology, Faculty of Nature Science, Vytautas Magnus University, 28 Daukanto str., Kaunas 44246, Lithuania
tel: +370612-82773, fax: +370-37-406572, e-mail: sg@kaunas.omnitel.net
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Volume 10 (2005) pp 37-47
Authors Kamila Środa1,3*, Janusz Rydlewski2, Marek Langner1, Arkadiusz Kozubek3, Michał Grzybek3 and Aleksander F. Sikorski1,3**
Abstract Liposomes containing the polyethylene glycol (PEG) derivative of phosphatidyl ethanolamine (PE) have recently been found to be promising drug carriers, as they facilitate controlled and target-oriented release of therapeutics. They also reduce the side effects of many drugs. Here, we present the results of a study on antiliposomal properties of rabbit sera obtained after weekly injections of small liposomes containing 20% PEG-PE. The effect was analysed as the level of induced carboxyfluorescein release from these liposomes in vitro. The incubation of liposomes with rabbit serum taken after the injections induced the release of carboxyfluorescein at a higher level than was seen for incubation with untreated animal’s serum. The strongest effect was observed for serum obtained after the second injection, i.e. during the second week of the study. The effect was much smaller after the serum samples were preheated at 56oC. The binding of serum proteins by PEGylated liposomes was analysed via gel filtration and via the immunoblot technique using goat anti-rabbit IgG; this revealed that the serum protein which bound to the liposomes in vitro had a molecular weight of ~55 kD and reacted with the anti-IgG antibody. Competition with PEG or lipids indicate that this IgG has an anti-PEG activity. We therefore assume that these antibodies are responsible for the activation of complement and leakage induction of PEG-liposomes. Such antibodies could be responsible for increased phagocytosis by RES macrophages (in particular liver macrophages) and decreased circulation time.
Address and Contact Information 1Academic Centre for the Biotechnology of Lipid Aggregates, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
2Department and Clinic of Internal Veterinary Medicine, Agricultural University of Wrocław, pl. Grunwaldzki 47, Wrocław, Poland,
3Institute of Biochemistry and Molecular Biology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
4Institute of Physics, Wrocław University of Technology, Wybrzeże Wyspiałskiego 27, 50-370 Wrocław, Poland
*Present address: Department of Biophysics, Wrocław Medical University, Chałubiłskiego 10, 50-368 Wrocław, Poland
**Corresponding author; tel: +4871 375 6233, fax: +4871 375 6208, e-mail: afsbc@ibmb.uni.wroc.pl
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Volume 10 (2005) pp 49-59
Authors Tatjana Sjakste1,2, Marion Röder1, Danute Labeikytė3, Lida Bagdoniene3, Anna Levina4, Benediktas Juodka3 and Nikolajs Sjakste4
Abstract The distribution of DNA complexes with proteins resistant to routine deproteinisation procedures (tightly bound proteins, TBP) was studied on the barley chromosome 1H by means of microsatellite analysis. The polypeptide spectrum of the barley shoot TBP was similar to that formerly described for other organisms. In order to reveal developmental changes in the distribution of the TBP, DNA was extracted from dry grains, coleoptiles, root tips, and young and old leaves. In the seeds, all the studied DNA sites were evenly distributed between free DNA and DNA containing the tight DNA-protein complexes. Germination made the interaction between TBP and chromosomal loci specific. In coleoptile DNA, sites containing microsatellites located in the distal part of the long arm of the chromosome were not bound to the TBP anymore, however, the centromeric markers were found exclusively in the tight DNA-protein complexes. A similar but not identical distribution of markers was observed in the root tips and young leaves. Leaf senescence was accompanied by a loss in interaction specificity between chromosomal loci and tightly bound proteins. These results are considered to reflect changes in chromatin domain interaction with the nuclear matrix during plant development.
Address and Contact Information 1Institute for Plant Genetics and Crop Research, Correnstrasse 3, Gatersleben, Germany,
2Laboratory of Plant Genetics, Institute of Biology, University of Latvia, Miera iela 3, Salaspils LV2169, Latvia,
3Department of Biochemistry and Biophysics, Vilnius University, M. K. Čiurlionio 21, LT-2009 Vilnius, Lithuania,
4Faculty of Medicine, University of Latvia, Šarlotes iela 1a, Riga LV-1001, Latvia
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Volume 10 (2005) pp 61-72
Authors Rizwan Hasan Khan1*, Aabgeena Naeem1 and Masroor Alam Baig2
Abstract A Systematic investigation of the effect of pH on concanavalin A in the presence of specific and non-specific sugars is made using CD (circular dichroism) and fluorescence. The specific and non-specific sugars for concanavalin A were methyl α-D-glucopyranoside and methyl α-Dgalactopyranoside respectively. Far-UV CD showed changes in the MRE value at 217 nm in the presence of the above-mentioned sugars. At pH 7, the CD and fluorescence spectra obtained in the presence of methyl α-D-glucopyranoside were slightly different from those for the native state and a significant difference was obtained in the presence of methyl α-D-galactopyranoside. Near-UV CD spectra showed the retention of a native-like tertiary structure in the presence of the specific sugar upon pH denaturation. Tryptophan fluorescence studies indicated a change in the tryptophan enviornment. The results obtained from our CD data are consistent with those obtained from fluorescence studies. Upon pH exposure of concanavalin A in the presence of methyl α-Dglucopyranoside and methyl α-D-galactopyranoside, the former acted as a protector preventing conformational alteration at different pH while the presence of latter induced a different stable conformational state and this state persists over the pH range from 2 to 10.
Address and Contact Information 1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh- 202002, India,
2Jamia Hamdard University, Hamdard Nagar, New Delhi 110 062, India
*Corresponding author; fax: 91-571-2701766, tel: 91-571-27020388, E-mail: rizwanhkhan@hotmail.com, rizwanhkhan1@yahoo.com
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Volume 10 (2005) pp73-89
Authors Dariusz Plewczyński1,2*, Adrian Tkacz3, Adam Godzik4,5 and Leszek Rychlewski1
Abstract We describe a bioinformatics tool that can be used to predict the position of phosphorylation sites in proteins based only on sequence information. The method uses the support vector machine (SVM) statistical learning theory. The statistical models for phosphorylation by various types of kinases are built using a dataset of short (9-amino acid long) sequence fragments. The sequence segments are dissected around post-translationally modified sites of proteins that are on the current release of the Swiss-Prot database, and that were experimentally confirmed to be phosphorylated by any kinase. We represent them as vectors in a multidimensional abstract space of short sequence fragments. The prediction method is as follows. First, a given query protein sequence is dissected into overlapping short segments. All the fragments are then projected into the multidimensional space of sequence fragments via a collection of different representations. Those points are classified with pre-built statistical models (the SVM method with linear, polynomial and radial kernel functions) either as phosphorylated or inactive ones. The resulting list of plausible sites for phosphorylation by various types of kinases in the query protein is returned to the user. The efficiency of the method for each type of phosphorylation is estimated using leave-one-out tests and presented here. The sensitivities of the models can reach over 70%, depending on the type of kinase. The additional information from profile representations of short sequence fragments helps in gaining a higher degree of accuracy in some phosphorylation types. The further development of an automatic phosphorylation site annotation predictor based on our algorithm should yield a significant improvement when using statistical algorithms in order to quantify the results.
Address and Contact Information 1BioInfoBank Institute, ul. Limanowskiego 24A/16, 60-744 Poznań, Poland,
2Interdisciplinary Centre for Mathematical and Computational Modeling, University of Warsaw, ul. Pawiłskiego 5a, 02-106 Warsaw, Poland,
3Bioinformatics Unit, Department of Physics, Adam Mickiewicz University, ul. Umultowska 85, 61-614 Poznań, Poland,
4The Burnham Institute, La Jolla, CA, USA,
5Bioinformatics Core JCSG, University of California San Diego, La Jolla, CA, USA
*Corresponding author; tel: +48-61-8653520, fax: +48-61-8643350, e-mail: darman@bioinfo.pl
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Volume 10 (2005) pp 91-99
Authors Hanna M. Kowalczyńska*, Małgorzata Nowak-Wyrzykowska, Marcin Inkielman, Liliana Stołowska and Ewa Marciniak
Abstract We studied the shape of L1210 leukaemia cells adhering in a proteinfree medium to sulfonated (styrene/methyl methacrylate) copolymer surfaces of two sulfonic group densities, and thus of differing wettability. The use of our image analysis method and the mathematical procedure [1] allowed us to calculate the values of the so-called shape parameter, which quantitatively determines the three-dimensional cell shape. Here, we show that the values of the shape parameter of the adhering cells and the F-actin concentration, in the region near the cell-substratum interface, depend on the density of sulfonic groups present on the substratum surface..
Address and Contact Information Department of Biophysics and Biomathematics, Medical Centre for Postgraduate Education, ul. Marymoncka 99, 01-813 Warszawa, Poland
*Corresponding author; fax: +48 22 8640834, e-mail: hmkowal@cmkp.edu.pl
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Volume 10 (2005) pp 101-121
Authors Sathyanarayana N. Gummadi* and Krishna S. Kumar
Abstract Phospholipid flip-flop is required for bilayer assembly and the maintenance of biogenic (self-synthesizing) membranes such as the eukaryotic endoplasmic reticulum and the bacterial cytoplasmic membrane. Due to the membrane topology of phospholipid biosynthesis, newly synthesized phospholipids are initially located in the cytoplasmic leaflet of biogenic membranes and must be translocated to the exoplasmic leaflet to give uniform bilayer growth. It is clear from many studies that phospholipid flip-flop in biogenic membranes occurs very rapidly, within a period of a few minutes. These studies also reveal that phospholipid translocation in biogenic membranes occurs bi-directionally, independently of the phospholipid head group, via a facilitated diffusion process in the absence of metabolic energy input, and that this type of transport requires specific membrane proteins. These translocators have been termed biogenic membrane flippases, and they differ from metabolic energy-dependent transporters (ABC transporters and MDR proteins). No biogenic membrane flippases have been characterized. This review briefly discusses the importance of biogenic membrane flippases, the various assay methods used for measuring the rate of phospholipid flip-flop, and the progress that has been made towards identifying these proteins.
Address and Contact Information Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India
*Corresponding author, e-mail: gummadi@iitm.ac.in
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Volume 10 (2005) pp 123-134
Authors Mingpei You1, Jeffrey G. Boersma2, Bevan J. Buirchell1,2, Mark W. Sweetingham1,2, Kadambot H. M. Siddique1 and Huaan Yang1,2*
Abstract Selection for anthracnose disease resistance is one of the major objectives in lupin breeding programs. The aim of this study was to develop a molecular marker linked to a gene conferring anthracnose resistance in narrowleafed lupin (Lupinus angustifolius L.), which can be widely used for MAS in lupin breeding. A F8 derived RIL population from a cross between cultivar Tanjil (resistant to anthracnose) and Unicrop (susceptible) was used for marker development. DNA fingerprinting was conducted on 12 representative plants by combining the AFLP method with primers designed based on conserved sequences of plant disease resistance genes. A co-dominant candidate marker was detected from a DNA fingerprint. The candidate marker was cloned, sequenced, and converted into a sequence-specific, simple PCR based marker. Linkage analysis based on a segregating population consisting of 184 RILs suggested that the marker, designated as AntjM2, is located 2.3 cM away from the R gene conferring anthracnose resistance in L. angustifolius. The marker has now being implemented for MAS in the Australian national lupin breeding program.
Address and Contact Information 1Centre for Legumes in Mediterranean Agriculture (CLIMA), Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, WA 6009, Australia,
2Department of Agriculture Western Australia, 3Baron-Hay Court, South Perth, WA 6151, Australia
* Corresponding author, e-mail: hyang@agric.wa.gov.au
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Volume 10 (2005) pp 135-149
Authors Pamela M. Taylor-Harris1*, Leanne E. Felkin2*, Emma J. Birks2, Rodney C.G. Franklin3, Magdi H. Yacoub2, Anthony J. Baines4, Paul J.R. Barton2 and Jennifer C. Pinder1**
Abstract The proteins, spectrin and 4.1 confer support and resilience to animal cell membranes, and promote assembly of multimeric, membrane-bound signalling complexes. Protein 4.1 also plays important roles in tumour suppression and the regulation of cell proliferation. To assess relative tissue expression of the four genes encoding human protein 4.1, we measured mRNA levels using quantitative real-time polymerase chain reaction. We compared 4.1 expression with that of a major splice variant of spectrin, bIIS2 that has a shortened C-terminus lacking a pleckstrin homology domain. mRNA for 4.1R is four-fold higher in bone marrow than in tissues with the next highest prevalence: cerebellum, lung, testis and thymus. 4.1G mRNA is highly expressed in brain, spinal cord and testis; 4.1N in brain, spinal cord and adrenal gland; 4.1B in testis, brain, spinal cord, and kidney. Thus, 4.1N, 4.1B and 4.1G all show high accumulation in nervous tissues. mRNA for bIIÎŁ2-spectrin is ubiquitous, but most abundant in cardiac and nervous tissues. Comparative transcript abundance was analysed in heart and brain. bIIS2-spectrin was the most abundant transcript in heart with levels 5 fold greater than 4.1G or 4.1N and at least 9 fold greater than 4.1B. In brain, 4.1N was the most abundant transcript, with levels 2.4 fold greater than 4.1B and at least 4 fold greater than 4.1G or bIIS2-spectrin. 4.1R abundance was very low in both tissues. Whilst we expected that 4.1 mRNAs would feature highly in muscle and nerve, we note their high abundance in testis, indicating previously unsuspected functions in reproduction.
Address and Contact Information 1Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK,
2Heart Science Centre, Imperial College London, Harefield, Middlesex UB9 6JH, UK,3Royal Brompton & Harefield NHS Trust, Harefield, Middlesex UB9 6JH, UK
4Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, UK
# Contribution to the Conference “Emerging Pathways in Cytoskeletal Communication”, Umea, Sweden 2004, organised by CytoNET
* These authors made equal contributions to the work
**Corresponding author; fax: +44-20-7848-6435, e-mail: jennifer.fordham@kcl.ac.uk
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Volume 10 (2005) pp 151-161
Title RAPD ANALYSIS OF THE INTERSPECIFIC SOMATIC HYBRIDS Solanum bulbocastanum (+) S. tuberosum
Authors Danuta Bołtowicz*, Anna Szczerbakowa and Bernard Wielgat
Abstract The diploid Mexican species S. bulbocastanum (blb) was used as a source of late blight resistance in somatic hybridization with the potato (S. tuberosum, tbr) dihaploid H-8105. The produced 2x blb (+) 2x tbr H-8105 somatic hybrids did not retain the blb parent's characteristic high resistance to P. infestans. The revealed aneuploidy of blb (+) tbr H-8105 hybrids indicated a possible loss of individual blb chromosome(s) carrying the resistance genes. Four hybrid clones differing in terms of their ploidy, morphology and growth potential were analysed for the presence of all twelve blb chromosomes (linkage groups). The RAPD markers assigned to particular chromosomes were selected on the basis of the linkage map of S. bulbocastanum constructed by Naess et al., Mol. Gen. Genom. 265 (2001) 694-704. Of the 86 markers analysed, twelve (14%) were common for blb and H-8105, while 34 (40%) and 40 (46%) markers were specific for the blb and H-8105 genome, respectively; this confirms the differences between the nuclear genomes of the two species. Seventeen markers (20%) present in one or the other parent were absent in the hybrids, and only one new marker was found in the hybrids. The poorly growing, aneuploid and chimeric hybrids had the same band profiles as the well growing, morphologically normal hybrids, except for two bands that were present only in normal hybrids. The presence of eleven blb linkage groups in the blb (+) tbr H- 8105 hybrids was confirmed. The markers specific for the second linkage group (chromosome 2) of blb were not present in the RAPD patterns of the somatic hybrids analysed, suggesting a loss or rearrangement of this chromosome in the combined nuclear genome of the hybrids.
Address and Contact Information Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawiłskiego 5A, 02-106 Warsaw, Poland
*Corresponding author, e-mail: danabo@ibb.waw.pl
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Volume 10 (2005) pp 163-171
Authors Fedor Konovalov, Eugenia Toshchakova and Sergei Gostimsky
Abstract A set of twelve CAPS markers was mapped for linkage group III of pea (Pisum sativum L.). New primers were designed to use a polymerase chain reaction to amplify fragments of sequenced pea genes containing at least one large intron. Amplification products were tested for polymorphism across three pea lines (Chi115, Flagman and WL1238) using eleven four-base restriction endonucleases. Nine STS markers for linkage group III from the literature were also tested for polymorphism, and five of these were used in this mapping study as anchor points. All polymorphic loci were located by genetic analysis of the F2 population from the cross Chi115 x WL1238, and a map of linkage group III consisting of one morphological and twelve CAPS markers was created. The map covers the full length of the chromosome and is about 162 cM long. All the CAPS markers in a set were used to test for polymorphism among 10 additional pea DNA samples extracted from different marker lines and cultivars.
Address and Contact Information Genetics Department, Moscow State University, Vorobjovy Gory, Moscow, 119899, Russia
*Corresponding author, e-mail: konovalov@pisumsativum.org
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Volume 10 (2005) pp 173-183
Authors Dimitrios G. Fatouros1,*, Nikolaos Bouropoulos2, Panayiotis V. Ioannou3 and Sophia G. Antimisiaris1
Abstract In this study, we investigated non-sonicated arsonolipid-containing liposomes (arsonoliposomes) in terms of the influence of lipid composition on their stability, assessed as membrane integrity and physical stability [size]. Vesicles consisting of plain arsonolipids or mixtures of arsonolipids with cholesterol [Chol] or with distearoyl-phospatidylcholine [DSPC] were studied. Membrane integrity was evaluated by measuring the retention of incorporated 5-(6)carboxyfluorescein [CF] during incubation of the vesicles in Tris buffer, pH 7.4. Photon correlation spectroscopy was used to investigate the time-dependent aggregation of arsonoliposomes in the absence and presence of Ca2+ ions. Vesicles composed of plain C18 (acyl fatty chain) arsonolipids were found to be unstable, with only 15% of the initially incorporated CF remaining in the vesicles after 24 hours. The addition of Chol to the membrane (1:1 mol/mol) significantly increased the stability of arsonoliposomes, while the addition of DSPC to the lipid bilayer (1:1 mol/mol) increased vesicle stability to a lower extent. The results of particle size analysis showed that non-sonicated arsonoliposomes consisting of plain arsonolipid Ars/Stearic are highly and rapidly aggregated, while calcium-induced aggregation is also significant, but slower. Aggregation could not be always explained on the basis of zeta potential changes, indicating that the process is complex.
Address and Contact Information 1Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, 26504, Greece,
2Department of Materials Science, University of Patras, 26504, Greece,
3Department of Chemistry, University of Patras, Patras, 26504, Greece
*Corresponding author; current address: Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, 2100, Copenhagen, Denmark, tel: +45 35306288, fax: +45 35306030, e-mail:df@dfuni.dk
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Volume 10 (2005) pp 185-193
Authors Jixi Li, Chaoneng Ji, Huarui Zheng, Xiangwei Fei, Mei Zheng, Jianfeng Dai, Shaohua Gu, Yi Xie and Yumin Mao*
Abstract Ankyrin repeat, one of the most important protein motifs, plays a wide variety of roles in protein-protein interactions and in the signal pathways. Via large-scale sequencing, a novel 941-bp gene was isolated from an 18-week old human fetal brain cDNA library. It encodes a putative protein of 158 amino acid residues with four conserved ankyrin repeat domains. It displays a high degree of homology with rat low-density lipoprotein receptor-related protein 2- binding protein (Lrp2bp), and was therefore was named hLrp2bp (human Lrp2bp). The hLrp2bp gene was located in chromosome 4q35 and the conserved ankyrin repeat domains were located between amino acid residues 10 and 116. RT-PCR revealed that hLrp2bp was mainly expressed in the human testis, small intestine, colon and blood leukocytes, and in human pancreatic adenocarcinoma cells. A HEK293 cell was transfected with the ORF of hLrp2bp, and analyses showed that the protein was distributed both in the cytoplasm and nucleus.
Address and Contact Information State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China
*Corresponding author; tel: +86-21-65643573, fax: +86-21-65642502, e-mail: ymmao@fudan.edu.cn
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