• QSAR and molecular docking for the search of AOX inhibitors: a rational drug discovery approach

      Rosell-Hidalgo, Alicia; Young, Luke; Moore, Anthony L.; Ghafourian, Taravat; University of Sussex; University of Bedfordshire (Springer Science and Business Media Deutschland GmbH, 2020-12-08)
      The alternative oxidase (AOX) is a monotopic diiron carboxylate protein that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. Although a number of AOX inhibitors have been discovered, little is still known about the ligand–protein interaction and essential chemical characteristics of compounds required for a potent inhibition. Furthermore, owing to the rapidly growing resistance to existing inhibitors, new compounds with improved potency and pharmacokinetic properties are urgently required. In this study we used two computational approaches, ligand–protein docking and Quantitative Structure–Activity Relationships (QSAR) to investigate binding of AOX inhibitors to the enzyme and the molecular characteristics required for inhibition. Docking studies followed by protein–ligand interaction fingerprint (PLIF) analysis using the AOX enzyme and the mutated analogues revealed the importance of the residues Leu 122, Arg 118 and Thr 219 within the hydrophobic cavity. QSAR analysis, using stepwise regression analysis with experimentally obtained IC50 values as the response variable, resulted in a multiple regression model with a good prediction accuracy. The model highlighted the importance of the presence of hydrogen bonding acceptor groups on specific positions of the aromatic ring of ascofuranone derivatives, acidity of the compounds, and a large linker group on the compounds on the inhibitory effect of AOX.
    • A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure

      Zhou, Shaobo; Bailey, Matthew J.; Dunn, Michael J.; Preedy, Victor R.; Emery, Peter W.; (Wiley, 2005-08-01)
      We report the results of a systematic investigation to quantify the losses of protein during a well-established two-dimensional polyacrylamide gel electrophoresis (2-DE) procedure. Radioactively labelled proteins ([(14)C]bovine serum albumin and a homogenate prepared from the liver of a rat that had been injected with [(35)S]methionine) were used, and recovery was quantified by digesting pieces of gel in H(2)O(2) and subjecting the digests to liquid scintillation counting. When samples were loaded onto the first dimension immobilised pH gradient strips by in-gel rehydration, recovery of protein from the strips was 44-80% of the amount of protein loaded, depending on the amount of protein in the sample. Most of the unrecovered protein appeared to have adhered to the reswelling tray. Losses during isoelectric focusing (IEF) were much smaller (7-14%), although approximately 2% of the protein appeared to migrate from sample strips to adjacent blank strips in the focussing apparatus. A further 17-24% of the proteins were lost into the buffers during equilibration prior to running in the second dimension. Losses during the second dimension run and subsequent staining with SYPRO Ruby amounted to less than 10%. The overall loss during 2-DE was reduced by approximately 25% when proteins were loaded onto the IEF strips using sample cups instead of by in-gel rehydration. These extensive and variable losses during the 2-DE procedure mean that spot intensities on 2-DE gels cannot be used to derive reliable, quantitative information on the amounts of proteins present in the original sample.
    • Reconstructing phylogenetic relationships based on repeat sequence similarities

      Vitales, Daniel; Garcia, Sonia; Dodsworth, Steven; Institut Botànic de Barcelona; Universitat de Barcelona; University of Bedfordshire (Elsevier, 2020-02-28)
      A recent phylogenetic method based on genome-wide abundance of different repeat types proved to be useful in reconstructing the evolutionary history of several plant and animal groups. Here, we demonstrate that an alternative information source from the repeatome can also be employed to infer phylogenetic relationships among taxa. Specifically, this novel approach makes use of the repeat sequence similarity matrices obtained from the comparative clustering analyses of RepeatExplorer 2, which are subsequently transformed to between-taxa distance matrices. These pairwise matrices are used to construct neighbour-joining trees for each of the top most-abundant clusters and they are finally summarized in a consensus network. This methodology was tested on three groups of angiosperms and one group of insects, resulting in congruent evolutionary hypotheses compared to more standard systematic analyses based on commonly used DNA markers. We propose that the combined application of these phylogenetic approaches based on repeat abundances and repeat sequence similarities could be helpful to understand mechanisms governing genome and repeatome evolution.
    • Regulation of fatty acid binding proteins by hypoxia inducible factors 1α and 2α in the placenta: Relevance to pre-eclampsia

      Jadoon, Ayesha; Cunningham, Phil; McDermott, Lindsay C. (Elsevier, 2014-09-25)
      Pre-eclampsia is characterized by placental hypoxia and dyslipidemia. Arachidonic and docosahexanoic acids are essential maternal nutrients for fetal development. They are transported via placental trophoblast cells by membrane and cytosolic fatty acid binding proteins. Others report the expressions of these proteins which are increased in hypoxic trophoblasts. Using bioinformatics, BeWo cells, reporter assays, quantitative real-time PCR and immunoblotting we tested the hypothesis that hypoxia inducible factors 1α (HIF-1α) and/ or 2α (HIF-2α) regulate the expressions of FABP1, FABP3, FABP4 and FATP2 proteins. Three hypoxia responsive elements (HRE) were identified in FABP1 which cumulatively responded strongly to HIF-1α and weakly to HIF-2α. FABP3 expression partially responded to HIF-1α. Two putative HRE were validated in FABP4 both of which responded weakly toHIF-1α and HIF-2α. FATP2 protein expression reacted positively to hypoxia. Thus, fetal essential fatty acid supply via the placenta is protected under hypoxia. It will be interesting to determine if our findings are replicated in human pre-eclamptic placenta.
    • Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog

      Lau, Ching-In; Outram, Susan V.; Saldaña, José Ignacio; Furmanski, Anna L.; Dessens, Johannes T.; Crompton, Tessa (American Society of Hematology, 2012-05-17)
      The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.
    • Repeated parallel losses of inflexed stamens in Moraceae: phylogenomics and generic revision of the tribe Moreae and the reinstatement of the tribe Olmedieae (Moraceae)

      Gardner, Elliot M.; Garner, Mira; Cowan, Robyn S.; Dodsworth, Steven; Epitawalage, Niroshini; Arifiani, Deby; Baker, William J.; Forest, Felix; Maurin, Olivier; Zerega, Nyree JC; et al. (Wiley, 2021-07-06)
      We present a densely sampled phylogenomic study of the mulberry tribe (Moreae, Moraceae), an economically important clade with a global distribution, revealing multiple losses of inflexed stamens, a character traditionally used to circumscribe Moreae. Inflexed stamens facilitate ballistic pollen release and are associated with wind pollination, and the results presented here suggest that losses of this character state may have evolved repeatedly in Moraceae. Neither Moreae nor several of its major genera (Morus, Streblus, Trophis) were found to be monophyletic. A revised system for a monophyletic Moreae is presented, including the reinstatement of the genera Ampalis, Maillardia, Taxotrophis, and Paratrophis, and the recognition of the new genus Afromorus. Pseudostreblus is reinstated and transferred to the Parartocarpeae, and Sloetiopsis is reinstated and transferred to the Dorstenieae. The tribe Olmedieae is reinstated, replacing the Castilleae, owing to the reinstatement of the type Olmedia and its exclusion from Moreae. Streblus s.str. is excluded from Moreae and transferred to the Olmedieae, which is characterized primarily by involucrate inflorescences without regard to stamen position. Nine new combinations are made.
    • Repetitive DNA restructuring across multiple Nicotiana allopolyploidisation events shows a lack of strong cytoplasmic bias in influencing repeat turnover

      Dodsworth, Steven; Guignard, Maite S.; Pérez-Escobar, Oscar A.; Struebig, Monika; Chase, Mark W.; Leitch, Andrew R.; ; University of Bedfordshire; Queen Mary University of London; Royal Botanic Gardens, Kew; et al. (MDPI, 2020-02-19)
      Allopolyploidy is acknowledged as an important force in plant evolution. Frequent allopolyploidy in Nicotiana across different timescales permits the evaluation of genome restructuring and repeat dynamics through time. Here we use a clustering approach on high-throughput sequence reads to identify the main classes of repetitive elements following three allotetraploid events, and how these are inherited from the closest extant relatives of the maternal and paternal subgenome donors. In all three cases, there was a lack of clear maternal, cytoplasmic bias in repeat evolution, i.e., lack of a predicted bias towards maternal subgenome-derived repeats, with roughly equal contributions from both parental subgenomes. Different overall repeat dynamics were found across timescales of <0.5 (N. rustica L.), 4 (N. repanda Willd.) and 6 (N. benthamiana Domin) Ma, with nearly additive, genome upsizing, and genome downsizing, respectively. Lower copy repeats were inherited in similar abundance to the parental subgenomes, whereas higher copy repeats contributed the most to genome size change in N. repanda and N. benthamiana. Genome downsizing post-polyploidisation may be a general long-term trend across angiosperms, but at more recent timescales there is species-specific variance as found in Nicotiana.
    • Repression of hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation

      Rowbotham, Nicola J.; Furmanski, Anna L.; Hager-Theodorides, Ariadne L.; Ross, Susan; Drakopoulou, Ekati; Koufaris, Costas; Outram, Susan V.; Crompton, Tessa (Taylor & Frances, 2008-04-16)
      Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2DeltaC2), increased T-cell differentiation and activationin response to TCR signalling. Expression of the Gli2DeltaC2 transgene increased differentiation from CD4(+)CD8(+) to single positive thymocyte, and increased peripheral T cell populations. Gli2DeltaC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.
    • Reservoirs of faecal indicator bacteria in well-head hand pumps in Bangladesh

      Osborne, Thomas H.; Ward, Seamus A.; Ahmed, Kazi M.; Santini, Joanne M. (IWA publishing, 2018-04-20)
      The majority of the population of Bangladesh (90%) rely on untreated groundwater for drinking and domestic use. At the point of collection, 40% of these supplies are contaminated with faecal indicator bacteria (FIB). Recent studies have disproved the theory that latrines discharging to shallow aquifers are the major contributor to this contamination. In this study, we tested the hypothesis that hand pumps are a reservoir of FIB. We sampled the handle, spout, piston and seal from 19 wells in Araihazar Upazila, Bangladesh and identified that the spout and seal were reservoirs of FIB. These findings led to our recommendation that well spouts be regularly cleaned, including the removal of precipitated deposits, and that the seals be regularly changed. It is envisaged that one or both of these interventions will reduce the numbers of FIB in drinking water, thereby reducing the burden of diarrhoeal disease in Bangladesh.
    • Resolving species boundaries in a recent radiation with the Angiosperms353 probe set: the Lomatium packardiae/L. anomalum clade of the L. triternatum (Apiaceae) complex

      Ottenlips, Michael V.; Mansfield, Donald H.; Buerki, Sven; Feist, Mary Ann E.; Downie, Stephen R.; Dodsworth, Steven; Forest, Felix; Plunkett, Gregory M.; Smith, James F.; Boise State University; et al. (Wiley, 2021-06-08)
      Speciation not associated with morphological shifts is challenging to detect unless molecular data are employed. Using Sanger-sequencing approaches, the Lomatium packardiae/L. anomalum subcomplex within the larger Lomatium triternatum complex could not be resolved. Therefore, we attempt to resolve these boundaries here. The Angiosperms353 probe set was employed to resolve the ambiguity within Lomatium triternatum species complex using 48 accessions assigned to L. packardiae, L. anomalum, or L. triternatum. In addition to exon data, 54 nuclear introns were extracted and were complete for all samples. Three approaches were used to estimate evolutionary relationships and define species boundaries: STACEY, a Bayesian coalescent-based species tree analysis that takes incomplete lineage sorting into account; ASTRAL-III, another coalescent-based species tree analysis; and a concatenated approach using MrBayes. Climatic factors, morphological characters, and soil variables were measured and analyzed to provide additional support for recovered groups. The STACEY analysis recovered three major clades and seven subclades, all of which are geographically structured, and some correspond to previously named taxa. No other analysis had full agreement between recovered clades and other parameters. Climatic niche and leaflet width and length provide some predictive ability for the major clades. The results suggest that these groups are in the process of incipient speciation and incomplete lineage sorting has been a major barrier to resolving boundaries within this lineage previously. These results are hypothesized through sequencing of multiple loci and analyzing data using coalescent-based processes.
    • Risk management analysis for novel Coronavirus in Wuhan, China

      Yue, Xiao-Guang; Shao, Xue-Feng; Li, Rita Yi Man; Crabbe, M. James C.; Mi, Lili; Hu, Siyan; Baker, Julien S.; Liang, Gang; European University Cyprus; Polytechnic Institute of Porto; et al. (MDPI, 2020-02-03)
      Recently, a novel coronavirus pneumonia (2019–nCoV) outbreak occurred in Wuhan, China, rapidly spreading first to the whole country, and then globally, causing widespread concern. From the perspectives of early warning and identification of risk, risk monitoring, and analysis, as well as risk management and handling, we propose corresponding solutions and recommendations, which include institutional cooperation, and to inform national and international policy-makers.
    • Risk prediction and assessment: duration, infections, and death toll of the COVID-19 and its impact on China’s economy

      Yue, Xiao-Guang; Shao, Xue-Feng; Li, Rita Yi Man; Crabbe, M. James C.; Mi, Lili; Hu, Siyan; Baker, Julien S.; Liu, Liting; Dong, Kechen; University of Bedfordshire; et al. (MDPI, 2020-04-03)
      This study first analyzes the national and global infection status of the Coronavirus Disease that emerged in 2019 (COVID-19). It then uses the trend comparison method to predict the inflection point and Key Point of the COVID-19 virus by comparison with the severe acute respiratory syndrome (SARS) graphs, followed by using the Autoregressive Integrated Moving Average model, Autoregressive Moving Average model, Seasonal Autoregressive Integrated Moving-Average with Exogenous Regressors, and Holt Winter’s Exponential Smoothing to predict infections, deaths, and GDP in China. Finally, it discusses and assesses the impact of these results. This study argues that even if the risks and impacts of the epidemic are significant, China’s economy will continue to maintain steady development.
    • A roadmap for global synthesis of the plant tree of life

      Eiserhardt, Wolf L.; Antonelli, Alexandre; Bennett, Dominic J.; Botigue, Laura R.; Burleigh, J. Gordon; Dodsworth, Steven; Enquist, Brian J.; Forest, Felix; Kim, Jan T.; Kozlov, Alexey M.; et al. (Wiley, 2018-03-31)
      Providing science and society with an integrated, up-to-date, high quality, open, reproducible and sustainable plant tree of life would be a huge service that is now coming within reach. However, synthesizing the growing body of DNA sequence data in the public domain and disseminating the trees to a diverse audience are often not straightforward due to numerous informatics barriers. While big synthetic plant phylogenies are being built, they remain static and become quickly outdated as new data are published and tree-building methods improve. Moreover, the body of existing phylogenetic evidence is hard to navigate and access for non-experts. We propose that our community of botanists, tree builders, and informaticians should converge on a modular framework for data integration and phylogenetic analysis, allowing easy collaboration, updating, data sourcing and flexible analyses. With support from major institutions, this pipeline should be re-run at regular intervals, storing trees and their metadata long-term. Providing the trees to a diverse global audience through user-friendly front ends and application development interfaces should also be a priority. Interactive interfaces could be used to solicit user feedback and thus improve data quality and to coordinate the generation of new data. We conclude by outlining a number of steps that we suggest the scientific community should take to achieve global phylogenetic synthesis.
    • Role of endogenous annexin-A1 in the regulation of thymocyte positive and negative selection

      Paschalidis, Nikolaos; Huggins, A.; Rowbotham, Nicola J.; Furmanski, Anna L.; Crompton, Tessa; Flower, R.J.; Perretti, M.; D'Acquisto, Fulvio; Queen Mary University of London (Taylor & Francis, 2010-02-15)
      We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1(-/-) thymocyte populations at the immature CD4(-)CD8(-) double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4(+) and CD8(+) single positive (SP) populations between Anx1(-/-) and AnxA1(+/+) mice. However, when we crossed AnxA1(-/-) mice onto HY-TCR transgenic mice, we observed an increase in CD4(+)CD8(+) double positive (DP) and CD4 SP cells in male AnxA1(-/-)/HY-TCR compared to AnxA1(+/+)/HY-TCR. Conversely, female AnxA1(-/-)/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1(+/+)/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NFkappaB activation in AnxA1(-/-) thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.
    • Role of Hedgehog signalling at the transition from double-positive to single-positive thymocyte

      Furmanski, Anna L.; Saldana, Jose Ignacio; Rowbotham, Nicola J.; Ross, Susan; Crompton, Tessa (Wiley, 2011-11-21)
      In the thymus, developing T cells receive signals that determine lineage choice, specificity, MHC restriction and tolerance to self‐antigen. One way in which thymocytes receive instruction is by secretion of Sonic hedgehog (Shh) from thymic epithelial cells. We have previously shown that Hedgehog (Hh) signalling in the thymus decreases the CD4:CD8 single‐positive (SP) thymocyte ratio. Here, we present data indicating that double‐positive (DP) thymocytes are Hh‐responsive and that thymocyte‐intrinsic Hh signalling plays a role in modulating the production of CD4+ (SP4), CD8+ (SP8) and unconventional T‐cell subsets. Repression of physiological Hh signalling in thymocytes altered the proportions of DP and SP4 cells. Thymocyte‐intrinsic Hh‐dependent transcription also attenuated both the production of mature SP4 and SP8 cells, and the establishment of peripheral T‐cell compartments in TCR‐transgenic mice. Additionally, stimulation or withdrawal of Hh signals in the WT foetal thymus impaired or enhanced upregulation of the CD4 lineage‐specific transcription factor Gata3 respectively. These data together suggest that Hh signalling may play a role in influencing the later stages of thymocyte development.
    • The role of histone protein modifications and mutations in histone modifiers in pediatric b-cell progenitor acute lymphoblastic leukemia

      Janczar, Szymon; Janczar, Karolina; Pastorczak, Agata; Harb, Hani; Paige, Adam J.W.; Zalewska-Szewczyk, Beata; Danilewicz, Marian; Mlynarski, Wojciech; Medical University of Lodz; Philipps University Marburg; et al. (MDPI, 2017-01-03)
      While cancer has been long recognized as a disease of the genome, the importance of epigenetic mechanisms in neoplasia was acknowledged more recently. The most active epigenetic marks are DNA methylation and histone protein modifications and they are involved in basic biological phenomena in every cell. Their role in tumorigenesis is stressed by recent unbiased large-scale studies providing evidence that several epigenetic modifiers are recurrently mutated or frequently dysregulated in multiple cancers. The interest in epigenetic marks is especially due to the fact that they are potentially reversible and thus druggable. In B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) there is a relative paucity of reports on the role of histone protein modifications (acetylation, methylation, phosphorylation) as compared to acute myeloid leukemia, T-cell ALL, or other hematologic cancers, and in this setting chromatin modifications are relatively less well studied and reviewed than DNA methylation. In this paper, we discuss the biomarker associations and evidence for a driver role of dysregulated global and loci-specific histone marks, as well as mutations in epigenetic modifiers in BCP-ALL. Examples of chromatin modifiers recurrently mutated/disrupted in BCP-ALL and associated with disease outcomes include MLL1, CREBBP, NSD2, and SETD2. Altered histone marks and histone modifiers and readers may play a particular role in disease chemoresistance and relapse. We also suggest that epigenetic regulation of B-cell differentiation may have parallel roles in leukemogenesis.
    • The role of zinc in renal pathological changes in diabetic status

      Fernando, Julie; Zhou, Shaobo; University of Bedfordshire (OMICS International, 2015-06-13)
      Diabetes mellitus (DM) was the 8th leading cause of death in 2011, resulting in 1.4 million deaths worldwide. One of the complications of DM is chronic kidney disease, which accounts for nearly 44% of all new cases of kidney failure in the US in 2011. Zinc (Zn), an essential trace element, plays an important role in regulating carbohydrate metabolism. Several studies have shown the beneficial effects of Zn on renal pathological changes in Type 1 and Type 2 diabetic subjects, by reducing levels of oxidative stress, glomerular damage, and urinary albumin excretion after Zn supplementation. In contrast, other studies have shown little effects of Zn supplementation on renal damage. This paper reviewed recent research developments and found promising results of Zn in reducing, and in some cases, completely preventing renal damage. Nevertheless, the use of Zn as a potential treatment and especially its long-term impact against renal pathological symptoms in DM patients needs to be further studied.
    • Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization

      Lee, Yung-I; Yap, Jing Wei; Izan, Shairul; Leitch, Ilia J.; Fay, Michael F.; Lee, Yi-Ching; Hidalgo, Oriane; Dodsworth, Steven; Smulders, Marinus J.M.; Gravendeel, Barbara; et al. (BioMed Central Ltd., 2017-11-24)
      Background: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA. Results: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26–28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific. Conclusions: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.
    • Separation and purification of the bovine milk fat globule membrane protein and its effect on improvement of C2C12 mouse skeletal muscle cell proliferation

      Li, He; Xu, Weili; Ma, Ying; Zhou, Shaobo; Harbin Institute of Technology; University of Bedfordshire (Royal Society of Chemistry, 2017-06-07)
      A novel method to improve the proliferation activity of C2C12 cells by the bovine milk fat globule membrane (MFGM) protein was established in this study. The MFGM protein was extracted and isolated into 4 fractions using an electric cream separator, and purified by a cellulose DEAE-52 column. Fraction 2 accounted for 57.8% of the total MFGM protein, and was used in the following study. The MTT assay showed that it induced cell proliferation activity, increased the cell survival rate and the cell number using flow cytometry and fluorescence microscopy analysis. There were only subtle changes in the morphology as observed using confocal scanning laser microscopy, but the number of mitochondria was significantly increased as observed using transmission electron microscopy analysis. Furthermore, the mRNA expression of MyoD, cyclin D1, p70S6K and mTOR was up-regulated as determined utilizing the quantitative real-time PCR assay, and the activation of Akt and mTOR phosphorylation was up regulated as determined using the Western blot assay. The main protein in fraction 2, assayed by 1-D gel electrophoresis and MALDI TOF-TOF, was identified as milk fat globule-EGF factor 8, the content was 65.6% of the total protein in fraction 2. The results elucidate a new molecular mechanism of the MFGM protein fraction 2: the activation of the Akt signal pathway in promoting cell proliferation.
    • Sequence-divergent units of the ABA-1 polyprotein array of the nematode Ascaris suum have similar fatty-acid- and retinol-binding properties but different binding-site environments

      Moore, Joyce; McDermott, Lindsay C.; Price, Nicholas C.; Kelly, Sharon M.; Cooper, Alan; Kennedy, Malcolm W. (Portland Press, 1999-05-15)
      Polyproteins comprise long polypeptides that are post-translationally cleaved into proteins of different function, or tandemly repetitive polypeptides which are processed into multiple versions of proteins which are presumed to have the same function. In the latter case the individual units of the polyprotein can differ substantially in sequence. Identity of function between the different units therefore cannot be assumed. Here we have examined the ABA-1 polyprotein allergen of the parasitic nematode Ascaris suum and found it to contain units which show a 50% difference in amino acid sequence. The parasite therefore produces at least two radically different forms of the allergen encoded within the polyprotein array. In fluorescence-based ligand-binding assays, recombinant polypeptides representing the two forms (designated ABA-1A1 and ABA-1B1) showed similar binding affinities for a range of fluorescent active-site probes [retinol, dansylundecanoic acid, dansyl-DL-alpha-amino-octanoic acid, cis-parinaric acid (cPnA)] and for the non-specific hydrophobic surface probe 8-anilinonaphthalene-1-sulphonic acid. However, the molecular environments in the active sites are markedly different, as indicated by disparate fluorescence emission peaks and intensities of bound probes. CD showed that the proteins have similar secondary structures but differ in susceptibility to chemical denaturation/unfolding by guanidinium chloride. Both retain a single conserved tryptophan residue in a characteristic non-polar environment, as revealed by extreme fluorescence blue shift. Thus the gross differences in sequence of the two proteins are not reflected in their ligand-binding specificities but in their binding-site environments.