Cba08129_prn 191.197

Comparative Biochemistry and Physiology, Part A 146 (2007) S191 – S197 Society for Experimental Biology Annual Main Meeting 31st March – 4th April 2007, Glasgow, Scotland noislands increased HOB spreading relative to planar controls The influence of nanoscale structures on osteoblast and induced mature adhesion formation.
M. Biggs, M. Dalby, C. Wilkinson, N. Gadegaard,(University of Glasgow); G. Richards, (AO ResearchInstitute, Switzerland) Topographical modification may be exploited to enhanced Interactions between cancer cell glycans and endothelial cytocompatibility at the cell–substrate interface. Regulation of cells during adhesion and transmigration events in cell adhesion and osteosynthesis in orthopaedic implant design can lead to improved osseoinduction and device function.
Cellular interactions with a biomaterial are mediated through H. Lomax-Browne, S. Brooks, C. Kinch, (Oxford Brookes Univer- transmembrane integrin receptors coupled to the extracellular sity); C. Kieda, (Centre National de la Rescherche Scientifique) matrix. These adhesion complexes contain structural and secon-dary signalling molecules crucial to cell adhesion and function.
Aberrant glycosylation is an established characteristic of cancer Here, nanoscale topographies have been in embossed into cells, and appears to have a functional role in metastasis – the polymethylmethacrylate so their effect on cell behaviour can be process by which cancer cells spread around the body. The lectin evaluated. Nanopits fabricated by electron-beam lithography from Helix pomatia, the Roman snail (Helix pomatia agglutinin, were 100 nm deep, 120 nm in diameter and 300 nm in pitch.
HPA) recognises cancer-associated aberrant glycans terminating Grooved substrates produced by photolithography were 327 nm in the monosaccharide α-N-acetylgalactosamine (GalNAc). The deep and 10, 25 and 100 μm wide. Irregular nanoislands and presence of these glycans, detected by HPA binding, is nanolacuni approximately 30 nm high/deep were fabricated by associated with metastasis and consequent poor survival. We are investigating the putative functional role of these glycans in This study was concerned with the effects these nanotopogra- cancer cell adhesion to, and migration through, the endothelium phies have on adhesion formation in S-phase primary human (lining blood vessels) during metastasis. Eight breast cell lines, osteoblasts (HOBs), as cells within S-phase have been shown to characterised for synthesis of GalNAc-glycans of interest, have exhibit increased cell spreading and more homogenous dis- been employed in investigations; HMT 3522, is derived from tribution of adhesion compared to cells within other phases of normal breast, and stably synthesises negligible levels of the cycle (as a whole). S-phase synchronisation was induced by GalNAc-glycans, and the other cell lines; BT474, MDA MB serum starvation and bromodeoxyuridine used to identify S- 435, MDA MB 468, ZR751, MCF7, T47D and DU4475, are derived from breast cancer and stably synthesise increasingly Nanopits were seen to prevented adhesion formation and greater amounts of GalNAc-glycans, consistent with their in- disrupted cytoskeletal organisation and cellular adhesion rela- tive to planar controls. Grooved substrates influenced adhesion A rocking adhesion assay was used to investigate the adhesion of orientation and induced contact guidance, which was correlated the breast cell lines to an endothelial cell monolayer derived with a reduction in adhesion formation. Polymer demixed na- from human brain microvasculature, a site physiologically Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 relevant to breast cancer metastasis. The effect of masking the polycaprolactone (bioresorbable) have been used as a bioactive GalNAc-glycans with HPA was investigated. The results from scaffold material and the cells/scaffolds have been cultured in a these assays provide novel evidence that GalNAc-glycans have a flow bioreactor. The osteoblast progenitor cells were seeded functional role in cancer cell adhesion to endothelial cells. It was onto microgrooved and flat PCL sheets and then cultured at 37 ° also found that the HPA binding profiles of the breast cell lines C in static environment for 1 week before moving into the flow appear to correlate with their abilities to adhere to endothelial chambers. The flow system was maintained for 4 weeks at 37 ° C and protein extraction was performed before analysis withdifferential in gel electrophoresis (DIGE). Proteins of interest were picked from the resulting gel and analyzed by massspectroscopy (MS). Identification of proteins that are modulatedwhen cells are grown on grooved materials will highlight path- ways that are involved in the cellular response to a patterned An investigation into the biological properties of spore culture surface. A number of cytoskeletal protein changes have surface component(s) from the opportunistic pathogen been noted which suggests that the cells are responding to mechanotransductive stimuli from the topographies. Thisinformation will contribute to understanding the factors that P. Little, K. Guy, L. Proudfoot, (Napier University) are important in scaffold design and culture conditions.
Aspergillus fumigatus is a saprophytic fungus responsible for 90% of aspergillosis in humans. This study focused on the effectof diffusible spore surface component(s) from Aspergillusfumigatus (termed AfD) on the function of human neutrophils.
Neutrophils are an important first line of defence and have a Observation of durotaxis on a well-defined continuous short life-span that involves an orchestrated process of migra- tion, phagocytosis and killing. Human neutrophils were treatedwith Af D and migration was assessed by the ability of P. Kuntanawat, C. Wilkinson, M. Riehle, (University of Glasgow) neutrophils to change shape (polarise) and transverse a poly-carbonate filter. Phagocytosis was measured by the ability of Subsequently it has been shown that durotaxis can also be neutrophils to ingest FITC-labeled E. coli and the respiratory observed on gradient-compliant substrata (Lo et al., 2000). The burst was measured by the release of superoxide anion. Further fabrication of such gradient gels is in principle based on a investigations looked at Af D-induced programmed cell death variation of: either the amount of photocatalytic initiators of the (PCD). Results showed Af D to reduce neutrophil polarisation polymerisation reaction created by varying the local illumination (76 ± 6%), migration (57 ± 29%), phagocytosis (31 ± 6%) and intensity (Wong et al., 2003), or by varying the precursor con- respiratory burst (95 ± 2%). Af D also induced PCD through a centration using a microfluidic device (Zaari et al., 2004). Here, heat-sensitive component(s); therefore, it is likely that the anti- we demonstrate a novel method to create such gradients to be inflammatory properties are explained by the Af D-induced used in cellular durotaxis studies. We create these gradients by PCD. Furthermore, Af D has heat-sensitive and heat-stable ac- exploiting the principle that a variation in height of a thin tive compounds suggesting multi-component activity.
polyacrylamide gel results in a variation of the apparent surfacestiffness. The thicker the gel, the less influence has the mecha- nical property of the (hard) substrate, thus the softer the surface.
To fabricate structures with a reliably similar slope, and thus asimilar change in surface properties we developed four different approaches. To measure the changes in mechanical properties Differential proteomics evaluation of osteoprogenitor we used AFM indentation, local indentation, as well as growth on microgrooved surfaces using bioreactor culture measurements of the bulk properties of the gels. The gels werecoated with fibronectin, and the cellular response of human F. Kantawong, M. Dalby, R. Burchmore, (University of fibroblasts to such gradients tested. The cell path were recorded, and mathematically analysed. To study gravitational effects dueto the slope we tilted the microscope. We observed cells migra- Bone tissue engineering is considered to hold potential for the production of bone fragments for use in repair of bone defects.
Here, human osteoprogenitor cells isolated from femoral heads 1. Lo et al., 2000. Biophys. J. 144.
have been used to represent the osteogenic cell types found 2. Wong et al., 2003. Langmuir. 1908.
within the bone marrow. In tissue engineering, the use of 3. Zaari et al., 2004. Adv. Mat. 2133.
bioresorbable scaffolds is attractive as over time the scaffoldwill be resorbed and bone tissue will form and replace the degraded material. In this study, microgrooved embossed in Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 nuclear localization signal and retention in the INE is Characterization and expression of cee, a highly conserved achieved by LBR binding mainly to chromatin and lamins.
In this study the targeting and retention of LBR–GFP in theplant NE has been investigated by introducing mutations in D. Macqueen, J. Fernandes, I. Johnston, (University of St key domains of LBR and employing fluorescence recovery after photobleaching experiments. Mutation of the chromatinbinding domain caused LBR to accumulate in nuclear Cee (conserved edge expressed protein) is conserved in a inclusions in which it was immobile. Deletion of the lamin striking range of eukaryotes, including yeast, insects, fish binding domain resulted in the construct being localized not and mammals. We have cloned cee orthologues in three only to the NE but also ER and to be significantly more teleost species and investigated its developmental expression mobile then the wild type LBR–GFP in the NE. In the case of pattern in Atlantic salmon embryos using in situ hybridisa- both the lamin binding deletion and wild type LBR–GFP, tion. Cee mRNA was not detected until approximately 25 mobility was found to be much greater than previously (out of ∼65) somites were formed. The most prominent staining during the segmentation period was a triple stripemaintained along the embryos' cranial–caudal axis, at the lateral and midline borders of the brain and neural tube. Ceewas also expressed at several structural levels in the eye andon the borders of the branchial arches and otolith nuclei. Inthe developing somites cee was expressed at each border and remarkably, along a line that divided them into rostral and caudal compartments. When segmentation was completeand the eye was darkly pigmented, cee mRNA was detected as a broadly diffuse and unrestricted band at the lateralborders of the whole embryo. At this stage cee was also The term epigenetics refers to variations in phenotype which expressed at the lateral myotome edge in both slow/fast are not represented by variations in genotype. These fibres and skin. We are currently examining the tissue epigenetic regulation mechanisms include changes to chro- distribution of cee in adult salmon by RT–PCR. Real-time matin structure, and alterations are brought about by PCR is also being used to test the hypothesis that cee is modifications to DNA and histone proteins associated with upregulated concomitant with the end of myotube production DNA in chromatin. Nucleosomes, the building blocks of in adult fast muscle as reported in the tiger pufferfish chromatin, consist of approximately 147 bp DNA wrapped around a histone octamer, separated by linker DNA and a linker-associated histone. While no consensus DNA sequence 1. Fernandes et al., 2005. Physiol. Genomics. 22, 327–338.
exists for nucleosome positioning, sequence preferencesinfluence nucleosome position. Repeating dinucleotide sequences with 10 bp periodicity confer curvature andflexibility to a DNA molecule and are often found innucleosomal DNA sequences. As genome architecture differsbetween species, it is likely that nucleosome positioning Dynamics of the lamin B receptor in the plant nuclear While positioning data exist for many species, plant-based data are unrepresented within the data pool. Therefore, theaims of this study are to determine any sequence biases for K. Graumann, D. Evans, S. Irons, J. Runions, (Oxford Brookes nucleosome positioning in Arabidopsis and if these differ from those discovered so far in mammalian and yeastsystems. To achieve this, libraries of tissue-specific nucleo- The nuclear envelope (NE) is a double membrane system somal DNA isolated by micrococcal nuclease digestion of consisting of the inner nuclear envelope (INE), the outer chromatin, are being constructed. Sequences will be analysed nuclear envelope (ONE) and nuclear pore complexes (NPCs).
using existing computer algorithms to identify known Most of our knowledge about the NE proteome comes from positioning motifs. Predicted nucleosome positions will be studies in animal systems. Recent investigations in plant compared to experimentally determined positions to determine systems have shown that plants do not have homologues for a ‘best fit’ model for Arabidopsis. Global occupancy by the majority of animal NE proteins. In a previous study in our nucleosomes is also being investigated using the Arabidopsis laboratory, a construct consisting of the N-terminus of the human lamin B receptor (LBR) fused to GFP was shown totarget the plant INE. In mammalian cells, LBR is an intrinsic INE protein, whose targeting to the INE is facilitated by a Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 the muscle and fibrosis, both of which are detectable by MRI SNARE-mediated regulation of plant K+ channels and thus widely used as a main diagnostic feature of ruptures.
However, these changes are also typical of the normal S. Sokolovski, A. Honsbein, P. Campanoni, R. Pratelli, I.
physiological ageing process. Thus, we hypothesize that such Johansson, M. Paneque, M. Blatt, (Laboratory of Plant symptoms are not exclusively indicative of a rotator cuff Physiology and Biophysics, Institute of Biomedical and Life rupture. To test this, biopsy muscle samples of the M.
Sciences, Bower Building, University of Glasgow, Glasgow, supraspinatus were taken from older patient with a rupture and from control patients in young and older age classes withoutrupture but having undergone other shoulder surgery. The Vesicle traffic underpins cell homeostasis, growth and amounts of fatty, connective and muscle tissue were assessed by development in plants, and is facilitated by a superfamily means of morphometrical and stereological methods using light of proteins known as SNAREs (soluble N-ethylmaleimide- microscopy. Stereology of electron micrographs was employed sensitive fusion protein attachment protein receptors). Several to determine volume densities of muscle fibre mitochondria and recent findings now indicate that SNAREs in plants are also essential for cellular signalling, although details of any Preliminary results indicate that the supraspinatus muscles of mechanisms are lacking. Previously, we identified SNAREs, old control subjects contain similar amounts of fatty and NtSyp121 (= NtSyr1) from tobacco and the homolog connective tissue, and of muscle fibre intracellular lipid, to AtSyp121 from Arabidopsis, as elements required for plant those of patients with a rupture. We conclude that detection of response to abscisic acid and drought stress. We are now fatty degeneration by MRI, as has previously been the accepted exploring SNARE interactions with selected ion channels to practice, is alone insufficient to estimate supraspinatus muscle address the question of their role(s) in signal processing at damage induced by rotator cuff ruptures.
several levels. Among these, analysis of K+ channel traffichas indicated their mobilization from the plasma membrane evoked by abscisic acid. More surprising, however, a dual-tagging strategy has shown that localization and mobility K+channels within the plane of the plasma membrane areprofoundly sensitive in vivo to the SNAREs, and point to the importance of anchoring of channel complexes in ABA Patterns of angiogenic gene expression during embryonic signalling. Combined molecular and biochemical studies have yielded partner proteins that interact with the SNAREs to regulate plasma membrane K+ channels. These data giveus an outline of the nature of the interactions and pave the J. Marschallinger, P. Steinbacher, J. Haslett, A. Sänger, W.
way for characterizing additional proteins important in scaffolding for effective signal transmission and ion channelcontrol.
The present work examines early blood vessel and blood celldevelopment in embryos of the brown trout Salmo trutta. Initial blood vessel formation during vertebrate embryogenesis is wellestablished for terrestrial vertebrates (mammals, birds) and inteleost fish (the zebrafish, Danio rerio, is the most commonmodel species for developmental research in this group of animals). However, in comparison to the zebrafish, the brown Fatty degeneration and fibrosis in M. supraspinatus – Can trout has bigger embryos and a longer developmental time.
this be seen as a diagnostic parameter for rotator cuff Considering these qualities, Salmo trutta is an ideal model organism for researches on embryonic blood vessel formation,as it provides a better overall picture in both time and space axes.
S. Kogler, P. Steinbacher, W. Stoiber, A. Obermayer, (Uni- In the present work, whole mount in situ hybridisation for the versity of Salzburg); M. Tauber, J. Haslett, A. Sänger; mRNAs of regulatory transcription factors (fli-1, GATA-1, GATA-2) and the VEGF receptor protein flk-1 is used toelucidate the patterns of angiogenic gene expression during The rotator cuff of the human shoulder is that group of muscles and their tendons that stabilize this joint. The muscles involved Results demonstrate that patterns of angiogenic and haemato- include the M. subscapularis, M. infraspinatus, M. supraspina- poietic marker gene expression in the brown trout embryo are tus and the M. teres minor. The most frequently ruptured tendon broadly similar to those in the zebrafish and provide the first within the rotator cuff is that of the M. supraspinatus, resulting confirmation of these patterns at the molecular level in a species in significant pain and functional impairment of the joint.
Consequences of rotator cuff ruptures are muscle fibre atrophy These observations will aid the elucidation of a comprehensive and loss of muscle flexibility. There is also fatty degeneration of general scheme of angiogenic stem cell activation and Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 circulatory system formation in the teleost embryo, and should and animals. Members of this protein family share a region of also guide future studies of angiogenic patterning and its mole- high homology in their central parts and are known to be cular regulators in vertebrates generally.
involved in various cellular processes ranging from the regu-lation of ion channels, control of membrane-bound proteases and functions in cellular senescence. Human stomatin has beenshown to directly interact with and modulate acid-sensing ionchannels from neuronal tissue; a related murine stomatin hasrecently been discovered to affect mammalian mechano- sensation. Additionally, various other functions concerning its Slow fibre heterogeneity in brown trout muscle: association with lipid rafts and the cytoskeleton have been Immunostaining and fine structure morphometry are not The Arabidopsis genome encodes for two stomatin-likeproteins (AtSlp-1 and AtSlp-2) that display common char- A. Obermayer, W. Stoiber, P. Steinbacher, J. Haslett, A. Sänger acteristic SPFH domain features. Like other SPFH proteinsfrom Arabidopsis, little is known about their subcellular The present work examines the fine structural development of the localization, tissue distribution and physiological significance muscle fibres in two slow muscle areas of the brown trout, Salmo in plants. The work presented here focuses on these issues by trutta lacustris. Animals from just prior to hatching to the onset employing a range of techniques including promoter activity of free swimming are used. Previous immunohistochemistry studies, biochemical characterisation, work with transgenic work has shown that a specific population of slow muscle fibres mutants and gene expression analysis. Arabidopsis stomatins in salmonid fish (rainbow trout, brown trout) become unreactive are expressed throughout the plant development in various to anti-slow myosin heavy chain (MHC) antibodies in the period organs and seem to be regulated by hormone action.
after hatching, thus indicating a change of MHC isoforms.
Additionally, they might function in developmental control of Here, transmission electron microscopy and stereology methods meristems at the basal rosette and are crucial for normal plant are used to establish whether the known change at the protein level is accompanied by a transition of fibre type at the finestructural level. Thus the area of slow fibres presumed to have undergone isoform change is compared with a control area ofunaffected slow fibres. Using randomised photosampling,volume densities of myofibrils, mitochondria, sarcoplasm,intracellular lipid and sarcotubular system components were determined. Results show that the slow fibres affected by the Involvement of the Arabidopsis plasma membrane SNARE immunological change do not undergo a fibre type change at the fine structural level. Any differences between fibres of thecompared areas reflected in the measured parameters must be A. Honsbein, I. Johansson, S. Sokolovski, P. Campanoni, M.
interpreted as developmental variations of the slow fibre type Paneque, (University of Glasgow); R. Pratelli, (Universiteat and are certainly not an indication of a switch to the intermediate or the fast fibre type. Possible alternativeexplanations for the immunological change are discussed, SNARE (soluble N-ethyl-maleimide sensitive factor attachment including aspects of fibre age, force transduction within the protein receptor) proteins play a central role in the process of intracellular membrane fusion in eukaryotic cells. Severalrecent findings indicate that SNARE functions are not limited to a role in vesicle trafficking alone. Studies on the AtSyp121tobacco homologue (NtSyr1) demonstrated that this SNARE isinvolved in ABA-dependent regulation of the potassium andchloride channel currents in Nicotiana guard cells.
Using a Yeast Mating based split-ubiquitin approach, we Functional and molecular characterisation of stomatin-like observed a direct interaction between the Arabidopsis thaliania plasma membrane SNARE AtSyp121 and a regulatory subunitof Arabidopsis Kv channels. We are currently undertaking co- B. Gehl, M. Blatt, (University of Glasgow, IBLS, Bower immunoprecipitation experiments in SF9 insect cells to provide Building, Laboratory Plant Physiology and Biophysics, supporting biochemical evidence. Electrophysiological mea- surements in oocytes showed that the presence of these twoproteins alters the gating properties of the Arabiodopsis Shaker Stomatins belong to the SPFH (Stomatin Prohibitin Flotillin channels. Patch-clamp data obtained from root epidermal pro- HflK/C) family of highly conserved integral membrane pro- toplasts of Syp121-knockout plants indicated that inward teins, some of which have been studied extensively in humans current was reduced, and the mutants showed altered root Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 growth in potassium-limited conditions. We are currently been proposed that DS inhibits ALDH by forming an inter- investigating whether these proteins interact in vivo and their molecular mixed disulfide, presumably with the active site influence on plant potassium nutrition.
thiols. It has also been shown that reactive sulfur species areproduced during DS and H2S metabolism. Here, we have shown that H2S, at or above 50 μM inhibits yeast ALDH, in an in vitrosystem. There was a dose–response relationship for this effectas increasing H2S concentration further decreased ALDHactivity. The decrease in ALDH activity is enhanced if H2S is incubated with ALDH for 30 min. Cytochrome C, a hemo- Comparative evaluation of amylase refolding through two protein and/or some metal cations increased ALDH inactivation by H2S. H2S is, therefore, a weak inhibitor of ALDH andrequires a catalyst to convert it into a more active species. It is B. Eftekharzadeh, (Institute of Biochemistry and Biophysics) postulated that heme or metal cations catalyse the oxidation ofH2S to reactive sulfur species which can form persulfides with Two different artificial chaperone systems were evaluated in the ALDH cystein at the catalytic site and inhibit its activity.
this work using either detergents or CDs as the stripping ALDH activity was restored by adding reducing agents such as agents. Upon dilution of urea-denatured α-amylase to non dithiothreitol and glutathione to reduce the cysteine persulfide denaturing urea concentration in the presence of the capturing moiety of the enzyme. H2S also increased the cytotoxicity of agent, complexes of the detergent and non-native protein acetaldehyde and/or 2-chloroacetaldehyde in the presence of the molecules are formed and thereby the formation of protein alcohol dehydrogenase inhibitor 4-methylpyrazole towards aggregates is prevented. The so-called captured protein is freshly isolated rat hepatocytes. ALDH inhibition by H2S unable to refold from the detergent-protein complex states indicates that the H2S must have an “antabuse” effect which unless a stripping agent is used to remove the detergent would suggest unpleasant reactions to alcohol in the people molecules. Therefore we used either β-CD and/or detergents to strip the detergent out of the detergent-protein complexes. Ourresults by fluorescence, UV, turbidity measurement, circular dichroism, surface tension and activity assay indicated that theextent of refolding assistance was different due to differentinter- and intra-molecular interactions in these two differentsystems. However, the high activity recovery in the presence of detergents, as the stripping agent, suggests that they can Parallel analysis of deletion mutants in Saccharomyces constitute suitable replacement for the more expensive and common stripping agent of cyclodextrins. Regarding our mainobjective to uncover conditions that favor high refolding F. Khan, C. Hogstrand, (Kings College London); W. Jo, A.
yields, we additionally evaluated the efficiency of different Loguinov, H. Wintz, C. Vulpe, (University of California at artificial chaperone-assisted refolding at low (4 °C) tempera- ture as opposed to the same process at (25 °C). Based on thisnew approach, the artificial chaperone-assisted refolding of As an essential micronutrient, zinc is required by all urea-denatured α-amylase using β-CD as the stripping agent organisms for a variety of vital biological processes, serving raised from 80% at (25 °C) to 94%, while the extent of the as a catalytic component of numerous enzymes and a accompanied aggregation decreased from 25% to 7%.
structural component of the ubiquitous zinc finger motifs Keywords: α-Amylase; Anionic detergents; Artificial chaper- found in many transcription factors. For an organism's one; Cationic detergents; Cyclodextrins; Refolding wellbeing, intracellular zinc concentrations must be tightlyregulated, as both deficiency and excess could lead to severe pathologies. During the last ten years, the baker's yeastSaccharomyces cerevisiae has established itself as a modelorganism for studying eukaryotic cells. The purpose of thisstudy was to conduct a parallel deletion analysis (PDA) of the non-essential genes in S. cerevisiae under zinc deficient conditions to identify potential zinc regulator genes and zincdependant cellular processes. In PDA or fitness profiling, M. Anoush, M. Eghbal, H. Hamzeyi, (Tabriz University of yeast strains containing single deletions for almost every gene Medical Sciences); P.O. Brien, (University of Toronto) in the genome and identified with unique molecular barcodes,are pooled and exposed to an experimental condition of Disulfiram (DS) has been clinically used to treat recovering interest. Microarrays spotted with complementary barcodes alcoholics for over 50 years. It exerts its pharmacologic effect are used to monitor the growth of each individual mutant.
by inhibiting hepatic aldehyde dehydrogenase (ALDH). It has Depending on the function of a deleted gene in the condition Abstracts / Comparative Biochemistry and Physiology, Part A 146 (2007) S191–S197 under study, a mutant strain survives and competes with Comparative analyses revealed that cee is composed of 9 exons others or is depleted from the pool. In this study, pools of and 8 introns and its splice sites are conserved amongst yeast homozygous diploid deletion strains were grown for vertebrates. Cee was present in the same chromosomal segment five and fifteen generations in zinc deficient media.
in teleost fish, tunicates, insects and yeast but synteny was Comparison to strain growth in zinc replete media revealed disrupted between teleosts and higher vertebrates. Phylogenetic a number of treatment-sensitive strains, suggesting that the footprinting did not reveal any evolutionary conserved regions deleted genes in these strains could be involved in zinc in the 10 kb region upstream of the cee transcription start site.
Salmon cee coded for a putative protein of 324 residues thatcontained the DUF410 conserved domain of unknown function.
The overall degree of Cee similarity across different phyla wasremarkable; for example, salmon Cee had approximately 85%identity with mammalian sequences. Phylogenetic analysesusing maximum likelihood and Bayesian posterior probabilitymethods showed that, as expected, salmon Cee clusters with other teleost species between the Otophysi and Acanthomorpha Phylogeny and evolution of cee, an ancient gene of clades. A codon-based Fisher's test of selection using the Nej and Gojobori method revealed that Cee is under purifyingselection to resist amino acid changes, suggesting that it is likely J. Fernandes, D. Macqueen, I. Johnston, (University of St to have a conserved function across several phyla. We are developing a myoblast tissue culture system to performfunctional studies that will elucidate the role of cee in myotube We have recently described a novel gene that is markedly up- regulated concomitant with cessation of myotube production in adult fast muscle of the tiger pufferfish (Fernandes et al., 2005).
1. Fernandes et al., 2005. Physiol. Genomics 22, 327–338.
Here we report the complete coding sequence and partial 5′-untranslated region of the Atlantic salmon orthologue, which we have named cee (GenBank Accession Number EF036472).

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