The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

PubMed

Meßling, Susanne; Matthias, Jan; Xiong, Qiuhong; Fischer, Sarah; Eichinger, Ludwig

2017-06-01

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Enantioselective cellular localisation of europium(iii) coordination complexes† †Electronic supplementary information (ESI) available: Complex synthesis and characterisation, selected cell images showing correspondence with commercial lysosomal or mitochondrial stains and spectral imaging profiles. See DOI: 10.1039/c7sc04422d

PubMed Central

Frawley, Andrew T.; Linford, Holly V.; Starck, Matthieu; Pal, Robert

2017-01-01

The selective mitochondrial localisation of the Λ enantiomer of three different emissive europium(iii) complexes in NIH 3T3 and MCF7 cells contrasts with the behaviour of the Δ enantiomer, for which a predominant lysosomal localisation was observed by confocal microscopy. In each case, cell uptake occurs via macropinocytosis. PMID:29675151

Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

PubMed

Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

2017-09-01

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Erenpreisa, Jekaterina; Cragg, Mark S.; Salmina, Kristine

Escape from mitotic catastrophe and generation of endopolyploid tumour cells (ETCs) represents a potential survival strategy of tumour cells in response to genotoxic treatments. ETCs that resume the mitotic cell cycle have reduced ploidy and are often resistant to these treatments. In search for a mechanism for genome reduction, we previously observed that ETCs express meiotic proteins among which REC8 (a meiotic cohesin component) is of particular interest, since it favours reductional cell division in meiosis. In the present investigation, we induced endopolyploidy in p53-dysfunctional human tumour cell lines (Namalwa, WI-L2-NS, HeLa) by gamma irradiation, and analysed the sub-cellular localisationmore » of REC8 in the resulting ETCs. We observed by RT-PCR and Western blot that REC8 is constitutively expressed in these tumour cells, along with SGOL1 and SGOL2, and that REC8 becomes modified after irradiation. REC8 localised to paired sister centromeres in ETCs, the former co-segregating to opposite poles. Furthermore, REC8 localised to the centrosome of interphase ETCs and to the astral poles in anaphase cells where it colocalised with the microtubule-associated protein NuMA. Altogether, our observations indicate that radiation-induced ETCs express features of meiotic cell divisions and that these may facilitate chromosome segregation and genome reduction.« less

Capturing novel mouse genes encoding chromosomal and other nuclear proteins.

PubMed

Tate, P; Lee, M; Tweedie, S; Skarnes, W C; Bickmore, W A

1998-09-01

The burgeoning wealth of gene sequences contrasts with our ignorance of gene function. One route to assigning function is by determining the sub-cellular location of proteins. We describe the identification of mouse genes encoding proteins that are confined to nuclear compartments by splicing endogeneous gene sequences to a promoterless betageo reporter, using a gene trap approach. Mouse ES (embryonic stem) cell lines were identified that express betageo fusions located within sub-nuclear compartments, including chromosomes, the nucleolus and foci containing splicing factors. The sequences of 11 trapped genes were ascertained, and characterisation of endogenous protein distribution in two cases confirmed the validity of the approach. Three novel proteins concentrated within distinct chromosomal domains were identified, one of which appears to be a serine/threonine kinase. The sequence of a gene whose product co-localises with splicesome components suggests that this protein may be an E3 ubiquitin-protein ligase. The majority of the other genes isolated represent novel genes. This approach is shown to be a powerful tool for identifying genes encoding novel proteins with specific sub-nuclear localisations and exposes our ignorance of the protein composition of the nucleus. Motifs in two of the isolated genes suggest new links between cellular regulatory mechanisms (ubiquitination and phosphorylation) and mRNA splicing and chromosome structure/function.

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity

PubMed Central

Heraud-Farlow, Jacki E.; Kiebler, Michael A.

2014-01-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. PMID:25012293

Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy.

PubMed

Carlsson, Emil; Thwaite, Joanne E; Jenner, Dominic C; Spear, Abigail M; Flick-Smith, Helen; Atkins, Helen S; Byrne, Bernadette; Ding, Jeak Ling

2016-01-01

Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence.

Tissue-specific promoter utilisation of the kallikrein-related peptidase genes, KLK5 and KLK7, and cellular localisation of the encoded proteins suggest roles in exocrine pancreatic function.

PubMed

Dong, Ying; Matigian, Nick; Harvey, Tracey J; Samaratunga, Hemamali; Hooper, John D; Clements, Judith A

2008-02-01

Abstract Tissue kallikrein (kallikrein 1) was first identified in pancreas and is the namesake of the kallikrein-related peptidase (KLK) family. KLK1 and the other 14 members of the human KLK family are encoded by 15 serine protease genes clustered at chromosome 19q13.4. Our Northern blot analysis of 19 normal human tissues for expression of KLK4 to KLK15 identified pancreas as a common expression site for the gene cluster spanning KLK5 to KLK13, as well as for KLK15 which is located adjacent to KLK1. Consistent with previous reports detailing the ability of KLK genes to generate organ- and disease-specific transcripts, detailed molecular and in silico analyses indicated that KLK5 and KLK7 generate transcripts in pancreas variant from those in skin or ovary. Consistently, we identified in the promoters of these KLK genes motifs which conform with consensus binding sites for transcription factors conferring pancreatic expression. In addition, immunohistochemical analysis revealed predominant localisation of KLK5 and KLK7 in acinar cells of the exocrine pancreas, suggesting roles for these enzymes in digestion. Our data also support expression patterns derived from gene duplication events in the human KLK cluster. These findings suggest that, in addition to KLK1, other related KLK enzymes will function in the exocrine pancreas.

Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

PubMed

Chen, Mao Xiang; Gorman, Shelby A; Benson, Bill; Singh, Kuljit; Hieble, J Paul; Michel, Martin C; Tate, Simon N; Trezise, Derek J

2004-06-01

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

PubMed

Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

2016-04-14

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback

PubMed Central

Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

2016-01-01

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress. PMID:27077813

The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity.

PubMed

Heraud-Farlow, Jacki E; Kiebler, Michael A

2014-09-01

Staufen (Stau) proteins belong to a family of RNA-binding proteins (RBPs) that are important for RNA localisation in many organisms. In this review we discuss recent findings on the conserved role played by Stau during both the early differentiation of neurons and in the synaptic plasticity of mature neurons. Recent molecular data suggest mechanisms for how Stau2 regulates mRNA localisation, mRNA stability, translation, and ribonucleoprotein (RNP) assembly. We offer a perspective on how this multifunctional RBP has been adopted to regulate mRNA localisation under several different cellular and developmental conditions. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

You, Jae-Hwan; Reed, Mark L.; Hiscox, Julian A.

The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in themore » C-terminal region of the protein.« less

Fibroblast growth factor 20 is protective towards dopaminergic neurons in vivo in a paracrine manner.

PubMed

Boshoff, Eugene L; Fletcher, Edward J R; Duty, Susan

2018-04-23

Neuroprotective strategies are an unmet medical need for Parkinson's disease. Fibroblast growth factor 20 (FGF20) enhances survival of cultured dopaminergic neurons but little is known about its in vivo potential. We set out to examine whether manipulation of the FGF20 system affected nigrostriatal tract integrity in rats, to identify which fibroblast growth factor receptors (FGFRs) might reside on dopaminergic neurons and to discover the source of endogenous FGF20 in the substantia nigra (SN). Male Sprague Dawley rats were subject to a partial 6-OHDA lesion alongside treatment with exogenous FGF20 or an FGFR antagonist. Behavioural readouts and tyrosine-hydroxylase (TH) immunohistochemistry were used to evaluate nigrostriatal tract integrity. Fluorescent immunohistochemistry was used to examine FGFR subtype expression on TH-positive dopamine neurons and FGF20 cellular localisation within the SN. FGF20 (2.5 μg/day) significantly protected TH-positive cells in the SN and terminals in the striatum, while reducing the development of motor asymmetry at 5, 8 and 11 days post lesion. Conversely, the FGFR antagonist PD173074 (2 mg/kg) significantly worsened both the 6-OHDA lesion and resultant motor asymmetry. Within the SN, TH-positive cells expressed FGFR1, 3 and 4 while FGF20 co-localised with GFAP-positive astrocytes. In conclusion, FGF20 protects dopaminergic neurons in vivo, an action likely mediated through activation of FGFRs1, 3 or 4 found on these neurons. Given FGF20 is localised to astrocytes in the adult SN, endogenous FGF20 provides its protection of dopamine neurons through a paracrine action. Boosting the endogenous FGF20 production might offer potential as a future therapeutic strategy in Parkinson's disease. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

PubMed

Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

2010-07-16

Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system.

PubMed

Winterhoff, Boris J N; Arlt, Alexander; Duttmann, Angelika; Ungefroren, Hendrik; Schäfer, Heiner; Kalthoff, Holger; Kruse, Marie-Luise

2012-03-01

The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB.

PubMed

Guadagno, Noemi A; Moriconi, Claudia; Licursi, Valerio; D'Acunto, Emanuela; Nisi, Paola S; Carucci, Nicoletta; De Jaco, Antonella; Cacci, Emanuele; Negri, Rodolfo; Lupo, Giuseppe; Miranda, Elena

2017-07-01

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif.

PubMed

Jespersen, Thomas; Rasmussen, Hanne B; Grunnet, Morten; Jensen, Henrik S; Angelo, Kamilla; Dupuis, Delphine S; Vogel, Lotte K; Jorgensen, Nanna K; Klaerke, Dan A; Olesen, Søren-Peter

2004-09-01

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE beta-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38-40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel.

Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos

PubMed Central

Lye, Claire M.; Naylor, Huw W.; Sanson, Bénédicte

2014-01-01

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis. PMID:25294944

Generation of shape complexity through tissue conflict resolution

PubMed Central

Rebocho, Alexandra B; Southam, Paul; Kennaway, J Richard; Coen, Enrico

2017-01-01

Out-of-plane tissue deformations are key morphogenetic events during plant and animal development that generate 3D shapes, such as flowers or limbs. However, the mechanisms by which spatiotemporal patterns of gene expression modify cellular behaviours to generate such deformations remain to be established. We use the Snapdragon flower as a model system to address this problem. Combining cellular analysis with tissue-level modelling, we show that an orthogonal pattern of growth orientations plays a key role in generating out-of-plane deformations. This growth pattern is most likely oriented by a polarity field, highlighted by PIN1 protein localisation, and is modulated by dorsoventral gene activity. The orthogonal growth pattern interacts with other patterns of differential growth to create tissue conflicts that shape the flower. Similar shape changes can be generated by contraction as well as growth, suggesting tissue conflict resolution provides a flexible morphogenetic mechanism for generating shape diversity in plants and animals. DOI: http://dx.doi.org/10.7554/eLife.20156.001 PMID:28166865

Placental expression of EG-VEGF and its receptors PKR1 (prokineticin receptor-1) and PKR2 throughout mouse gestation.

PubMed

Hoffmann, P; Feige, J-J; Alfaidy, N

2007-10-01

Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be disregulated in pre-eclampsia (PE). Recently, we characterised the expression of EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK1) in human placenta during the first trimester of pregnancy and showed that this factor is likely to play an important role in human placentation. However, because it is impossible to prospectively study placentation in humans, it has been impossible to further characterise EG-VEGF expression throughout complete gestation and especially at critical gestational ages for PE development. In the present study, we used mouse placenta to further characterise EG-VEGF expression throughout gestation. We investigated the pattern of expression of EG-VEGF and its receptors, PKR1 and PKR2 at the mRNA and protein levels. Our results show that EG-VEGF and VEGF exhibit different patterns of expression and different localisations in the mouse placenta. EG-VEGF was mainly localised in the labyrinth whereas VEGF was mainly present in glycogen and giant cells. EG-VEGF mRNA and protein levels were highest before 10.5days post coitus (dpc) whereas those of VEGF showed stable expression throughout gestation. PKR1 protein was localised to the labyrinth layer and showed the same pattern of expression as EG-VEGF whereas PKR2 expression was maintained over 10.5dpc with both trophoblastic and endothelial cell localisations. Altogether these findings suggest that EG-VEGF may have a direct effect on both endothelial and trophoblastic cells and is likely to play an important role in mouse placentation.

Human β-Defensin 3 [corrected] Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid.

PubMed

Semple, Fiona; MacPherson, Heather; Webb, Sheila; Kilanowski, Fiona; Lettice, Laura; McGlasson, Sarah L; Wheeler, Ann P; Chen, Valerie; Millhauser, Glenn L; Melrose, Lauren; Davidson, Donald J; Dorin, Julia R

2015-12-01

Human β-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six β-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-β in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-β. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-β, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans.

Human β-D-3 Exacerbates MDA5 but Suppresses TLR3 Responses to the Viral Molecular Pattern Mimic Polyinosinic:Polycytidylic Acid

PubMed Central

Semple, Fiona; MacPherson, Heather; Webb, Sheila; Kilanowski, Fiona; Lettice, Laura; McGlasson, Sarah L.; Wheeler, Ann P.; Chen, Valerie; Millhauser, Glenn L.; Melrose, Lauren; Davidson, Donald J.; Dorin, Julia R.

2015-01-01

Human β-defensin 3 (hBD3) is a cationic host defence peptide and is part of the innate immune response. HBD3 is present on a highly copy number variable block of six β-defensin genes, and increased copy number is associated with the autoimmune disease psoriasis. It is not known how this increase influences disease development, but psoriasis is a T cell-mediated disease and activation of the innate immune system is required for the initial trigger that leads to the amplification stage. We investigated the effect of hBD3 on the response of primary macrophages to various TLR agonists. HBD3 exacerbated the production of type I Interferon-β in response to the viral ligand mimic polyinosinic:polycytidylic acid (polyI:C) in both human and mouse primary cells, although production of the chemokine CXCL10 was suppressed. Compared to polyI:C alone, mice injected with both hBD3 peptide and polyI:C also showed an enhanced increase in Interferon-β. Mice expressing a transgene encoding hBD3 had elevated basal levels of Interferon-β, and challenge with polyI:C further increased this response. HBD3 peptide increased uptake of polyI:C by macrophages, however the cellular response and localisation of polyI:C in cells treated contemporaneously with hBD3 or cationic liposome differed. Immunohistochemistry showed that hBD3 and polyI:C do not co-localise, but in the presence of hBD3 less polyI:C localises to the early endosome. Using bone marrow derived macrophages from knockout mice we demonstrate that hBD3 suppresses the polyI:C-induced TLR3 response mediated by TICAM1 (TRIF), while exacerbating the cytoplasmic response through MDA5 (IFIH1) and MAVS (IPS1/CARDIF). Thus, hBD3, a highly copy number variable gene in human, influences cellular responses to the viral mimic polyI:C implying that copy number may have a significant phenotypic effect on the response to viral infection and development of autoimmunity in humans. PMID:26646717

Expression and Activity of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Distal Lung Epithelial Cells In Vitro.

PubMed

Nickel, Sabrina; Selo, Mohammed Ali; Fallack, Juliane; Clerkin, Caoimhe G; Huwer, Hanno; Schneider-Daum, Nicole; Lehr, Claus-Michael; Ehrhardt, Carsten

2017-12-01

Breast cancer resistance protein (BCRP/ABCG2) has previously been identified with high expression levels in human lung. The subcellular localisation and functional activity of the transporter in lung epithelia, however, remains poorly investigated. The aim of this project was to study BCRP expression and activity in freshly isolated human alveolar epithelial type 2 (AT2) and type 1-like (AT1-like) cells in primary culture, and to compare these findings with data obtained from the NCI-H441 cell line. BCRP expression levels in AT2 and AT1-like cells and in different passages of NCI-H441 cells were determined using q-PCR and immunoblot. Transporter localisation was confirmed by confocal laser scanning microscopy. Efflux and transport studies using the BCRP substrate BODIPY FL prazosin and the inhibitor Ko143 were carried out to assess BCRP activity in the different cell models. BCRP expression decreased during transdifferentiation from AT2 to AT1-like phenotype. Culturing NCI-H441 cells at an air-liquid interface or submersed did not change BCRP abundance, however, BCRP levels increased with passage number. BCRP was localised to the apical membrane and cytosol in NCI-H441 cells. In primary cells, the protein was found predominantly in the nucleus. Functional studies were consistent with expression data. BCRP is differently expressed in AT2 and AT1-like cells with lower abundance and activity in the latter ones. Nuclear BCRP might play a transcriptional role in distal lung epithelium. In NCI-H441 cells, BCRP is expressed in apical cell membranes and its activity is consistent with the localisation pattern.

Tetracycline-inducible protein expression in pancreatic cancer cells: Effects of CapG overexpression

PubMed Central

Tonack, Sarah; Patel, Sabina; Jalali, Mehdi; Nedjadi, Taoufik; Jenkins, Rosalind E; Goldring, Christopher; Neoptolemos, John; Costello, Eithne

2011-01-01

AIM: To establish stable tetracycline-inducible pancreatic cancer cell lines. METHODS: Suit-2, MiaPaca-2, and Panc-1 cells were transfected with a second generation reverse tetracycline-controlled transactivator protein (rtTA2S-M2), under the control of either a cytomegalovirus (CMV) or a chicken β-actin promoter, and the resulting clones were characterised. RESULTS: Use of the chicken (β-actin) promoter proved superior for both the production and maintenance of doxycycline-inducible cell lines. The system proved versatile, enabling transient inducible expression of a variety of genes, including GST-P, CYP2E1, S100A6, and the actin capping protein, CapG. To determine the physiological utility of this system in pancreatic cancer cells, stable inducible CapG expressors were established. Overexpressed CapG was localised to the cytoplasm and the nuclear membrane, but was not observed in the nucleus. High CapG levels were associated with enhanced motility, but not with changes to the cell cycle, or cellular proliferation. In CapG-overexpressing cells, the levels and phosphorylation status of other actin-moduating proteins (Cofilin and Ezrin/Radixin) were not altered. However, preliminary analyses suggest that the levels of other cellular proteins, such as ornithine aminotransferase and enolase, are altered upon CapG induction. CONCLUSION: We have generated pancreatic-cancer derived cell lines in which gene expression is fully controllable. PMID:21528072

Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation

NASA Astrophysics Data System (ADS)

Wang, Szu-Chieh; Hsu, Hao-Jen; Lin, Gee-Way; Wang, Ting-Fang; Chang, Chun-Che; Lin, Ming-Der

2015-09-01

Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12.

PubMed

Sellars, Laura E; Bryant, Jack A; Sánchez-Romero, María-Antonia; Sánchez-Morán, Eugenio; Busby, Stephen J W; Lee, David J

2017-08-03

In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

Connexin Communication Compartments and Wound Repair in Epithelial Tissue.

PubMed

Chanson, Marc; Watanabe, Masakatsu; O'Shaughnessy, Erin M; Zoso, Alice; Martin, Patricia E

2018-05-03

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

Subcellular mRNA localisation at a glance

PubMed Central

Parton, Richard M.; Davidson, Alexander; Davis, Ilan; Weil, Timothy T.

2014-01-01

ABSTRACT mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms. PMID:24833669

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

PubMed

Assier, E; Bouzinba-Segard, H; Stolzenberg, M C; Stephens, R; Bardos, J; Freemont, P; Charron, D; Trowsdale, J; Rich, T

1999-04-16

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels.

PubMed

O'Callaghan, Dermott W; Hasdemir, Burcu; Leighton, Mark; Burgoyne, Robert D

2003-12-01

KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and beta-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

PubMed

Ruane, Peter T; Gumy, Laura F; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J; Hoogenraad, Casper C; Allan, Victoria J

2016-06-07

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.

An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

PubMed

Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

2015-03-01

As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Immunohistochemical characterisation of the hepatic stem cell niche in feline hepatic lipidosis: a preliminary morphological study.

PubMed

Valtolina, Chiara; Robben, Joris H; Favier, Robert P; Rothuizen, Jan; Grinwis, Guy Cm; Schotanus, Baukje A; Penning, Louis C

2018-05-01

Objectives The aim of this study was to describe the cellular and stromal components of the hepatic progenitor cell niche in feline hepatic lipidosis (FHL). Methods Immunohistochemical staining for the progenitor/bile duct marker (K19), activated Kupffer cells (MAC387), myofibroblasts (alpha-smooth muscle actin [α-SMA]) and the extracellular matrix component laminin were used on seven liver biopsies of cats with FHL and three healthy cats. Double immunofluorescence stainings were performed to investigate co-localisation of different cell types in the hepatic progenitor cell (HPC) niche. Results HPCs, Kupffer cells, myofibroblasts and laminin deposition were observed in the liver samples of FHL, although with variability in the expression and positivity of the different immunostainings between different samples. When compared with the unaffected cats where K19 positivity and minimal α-SMA and laminin positivity were seen mainly in the portal area, in the majority of FHL samples K19 and α-SMA-positive cells and laminin positivity were seen also in the periportal and parenchymatous area. MAC387-positive cells were present throughout the parenchyma. Conclusions and relevance This is a preliminary morphological study to describe the activation and co-localisation of components of the HPC niche in FHL. Although the HPC niche in FHL resembles that described in hepatopathies in dogs and in feline lymphocytic cholangitis, the expression of K19, α-SMA, MAC387 and lamin is more variable in FHL, and a common pattern of activation could not be established. Nevertheless, when HPCs were activated, a spatial association between HPCs and their niche could be demonstrated.

Strength training alters MCT1-protein expression and exercise-induced translocation in erythrocytes of men with non-insulin-dependent type-2 diabetes.

PubMed

Opitz, David; Kreutz, Thorsten; Lenzen, Edward; Dillkofer, Benedict; Wahl, Patrick; Montiel-Garcia, Gracia; Graf, Christine; Bloch, Wilhelm; Brixius, Klara

2014-03-01

We investigated the cellular distribution of lactate transporter (MCT1) and its chaperone CD147 (using immunohistochemistry and fluorescence-activated cell sorting) in the erythrocytes of men with non-insulin-dependent type-2 diabetes (NIDDM, n = 11, 61 ± 8 years of age) under acute exercise (ergometer cycling test, World Health Organisation scheme) performed before and after a 3-month strength training program. Cytosolic MCT1 distribution and membraneous CD147 density did not change after acute exercise (ergometer). After the 3-month strength training, MCT1-density was increased and the reaction of MCT1 (but not that of CD147) towards acute exercise (ergometer) was altered. MCT1 localisation was shifted from the centre to the cellular membrane. This resulted in a decrease in the immunohistochemically measured cytosolic MCT1-density. We conclude that strength training alters the acute exercise reaction of MCT1 but not that of CD147 in erythrocytes in patients with NIDDM. This reaction may contribute to long-term normalisation and stabilisation of the regulation of lactate plasma concentration in NIDDM.

Intracellular activity of clinical concentrations of phenothiazines including thioridiazine against phagocytosed Staphylococcus aureus.

PubMed

Ordway, Diane; Viveiros, Miguel; Leandro, Clara; Arroz, Maria Jorge; Amaral, Leonard

2002-07-01

The effect of thioridazine (TZ) was studied on the killing activity of human peripheral blood monocyte derived macrophages (HPBMDM) and of human macrophage cell line THP-1 at extracellular concentrations below those achievable clinically. These macrophages have nominal killing activity against bacteria and therefore, would not influence any activity that the compounds may have against intracellular localised Staphylococcus aureus. The results indicated that whereas TZ has an in vitro minimum inhibitory concentration (MIC) against the strains of S. aureus of 18, 0.1 mg/l of TZ in the medium completely inhibits the growth of S. aureus that has been phagocytosed by macrophages. The latter concentration was non-toxic to macrophages, did not cause cellular expression of activation marker CD69 nor induction of CD3+ T cell production of IFN-gamma, but blocked cellular proliferation and down-regulated the production of T cell-derived cytokines (IFN-gamma, IL-5). These results suggest that TZ induces intracellular bactericidal activities independent of the capacity to generate Type 1 responses against S. aureus.

Gamma-enolase: a well-known tumour marker, with a less-known role in cancer

PubMed Central

Vizin, Tjasa; Kos, Janko

2015-01-01

Background Gamma-enolase, known also as neuron-specific enolase (NSE), is an enzyme of the glycolytic pathway, which is expressed predominantly in neurons and cells of the neuroendocrine system. As a tumour marker it is used in diagnosis and prognosis of cancer; however, the mechanisms enrolling it in malignant progression remain elusive. As a cytoplasmic enzyme gamma-enolase is involved in increased aerobic glycolysis, the main source of energy in cancer cells, supporting cell proliferation. However, different cellular localisation at pathophysiological conditions, proposes other cellular engagements. Conclusions The C-terminal part of the molecule, which is not related to glycolytic pathway, was shown to promote survival of neuronal cells by regulating neuronal growth factor receptor dependent signalling pathways, resulting also in extensive actin cytoskeleton remodelling. This additional function could be important also in cancer cells either to protect cells from stressful conditions and therapeutic agents or to promote tumour cell migration and invasion. Gamma-enolase might therefore have a multifunctional role in cancer progression: it supports increased tumour cell metabolic demands, protects tumour cells from stressful conditions and promotes their invasion and migration. PMID:26401126

Plants and pathogens: putting infection strategies and defence mechanisms on the map.

PubMed

Faulkner, Christine; Robatzek, Silke

2012-12-01

All plant organs are vulnerable to colonisation and molecular manipulation by microbes. When this interaction allows proliferation of the microbe at the expense of the host, the microbe can be described as a pathogen. In our attempts to understand the full nature of the interactions that occur between a potential pathogen and its host, various aspects of the molecular mechanisms of infection and defence have begun to be characterised. There is significant variation in these mechanisms. While previous research has examined plant-pathogen interactions with whole plant/organ resolution, the specificity of infection strategies and changes in both gene expression and protein localisation of immune receptors upon infection suggest there is much to be gained from examination of plant-microbe interactions at the cellular level. Copyright © 2012 Elsevier Ltd. All rights reserved.

Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

PubMed

Prieto-Martínez, Noelia; Vilagran, Ingrid; Morató, Roser; Rodríguez-Gil, Joan E; Yeste, Marc; Bonet, Sergi

2016-04-01

Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (P<0.05) with sperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

Neuronal NOS localises to human airway cilia.

PubMed

Jackson, Claire L; Lucas, Jane S; Walker, Woolf T; Owen, Holly; Premadeva, Irnthu; Lackie, Peter M

2015-01-30

Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air-liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function. Copyright © 2014 Elsevier Inc. All rights reserved.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

PubMed Central

Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

2016-01-01

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

PubMed Central

Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

2015-01-01

A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

Heat Shock Factor 1 Deficiency Affects Systemic Body Temperature Regulation.

PubMed

Ingenwerth, Marc; Noichl, Erik; Stahr, Anna; Korf, Horst-Werner; Reinke, Hans; von Gall, Charlotte

2016-01-01

Heat shock factor 1 (HSF1) is a ubiquitous heat-sensitive transcription factor that mediates heat shock protein transcription in response to cellular stress, such as increased temperature, in order to protect the organism against misfolded proteins. In this study, we analysed the effect of HSF1 deficiency on core body temperature regulation. Body temperature, locomotor activity, and food consumption of wild-type mice and HSF1-deficient mice were recorded. Prolactin and thyroid-stimulating hormone levels were measured by ELISA. Gene expression in brown adipose tissue was analysed by quantitative real-time PCR. Hypothalamic HSF1 and its co-localisation with tyrosine hydroxylase was analysed using confocal laser scanning microscopy. HSF1-deficient mice showed an increase in core body temperature (hyperthermia), decreased overall locomotor activity, and decreased levels of prolactin in pituitary and blood plasma reminiscent of cold adaptation. HSF1 could be detected in various hypothalamic regions involved in temperature regulation, suggesting a potential role of HSF1 in hypothalamic thermoregulation. Moreover, HSF1 co-localises with tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, suggesting a potential role of HSF1 in the hypothalamic control of prolactin release. In brown adipose tissue, levels of prolactin receptor and uncoupled protein 1 were increased in HSF1-deficient mice, consistent with an up-regulation of heat production. Our data suggest a role of HSF1 in systemic thermoregulation. © 2015 S. Karger AG, Basel.

Etoposide Induces Nuclear Re-Localisation of AID

PubMed Central

Lambert, Laurens J.; Walker, Simon; Feltham, Jack; Lee, Heather J.; Reik, Wolf; Houseley, Jonathan

2013-01-01

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming. PMID:24324754

Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method.

PubMed

Barnard, Emma; McFerran, Neil V; Trudgett, Alan; Nelson, John; Timson, David J

2008-05-01

An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.

CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malizia, Andrea P.; Lacey, Noreen; Walls, Dermot

Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonicalmore » Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.« less

The temporal expression of estrogen receptor alpha-36 and runx2 in human bone marrow derived stromal cells during osteogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Francis, W.R., E-mail: w.francis@swansea.ac.uk; Owens, S.E.; Wilde, C.

2014-10-24

Highlights: • ERα36 is the predominant ERα isoform involved in bone regulation in human BMSC. • ERα36 mRNA is significantly upregulated during the process of osteogenesis. • The pattern of ERα36 and runx2 mRNA expression is similar during osteogenesis. • ERα36 appears to be co-localised with runx2 during osteogenesis. - Abstract: During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2),more » a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments.« less

Imaging cellular structures in super-resolution with SIM, STED and Localisation Microscopy: A practical comparison.

PubMed

Wegel, Eva; Göhler, Antonia; Lagerholm, B Christoffer; Wainman, Alan; Uphoff, Stephan; Kaufmann, Rainer; Dobbie, Ian M

2016-06-06

Many biological questions require fluorescence microscopy with a resolution beyond the diffraction limit of light. Super-resolution methods such as Structured Illumination Microscopy (SIM), STimulated Emission Depletion (STED) microscopy and Single Molecule Localisation Microscopy (SMLM) enable an increase in image resolution beyond the classical diffraction-limit. Here, we compare the individual strengths and weaknesses of each technique by imaging a variety of different subcellular structures in fixed cells. We chose examples ranging from well separated vesicles to densely packed three dimensional filaments. We used quantitative and correlative analyses to assess the performance of SIM, STED and SMLM with the aim of establishing a rough guideline regarding the suitability for typical applications and to highlight pitfalls associated with the different techniques.

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture.

PubMed

Penrad-Mobayed, May; Perrin, Caroline; Lepesant, Jean-Antoine

2012-12-01

Subterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART).

A Novel Type III Endosome Transmembrane Protein, TEMP

PubMed Central

Aturaliya, Rajith N.; Kerr, Markus C.; Teasdale, Rohan D.

2012-01-01

As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP’s plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport. PMID:24710541

Expression and functional characterisation of System L amino acid transporters in the human term placenta.

PubMed

Gaccioli, Francesca; Aye, Irving L M H; Roos, Sara; Lager, Susanne; Ramirez, Vanessa I; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2015-06-09

System L transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) mediate the uptake of large, neutral amino acids in the human placenta. Many System L substrates are essential amino acids, thus representing crucial nutrients for the growing fetus. Both LAT isoforms are expressed in the human placenta, but the relative contribution of LAT1 and LAT2 to placental System L transport and their subcellular localisation are not well established. Moreover, the influence of maternal body mass index (BMI) on placental System L amino acid transport is poorly understood. Therefore the aims of this study were to determine: i) the relative contribution of the LAT isoforms to System L transport activity in primary human trophoblast (PHT) cells isolated from term placenta; ii) the subcellular localisation of LAT transporters in human placenta; and iii) placental expression and activity of System L transporters in response to maternal overweight/obesity. System L mediated leucine uptake was measured in PHT cells after treatment with si-RNA targeting LAT1 and/or LAT2. The localisation of LAT isoforms was studied in isolated microvillous plasma membranes (MVM) and basal membranes (BM) by Western blot analysis. Results were confirmed by immunohistochemistry in sections of human term placenta. Expression and activity System L transporters was measured in isolated MVM from women with varying pre-pregnancy BMI. Both LAT1 and LAT2 isoforms contribute to System L transport activity in primary trophoblast cells from human term placenta. LAT1 and LAT2 transporters are highly expressed in the MVM of the syncytiotrophoblast layer at term. LAT2 is also localised in the basal membrane and in endothelial cells lining the fetal capillaries. Measurements in isolated MVM vesicles indicate that System L transporter expression and activity is not influenced by maternal BMI. LAT1 and LAT2 are present and functional in the syncytiotrophoblast MVM, whereas LAT2 is also expressed in the BM and in the fetal capillary endothelium. In contrast to placental System A and beta amino acid transporters, MVM System L activity is unaffected by maternal overweight/obesity.

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells.

PubMed

Williams, Michael J

2009-03-25

When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

PROTEIN TARGETING TO STARCH Is Required for Localising GRANULE-BOUND STARCH SYNTHASE to Starch Granules and for Normal Amylose Synthesis in Arabidopsis

PubMed Central

Seung, David; Soyk, Sebastian; Coiro, Mario; Maier, Benjamin A.; Eicke, Simona; Zeeman, Samuel C.

2015-01-01

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis. PMID:25710501

The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells

PubMed Central

Williams, Michael J

2009-01-01

Background When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. Results Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) [1]. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface. Conclusion The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1. PMID:19320973

Developing and validating a localised, self-training mindfulness programme for older Singaporean adults: effects on cognitive functioning and implications for healthcare

PubMed Central

Tam, Bryan Wei Hoe; Lo, Dana Rui Ting; Seah, Daniel Wen Hao; Lee, Jun Xian; Foo, Zann Fang Ying; Poh, Zoe Yu Yah; Thong, Fionna Xiu Jun; Sim, Sam Kim Yang; Chee, Chew Sim

2017-01-01

There is a paucity of research available on the effect of mindfulness on cognitive function. However, the topic has recently gained more attention due to the ageing population in Singapore, catalysed by recent findings on brain function and cellular ageing. Recognising the potential benefits of practising mindfulness, we aimed to develop a localised, self-training mindfulness programme, guided by expert practitioners and usability testing, for older Singaporean adults. This was followed by a pilot study to examine the potential cognitive benefits and feasibility of this self-training programme for the cognitive function of older adults in Singapore. We found that the results from the pilot study were suggestive but inconclusive, and thus, merit further investigation. PMID:27868134

Developing and validating a localised, self-training mindfulness programme for older Singaporean adults: effects on cognitive functioning and implications for healthcare.

PubMed

Tam, Bryan Wei Hoe; Lo, Dana Rui Ting; Seah, Daniel Wen Hao; Lee, Jun Xian; Foo, Zann Fang Ying; Poh, Zoe Yu Yah; Thong, Fionna Xiu Jun; Sim, Sam Kim Yang; Chee, Chew Sim

2017-03-01

There is a paucity of research available on the effect of mindfulness on cognitive function. However, the topic has recently gained more attention due to the ageing population in Singapore, catalysed by recent findings on brain function and cellular ageing. Recognising the potential benefits of practising mindfulness, we aimed to develop a localised, self-training mindfulness programme, guided by expert practitioners and usability testing, for older Singaporean adults. This was followed by a pilot study to examine the potential cognitive benefits and feasibility of this self-training programme for the cognitive function of older adults in Singapore. We found that the results from the pilot study were suggestive but inconclusive, and thus, merit further investigation. Copyright: © Singapore Medical Association.

Determinants of GPI-PLC Localisation to the Flagellum and Access to GPI-Anchored Substrates in Trypanosomes

PubMed Central

Sunter, Jack; Webb, Helena; Carrington, Mark

2013-01-01

In Trypanosoma brucei, glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor that releases variant surface glycoprotein (VSG) from dying cells. In live cells, GPI-PLC is localised to the plasma membrane where it is concentrated on the flagellar membrane, so activity or access must be tightly regulated as very little VSG is shed. Little is known about regulation except that acylation within a short internal motif containing three cysteines is necessary for GPI-PLC to access VSG in dying cells. Here, GPI-PLC mutants have been analysed both for subcellular localisation and for the ability to release VSG from dying cells. Two sequence determinants necessary for concentration on the flagellar membrane were identified. First, all three cysteines are required for full concentration on the flagellar membrane. Mutants with two cysteines localise predominantly to the plasma membrane but lose some of their flagellar concentration, while mutants with one cysteine are mainly localised to membranes between the nucleus and flagellar pocket. Second, a proline residue close to the C-terminus, and distant from the acylated cysteines, is necessary for concentration on the flagellar membrane. The localisation of GPI-PLC to the plasma but not flagellar membrane is necessary for access to the VSG in dying cells. Cellular structures necessary for concentration on the flagellar membrane were identified by depletion of components. Disruption of the flagellar pocket collar caused loss of concentration whereas detachment of the flagellum from the cell body after disruption of the flagellar attachment zone did not. Thus, targeting to the flagellar membrane requires: a titratable level of acylation, a motif including a proline, and a functional flagellar pocket. These results provide an insight into how the segregation of flagellar membrane proteins from those present in the flagellar pocket and cell body membranes is achieved. PMID:23990786

Domains involved in calcineurin phosphatase inhibition and nuclear localisation in the African swine fever virus A238L protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abrams, Charles C.; Chapman, Dave A.G.; Silk, Rhiannon

2008-05-10

The African swine fever virus A238L protein inhibits calcineurin phosphatase activity and activation of NF-{kappa}B and p300 co-activator. An 82 amino acid domain containing residues 157 to 238 at the C-terminus of A238L was expressed in E. coli and purified. This purified A238L fragment acted as a potent inhibitor of calcineurin phosphatase in vitro with an IC{sub 50} of approximately 70 nM. Two putative nuclear localisation signals were identified between residues 80 to 86 (NLS-1) and between residues 203 to 207 overlapping with the N-terminus of the calcineurin docking motif (NLS-2). Mutation of these motifs independently did not reduce nuclearmore » localisation compared to the wild type A238L protein, whereas mutation of both motifs significantly reduced nuclear localisation of A238L. Mutation of the calcineurin docking motif resulted in a dramatic increase in the nuclear localisation of A238L provided an intact NLS was present. We propose that binding of calcineurin to A238L masks NLS-2 contributing to the cytoplasmic retention of A238L.« less

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

PubMed

Calabro, Sarah R; Maczurek, Annette E; Morgan, Alison J; Tu, Thomas; Wen, Victoria W; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A; McCaughan, Geoffrey W; Warner, Fiona J; McLennan, Susan V; Shackel, Nicholas A

2014-01-01

The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tunbak, Hale, E-mail: h.tunbak@ucl.ac.uk; Georgiou, Christiana, E-mail: christiana.georgiou.10@ucl.ac.uk; Guan, Cui, E-mail: c.guan@qmul.ac.uk

PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclearmore » localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5–6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. -- Highlights: •Zinc fingers 1, 2, 5, and 6 are necessary for efficient nuclear localisation of PRDM4. •Zinc fingers 3 and 4 are dispensable for nuclear localisation of PRDM4. •Zinc knuckle is dispensable for nuclear localisation of PRDM4. •PRDM4 nuclear transport is independent of importin α/β-mediated pathway of nuclear import.« less

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

PubMed

Schaletzki, Yvonne; Kromrey, Marie-Luise; Bröderdorf, Susanne; Hammer, Elke; Grube, Markus; Hagen, Paul; Sucic, Sonja; Freissmuth, Michael; Völker, Uwe; Greinacher, Andreas; Rauch, Bernhard H; Kroemer, Heyo K; Jedlitschky, Gabriele

2017-01-05

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma.

PubMed

Christiansen, H; Sahin, K; Berthold, F; Hero, B; Terpe, H J; Lampert, F

1995-01-01

A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN.

Differential co-localisation of the P2X7 receptor subunit with vesicular glutamate transporters VGLUT1 and VGLUT2 in rat CNS.

PubMed

Atkinson, L; Batten, T F C; Moores, T S; Varoqui, H; Erickson, J D; Deuchars, J

2004-01-01

Presynaptic P2X(7) receptors are thought to play a role in the modulation of transmitter release and have been localised to terminals with the location and morphology typical of excitatory boutons. To test the hypothesis that this receptor is preferentially associated with excitatory terminals we combined immunohistochemistry for the P2X(7) receptor subunit (P2X(7)R) with that for two vesicular glutamate transporters (VGLUT1 and VGLUT2) in the rat CNS. This confirmed that P2X(7)R immunoreactivity (IR) is present in glutamatergic terminals; however, whether it was co-localised with VGLUT1-IR or VGLUT2-IR depended on the CNS region examined. In the spinal cord, P2X(7)R-IR co-localised with VGLUT2-IR. In the brainstem, co-localisation of P2X(7)R-IR with VGLUT2-IR was widespread, but co-localisation with VGLUT1-IR was seen only in the external cuneate nucleus and spinocerebellar tract region of the ventral medulla. In the cerebellum, P2X(7)R-IR co-localised with both VGLUT1 and VGLUT2-IR in the granular layer. In the hippocampus it was co-localised only with VGLUT1-IR, including in the polymorphic layer of the dentate gyrus and the substantia radiatum of the CA3 region. In other forebrain areas, P2X(7)R-IR co-localised with VGLUT1-IR throughout the amygdala, caudate putamen, striatum, reticular thalamic nucleus and cortex and with VGLUT2-IR in the dorsal lateral geniculate nucleus, amygdala and hypothalamus. Dual labelling studies performed using markers for cholinergic, monoaminergic, GABAergic and glycinergic terminals indicated that in certain brainstem and spinal cord nuclei the P2X(7)R is also expressed by subpopulations of cholinergic and GABAergic/glycinergic terminals. These data support our previous hypothesis that the P2X(7)R may play a role in modulating glutamate release in functionally different systems throughout the CNS but further suggest a role in modulating release of inhibitory transmitters in some regions.

Affimer proteins are versatile and renewable affinity reagents

PubMed Central

Tiede, Christian; Bedford, Robert; Heseltine, Sophie J; Smith, Gina; Wijetunga, Imeshi; Ross, Rebecca; AlQallaf, Danah; Roberts, Ashley PE; Balls, Alexander; Curd, Alistair; Hughes, Ruth E; Martin, Heather; Needham, Sarah R; Zanetti-Domingues, Laura C; Sadigh, Yashar; Peacock, Thomas P; Tang, Anna A; Gibson, Naomi; Kyle, Hannah; Platt, Geoffrey W; Ingram, Nicola; Taylor, Thomas; Coletta, Louise P; Manfield, Iain; Knowles, Margaret; Bell, Sandra; Esteves, Filomena; Maqbool, Azhar; Prasad, Raj K; Drinkhill, Mark; Bon, Robin S; Patel, Vikesh; Goodchild, Sarah A; Martin-Fernandez, Marisa; Owens, Ray J; Nettleship, Joanne E; Webb, Michael E; Harrison, Michael; Lippiat, Jonathan D; Ponnambalam, Sreenivasan; Peckham, Michelle; Smith, Alastair; Ferrigno, Paul Ko; Johnson, Matt; McPherson, Michael J; Tomlinson, Darren Charles

2017-01-01

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001 PMID:28654419

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

PubMed

Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

2013-01-01

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

A Cul-3-BTB ubiquitylation pathway regulates junctional levels and asymmetry of core planar polarity proteins

PubMed Central

Strutt, Helen; Searle, Elizabeth; Thomas-MacArthur, Victoria; Brookfield, Rosalind; Strutt, David

2013-01-01

The asymmetric localisation of core planar polarity proteins at apicolateral junctions is required to specify cell polarity in the plane of epithelia. This asymmetric distribution of the core proteins is proposed to require amplification of an initial asymmetry by feedback loops. In addition, generation of asymmetry appears to require the regulation of core protein levels, but the importance of such regulation and the underlying mechanisms is unknown. Here we show that ubiquitylation acts through more than one mechanism to control core protein levels in Drosophila, and that without this regulation cellular asymmetry is compromised. Levels of Dishevelled at junctions are regulated by a Cullin-3-Diablo/Kelch ubiquitin ligase complex, the activity of which is most likely controlled by neddylation. Furthermore, activity of the deubiquitylating enzyme Fat facets is required to maintain Flamingo levels at junctions. Notably, ubiquitylation does not alter the total cellular levels of Dishevelled or Flamingo, but only that of the junctional population. When junctional core protein levels are either increased or decreased by disruption of the ubiquitylation machinery, their asymmetric localisation is reduced and this leads to disruption of planar polarity at the tissue level. Loss of asymmetry by altered core protein levels can be explained by reference to feedback models for amplification of asymmetry. PMID:23487316

Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

PubMed

Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Del Angel, Rosa María; Medina-Ramírez, Fernando; Santos-Argumedo, Leopoldo; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

2017-08-01

Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.

AarF Domain Containing Kinase 3 (ADCK3) Mutant Cells Display Signs of Oxidative Stress, Defects in Mitochondrial Homeostasis and Lysosomal Accumulation

PubMed Central

Cullen, Jason K.; Abdul Murad, Norazian; Yeo, Abrey; McKenzie, Matthew; Ward, Micheal; Chong, Kok Leong; Schieber, Nicole L.; Parton, Robert G.; Lim, Yi Chieh; Wolvetang, Ernst; Maghzal, Ghassan J.; Stocker, Roland; Lavin, Martin F.

2016-01-01

Autosomal recessive ataxias are a clinically diverse group of syndromes that in some cases are caused by mutations in genes with roles in the DNA damage response, transcriptional regulation or mitochondrial function. One of these ataxias, known as Autosomal Recessive Cerebellar Ataxia Type-2 (ARCA-2, also known as SCAR9/COQ10D4; OMIM: #612016), arises due to mutations in the ADCK3 gene. The product of this gene (ADCK3) is an atypical kinase that is thought to play a regulatory role in coenzyme Q10 (CoQ10) biosynthesis. Although much work has been performed on the S. cerevisiae orthologue of ADCK3, the cellular and biochemical role of its mammalian counterpart, and why mutations in this gene lead to human disease is poorly understood. Here, we demonstrate that ADCK3 localises to mitochondrial cristae and is targeted to this organelle via the presence of an N-terminal localisation signal. Consistent with a role in CoQ10 biosynthesis, ADCK3 deficiency decreased cellular CoQ10 content. In addition, endogenous ADCK3 was found to associate in vitro with recombinant Coq3, Coq5, Coq7 and Coq9, components of the CoQ10 biosynthetic machinery. Furthermore, cell lines derived from ARCA-2 patients display signs of oxidative stress, defects in mitochondrial homeostasis and increases in lysosomal content. Together, these data shed light on the possible molecular role of ADCK3 and provide insight into the cellular pathways affected in ARCA-2 patients. PMID:26866375

Chromosome-specific physical localisation of expressed sequence tag loci in Corchorus olitorius L.

PubMed

Joshi, A; Das, S K; Samanta, P; Paria, P; Sen, S K; Basu, A

2014-11-01

Jute (Corchorus spp.), as a natural fibre-producing species, ranks next only to cotton. Inadequate understanding of its genetic architecture is a major lacuna for genetic improvement of this crop in terms of yield and quality. Establishment of a physical map provides a genomic tool that helps in positional cloning of valuable genes. In this report, an attempt was initiated to study association and localisation of single copy expressed sequence tag (EST) loci in the genome of Corchorus olitorius. The chromosome-specific association of EST was determined based on the appearance of an extra signal for a single copy cDNA probe in mitotic interphase nuclei of specific trisomic(s) for fluorescence in situ hybridisation, and validated using a cDNA fragment of the 26S rRNA gene (600 bp) as molecular probe. The probe exhibited three signals in meiotic interphase nuclei of trisomic 5, instead of two as observed in diploids and other trisomics, indicating its association with chromosome 5. Subsequent hybridisation of the same probe on the pachytene chromosomes of diploids confirmed that 26S rRNA occupies the terminal end of the short arm of chromosome 5 in C. olitorius. Subsequently, chromosome-specific association of 63 single copy EST and their physical localisation were determined on chromosomes 2, 4, 5 and 7. The study describes chromosome-specific physical localisation of genes in jute. The approach used here could be a step towards construction of genome-wide physical maps for any recalcitrant plant species like jute. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

PubMed Central

de Jong, Femke; Hanley, Steven J.; Beale, Michael H.; Karp, Angela

2015-01-01

Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow. PMID:26070140

Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells.

PubMed

Owen, Sioned; Zhao, Huishan; Dart, Alwyn; Wang, Yamei; Ruge, Fiona; Gao, Yong; Wei, Cong; Wu, Yiling; Jiang, Wen G

2016-11-01

Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

PubMed

Guerra, D M; Giometti, I C; Price, C A; Andrade, P B; Castilho, A C; Machado, M F; Ripamonte, P; Papa, P C; Buratini, J

2008-01-01

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Hepatocyte Produced Matrix Metalloproteinases Are Regulated by CD147 in Liver Fibrogenesis

PubMed Central

Morgan, Alison J.; Tu, Thomas; Wen, Victoria W.; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A.; McCaughan, Geoffrey W.; Warner, Fiona J.; McLennan, Susan V.; Shackel, Nicholas A.

2014-01-01

Background The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Methods Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. Results In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. Conclusion We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents. PMID:25076423

Differential expression and localisation of gasdermin-like (GSDML), a novel member of the cancer-associated GSDMDC protein family, in neoplastic and non-neoplastic gastric, hepatic, and colon tissues.

PubMed

Carl-McGrath, Stacy; Schneider-Stock, Regine; Ebert, Matthias; Röcken, Christoph

2008-01-01

Gasdermin-like (GSDML) is a novel member of the cancer associated gasdermin-domain containing (GSDMDC) protein family. The GSDMDC family has been linked to cancer development and progression, and this is the first study analysing the expression and intracellular localisation of GSDML. GSDML gene transcription was analysed using quantitative real-time RT-PCR. Anti-peptide antibodies against GSDML were developed in rabbits, and an in vitro transcription-translation reaction was used to verify specificity. The Protein-G affinity purified antibodies were used in immunohistochemistry and immunoblotting on hepatocellular, gastric, and colorectal carcinomas and non-lesional tissues. The GSDML gene was transcribed in human gastric, liver and colon cell lines, carcinomas and non-lesional tissues. The GSDML protein was localised to the cytoplasm of cells in both tumour and non-lesional tissues. The GSDML protein splicing variants range in molecular weight from 35 to 50 kDa, and the expression profile varies between tumour and non-tumour. A distinctive vesicular staining pattern was exhibited by GSDML in the apical region of gastric chief cells and colonic surface mucous cells, and the basal region of neuroendocrine cells. GSDML may be a secretory or metabolic product involved in a secretory pathway, and changes in the regulation of GSDML splicing variant transcription and translation may be seen in the development and/or progression of gastrointestinal and hepatic cancers.

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer.

PubMed

Frietsch, J J; Grunewald, T G P; Jasper, S; Kammerer, U; Herterich, S; Kapp, M; Honig, A; Butt, E

2010-05-25

LIM and SH3 protein 1 (LASP-1) is a nucleo-cytoplasmatic signalling protein involved in cell proliferation and migration and is upregulated in breast cancer in vitro studies have shown that LASP-1 might be regulated by prostate-derived ETS factor (PDEF), p53 and/or LASP1 gene amplification. This current study analysed the prognostic significance of LASP-1 on overall survival (OS) in 177 breast cancer patients and addressed the suggested mechanisms of LASP-1-regulation. Nucleo-cytoplasmatic LASP-1-positivity of breast carcinoma samples was correlated with long-term survival, clinicopathological parameters, Ki67-positivity and PDEF expression. Rate of LASP1 amplification was determined in micro-dissected primary breast cancer cells using quantitative RT-PCR. Cell-phase dependency of nuclear LASP-1-localisation was studied in synchronised cells. In addition, LASP-1, PDEF and p53 expression was compared in cell lines of different tumour entities to define principles for LASP-1-regulation. We showed that LASP-1 overexpression is not due to LASP1 gene amplification. Moreover, no correlation between p53-mutations or PDEF-expression and LASP-1-status was observed. However, nuclear LASP-1-localisation in breast carcinomas is increased during proliferation with peak in G2/M-phase and correlated significantly with Ki67-positivity and poor OS. Our results provide evidence that nuclear LASP-1-positivity may serve as a negative prognostic indicator for long-term survival of breast cancer patients.

Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces.

PubMed

Moffatt, Pierre; Wazen, Rima M; Dos Santos Neves, Juliana; Nanci, Antonio

2014-12-01

Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces.

Expression and effects of modulation of the K2P potassium channels TREK-1 (KCNK2) and TREK-2 (KCNK10) in the normal human ovary and epithelial ovarian cancer.

PubMed

Innamaa, A; Jackson, L; Asher, V; van Schalkwyk, G; Warren, A; Keightley, A; Hay, D; Bali, A; Sowter, H; Khan, R

2013-11-01

Aberrant expression of potassium (K(+)) channels contributes to cancer cell proliferation and apoptosis, and K(+) channel blockers can inhibit cell proliferation. TREK-1 and -2 belong to the two-pore domain (K2P) superfamily. We report TREK-1 and -2 expression in ovarian cancer and normal ovaries, and the effects of TREK-1 modulators on cell proliferation and apoptosis. The cellular localisation of TREK-1 and -2 was investigated by immunofluorescence in SKOV-3 and OVCAR-3 cell lines and in cultured ovarian surface epithelium and cancer. Channel expression in normal ovaries and cancer was quantified by western blotting. Immunohistochemical analysis demonstrated the association between channel expression and disease prognosis, stage, and grade. TREK-1 modulation of cell proliferation in the cell lines was investigated with the MTS-assay and the effect on apoptosis determined using flow cytometry. Expression was identified in both cell lines, ovarian cancer (n = 22) and normal ovaries (n = 6). IHC demonstrated positive staining for TREK-1 and -2 in 95.7 % of tumours (n = 69) and 100 % of normal ovaries (n = 9). A reduction in cell proliferation (P < 0.05) was demonstrated at 96 h in SKOV-3 and OVCAR-3 cells incubated TREK-1 modulating agents. Curcumin caused a significant reduction in early apoptosis in SKOV-3 (P < 0.001) and OVCAR-3 (P < 0.0001) cells and a significant increase in late apoptosis in SKOV-3 (P < 0.01) and OVCAR-3 cells (P < 0.0001). TREK-1 and -2 are expressed in normal ovaries and ovarian cancer. TREK-1 modulators have a significant effect on cell proliferation and apoptosis. We propose investigation of the therapeutic potential of TREK-1 blockers is warranted.

Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol

NASA Astrophysics Data System (ADS)

McNamara, A. L.; Kam, W. W. Y.; Scales, N.; McMahon, S. J.; Bennett, J. W.; Byrne, H. L.; Schuemann, J.; Paganetti, H.; Banati, R.; Kuncic, Z.

2016-08-01

Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies  ˜70 eV, substantially lower than that of liquid water  ˜78 eV. Monte Carlo simulations for 10-50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of  ˜1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol.

Dose enhancement effects to the nucleus and mitochondria from gold nanoparticles in the cytosol

PubMed Central

McNamara, AL; Kam, WW-Y; Scales, N; McMahon, SJ; Bennett, JW; Byrne, HL; Schuemann, J; Paganetti, H; Banati, R; Kuncic, Z

2016-01-01

Gold nanoparticles (GNPs) have shown potential as dose enhancers for radiation therapy. Since damage to the genome affects the viability of a cell, it is generally assumed that GNPs have to localise within the cell nucleus. In practice, however, GNPs tend to localise in the cytoplasm yet still appear to have a dose enhancing effect on the cell. Whether this effect can be attributed to stress-induced biological mechanisms or to physical damage to extra-nuclear cellular targets is still unclear. There is however growing evidence to suggest that the cellular response to radiation can also be influenced by indirect processes induced when the nucleus is not directly targeted by radiation. The mitochondrion in particular may be an effective extra-nuclear radiation target given its many important functional roles in the cell. To more accurately predict the physical effect of radiation within different cell organelles, we measured the full chemical composition of a whole human lymphocytic JURKAT cell as well as two separate organelles; the cell nucleus and the mitochondrion. The experimental measurements found that all three biological materials had similar ionisation energies ~ 70 eV, substantially lower than that of liquid water ~ 78 eV. Monte Carlo simulations for 10 – 50 keV incident photons showed higher energy deposition and ionisation numbers in the cell and organelle materials compared to liquid water. Adding a 1% mass fraction of gold to each material increased the energy deposition by a factor of ~ 1.8 when averaged over all incident photon energies. Simulations of a realistic compartmentalised cell show that the presence of gold in the cytosol increases the energy deposition in the mitochondrial volume more than within the nuclear volume. We find this is due to sub-micron delocalisation of energy by photoelectrons, making the mitochondria a potentially viable indirect radiation target for GNPs that localise to the cytosol. PMID:27435339

Two COWP-like cysteine rich proteins from Eimeria nieschulzi (coccidia, apicomplexa) are expressed during sporulation and involved in the sporocyst wall formation.

PubMed

Jonscher, Ernst; Erdbeer, Alexander; Günther, Marie; Kurth, Michael

2015-07-25

The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation. In this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo. Both investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony. Our results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.

Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family

PubMed Central

2013-01-01

Background Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution. PMID:23433225

Dynamic Localisation of Mature MicroRNAs in Human Nucleoli is Influenced by Exogenous Genetic Materials

PubMed Central

Li, Zhou Fang; Liang, Yi Min; Lau, Pui Ngan; Shen, Wei; Wang, Dai Kui; Cheung, Wing Tai; Xue, Chun Jason; Poon, Lit Man; Lam, Yun Wah

2013-01-01

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials. PMID:23940654

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)☆

PubMed Central

Duke, Elizabeth M.H.; Razi, Minoo; Weston, Anne; Guttmann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Tooze, Sharon A.; Collinson, Lucy M.

2014-01-01

Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities. PMID:24238600

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

PubMed

Massimi, Mara; Devirgiliis, Laura Conti

2007-02-01

In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.

Neuronal control of localized inflammation through expressed nicotinic acetylcholine receptors: a study carried out in mice.

PubMed

Thayabaran, M; Yasawardene, S G

2015-12-01

Although the local inflammatory reactions are known to be regulated through cholinergic anti-inflammatory pathways, the exact subtypes of nicotinic acetylcholine receptors involved in neuroimmune modulation are not well identified. Immunohistochemical localisation of a1 subunit of nicotinic acetylcholine receptors (a1nAChR) in sites of localised inflammation induced by injecting turpentine to the hind limbs of Balb/C mice. Localised inflammation and subsequent development of sterile abscesses was induced by injecting sterile turpentine subcutaneously into thighs of Balb/C mice. Sterile saline was used in the control.. Skin and muscle tissues of inflammatory sites were recovered from the animals after 48 hours and were stained with hematoxylin and eosin. Indirect immunohistochemistry was done using anti-a1nAChR as the primary antibody and biotinylated anti-rat IgG as the secondary antibody. Labeled streptavidin biotin (LSAB) technique was used with diaminobenzedene to detect the immunoreactivity (IR). Intensity of immunostaining was determined based upon a score of 0 - 3+ by qualitative computerised image analysis using FSX 100 Olympus microscope. H and E stained slides showed polymorphonuclear leukocytes (PNL) infiltration at the abscess sites while the saline injected control tissue sections did not show PNL infiltration. A 2+ immunoreactivity (IR) of a1nAChRs was visible at peripheral zones of sterile abscesses where PNL infiltrations were high while the central area with necrotic tissue did not show IR. A subcutaneous lymph node found within the inflammatory region expressed IR of a1nAChR in its capsular sinuses, subcapsular sinuses and trabecular regions. The findings suggest the possible role of controlling localised inflammatory response by parasympathetic cholinergic nerves through a1nAChRs of inflammation sites.

Hydroxysteroid dehydrogenase HSD1L is localised to the pituitary–gonadal axis of primates

PubMed Central

Bird, A Daniel; Greatorex, Spencer; Reser, David; Lavery, Gareth G

2017-01-01

Steroid hormones play clinically important and specific regulatory roles in the development, growth, metabolism, reproduction and brain function in human. The type 1 and 2 11-beta hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 2) have key roles in the pre-receptor modification of glucocorticoids allowing aldosterone regulation of blood pressure, control of systemic fluid and electrolyte homeostasis and modulation of integrated metabolism and brain function. Although the activity and function of 11β-HSDs is thought to be understood, there exists an open reading frame for a distinct 11βHSD-like gene; HSD11B1L, which is present in human, non-human primate, sheep, pig and many other higher organisms, whereas an orthologue is absent in the genomes of mouse, rat and rabbit. We have now characterised this novel HSD11B1L gene as encoded by 9 exons and analysis of EST library transcripts indicated the use of two alternate ATG start sites in exons 2 and 3, and alternate splicing in exon 9. Relatively strong HSD11B1L gene expression was detected in human, non-human primate and sheep tissue samples from the brain, ovary and testis. Analysis in non-human primates and sheep by immunohistochemistry localised HSD11B1L protein to the cytoplasm of ovarian granulosa cells, testis Leydig cells, and gonadatroph cells in the anterior pituitary. Intracellular localisation analysis in transfected human HEK293 cells showed HSD1L protein within the endoplasmic reticulum and sequence analysis suggests that similar to 11βHSD1 it is membrane bound. The endogenous substrate of this third HSD enzyme remains elusive with localisation and expression data suggesting a reproductive hormone as a likely substrate. PMID:28871060

The nuclear proteome of Trypanosoma brucei

PubMed Central

Goos, Carina; Dejung, Mario; Janzen, Christian J.; Butter, Falk

2017-01-01

Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. PMID:28727848

Prediction of gene expression in embryonic structures of Drosophila melanogaster.

PubMed

Samsonova, Anastasia A; Niranjan, Mahesan; Russell, Steven; Brazma, Alvis

2007-07-01

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms.

Prediction of Gene Expression in Embryonic Structures of Drosophila melanogaster

PubMed Central

Samsonova, Anastasia A; Niranjan, Mahesan; Russell, Steven; Brazma, Alvis

2007-01-01

Understanding how sets of genes are coordinately regulated in space and time to generate the diversity of cell types that characterise complex metazoans is a major challenge in modern biology. The use of high-throughput approaches, such as large-scale in situ hybridisation and genome-wide expression profiling via DNA microarrays, is beginning to provide insights into the complexities of development. However, in many organisms the collection and annotation of comprehensive in situ localisation data is a difficult and time-consuming task. Here, we present a widely applicable computational approach, integrating developmental time-course microarray data with annotated in situ hybridisation studies, that facilitates the de novo prediction of tissue-specific expression for genes that have no in vivo gene expression localisation data available. Using a classification approach, trained with data from microarray and in situ hybridisation studies of gene expression during Drosophila embryonic development, we made a set of predictions on the tissue-specific expression of Drosophila genes that have not been systematically characterised by in situ hybridisation experiments. The reliability of our predictions is confirmed by literature-derived annotations in FlyBase, by overrepresentation of Gene Ontology biological process annotations, and, in a selected set, by detailed gene-specific studies from the literature. Our novel organism-independent method will be of considerable utility in enriching the annotation of gene function and expression in complex multicellular organisms. PMID:17658945

Location service for wireless network using improved RSS-based cellular localisation

NASA Astrophysics Data System (ADS)

Fayaz, Sara; Sarrafian, Sara

2014-06-01

Value-added services, especially in mobile environments, have recently become the key component of making more profit and attracting more subscribers. One of the most commonly used such service is location-based advertiser services. The main issue which should be considered in providing such services is determining the position of the mobile terminals precisely. In this paper, one pattern recognition localisation method based on the signal strength appropriated for implementing a location-based service is presented. The main aim is to introduce some practical solutions to decrease error and computational load and also eliminate the necessity of updating the database. Practical results illustrate high accuracy of this technique and its suitability to apply in such services. The mean error declines to 9.7 m and mean error corresponding to CDF = 67% and CDF = 95% are less than 11 m and 23 m, respectively. We also present a location-based advertising service, in which the customer's interests and local time are considered, in order to enhance the efficiency and individualism of this service.

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Lipoteichoic acids are embedded in cell walls during logarithmic phase, but exposed on membrane vesicles in Lactobacillus gasseri JCM 1131T.

PubMed

Shiraishi, T; Yokota, S; Sato, Y; Ito, T; Fukiya, S; Yamamoto, S; Sato, T; Yokota, A

2018-06-15

Lipoteichoic acid (LTA) is a cell surface molecule specific to Gram-positive bacteria. How LTA localises on the cell surface is a fundamental issue in view of recognition and immunomodulation in hosts. In the present study, we examined LTA localisation using strain JCM 1131T of Lactobacillus gasseri, which is a human intestinal lactic acid bacterium, during various growth phases by immunoelectron microscopy. We first evaluated the specificity of anti-LTA monoclonal antibody clone 55 used as a probe. The glycerophosphate backbone comprising almost intact size (20 to 30 repeating units) of LTA was required for binding. The antibody did not bind to other cellular components, including wall-teichoic acid. Immunoelectron microscopy indicated that LTA was embedded in the cell wall during the logarithmic phase, and was therefore not exposed on the cell surface. Similar results were observed for Lactobacillus fermentum ATCC 9338 and Lactobacillus rhamnosus ATCC 7469T. By contrast, membrane vesicles were observed in the logarithmic phase of L. gasseri with LTA exposed on their surface. In the stationary and death phases, LTA was exposed on cell wall-free cell membrane generated by autolysis. The dramatic alternation of localisation in different growth phases and exposure on the surface of membrane vesicles should relate with complicated interaction between bacteria and host.

Families of phosphoinositide-specific phospholipase C: structure and function.

PubMed

Katan, M

1998-12-08

A large number of extracellular signals stimulate hydrolysis of phosphatidylinositol 4,5-bisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). PI-PLC isozymes have been found in a broad spectrum of organisms and although they have common catalytic properties, their regulation involves different signalling pathways. A number of recent studies provided an insight into domain organisation of PI-PLC isozymes and contributed towards better understanding of the structural basis for catalysis, cellular localisation and molecular changes that could underlie the process of their activation.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

PubMed

Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Nuclear localisation and epigenetic inactivation of the ras effector/tumour suppressor RASSF2A in multiple human cancers

PubMed Central

Cooper, Wendy N.; Dickinson, Rachel E.; Dallol, Ashraf; Grigorieva, Elvira V.; Pavlova, Tatiana V.; Hesson, Luke B.; Bieche, Ivan; Broggini, Massimo; Maher, Eamonn R; Zabarovsky, Eugene R.; Clark, Geoffrey J; Latif, Farida

2010-01-01

RASSF2 is a recently identified member of a class of novel tumour suppressor genes, all containing a ras association domain. We previously demonstrated that the A isoform of RASSF2, is frequently inactivated by promoter region hypermethylation in colorectal tumours and adenomas, methylation was tumour specific and that expression in methylated tumour lines could be reactivated by treatment with 5-aza-2dc. RASSF2 resides at 20p13, this region has been demonstrated to be frequently lost in human cancers. In this report we investigated methylation status of the RASSF2A promoter CpG island in a series of breast, ovarian and non-small cell lung cancers (NSCLC). RASSF2A was frequently methylated in breast tumour cell lines 65% (13/20) and in primary breast tumours 38% (15/40). RASSF2A gene expression could be switched back on in methylated breast tumour cell lines after treatment with 5-aza-2dC, whilst unmethylated lines showed no difference in level of expression before and after 5-aza-2dC treatment. RASSF2A was also frequently methylated in NSCLC tumours 44% (22/50). Methylation in breast tumours and NSCLC was tumour specific. We did not detect RASSF2A methylation in ovarian tumours (0/17). Furthermore no mutations were found in the coding region of RASSF2A in these ovarian tumours. RASSF2A suppressed breast tumour cell growth in vitro (through colony formation and soft agar assays) and in vivo. We identified a highly conserved putative bipartite nuclear localisation signal (NLS) between amino acids 151 and 167 in the RASSF2A sequence and demonstrated that endogenous RASSF2A localised to the nucleus. Mutation of the putative nuclear localisation signal abolished the nuclear localisation so RASSF2A became predominantly cytoplasmic. Our data indicates that RASSF2A is frequently methylated in colorectal, breast and NSCLC tumours, furthermore, the methylation is tumour specific. Hence we have identified RASSF2A as a novel methylation marker for multiple malignancies and it has the potential to be developed into a valuable marker for screening several cancers in parallel using promoter hypermethylation profiles. We also demonstrate that RASSF2 has a functional NLS signal. Furthermore this is the first report demonstrating that RASSF2 suppresses growth of cancer cells in vivo. Hence providing further evidence for its role as a tumour suppressor gene located at 20p13. PMID:17891178

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.

2008-08-15

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicatedmore » that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.« less

The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin.

PubMed

Menges, R; Muth, G; Wohlleben, W; Stegmann, E

2007-11-01

All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host

PubMed Central

Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai

2017-01-01

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane–bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo. PMID:28107409

Targeted Deletion of a Plasmodium Site-2 Protease Impairs Life Cycle Progression in the Mammalian Host.

PubMed

Koussis, Konstantinos; Goulielmaki, Evi; Chalari, Anna; Withers-Martinez, Chrislaine; Siden-Kiamos, Inga; Matuschewski, Kai; Loukeris, Thanasis G

2017-01-01

Site-2 proteases (S2P) belong to the M50 family of metalloproteases, which typically perform essential roles by mediating activation of membrane-bound transcription factors through regulated intramembrane proteolysis (RIP). Protease-dependent liberation of dormant transcription factors triggers diverse cellular responses, such as sterol regulation, Notch signalling and the unfolded protein response. Plasmodium parasites rely on regulated proteolysis for controlling essential pathways throughout the life cycle. In this study we examine the Plasmodium-encoded S2P in a murine malaria model and show that it is expressed in all stages of Plasmodium development. Localisation studies by endogenous gene tagging revealed that in all invasive stages the protein is in close proximity to the nucleus. Ablation of PbS2P by reverse genetics leads to reduced growth rates during liver and blood infection and, hence, virulence attenuation. Strikingly, absence of PbS2P was compatible with parasite life cycle progression in the mosquito and mammalian hosts under physiological conditions, suggesting redundant or dispensable roles in vivo.

Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian-dinoflagellate symbiosis.

PubMed

Dani, Vincent; Priouzeau, Fabrice; Mertz, Marjolijn; Mondin, Magali; Pagnotta, Sophie; Lacas-Gervais, Sandra; Davy, Simon K; Sabourault, Cécile

2017-10-01

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association. © 2017 John Wiley & Sons Ltd.

The immunohistochemical expression of calcitonin receptor-like receptor (CRLR) in human gliomas

PubMed Central

Benes, L; Kappus, C; McGregor, G P; Bertalanffy, H; Mennel, H D; Hagner, S

2004-01-01

Background: Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin. Aim: To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin. Methods: Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR. Results: All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels. Conclusions: This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours. PMID:14747444

The immunohistochemical expression of calcitonin receptor-like receptor (CRLR) in human gliomas.

PubMed

Benes, L; Kappus, C; McGregor, G P; Bertalanffy, H; Mennel, H D; Hagner, S

2004-02-01

Gliomas are the most common primary tumours of the central nervous system and exhibit rapid growth that is associated with neovascularisation. Adrenomedullin is an important tumour survival factor in human carcinogenesis. It has growth promoting effects on gliomas, and blockade of its actions has been experimentally shown to reduce the growth of glioma tissues and cell lines. There is some evidence that the calcitonin receptor-like receptor (CRLR) mediates the tumorigenic actions of adrenomedullin. To determine whether CRLR is expressed in human gliomas and the probable cellular targets of adrenomedullin. Biopsies from 95 human gliomas of varying grade were processed for immunohistochemical analysis using a previously developed and characterised antibody to CRLR. All tumour specimens were positive for CRLR. As previously found in normal peripheral tissues, CRLR immunostaining was particularly intense in the endothelial cells. This was evident in all the various vascular conformations that were observed, and which are typical of gliomas. In addition, clear immunostaining of tumour cells with astrocyte morphology was observed. These were preferentially localised around vessels. This study has shown for the first time that the CRLR protein is present in human glioma tissue. The expression of the receptor in endothelial cells and in astrocytic tumour cells is consistent with the evidence that its endogenous ligand, adrenomedullin, may influence glioma growth by means of both direct mitogenic and indirect angiogenic effects. CRLR may be a valuable target for effective therapeutic intervention in these malignant tumours.

Histopathological changes associated with high intensity focused ultrasound (HIFU) treatment for localised adenocarcinoma of the prostate

PubMed Central

Van Leenders, G J L H; Beerlage, H; Ruijter, E; de la Rosette, J J M C H; van de Kaa, C A

2000-01-01

Aims—Investigation of the histopathological changes in prostatectomy specimens of patients with prostate cancer after high intensity focused ultrasound (HIFU) and identification of immunohistochemical markers for tissue damage after HIFU treatment. Methods—Nine patients diagnosed with adenocarcinoma of the prostate underwent unilateral HIFU treatment seven to 12 days before radical prostatectomy. The prostatectomy specimens were analysed histologically. Immunohistochemical staining and electron microscopy were performed to characterise more subtle phenotypic changes. Results—All prostatectomy specimens revealed well circumscribed HIFU lesions at the dorsal side of the prostate lobe treated. Most epithelial glands in the centre of the HIFU lesions revealed signs of necrosis. Glands without apparently necrotic features were also situated in the HIFU lesions, raising the question of whether lethal destruction had occurred. This epithelium reacted with antibodies to pancytokeratin, prostate specific antigen (PSA), and Ki67, but did not express cytokeratin 8, which is indicative of severe cellular damage. Ultrastructural examination revealed disintegration of cellular membranes and cytoplasmic organelles consistent with cell necrosis. HIFU treatment was incomplete at the ventral, lateral, and dorsal sides of the prostate lobe treated. Conclusions—HIFU treatment induces a spectrum of morphological changes ranging from apparent light microscopic necrosis to more subtle ultrastructural cell damage. All HIFU lesions are marked by loss of cytokeratin 8. HIFU does not affect the whole area treated, leaving vital tissue at the ventral, lateral, and dorsal sides of the prostate. Key Words: prostate cancer • high intensity focused ultrasound treatment PMID:10889823

Overexpression of microRNA-1288 in oesophageal squamous cell carcinoma.

PubMed

Gopalan, Vinod; Islam, Farhadul; Pillai, Suja; Tang, Johnny Cheuk-On; Tong, Daniel King-Hung; Law, Simon; Chan, Kwok-Wah; Lam, Alfred King-Yin

2016-11-01

This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS was examined using immunofluorescence and western blot. Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. Copyright © 2016 Elsevier Inc. All rights reserved.

Increased expression of transforming growth factor beta s after acute oedematous pancreatitis in rats suggests a role in pancreatic repair.

PubMed Central

Riesle, E; Friess, H; Zhao, L; Wagner, M; Uhl, W; Baczako, K; Gold, L I; Korc, M; Büchler, M W

1997-01-01

BACKGROUND: Transforming growth factor beta isoforms (TGF beta s) belong to a family of multifunctional regulators of cellular growth and differentiation. They are mitogenic and chemotactic for fibroblasts and are potent stimulators of extracellular matrix production (collagen) and deposition. Upregulation of TGF beta transcription has been reported for several in vivo systems during repair after injury. AIMS: To study the expression of the three mammalian isoforms of TGF beta (TGF beta 1-3) and their relation to collagen expression as a marker for fibroblast response in acute oedematous pancreatitis in rats. METHODS: Using northern blot analysis and immunohistochemistry, the expression and localisation of TGF beta isoforms, collagen, and amylase were analysed during the course of acute oedematous pancreatitis in rats, experimentally induced by intravenous caerulein infusion. RESULTS: Induction of acute pancreatitis resulted in a biphasic peak pattern of expression of TGF beta 1, beta 2, and beta 3 mRNA, with a pronounced increase from day 1 to day 3 (sixfold, 2.5-fold, fivefold, respectively) and again from day 5 to day 7 (three-fold, 2.3-fold, 3.5-fold, respectively). The temporal changes in TGF beta mRNA identically paralleled the expression in collagen mRNA. In contrast, amylase mRNA expression, used as a general indicator of acinar cell integrity, was slightly decreased after induction of acute pancreatitis. Immunohistochemical analysis of pancreatitis tissue showed that increased expression of TGF beta s was mainly present in the pancreatic acinar and ductal cells; this was evident within one day after pancreatitis induction. CONCLUSION: Overexpression of TGF beta s after induction of acute pancreatitis suggests a role for these proteins in pancreatic repair and remodelling. The increased levels of TGF beta s may help suppress immune activation, and may contribute to the increase in the extracellular matrix including collagen and to the repair of the pancreatic parenchyma. Images PMID:9155579

Expression of FLNa in human melanoma cells regulates the function of integrin α1β1 and phosphorylation and localisation of PKB/AKT/ERK1/2 kinases.

PubMed

Krebs, Kristi; Ruusmann, Anu; Simonlatser, Grethel; Velling, Teet

2015-12-01

FLNa is a ubiquitous cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and functions as a signalling intermediate. We investigated FLNa's role in the function of integrin-type collagen receptors, EGF-EGFR signalling and regulation of PKB/Akt and ERK1/2. Using FLNa-deficient M2 human melanoma cells, and same cells expressing EGFP-FLNa (M2F) or its Ig-like repeats 1-8+24, 8-15+24 and 16-24, we found that in M2F and M2 8-15+24 cells, EGF induced the increased phosphorylation of PKB/Akt and ERK1/2. In M2F cells EGF induced the localisation of these kinases to cell nucleus and lamellipodia, respectively, and the ERK1/2 phosphorylation-dependent co-immunoprecipitation of FLNa with ERK1/2. Only M2F and M2 8-15+24 cells adhered to and spread on type I collagen whereas on fibronectin all cells behaved similarly. α1β1 and α2β1 were the integrin-type collagen receptors expressed on these cells with primarily α1β1 localising to focal contacts and affecting cell adhesion and migration in a manner dependent on FLNa or its Ig-like repeats 8-15. Our results suggest a role for FLNa repeats 8-15 in the α1-subunit-dependent regulation of integrin α1β1 function, EGF-EGFR signalling to PKB/Akt and ERK1/2, identify ERK1/2 in EGF-induced FLNa-associated protein complexes, and show that the function of different integrins is subjected to differential regulation by FLNa. Copyright © 2015. Published by Elsevier GmbH.

SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease.

PubMed

Singh, Preeti; Hanson, Peter S; Morris, Christopher M

2017-06-02

Sirtuins (SIRTs) are NAD + dependent lysine deacetylases which are conserved from bacteria to humans and have been associated with longevity and lifespan extension. SIRT1, the best studied mammalian SIRT is involved in many physiological and pathological processes and changes in SIRT1 have been implicated in neurodegenerative disorders, with SIRT1 having a suggested protective role in Parkinson's disease. In this study, we determined the effect of SIRT1 on cell survival and α-synuclein aggregate formation in SH-SY5Y cells following oxidative stress. Over-expression of SIRT1 protected SH-SY5Y cells from toxin induced cell death and the protection conferred by SIRT1 was partially independent of its deacetylase activity, which was associated with the repression of NF-кB and cPARP expression. SIRT1 reduced the formation of α-synuclein aggregates but showed minimal co-localisation with α-synuclein. In post-mortem brain tissue obtained from patients with Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies and Alzheimer's disease, the activity of SIRT1 was observed to be down-regulated. These findings suggests a negative effect of oxidative stress in neurodegenerative disorders and possibly explain the reduced activity of SIRT1 in neurodegenerative disorders. Our study shows that SIRT1 is a pro-survival protein that is downregulated under cellular stress.

Novel photosensitisers derived from pyropheophorbide-a: uptake by cells and photodynamic efficiency in vitro.

PubMed

Stamati, Ioanna; Kuimova, Marina K; Lion, Mattia; Yahioglu, Gokhan; Phillips, David; Deonarain, Mahendra P

2010-07-30

Photodynamic Therapy (PDT) is a minimally invasive procedure used for treating a range of neoplastic diseases, which utilises combined action of light and a PDT drug called a photosensitiser. The efficiency of this treatment depends crucially on the properties of the photosensitiser used, namely on its efficient uptake by cells or by the surrounding vasculature, intracellular localisation, minimal dark toxicity and substantial phototoxicity. In this report we compare the spectroscopic properties, cell uptake and in vitro phototoxicity of two novel hydrophilic photosensitisers derived from pyropheophorbide-a (PPa). Both new photosensitisers have the potential to form bioconjugates with antibody fragments for targeted PDT. We find that the photophysical properties of both new photosensitisers are favourable compared to the parent PPa, including enhanced absorption in the red spectral region and substantial singlet oxygen quantum yields. Both molecules show efficient cellular uptake, but display a different intracellular localisation. Both new photosensitisers exhibit no significant dark-toxicity at concentrations of up to 100 microM. The phototoxicity of the two photosensitisers is strikingly different, with one derivative being 13 times more efficient than the parent PPa and another derivative being 18 times less efficient in SKOV3 ovarian cancer cells. We investigate the reasons behind such drastic differences in phototoxicity using confocal fluorescence microscopy and conclude that intracellular localisation is a crucial factor in the photodynamic efficiency of pheophorbide derivatives. These studies highlight the underlying factors behind creating more potent photosensitisers through synthetic manipulation.

Collagen type IV at the fetal-maternal interface.

PubMed

Oefner, C M; Sharkey, A; Gardner, L; Critchley, H; Oyen, M; Moffett, A

2015-01-01

Extracellular matrix proteins play a crucial role in influencing the invasion of trophoblast cells. However the role of collagens and collagen type IV (col-IV) in particular at the implantation site is not clear. Immunohistochemistry was used to determine the distribution of collagen types I, III, IV and VI in endometrium and decidua during the menstrual cycle and the first trimester of pregnancy. Expression of col-IV alpha chains during the reproductive cycle was determined by qPCR and protein localisation by immunohistochemistry. The structure of col-IV in placenta was examined using transmission electron microscopy. Finally, the expression of col-IV alpha chain NC1 domains and collagen receptors was localised by immunohistochemistry. Col-IV alpha chains were selectively up-regulated during the menstrual cycle and decidualisation. Primary extravillous trophoblast cells express collagen receptors and secrete col-IV in vitro and in vivo, resulting in the increased levels found in decidua basalis compared to decidua parietalis. A novel expression pattern of col-IV in the mesenchyme of placental villi, as a three-dimensional network, was found. NC1 domains of col-IV alpha chains are known to regulate tumour cell migration and the selective expression of these domains in decidua basalis compared to decidua parietalis was determined. Col-IV is expressed as novel forms in the placenta. These findings suggest that col-IV not only represents a structural protein providing tissue integrity but also influences the invasive behaviour of trophoblast cells at the implantation site. Copyright © 2014 Elsevier Ltd. All rights reserved.

Early cell lineage specification in a marsupial: a case for diverse mechanisms among mammals.

PubMed

Frankenberg, Stephen; Shaw, Geoff; Freyer, Claudia; Pask, Andrew J; Renfree, Marilyn B

2013-03-01

Early cell lineage specification in eutherian mammals results in the formation of a pluripotent inner cell mass (ICM) and trophoblast. By contrast, marsupials have no ICM. Here, we present the first molecular analysis of mechanisms of early cell lineage specification in a marsupial, the tammar wallaby. There was no overt differential localisation of key lineage-specific transcription factors in cleavage and early unilaminar blastocyst stages. Pluriblast cells (equivalent to the ICM) became distinguishable from trophoblast cells by differential expression of POU5F1 and, to a greater extent, POU2, a paralogue of POU5F1. Unlike in the mouse, pluriblast-trophoblast differentiation coincided with a global nuclear-to-cytoplasmic transition of CDX2 localisation. Also unlike in the mouse, Hippo pathway factors YAP and WWTR1 showed mutually distinct localisation patterns that suggest non-redundant roles. NANOG and GATA6 were conserved as markers of epiblast and hypoblast, respectively, but some differences to the mouse were found in their mode of differentiation. Our results suggest that there is considerable evolutionary plasticity in the mechanisms regulating early lineage specification in mammals.

The biosynthetic gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor contains its co-expressed vacuolar MATE transporter

PubMed Central

Darbani, Behrooz; Motawia, Mohammed Saddik; Olsen, Carl Erik; Nour-Eldin, Hussam H.; Møller, Birger Lindberg; Rook, Fred

2016-01-01

Genomic gene clusters for the biosynthesis of chemical defence compounds are increasingly identified in plant genomes. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucoside biosynthesis in three plant lineages. Here we report that the gene cluster for the cyanogenic glucoside dhurrin in Sorghum bicolor additionally contains a gene, SbMATE2, encoding a transporter of the multidrug and toxic compound extrusion (MATE) family, which is co-expressed with the biosynthetic genes. The predicted localisation of SbMATE2 to the vacuolar membrane was demonstrated experimentally by transient expression of a SbMATE2-YFP fusion protein and confocal microscopy. Transport studies in Xenopus laevis oocytes demonstrate that SbMATE2 is able to transport dhurrin. In addition, SbMATE2 was able to transport non-endogenous cyanogenic glucosides, but not the anthocyanin cyanidin 3-O-glucoside or the glucosinolate indol-3-yl-methyl glucosinolate. The genomic co-localisation of a transporter gene with the biosynthetic genes producing the transported compound is discussed in relation to the role self-toxicity of chemical defence compounds may play in the formation of gene clusters. PMID:27841372

Uptake of leptin and albumin via separate pathways in proximal tubule cells.

PubMed

Briffa, Jessica F; Grinfeld, Esther; Poronnik, Philip; McAinch, Andrew J; Hryciw, Deanne H

2016-10-01

The adipokine leptin and oncotic protein albumin are endocytosed in the proximal tubule via the scavenger receptor megalin. Leptin reduces megalin expression and activates cell signalling pathways that upregulate fibrotic protein expression. The aim of this study was to investigate if leptin uptake in proximal tubule cells was via the albumin-megalin endocytic complex. In immortalised proximal tubule Opossum kidney cells (OK) fluorescent leptin and albumin co-localised following 5min exposure, however there was no co-localisation at 10, 20 and 30min exposure. In OK cells, acute exposure to leptin for 2h did not alter NHE3, ClC-5, NHERF1 and NHERF2 mRNA. However, acute leptin exposure increased NHERF2 protein expression in proximal tubule cells. In OK cells, immunoprecipitation experimentation indicated leptin did not bind to ClC-5. Leptin uptake in OK cells was enhanced by bafilomycin and ammonium chloride treatment, demonstrating that uptake was not dependent on lysosomal pH. Thus, it is likely that two pools of megalin exist in proximal tubule cells to facilitate separate uptake of leptin and albumin by endocytosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein.

PubMed

Cornejo, Isabel; Villanueva, Sandra; Burgos, Johanna; López-Cayuqueo, Karen I; Chambrey, Régine; Julio-Kalajzić, Francisca; Buelvas, Neudo; Niemeyer, María I; Figueiras-Fierro, Dulce; Brown, Peter D; Sepúlveda, Francisco V; Cid, L P

2018-01-01

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K + channel present in epithelia where it shares membrane localization with the Na + /K + -pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb + currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.

Characterisation of P-glycoprotein-9.1 in Haemonchus contortus.

PubMed

Godoy, Pablo; Che, Hua; Beech, Robin N; Prichard, Roger K

2016-01-28

The existence nematodes of veterinary importance such as Haemonchus contortus resistant to anthelmintic drugs, including the macrocyclic lactones, has become a major concern in animal health. Macrocyclic lactone resistance in H. contortus seems to be multigenic including the active efflux of these drugs by P-glycoproteins, members of the ABC transporter family, present in this parasite. The goals of the present work were to determine the activity of H. contortus P-glycoprotein 9.1 (Hco-PGP-9.1) and its interaction with the avermectins, ivermectin, abamectin, and also the milbemycin, moxidectin. Additionally, the localisation of Hco-PGP-9.1 was sought in adult worms. Hco-Pgp-9.1 was cloned and expressed in mammalian cells and its expression profile was determined at the transcriptional and protein level by qRT-PCR and Western-blot, respectively. The nematode transport activity was assessed using the tracer dye Rhodamine 123. A ligand competition assay between different macrocyclic lactones and Rhodamine 123 was used to establish whether or not there was interaction between Hco-PGP-9.1 and the avermectins (abamectin and ivermectin) or moxidectin. In addition, immunostaining was carried out to localise Hco-PGP-9.1 expression in the transgenic cells and in adult female parasites. Hco-PGP-9.1 was expressed in the cell membrane of the transfected host cells and was able to extrude Rhodamine 123. Ivermectin and abamectin, but not moxidectin, had a pronounced inhibitory effect on the ability of Hco-PGP-9.1 to transport Rhodamine 123. Antibodies raised against Hco-PGP-9.1 epitopes localised to the uterus of adult female H. contortus. These results suggest a strong interaction of the avermectins with Hco-PGP-9.1. However, possibly due to its physico-chemical properties, moxidectin had markedly less effect on Hco-PGP-9.1. Because of the greater interaction of the avermectins than moxidectin with this transporter, it is more likely to contribute to avermectin resistance than to moxidectin resistance in H. contortus. Possible over expression of Hco-PGP-9.1 in the female reproductive system in resistant worms could reduce paralysis of the uterus by macrocyclic lactones, allowing continued egg release in drug challenged resistant worms.

Promotion of the Unfolding Protein Response in Orexin/Dynorphin Neurons in Sudden Infant Death Syndrome (SIDS): Elevated pPERK and ATF4 Expression.

PubMed

Hunt, Nicholas J; Waters, Karen A; Machaalani, Rita

2017-11-01

We previously demonstrated that sudden infant death syndrome (SIDS) infants have decreased orexin immunoreactivity within the hypothalamus and pons compared to non-SIDS infants. In this study, we examined multiple mechanisms that may promote loss of orexin expression including programmed cell death, impaired maturation/structural stability, neuroinflammation and impaired unfolding protein response (UPR). Immunofluorescent and immunohistochemical staining for a number of markers was performed in the tuberal hypothalamus and pons of infants (1-10 months) who died from SIDS (n = 27) compared to age- and sex-matched non-SIDS infants (n = 19). The markers included orexin A (OxA), dynorphin (Dyn), cleaved caspase 3 (CC3), cleaved caspase 9 (CC9), glial fibrillary acid protein (GFAP), tubulin beta chain 3 (TUBB3), myelin basic protein (MBP), interleukin 1β (IL-1β), terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL), c-fos and the UPR activation markers: phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (pPERK), and activating transcription factor 4 (ATF4). It was hypothesised that pPERK and ATF4 would be upregulated in Ox neurons in SIDS compared to non-SIDS. Within the hypothalamus, OxA and Dyn co-localised with a 20 % decrease in expression in SIDS infants (P = 0.001). pPERK and ATF4 expression in OxA neurons were increased by 35 % (P = 0.001) and 15 % (P = 0.001) respectively, with linear relationships between the decreased OxA/Dyn expression and the percentages of co-localised pPERK/OxA and ATF4/OxA evident (P = 0.01, P = 0.01). No differences in co-localisation with CC9, CC3, TUNEL or c-fos, nor expression of MBP, TUBB3, IL-1β and GFAP, were observed in the hypothalamus. In the pons, there were 40 % and 20 % increases in pPERK expression in the locus coeruleus (P = 0.001) and dorsal raphe (P = 0.022) respectively; ATF4 expression was not changed. The findings that decreased orexin levels in SIDS infants may be associated with an accumulation of pPERK suggest decreased orexin translation. As pPERK may inhibit multiple neuronal groups in the pons in SIDS infants, it could also indicate that a common pathway promotes loss of protein expression and impaired functionality of multiple brainstem neuronal groups.

Evaluation of specimen preparation techniques for micro-PIXE localisation of elements in hyperaccumulating plants

NASA Astrophysics Data System (ADS)

Kachenko, Anthony G.; Siegele, Rainer; Bhatia, Naveen P.; Singh, Balwant; Ionescu, Mihail

2008-04-01

Hybanthus floribundus subsp. floribundus, a rare Australian Ni-hyperaccumulating shrub and Pityrogramma calomelanos var. austroamericana, an Australian naturalized As-hyperaccumulating fern are promising species for use in phytoremediation of contaminated sites. Micro-proton-induced X-ray emission (μ-PIXE) spectroscopy was used to map the elemental distribution of the accumulated metal(loid)s, Ca and K in leaf or pinnule tissues of the two plant species. Samples were prepared by two contrasting specimen preparation techniques: freeze-substitution in tetrahydrofuran (THF) and freeze-drying. The specimens were analysed to compare the suitability of each technique in preserving (i) the spatial elemental distribution and (ii) the tissue structure of the specimens. Further, the μ-PIXE results were compared with concentration of elements in the bulk tissue obtained by ICP-AES analysis. In H. floribundus subsp. floribundus, μ-PIXE analysis revealed Ni, Ca and K concentrations in freeze-dried leaf tissues were at par with bulk tissue concentrations. Elemental distribution maps illustrated that Ni was preferentially localised in the adaxial epidermal tissues (1% DW) and least concentration was found in spongy mesophyll tissues (0.53% DW). Conversely, elemental distribution maps of THF freeze-substituted tissues indicated significantly lower Ni, Ca and K concentrations than freeze-dried specimens and bulk tissue concentrations. Moreover, Ni concentrations were uniform across the whole specimen and no localisation was observed. In P. calomelanos var. austroamericana freeze-dried pinnule tissues, μ-PIXE revealed statistically similar As, Ca and K concentrations as compared to bulk tissue concentrations. Elemental distribution maps showed that As localisation was relatively uniform across the whole specimen. Once again, THF freeze-substituted tissues revealed a significant loss of As compared to freeze-dried specimens and the concentrations obtained by bulk tissue analysis. The results demonstrate that freeze-drying is a suitable sample preparation technique to study elemental distribution of ions in H. floribundus and P. calomelanos plant tissues using μ-PIXE spectroscopy. Furthermore, cellular structure was preserved in samples prepared using this technique.

A C-Repeat Binding Factor Transcriptional Activator (CBF/DREB1) from European Bilberry (Vaccinium myrtillus) Induces Freezing Tolerance When Expressed in Arabidopsis thaliana

PubMed Central

Oakenfull, Rachael J.; Baxter, Robert; Knight, Marc R.

2013-01-01

Freezing stress affects all plants from temperate zones to the poles. Global climate change means such freezing events are becoming less predictable. This in turn reduces the ability of plants to predict the approaching low temperatures and cold acclimate. This has consequences for crop yields and distribution of wild plant species. C-repeat binding factors (CBFs) are transcription factors previously shown to play a vital role in the acclimation process of Arabidopsis thaliana, controlling the expression of hundreds of genes whose products are necessary for freezing tolerance. Work in other plant species cements CBFs as key determinants in the trait of freezing tolerance in higher plants. To test the function of CBFs from highly freezing tolerant plants species we cloned and sequenced CBF transcription factors from three Vaccinium species (Vaccinium myrtillus, Vaccinium uliginosum and Vaccinium vitis-idaea) which we collected in the Arctic. We tested the activity of CBF transcription factors from the three Vaccinium species by producing transgenic Arabidopsis lines overexpressing them. Only the Vaccinium myrtillus CBF was able to substantially activate COR (CBF-target) gene expression in the absence of cold. Correspondingly, only the lines expressing the Vaccinium myrtillus CBF were constitutively freezing tolerant. The basis for the differences in potency of the three Vaccinium CBFs was tested by observing cellular localisation and protein levels. All three CBFs were correctly targeted to the nucleus, but Vaccinium uliginosum CBF appeared to be relatively unstable. The reasons for lack of potency for Vaccinium vitis-idaea CBF were not due to stability or targeting, and we speculate that this was due to altered transcription factor function. PMID:23349799

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

PubMed Central

Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini/, Fabricio K

2015-01-01

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community. PMID:26200713

TAT and HA2 Facilitate Cellular Uptake of Gold Nanoparticles but Do Not Lead to Cytosolic Localisation

PubMed Central

Free, Paul; Lévy, Raphaël

2015-01-01

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles. PMID:25836335

A placebo controlled observer blind immunocytochemical and histologic study of epithelium adjacent to anogenital warts in patients treated with systemic interferon alpha in combination with cryotherapy or cryotherapy alone.

PubMed Central

Handley, J M; Maw, R D; Horner, T; Lawther, H; Walsh, M; Dinsmore, W W

1992-01-01

OBJECTIVE--To examine biopsy specimens of tissue immediately adjacent to anogenital (AG) warts which had been treated with either cryotherapy plus subcutaneous interferon (IFN) alpha 2a or cryotherapy alone, for histological features of (a) human papilloma virus (HPV) infection (b) localised cellular immune responses, to further characterise any cellular immune infiltrates with tissue immunocytochemistry, and to relate any histological, immunocytochemical findings to the treatment response of nearby AG warts. DESIGN--A randomised placebo controlled observer blind study. SETTING--Genitourinary Medicine clinic, Department of Immunopathology, Royal Victoria Hospital, Belfast, N. Ireland. SUBJECTS--Thirty patients with AG warts; 16 treated with IFN alpha 2a plus cryotherapy, and 14 treated with cryotherapy alone. OUTCOME MEASURES--(1) Light microscopic features associated with HPV infection and local cellular immune responses. (2) Indirect immunofluorescence detection of the following cell surface markers: HLA DR, alpha one antitrypsin, CD1, CD3, CD4, CD8, CD22. (3) Clinical response of AG warts to treatment. RESULTS--In pre-treatment biopsies only non specific indicators of HPV infection (acanthosis, 29/30 biopsies, and hyperkeratosis, 7/30 biopsies) were seen on light microscopy. Mononuclear cells were seen both throughout the upper dermis and centred around dermal blood vessels in 19/30 (63.3%) biopsies, and infiltrating into the epidermis in 12/30 (40%) biopsies. On indirect immunofluorescence CD3, CD8, CD4 antigen was detected on the surface of cells throughout the upper dermis in 24/29 (82.7%), 15/29 (51.7%), and 3/29 (10.3%), of biopsy specimens respectively. CD3 antigen, CD8 antigen and CD4 antigen was detected on the surface of cells infiltrating into the epidermis in 18/29 (62%), 7/29 (24.1%), and 6/29 (20.7%) of biopsy specimens respectively. CD1 antigen was seen on the surface of dendritic cells throughout the epidermis in all specimens; CD1 positive cells infiltrated into the upper dermis in 5/29 (17.2%). HLA DR was detected on the surface of dendritic cells throughout the epidermis in 22/29 (75.9%) of specimens, and on the surface of cells scattered both diffusely throughout the upper dermis and centred around dermal blood vessels in all specimens. Alpha one antitrypsin (A1AT) antigen was seen on the surface of cells in the upper dermis in 6/29 (20.7%) of biopsy specimens; no cells expressing CD22 surface antigen were seen. The nature of this local cellular immune response was not altered by treatment of nearby warts with either cryotherapy alone or cryotherapy plus systemic IFN alpha 2a, or related to the therapeutic outcome of these warts. CONCLUSIONS--(1) No convincing histological evidence of HPV infection was seen in epithelium surrounding AG warts. (2) A predominantly T cell-mediated immune response (the target of which is uncertain) was seen in this perilesional epithelium. (3) In the dosage regimens used in this study, treatment of AG warts with either systemic IFN alpha 2a plus cryotherapy or cryotherapy alone did not appear to augment localised cellular immune responses (against any presumed subclinical HPV infection) in epithelium surrounding AG warts. Images PMID:1316307

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

PubMed

Kim, Dong-Hyun; Jarvis, Roger M; Allwood, J William; Batman, Gavin; Moore, Rowan E; Marsden-Edwards, Emma; Hampson, Lynne; Hampson, Ian N; Goodacre, Royston

2010-12-01

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Fibroblast growth factor receptor inhibition induces loss of matrix MCL1 and necrosis in cholangiocarcinoma.

PubMed

Kabashima, Ayano; Hirsova, Petra; Bronk, Steven F; Hernandez, Matthew C; Truty, Mark J; Rizvi, Sumera; Kaufmann, Scott H; Gores, Gregory J

2018-03-08

Myeloid cell leukemia 1 (MCL1), a prosurvival member of the BCL2 protein family, has a pivotal role in human cholangiocarcinoma (CCA) cell survival. We previously reported that fibroblast growth factor receptor (FGFR) signalling mediates MCL1-dependent survival of CCA cells in vitro and in vivo. However, the mode and mechanisms of cell death in this model were not delineated. Human CCA cell lines were treated with the pan-FGFR inhibitor LY2874455 and the mode of cell death examined by several complementary assays. Mitochondrial oxidative metabolism was examined using a XF24 extracellular flux analyser. The efficiency of FGFR inhibition in patient-derived xenografts (PDX) was also assessed. CCA cells expressed two species of MCL1, a full-length form localised to the outer mitochondrial membrane, and an N terminus-truncated species compartmentalised within the mitochondrial matrix. The pan-FGFR inhibitor LY2874455 induced non-apoptotic cell death in the CCA cell lines associated with cellular depletion of both MCL1 species. The cell death was accompanied by failure of mitochondrial oxidative metabolism and was most consistent with necrosis. Enforced expression of N terminus-truncated MCL1 targeted to the mitochondrial matrix, but not full-length MCL1 targeted to the outer mitochondrial membrane, rescued cell death and mitochondrial function. LY2874455 treatment of PDX-bearing mice was associated with tumour cell loss of MCL1 and cell necrosis. FGFR inhibition induces loss of matrix MCL1, resulting in cell necrosis. These observations support a heretofore unidentified, alternative MCL1 survival function, namely prevention of cell necrosis, and have implications for treatment of human CCA. Herein, we report that therapeutic inhibition of a cell receptor expressed by bile duct cancer cells resulted in the loss of a critical survival protein termed MCL1. Cellular depletion of MCL1 resulted in the death of the cancer cells by a process characterised by cell rupture. Cell death by this process can stimulate the immune system and has implications for combination therapy using receptor inhibition with immunotherapy. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Functional characterisation of Arabidopsis SPL7 conserved protein domains suggests novel regulatory mechanisms in the Cu deficiency response.

PubMed

Garcia-Molina, Antoni; Xing, Shuping; Huijser, Peter

2014-08-30

The Arabidopsis SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor SPL7 reprograms cellular gene expression to adapt plant growth and cellular metabolism to copper (Cu) limited culture conditions. Plant cells require Cu to maintain essential processes, such as photosynthesis, scavenging reactive oxygen species, cell wall lignification and hormone sensing. More specifically, SPL7 activity promotes a high-affinity Cu-uptake system and optimizes Cu (re-)distribution to essential Cu-proteins by means of specific miRNAs targeting mRNA transcripts for those dispensable. However, the functional mechanism underlying SPL7 activation is still to be elucidated. As SPL7 transcript levels are largely non-responsive to Cu availability, post-translational modification seems an obvious possibility. Previously, it was reported that the SPL7 SBP domain does not bind to DNA in vitro in the presence of Cu ions and that SPL7 interacts with a kin17 domain protein to raise SPL7-target gene expression upon Cu deprivation. Here we report how additional conserved SPL7 protein domains may contribute to the Cu deficiency response in Arabidopsis. Cytological and biochemical approaches confirmed an operative transmembrane domain (TMD) and uncovered a dual localisation of SPL7 between the nucleus and an endomembrane system, most likely the endoplasmic reticulum (ER). This new perspective unveiled a possible link between Cu deficit and ER stress, a metabolic dysfunction found capable of inducing SPL7 targets in an SPL7-dependent manner. Moreover, in vivo protein-protein interaction assays revealed that SPL7 is able to homodimerize, probably mediated by the IRPGC domain. These observations, in combination with the constitutive activation of SPL7 targets, when ectopically expressing the N-terminal part of SPL7 including the SBP domain, shed some light on the mechanisms governing SPL7 function. Here, we propose a revised model of SPL7 activation and regulation. According to our results, SPL7 would be initially located to endomembranes and activated during ER stress as a result of Cu deficiency. Furthermore, we added the SPL7 dimerization in the presence of Cu ions as an additional regulatory mechanism to modulate the Cu deficiency response.

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

PubMed

Whittington, Hannah J; Ostrowski, Philip J; McAndrew, Debra J; Cao, Fang; Shaw, Andrew; Eykyn, Thomas R; Lake, Hannah; Tyler, Jack; Schneider, Jurgen E; Neubauer, Stefan; Zervou, Sevasti; Lygate, Craig A

2018-03-02

Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesised that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Mice were created overexpressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localisation of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55±4% in WT vs 29±4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

Vascular Endothelial Growth Factor-A165b Restores Normal Glomerular Water Permeability in a Diphtheria-Toxin Mouse Model of Glomerular Injury.

PubMed

Stevens, Megan; Neal, Christopher R; Salmon, Andrew H J; Bates, David O; Harper, Steven J; Oltean, Sebastian

2018-01-01

Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b). The Pod-DTR mouse was crossed with a mouse that over-expressed VEGF-A165b specifically in the podocytes (Neph-VEGF-A165b). Wild type (WT), Pod-DTR, Neph-VEGF-A165b and Pod-DTR X Neph-VEGF-A165b mice were treated with several doses of DT (1, 5, 100, and 1,000 ng/g bodyweight). Urine was collected and the glomerular water permeability (LpA/Vi) was measured ex vivo after 14 days. Structural analysis and podocyte marker expression were also assessed. Pod-DTR mice developed an increased glomerular LpA/Vi 14 days after administration of DT (all doses), which was prevented when the mice over-expressed VEGF-A165b. No major structural abnormalities, podocyte ablation or albuminuria was observed in Pod-DTR mice, indicating this to be a mild model of podocyte disease. However, a change in expression and localisation of nephrin within the podocytes was observed, indicating disruption of the slit diaphragm in the Pod-DTR mice. This was prevented in the Pod-DTR X Neph-VEGF-A165b mice. Although only a mild model of podocyte injury, over-expression of the anti-permeability VEGF-A165b isoform in the podocytes of Pod-DTR mice had a protective effect. Therefore, this study further highlights the therapeutic potential of VEGF-A165b in glomerular disease. © 2018 The Author(s) Published by S. Karger AG, Basel.

Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos.

PubMed

Bošnjak, Ivana; Pleić, Ivana Lepen; Borra, Marco; Mladineo, Ivona

2013-12-01

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

Reduction in expression of the benign AR transcriptome is a hallmark of localised prostate cancer progression.

PubMed

Stuchbery, Ryan; Macintyre, Geoff; Cmero, Marek; Harewood, Laurence M; Peters, Justin S; Costello, Anthony J; Hovens, Christopher M; Corcoran, Niall M

2016-05-24

Despite the importance of androgen receptor (AR) signalling to prostate cancer development, little is known about how this signalling pathway changes with increasing grade and stage of the disease. To explore changes in the normal AR transcriptome in localised prostate cancer, and its relation to adverse pathological features and disease recurrence. Publically accessible human prostate cancer expression arrays as well as RNA sequencing data from the prostate TCGA. Tumour associated PSA and PSAD were calculated for a large cohort of men (n=1108) undergoing prostatectomy. We performed a meta-analysis of the expression of an androgen-regulated gene set across datasets using Oncomine. Differential expression of selected genes in the prostate TCGA database was probed using the edgeR Bioconductor package. Changes in tumour PSA density with stage and grade were assessed by Student's t-test, and its association with biochemical recurrence explored by Kaplan-Meier curves and Cox regression. Meta-analysis revealed a systematic decline in the expression of a previously identified benign prostate androgen-regulated gene set with increasing tumour grade, reaching significance in nine of 25 genes tested despite increasing AR expression. These results were confirmed in a large independent dataset from the TCGA. At the protein level, when serum PSA was corrected for tumour volume, significantly lower levels were observed with increasing tumour grade and stage, and predicted disease recurrence. Lower PSA secretion-per-tumour-volume is associated with increasing grade and stage of prostate cancer, has prognostic relevance, and reflects a systematic perturbation of androgen signalling.

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary.

PubMed

Hall, Sally E; Upton, Rose M O; McLaughlin, Eileen A; Sutherland, Jessie M

2017-09-26

The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.

Bodies that Shatter: Creativity, Culture and the New Pedagogical Imaginary

ERIC Educational Resources Information Center

Harris, Anne; Lemon, Andrea

2012-01-01

Taking Butler's notion of embodied performativity in relation to education and global flow, this article uses one example of Samoan cultural dance within a community arts education programme to argue the need for localised, context- and culture-specific arts pedagogies which incorporate diversity in all its expressions, suggest an emerging…

Neurotrophin gene therapy for sustained neural preservation after deafness.

PubMed

Atkinson, Patrick J; Wise, Andrew K; Flynn, Brianna O; Nayagam, Bryony A; Hume, Clifford R; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

2012-01-01

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.

Using Nano-mechanics and Surface Acoustic Wave (SAW) for Disease Monitoring and Diagnostics at a Cellular Level in Red Blood Cells

NASA Astrophysics Data System (ADS)

Sivanantha, Ninnuja; Ma, Charles; Collins, David J.; Sesen, Muhsincan; Brenker, Jason; Coppel, Ross L.; Neild, Adrian; Alan, Tuncay

A popular approach to monitoring diseases and their diagnosis is through biological, pathological or immunological characterization. However, at a cellular level progression of certain diseases manifests itself through mechanical effects as well. Here, we present a method which exploits localised flow; surface acoustic wave (SAW) induced acoustic streaming in a 9 μL droplet to characterize the adhesive properties of red blood cells (healthy, gluteraldehyde treated and malaria infected) in approximately 50 seconds. Our results show a 79% difference in cell mobilization between healthy malaria infected RBCs (and a 39% difference between healthy and treated ones), indicating that the method can serve as a platform for rapid clinical diagnosis; where separation of two or more different cell populations in a mixed solution is desirable. It can also act as a key biomarker for monitoring some diseases offering quantitative measures of disease progression and response to therapy.

Effects of variable thermal diffusivity on the structure of convection

NASA Astrophysics Data System (ADS)

Shcheritsa, O. V.; Getling, A. V.; Mazhorova, O. S.

2018-03-01

The structure of multiscale convection in a thermally stratified plane horizontal fluid layer is investigated by means of numerical simulations. The thermal diffusivity is assumed to produce a thin boundary sublayer convectively much more unstable than the bulk of the layer. The simulated flow is a superposition of cellular structures with three different characteristic scales. In contrast to the largest convection cells, the smaller ones are localised in the upper portion of the layer. The smallest cells are advected by the larger-scale convective flows. The simulated flow pattern qualitatively resembles that observed on the Sun.

Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

PubMed Central

Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

2016-01-01

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gopalan, Vinod; Islam, Farhadul; Pillai, Suja

Purpose: This study aims to examine the expression profiles miR-1288 in oesophageal squamous cell carcinoma (ESCC). The cellular implications and target interactions of ESCC cells following miR-1288 overexpression was also examined. Methods: In total, 120 oesophageal tissues (90 primary ESCCs and 30 non-neoplastic tissues) were recruited for miR-1288 expression analysis using qRT-PCR. An exogenous miR-1288 mimic and its inhibitor were used to explore the in-vitro effects of miR-1288 on ESCC cells by performing cell proliferation, colony formation, cell invasion and migration assays. Localisation and modulatory changes of various miR-1288 regulated proteins such as FOXO1, p53, TAB3, BCL2 and kRAS wasmore » examined using immunofluorescence and western blot. Results: Overexpression of miR-1288 was more often noted in ESCC tissues when compared to non-neoplastic oesophageal tissues. High expression was often noted in high grade carcinomas and with metastases. Patients with high levels of miR-1288 expression showed a slightly better survival compared to patients with low miR-1288 levels. Furthermore, overexpression of miR-1288 showed increased cell proliferation and colony formation, improved cell migration and enhanced cell invasion properties in ESCC cells. In addition, miR-1288 overexpression in ESCC cells showed repression of cytoplasmic tumour suppressor FOXO1 protein expression. Inversely, inhibition of miR-1288 expression exhibited remarkable upregulation of FOXO1 protein, while expressions of other tested proteins remain unchanged. Conclusions: Up regulation of miR-1288 expression in ESCC tissues and miR-1288 induced oncogenic features of ESCC cells in-vitro indicates the oncogenic roles of miR-1288 in ESCCs. Overexpression of miR-1288 play a key role in the pathogenesis of ESCCs and its modulation may have potential therapeutic value in patients with ESCC. - Highlights: • miR-1288 was more often noted in neoplastic than non-neoplastic tissue. • miR-1288 overexpression increased proliferative/invasive activities of ESCC. • miR-1288 overexpression showed repression of FOXO1 protein expression. • miR-1288 functions as an oncogenic miRNA in ESCCs.« less

Does advancing male age influence the expression levels and localisation patterns of phospholipase C zeta (PLCζ) in human sperm?

PubMed Central

Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Yelumalai, Suseela; Mounce, Ginny; da Silva, Sarah J. Martins; Child, Tim; Coward, Kevin

2016-01-01

Socio-economic factors have led to an increasing trend for couples to delay parenthood. However, advancing age exerts detrimental effects upon gametes which can have serious consequences upon embryo viability. While such effects are well documented for the oocyte, relatively little is known with regard to the sperm. One fundamental role of sperm is to activate the oocyte at fertilisation, a process initiated by phospholipase C zeta (PLCζ), a sperm-specific protein. While PLCζ deficiency can lead to oocyte activation deficiency and infertility, it is currently unknown whether the expression or function of PLCζ is compromised by advancing male age. Here, we evaluate sperm motility and the proportion of sperm expressing PLCζ in 71 males (22–54 years; 44 fertile controls and 27 infertile patients), along with total levels and localisation patterns of PLCζ within the sperm head. Three different statistical approaches were deployed with male age considered both as a categorical and a continuous factor. While progressive motility was negatively correlated with male age, all three statistical models concurred that no PLCζ–related parameter was associated with male age, suggesting that advancing male age is unlikely to cause problems in terms of the sperm’s fundamental ability to activate an oocyte. PMID:27270687

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

PubMed Central

Wilson, K.; Mole, D.J.; Binnie, M.; Homer, N.Z.M.; Zheng, X.; Yard, B.A.; Iredale, J.P.; Auer, M.; Webster, S.P.

2014-01-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington’s disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

Rac1 as a potential therapeutic target for chemo-radioresistant head and neck squamous cell carcinomas (HNSCC).

PubMed

Skvortsov, S; Dudás, J; Eichberger, P; Witsch-Baumgartner, M; Loeffler-Ragg, J; Pritz, C; Schartinger, V H; Maier, H; Hall, J; Debbage, P; Riechelmann, H; Lukas, P; Skvortsova, I

2014-05-27

In order to improve therapy for HNSCC patients, novel methods to predict and combat local and/or distant tumour relapses are urgently needed. This study has been dedicated to the hypothesis that Rac1, a Rho GTPase, is implicated in HNSCC insensitivity to chemo-radiotherapy resulting in tumour recurrence development. Parental and radiation-resistant (IRR) HNSCC cells were used to support this hypothesis. All cells were investigated for their sensitivity to ionising radiation and cisplatin, Rac1 activity, its intracellular expression and subcellular localisation. Additionally, tumour tissues obtained from 60 HNSCC patients showing different therapy response were evaluated for intratumoral Rac1 expression. Radiation-resistant IRR cells also revealed resistance to cisplatin accompanied by increased expression, activity and trend towards nuclear translocation of Rac1 protein. Chemical inhibition of Rac1 expression and activity resulted in significant improvement of HNSCC sensitivity to ionising radiation and cisplatin. Preclinical results were confirmed in clinical samples. Although Rac1 was poorly presented in normal mucosa, tumour tissues revealed increased Rac1 expression. The most pronounced Rac1 presence was observed in HNSCC patients with poor early or late responses to chemo-radiotherapy. Tissues taken at recurrence were characterised not only by enhanced Rac1 expression but also increased nuclear Rac1 content. Increased expression, activity and subcellular localisation of Rac1 could be associated with lower early response rate and higher risk of tumour recurrences in HNSCC patients and warrants further validation in larger independent studies. Inhibition of Rac1 activity can be useful in overcoming treatment resistance and could be proposed for HNSCC patients with primary or secondary chemo-radioresistance.

The nuclear protein Poly(ADP-ribose) polymerase 3 (AtPARP3) is required for seed storability in Arabidopsis thaliana.

PubMed

Rissel, D; Losch, J; Peiter, E

2014-11-01

The deterioration of seeds during prolonged storage results in a reduction of viability and germination rate. DNA damage is one of the major cellular defects associated with seed deterioration. It is provoked by the formation of reactive oxygen species (ROS) even in the quiescent state of the desiccated seed. In contrast to other stages of seed life, DNA repair during storage is hindered through the low seed water content; thereby DNA lesions can accumulate. To allow subsequent seedling development, DNA repair has thus to be initiated immediately upon imbibition. Poly(ADP-ribose) polymerases (PARPs) are important components in the DNA damage response in humans. Arabidopsis thaliana contains three homologues to the human HsPARP1 protein. Of these three, only AtPARP3 was very highly expressed in seeds. Histochemical GUS staining of embryos and endosperm layers revealed strong promoter activity of AtPARP3 during all steps of germination. This coincided with high ROS activity and indicated a role of the nuclear-localised AtPARP3 in DNA repair during germination. Accordingly, stored parp3-1 mutant seeds lacking AtPARP3 expression displayed a delay in germination as compared to Col-0 wild-type seeds. A controlled deterioration test showed that the mutant seeds were hypersensitive to unfavourable storage conditions. The results demonstrate that AtPARP3 is an important component of seed storability and viability. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal, newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker.

PubMed

Smith, Susan M; Shu, Cindy; Melrose, James

2010-09-01

We undertook a comparative immunolocalisation study on type II collagen, aggrecan and perlecan in a number of 12- to 14-week-old human foetal and postnatal (7-19 months) ovine joints including finger, toe, knee, elbow, hip and shoulder. This demonstrated that perlecan followed a virtually identical immunolocalisation pattern to that of type II collagen in the foetal tissues, but a slightly divergent localisation pattern in adult tissues. Aggrecan was also localised in the cartilaginous joint tissues, which were clearly delineated by toluidine blue staining and the type II collagen immunolocalisations. It was also present in the capsular joint tissues and in ligaments and tendons in the joint, which stained poorly or not at all with toluidine blue. In higher power microscopic views, antibodies to perlecan also stained small blood vessels in the synovial lining tissues of the joint capsule; however, this was not discernable in low power macroscopic views where the immunolocalisation of perlecan to pericellular regions of cells within the cartilaginous rudiments was a predominant feature. Perlecan was also evident in small blood vessels in stromal connective tissues associated with the cartilage rudiments and with occasional nerves in the vicinity of the joint tissues. Perlecan was expressed by rounded cells in the enthesis attachment points of tendons to bone and in rounded cells in the inner third of the meniscus, which stained prominently with type II collagen and aggrecan identifying the chondrogenic background of these cells and local compressive loads. Flattened cells within the tendon and in the surface laminas of articular cartilages and the meniscus did not express perlecan. Collected evidence presented herein, therefore, indicates that besides being a basement membrane component, perlecan is also a marker of chondrogenic cells in prenatal cartilages. In postnatal cartilages, perlecan displayed a pericellular localisation pattern rather than the territorial or interterritorial localisation it displayed in foetal cartilages. This may reflect processing of extracellular perlecan presumably as a consequence of intrinsic biomechanical loading on these tissues or to divergent functions for perlecan and type II collagen in adult compared to prenatal tissues.

Expression and in vitro regulation of integrins by normal human urothelial cells.

PubMed

Southgate, J; Kennedy, W; Hutton, K A; Trejdosiewicz, L K

1995-08-01

Integrins are thought to be essential adhesion receptors for the maintenance of tissue histioarchitecture. The purpose of this study was to determine integrin expression patterns in the human stratified transitional epithelium of the urinary tract (urothelium). In situ expression patterns were compared with in vitro expression, using a normal cell culture model system in which the effects of cell stratification can be studied independently of differentiation. By immunohistological criteria, the urothelia of bladder, ureter and renal pelvis expressed alpha 2 beta 1 and alpha 3 beta 1 integrins in all layers at intercellular junctions, and cytoplasmically in the lower strata. By contrast, alpha 6 beta 4 and occasionally alpha v beta 4 were expressed only by basal cells and localised to the basal lamina. These expression patterns were unaltered in specimens where an inflammatory cell infiltrate was present. In long-term cultures of normal urothelial cells maintained in a low-Ca++ serum-free medium, the monolayer cultures expressed alpha 2 beta 1, alpha 3 beta 1 and alpha 5 beta 1 integrins at intercellular junctions and in cytoplasmic inclusions, whereas alpha 6 beta 4 was distributed in a random pattern over the substratum. Increasing exogenous Ca++ concentrations induced cell stratification and desmosome formation, but not cytodifferentiation. Under these conditions, alpha 6 beta 4 became cell-, rather than substratum-associated, localising particularly to filopodia and lamellipodia. Quantitation of integrin expression by flow cytometry confirmed increased surface expression of alpha 6 beta 4 in high Ca++ media, and also of alpha 3 and alpha 5, but not alpha 2, subunits. These results suggest that alpha 2 beta 1 and alpha 3 beta 1 integrins, although differentially regulated, are mainly involved in homotypic cell-cell interactions and the maintenance of a stratified morphology, whereas alpha 6 beta 4 is the principal integrin involved in substratum adhesion.

Philanthropic Engagement in Education: Localised Expressions of Global Flows in India

ERIC Educational Resources Information Center

Srivastava, Prachi

2016-01-01

This article argues that the rise of domestic and international philanthropic engagement in education in India cannot be understood in isolation; rather, it is part of a broader trend of what is termed "new global philanthropy in education" in the Global South. Central to understanding the nature of this engagement is the localised…

Differential pattern of HIF-1α expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect.

PubMed

Wozny, Anne-Sophie; Lauret, Alexandra; Battiston-Montagne, Priscillia; Guy, Jean-Baptiste; Beuve, Michael; Cunha, Micaela; Saintigny, Yannick; Blond, Emilie; Magne, Nicolas; Lalle, Philippe; Ardail, Dominique; Alphonse, Gersende; Rodriguez-Lafrasse, Claire

2017-05-09

Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1α (HIF-1α) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1α. Under hypoxia, compared to non-CSCs, HIF-1α is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1α expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1α expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1α. Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1α inhibition. Hypoxia-inducible factor-1α plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1α targeting an attractive therapeutic challenge.

Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

PubMed

Bolte, Petra; Bleibaum, Florian; Einwich, Angelika; Günther, Anja; Liedvogel, Miriam; Heyers, Dominik; Depping, Anne; Wöhlbrand, Lars; Rabus, Ralf; Janssen-Bienhold, Ulrike; Mouritsen, Henrik

2016-01-01

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

[Expression of EphA2 in Metastatic and Non-Metastatic Primary Uveal Melanoma].

PubMed

Vukoja, V; Brandenbusch, T; Tura, A; Nassar, K; Rohrbach, D J M; Lüke, M; Grisanti, S; Lüke, J

2016-03-01

Little is known about how the expression of Ephrin type-A receptor 2 (EphA2) influences cell-cell adhesion, migration, angiogenesis, and the formation of vasculogenic mimicry (VM) channels in uveal melanomas or how this may be related to the rate of metastasis. Paraffin embedded sections of 50 histopathologically well characterised primary uveal melanomas (mean largest tumour diameter: 16.3 mm) were evaluated with respect to the expression of EphA2. Systemic metastasis was detected in 29 patients. The remaining 21 patients were followed for a mean of 10 years. Tumour angiogensis was analysed by endoglin expression (CD105), the activity of the mature vascular system (von Willebrand factor) and the presence of VM (CD31/PAS staining). All uveal melanomas expressed EphA2, with a mean of 95.93 % positive cells ± SD: 6.3 %. There was no significant association between EphA2 and the rate of metastases (p = 0.196), endoglin expression (p = 0.652), VM (p = 0.267) or with any other clinical or histopathological factors (p < 0.05). However, there was significant up-regulation of EphA2 in the nucleus of the metastatic uveal melanoma subgroup, while cytoplasmatic localisation in the subgroup was associated with better prognosis (p = 0.006). There were low levels of EphA2 expression in the specific retinal layers, the ciliary and corneal epithelium, and the choroidal and corneal endothelium. Nuclear expression of EphA2 in this series of large tumours was significantly associated with an increased rate of metastasis. On the other hand, cytoplasmic localisation was associated with a better prognosis. As there was no correlation between EphA2 expression and angiogenesis, the mature vasculature or VM, EphA2 appears to become less important in the advanced stages of the disease. Georg Thieme Verlag KG Stuttgart · New York.

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

PubMed Central

Schmied, Stéfanie; Affentranger, Sarah; Parvanova, Iana; Kang'a, Simon; Nene, Vishvanath; Katzer, Frank; McKeever, Declan; Müller, Joachim; Bishop, Richard; Pain, Arnab; Dobbelaere, Dirk A. E.

2009-01-01

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins. PMID:19325907

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

PubMed

Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

2014-03-01

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Xanthine oxido-reductase activity in ischemic human and rat intestine.

PubMed

Bianciardi, Paola; Scorza, Roberto; Ghilardi, Giorgio; Samaja, Michele

2004-09-01

We measured time course and extent of xanthine dehydrogenase (XD) to xanthine oxidase (XO) conversion in ischemic human and rat intestine. To model normothermic no-flow ischemia, we incubated fresh biopsies for 0, 2, 4, 8 and 16h. At t = 0h, XO was less in humans than in rats (P < 0.0004), while XD was essentially the same (P = NS). After 16h incubation at 37 degrees C, there was no appreciable XD-to-XO conversion and no change in neither XO nor XD activity in human intestine. In contrast, the rat intestine had XO/(XO + XD) ratio doubled in the first 2h and then maintained that value until t = 16 h. In conclusion, no XO-to-XD conversion was appreciable after 16 h no-flow normothermic ischemia in human intestine; in contrast, XO activity in rats increased sharply after the onset of ischemia. An immunohistochemical labelling study shows that, whereas XO + XD expression in liver tissue is localised in both hepatocytes and endothelial cells, in the intestine that expression is mostly localised in epithelial cells. We conclude that XO may be considered as a major source of reactive oxygen species in rats but not in humans.

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions

PubMed Central

Sunter, Jack D.; Benz, Corinna; Andre, Jane; Whipple, Sarah; McKean, Paul G.; Gull, Keith; Ginger, Michael L.; Lukeš, Julius

2015-01-01

ABSTRACT The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure – the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms. PMID:26148511

Dupuytren's: a systems biology disease

PubMed Central

2011-01-01

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder of the palm of the hands leading to digital contracture. DD commonly occurs in individuals of northern European extraction. Cellular components and processes associated with DD pathogenesis include altered gene and protein expression of cytokines, growth factors, adhesion molecules, and extracellular matrix components. Histology has shown increased but varying levels of particular types of collagen, myofibroblasts and myoglobin proteins in DD tissue. Free radicals and localised ischaemia have been suggested to trigger the proliferation of DD tissue. Although the existing available biological information on DD may contain potentially valuable (though largely uninterpreted) information, the precise aetiology of DD remains unknown. Systems biology combines mechanistic modelling with quantitative experimentation in studies of networks and better understanding of the interaction of multiple components in disease processes. Adopting systems biology may be the ideal approach for future research in order to improve understanding of complex diseases of multifactorial origin. In this review, we propose that DD is a disease of several networks rather than of a single gene, and show that this accounts for the experimental observations obtained to date from a variety of sources. We outline how DD may be investigated more effectively by employing a systems biology approach that considers the disease network as a whole rather than focusing on any specific single molecule. PMID:21943049

Multi-layered epigenetic mechanisms contribute to transcriptional memory in T lymphocytes.

PubMed

Dunn, Jennifer; McCuaig, Robert; Tu, Wen Juan; Hardy, Kristine; Rao, Sudha

2015-05-06

Immunological memory is the ability of the immune system to respond more rapidly and effectively to previously encountered pathogens, a key feature of adaptive immunity. The capacity of memory T cells to "remember" previous cellular responses to specific antigens ultimately resides in their unique patterns of gene expression. Following re-exposure to an antigen, previously activated genes are transcribed more rapidly and robustly in memory T cells compared to their naïve counterparts. The ability for cells to remember past transcriptional responses is termed "adaptive transcriptional memory". Recent global epigenome studies suggest that epigenetic mechanisms are central to establishing and maintaining transcriptional memory, with elegant studies in model organisms providing tantalizing insights into the epigenetic programs that contribute to adaptive immunity. These epigenetic mechanisms are diverse, and include not only classical acetylation and methylation events, but also exciting and less well-known mechanisms involving histone structure, upstream signalling pathways, and nuclear localisation of genomic regions. Current global health challenges in areas such as tuberculosis and influenza demand not only more effective and safer vaccines, but also vaccines for a wider range of health priorities, including HIV, cancer, and emerging pathogens such as Ebola. Understanding the multi-layered epigenetic mechanisms that underpin the rapid recall responses of memory T cells following reactivation is a critical component of this development pathway.

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed Central

Matamales, Miriam

2012-01-01

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance. PMID:24327840

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed

Matamales, Miriam

2012-12-19

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

PubMed

Matamales, Miriam

2012-01-01

Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

EphB4 localises to the nucleus of prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mertens-Walker, Inga, E-mail: inga.mertenswalker@qut.edu.au; Australian Prostate Cancer Research Centre—Queensland, Translational Research Institute, 37 Kent Street, Woolloongabba 4102, QLD; Lisle, Jessica E.

2015-04-10

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of bothmore » NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor. - Highlights: • The EphB4 protein can be found in the nucleus of prostate cancer cell lines. • EphB4 contains two functional nuclear localisation signals. • Chromatin immunoprecipitation has identified potential genome sequences to which EphB4 binds. • Lef1 is a confirmed target for EphB4-mediated gene regulation.« less

Underwater Acoustic Source Localisation Among Blind and Sighted Scuba Divers: Comparative study.

PubMed

Cambi, Jacopo; Livi, Ludovica; Livi, Walter

2017-05-01

Many blind individuals demonstrate enhanced auditory spatial discrimination or localisation of sound sources in comparison to sighted subjects. However, this hypothesis has not yet been confirmed with regards to underwater spatial localisation. This study therefore aimed to investigate underwater acoustic source localisation among blind and sighted scuba divers. This study took place between February and June 2015 in Elba, Italy, and involved two experimental groups of divers with either acquired (n = 20) or congenital (n = 10) blindness and a control group of 30 sighted divers. Each subject took part in five attempts at an under-water acoustic source localisation task, in which the divers were requested to swim to the source of a sound originating from one of 24 potential locations. The control group had their sight obscured during the task. The congenitally blind divers demonstrated significantly better underwater sound localisation compared to the control group or those with acquired blindness ( P = 0.0007). In addition, there was a significant correlation between years of blindness and underwater sound localisation ( P <0.0001). Congenital blindness was found to positively affect the ability of a diver to recognise the source of a sound in an underwater environment. As the correct localisation of sounds underwater may help individuals to avoid imminent danger, divers should perform sound localisation tests during training sessions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shaw, Debra J.; Morse, Robert; Todd, Adrian G.

The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import,more » they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.« less

Evolving adoptive cellular therapies in urological malignancies.

PubMed

Wong, Yien Ning Sophia; Joshi, Kroopa; Pule, Martin; Peggs, Karl S; Swanton, Charles; Quezada, Sergio A; Linch, Mark

2017-06-01

Immunotherapies have long been used to treat urological cancers but rarely lead to cure. In the past 5 years, success of immune checkpoint inhibition has led to a resurgence of enthusiasm for immunotherapy in the treatment of solid tumours. Increased understanding of tumour immune biology, technological advancements of gene transfer and cell culture, and improved clinical infrastructures for routine delivery of cell products, has made cell-based immunotherapeutics a real prospect for cancer therapy. These scientific and clinical activities, attempting to exploit the innate and adaptive immune systems for therapeutic gain, are well exemplified by the urological malignancies of renal, bladder, prostate, and penile cancer, a group of anatomically localised diseases, each with a distinct biology and different immunotherapeutic challenges. In this Review, we present the results of clinical studies investigating autologous cellular therapies in urological malignancies. Specifically, we discuss the rationale for upcoming studies, and how novel therapies and adoptive cell combinations can be used for personalised cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability.

PubMed

Myers, Katie N; Barone, Giancarlo; Ganesh, Anil; Staples, Christopher J; Howard, Anna E; Beveridge, Ryan D; Maslen, Sarah; Skehel, J Mark; Collis, Spencer J

2016-10-14

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.

Characterisation, analysis of expression and localisation of circadian clock genes from the perspective of photoperiodism in the aphid Acyrthosiphon pisum.

PubMed

Barberà, Miquel; Collantes-Alegre, Jorge Mariano; Martínez-Torres, David

2017-04-01

Aphids are typical photoperiodic insects that switch from viviparous parthenogenetic reproduction typical of long day seasons to oviparous sexual reproduction triggered by the shortening of photoperiod in autumn yielding an overwintering egg in which an embryonic diapause takes place. While the involvement of the circadian clock genes in photoperiodism in mammals is well established, there is still some controversy on their participation in insects. The availability of the genome of the pea aphid Acyrthosiphon pisum places this species as an excellent model to investigate the involvement of the circadian system in the aphid seasonal response. In the present report, we have advanced in the characterisation of the circadian clock genes and showed that these genes display extensive alternative splicing. Moreover, the expression of circadian clock genes, analysed at different moments of the day, showed a robust cycling of central clock genes period and timeless. Furthermore, the rhythmic expression of these genes was shown to be rapidly dampened under DD (continuous darkness conditions), thus supporting the model of a seasonal response based on a heavily dampened circadian oscillator. Additionally, increased expression of some of the circadian clock genes under short-day conditions suggest their involvement in the induction of the aphid seasonal response. Finally, in situ localisation of transcripts of genes period and timeless in the aphid brain revealed the site of clock neurons for the first time in aphids. Two groups of clock cells were identified: the Dorsal Neurons (DN) and the Lateral Neurons (LN), both in the protocerebrum. Copyright © 2017 Elsevier Ltd. All rights reserved.

Astrocyte activation and wound healing in intact-skull mouse after focal brain injury.

PubMed

Suzuki, Takayuki; Sakata, Honami; Kato, Chiaki; Connor, John A; Morita, Mitsuhiro

2012-12-01

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery. © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Underwater Acoustic Source Localisation Among Blind and Sighted Scuba Divers

PubMed Central

Cambi, Jacopo; Livi, Ludovica; Livi, Walter

2017-01-01

Objectives Many blind individuals demonstrate enhanced auditory spatial discrimination or localisation of sound sources in comparison to sighted subjects. However, this hypothesis has not yet been confirmed with regards to underwater spatial localisation. This study therefore aimed to investigate underwater acoustic source localisation among blind and sighted scuba divers. Methods This study took place between February and June 2015 in Elba, Italy, and involved two experimental groups of divers with either acquired (n = 20) or congenital (n = 10) blindness and a control group of 30 sighted divers. Each subject took part in five attempts at an under-water acoustic source localisation task, in which the divers were requested to swim to the source of a sound originating from one of 24 potential locations. The control group had their sight obscured during the task. Results The congenitally blind divers demonstrated significantly better underwater sound localisation compared to the control group or those with acquired blindness (P = 0.0007). In addition, there was a significant correlation between years of blindness and underwater sound localisation (P <0.0001). Conclusion Congenital blindness was found to positively affect the ability of a diver to recognise the source of a sound in an underwater environment. As the correct localisation of sounds underwater may help individuals to avoid imminent danger, divers should perform sound localisation tests during training sessions. PMID:28690888

Hydrodynamic gene delivery in human skin using a hollow microneedle device.

PubMed

Dul, M; Stefanidou, M; Porta, P; Serve, J; O'Mahony, C; Malissen, B; Henri, S; Levin, Y; Kochba, E; Wong, F S; Dayan, C; Coulman, S A; Birchall, J C

2017-11-10

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dawson, P., E-mail: philip.dawson@manchester.ac.uk; Schulz, S.; Oliver, R. A.

In this paper, we compare and contrast the experimental data and the theoretical predictions of the low temperature optical properties of polar and nonpolar InGaN/GaN quantum well structures. In both types of structure, the optical properties at low temperatures are governed by the effects of carrier localisation. In polar structures, the effect of the in-built electric field leads to electrons being mainly localised at well width fluctuations, whereas holes are localised at regions within the quantum wells, where the random In distribution leads to local minima in potential energy. This leads to a system of independently localised electrons and holes.more » In nonpolar quantum wells, the nature of the hole localisation is essentially the same as the polar case but the electrons are now coulombically bound to the holes forming localised excitons. These localisation mechanisms are compatible with the large photoluminescence linewidths of the polar and nonpolar quantum wells as well as the different time scales and form of the radiative recombination decay curves.« less

Robust imaging and gene delivery to study human lymphoblastoid cell lines.

PubMed

Jolly, Lachlan A; Sun, Ying; Carroll, Renée; Homan, Claire C; Gecz, Jozef

2018-06-20

Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.

Learning from Heterogeneous Data Sources: An Application in Spatial Proteomics

PubMed Central

Breckels, Lisa M.; Holden, Sean B.; Wojnar, David; Mulvey, Claire M.; Christoforou, Andy; Groen, Arnoud; Trotter, Matthew W. B.; Kohlbacher, Oliver; Lilley, Kathryn S.; Gatto, Laurent

2016-01-01

Sub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis. PMID:27175778

Proteomics profiling of interactome dynamics by colocalisation analysis (COLA).

PubMed

Mardakheh, Faraz K; Sailem, Heba Z; Kümper, Sandra; Tape, Christopher J; McCully, Ryan R; Paul, Angela; Anjomani-Virmouni, Sara; Jørgensen, Claus; Poulogiannis, George; Marshall, Christopher J; Bakal, Chris

2016-12-20

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein-protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.

The conserved Wdr8-hMsd1/SSX2IP complex localises to the centrosome and ensures proper spindle length and orientation

PubMed Central

Hori, Akiko; Morand, Agathe; Ikebe, Chiho; Frith, David; Snijders, Ambrosius P.; Toda, Takashi

2015-01-01

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, as one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation. PMID:26545777

Epstein-Barr virus growth/latency III program alters cellular microRNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cameron, Jennifer E.; Tulane Cancer Center, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL79, New Orleans, LA 70112; Fewell, Claire

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lowermore » in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.« less

Localised boundary air layer and clothing evaporative resistances for individual body segments.

PubMed

Wang, Faming; del Ferraro, Simona; Lin, Li-Yen; Sotto Mayor, Tiago; Molinaro, Vincenzo; Ribeiro, Miguel; Gao, Chuansi; Kuklane, Kalev; Holmér, Ingvar

2012-01-01

Evaporative resistance is an important parameter to characterise clothing thermal comfort. However, previous work has focused mainly on either total static or dynamic evaporative resistance. There is a lack of investigation of localised clothing evaporative resistance. The objective of this study was to study localised evaporative resistance using sweating thermal manikins. The individual and interaction effects of air and body movements on localised resultant evaporative resistance were examined in a strict protocol. The boundary air layer's localised evaporative resistance was investigated on nude sweating manikins at three different air velocity levels (0.18, 0.48 and 0.78 m/s) and three different walking speeds (0, 0.96 and 1.17 m/s). Similarly, localised clothing evaporative resistance was measured on sweating manikins at three different air velocities (0.13, 0.48 and 0.70 m/s) and three walking speeds (0, 0.96 and 1.17 m/s). Results showed that the wind speed has distinct effects on local body segments. In contrast, walking speed brought much more effect on the limbs, such as thigh and forearm, than on body torso, such as back and waist. In addition, the combined effect of body and air movement on localised evaporative resistance demonstrated that the walking effect has more influence on the extremities than on the torso. Therefore, localised evaporative resistance values should be provided when reporting test results in order to clearly describe clothing local moisture transfer characteristics. Localised boundary air layer and clothing evaporative resistances are essential data for clothing design and assessment of thermal comfort. A comprehensive understanding of the effects of air and body movement on localised evaporative resistance is also necessary by both textile and apparel researchers and industry.

Cytosolic glucocorticoid receptor in the testis of Bufo arenarum: seasonal changes in its binding parameters.

PubMed

Denari, Daniela; Ceballos, Nora R

2006-07-01

Glucocorticoids (GC) are the hormonal mediators of stress. In mammals, high levels of GC have negative effects on reproductive physiology. For instance, GC can inhibit testicular testosterone synthesis by acting via glucocorticoid receptors (GR), the extent of the inhibition being dependent on GC levels. However, the effect of GC on testicular function and even the presence of GR in amphibians are still unclear. The purpose of this work was to characterise testicular cytosolic GR in Bufo arenarum, determining the seasonal changes in its binding parameters as well as the intratesticular localisation. The binding assays were performed in testis cytosol with [3H]dexamethasone (DEX) and [3H]corticosterone (CORT). Binding kinetics of DEX and CORT fitted to a one-site model. Results were expressed as means +/- standard error. Apparent number of binding sites (Bapp) was similar for both steroids (Bapp DEX = 352.53 +/- 72.08 fmol/mg protein; Bapp CORT = 454.24 +/- 134.97 fmol/mg protein) suggesting that both hormones bind to the same site. Competition studies with different steroids showed that the order of displacement of [3H]DEX and [3H]CORT specific binding is: DEX approximately RU486 approximately deoxycorticosterone (DOC) > CORT > aldosterone > RU28362 > progesterone > 11-dehydroCORT. The affinity of GR for DEX (Kd = 11.2 +/- 1.5 nM) remained constant throughout the year while circulating CORT clearly increased during the reproductive season. Therefore, testis sensitivity to GC action would depend mainly on inactivating mechanisms (11beta-hydroxysteroid dehydrogenase type 2) and CORT plasma levels. Since total and free CORT are higher in the reproductive than in the non-reproductive period, the magnitude of GC actions could be higher during the breeding season. The intratesticular localisation of the GR was determined after separation of cells by a Percoll density gradient followed by binding assays in each fraction. DEX binds to two different fractions corresponding to Leydig and Sertoli cells. In conclusion, in the testis of B. arenarum GC could regulate the function of both cellular types particularly during breeding when CORT reaches the highest plasma concentration.

Breathing pulses in singularly perturbed reaction-diffusion systems

NASA Astrophysics Data System (ADS)

Veerman, Frits

2015-07-01

The weakly nonlinear stability of pulses in general singularly perturbed reaction-diffusion systems near a Hopf bifurcation is determined using a centre manifold expansion. A general framework to obtain leading order expressions for the (Hopf) centre manifold expansion for scale separated, localised structures is presented. Using the scale separated structure of the underlying pulse, directly calculable expressions for the Hopf normal form coefficients are obtained in terms of solutions to classical Sturm-Liouville problems. The developed theory is used to establish the existence of breathing pulses in a slowly nonlinear Gierer-Meinhardt system, and is confirmed by direct numerical simulation.

The accuracy of colonoscopic localisation of colorectal tumours: a prospective, multi-centred observational study.

PubMed

Johnstone, M S; Moug, S J

2014-05-01

Colonoscopy is essential for accurate pre-operative colorectal tumour localisation, but its accuracy for localisation remains undetermined due to limitations of previous work. This study aimed to establish the accuracy of colonoscopic localisation and to determine how frequently inaccuracy results in altered surgical management. A prospective, multi-centred, powered observational study recruited 79 patients with colorectal tumours that underwent curative surgical resection. Patient and colonoscopic factors were recorded. Pre-operative colonoscopic and radiological lesion localisations were compared to intra-operative localisation using pre-defined anatomical bowel segments to determine accuracy, with changes in planned surgical management documented. Colonoscopy accurately located the colorectal tumour in 64/79 patients (81%). Five out of 15 inaccurately located patients required on-table alteration in planned surgical management. Pre-operative imaging was unable to visualise the primary tumour in 23.1% of cases, a finding that was more prevalent amongst bowel screener patients compared to symptomatic patients (45.8% vs. 13%; p = 0.003). Colonoscopic lesion localisation is inaccurate in 19.0% of cases and occurred throughout the colon with a change in on-table surgical management in 6.3%. With CT unable to visualise lesions in just under a quarter of cases, particularly in the screening population, preoperative localisation is heavily reliant on colonoscopy.

Modulation of microRNA-mRNA Target Pairs by Human Papillomavirus 16 Oncoproteins

PubMed Central

Harden, Mallory E.; Prasad, Nripesh; Griffiths, Anthony

2017-01-01

ABSTRACT The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation. PMID:28049151

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.

PubMed

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-07-24

R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5-E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development

PubMed Central

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-01-01

Background R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Results Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5–E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. Conclusion These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway. PMID:18651984

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model

PubMed Central

Briant, Kit; Streit, Anne-Kathrin; Thomson, Steven; Koay, Yee Hui

2016-01-01

ABSTRACT Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1. PMID:27519691

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model.

PubMed

Uggenti, Carolina; Briant, Kit; Streit, Anne-Kathrin; Thomson, Steven; Koay, Yee Hui; Baines, Richard A; Swanton, Eileithyia; Manson, Forbes D

2016-11-01

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca 2+ -gated Cl - channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl - ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl - conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1. © 2016. Published by The Company of Biologists Ltd.

Elution of Labile Fluorescent Dye from Nanoparticles during Biological Use

PubMed Central

Tenuta, Tiziana; Monopoli, Marco P.; Kim, JongAh; Salvati, Anna; Dawson, Kenneth A.; Sandin, Peter; Lynch, Iseult

2011-01-01

Cells act as extremely efficient filters for elution of unbound fluorescent tags or impurities associated with nanoparticles, including those that cannot be removed by extensive cleaning. This has consequences for quantification of nanoparticle uptake and sub-cellular localization in vitro and in vivo as a result of the presence of significant amount of labile dye even following extensive cleaning by dialysis. Polyacrylamide gel electrophoresis (PAGE) can be used to monitor the elution of unbound fluorescent probes from nanoparticles, either commercially available or synthesized in-house, and to ensure their complete purification for biological studies, including cellular uptake and sub-cellular localisation. Very different fluorescence distribution within cells is observed after short dialysis times versus following extensive dialysis against a solvent in which the free dye is more soluble, due to the contribution from free dye. In the absence of an understanding of the presence of residual free dye in (most) labeled nanoparticle solutions, the total fluorescence intensity in cells following exposure to nanoparticle solutions could be mis-ascribed to the presence of nanoparticles through the cell, rather than correctly assigned to either a combination of free-dye and nanoparticle-bound dye, or even entirely to free dye depending on the exposure conditions (i.e. aggregation of the particles etc). Where all of the dye is nanoparticle-bound, the particles are highly localized in sub-cellular organelles, likely lysosomes, whereas in a system containing significant amounts of free dye, the fluorescence is distributed through the cell due to the free diffusion of the molecule dye across all cellular barriers and into the cytoplasm. PMID:21998668

Investigation of the distribution of localised and extended states in amorphous MoOx

NASA Astrophysics Data System (ADS)

Dizayee, Wala; Ying, Minju; Griffin, Jonathan; Alqahtani, Mohammed S.; Buckley, Alastair; Fox, A. Mark; Gehring, Gillian A.

2018-05-01

Amorphous films of MoOx have both structural disorder and also chemical disorder for x<3. We have shown that this disorder can introduce localised states in thin films and have shown that the existence of localised states can be deduced from the XPS data that identifies the relevant occupations of different ionisation states of the Mo ions. This effect, which depends on both the oxygen concentration and the method of fabrication, is more important than electron-electron interactions in producing the observed localisation. We have also shown that magneto-optical dichroism is also a powerful technique to determine the energy distribution of localised and delocalised states.

Visual detail about the body modulates tactile localisation biases.

PubMed

Margolis, Aaron N; Longo, Matthew R

2015-02-01

The localisation of tactile stimuli requires the integration of visual and somatosensory inputs within an internal representation of the body surface and is prone to consistent bias. Joints may play a role in segmenting such internal body representations, and may therefore influence tactile localisation biases, although the nature of this influence remains unclear. Here, we investigate the relationship between conceptual knowledge of joint locations and tactile localisation biases on the hand. In one task, participants localised tactile stimuli applied to the dorsum of their hand. A distal localisation bias was observed in all participants, consistent with previous results. We also manipulated the availability of visual information during this task, to determine whether the absence of this information could account for the distal bias observed here and by Mancini et al. (Neuropsychologia 49:1194-1201, 2011). The observed distal bias increased in magnitude when visual information was restricted, without a corresponding decrease in precision. In a separate task, the same participants indicated, from memory, knuckle locations on a silhouette image of their hand. Analogous distal biases were also seen in the knuckle localisation task. The accuracy of conceptual joint knowledge was not correlated with tactile localisation bias magnitude, although a similarity in observed bias direction suggests that both tasks may rely on a common, higher-order body representation. These results also suggest that distortions of conceptual body representation may be more common in healthy individuals than previously thought.

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

PubMed Central

Zwaenepoel, Karen; Louis, Justin V; Goris, Jozef; Janssens, Veerle

2008-01-01

Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. PMID:18715506

LIM domain protein TES changes its conformational states in different cellular compartments.

PubMed

Zhong, Yingli; Zhu, Jiaolian; Wang, Yan; Zhou, Jianlin; Ren, Kaiqun; Ding, Xiaofeng; Zhang, Jian

2009-01-01

The human TESTIN (TES) is a putative tumor suppressor and localizes to the cytoplasm as a component of focal adhesions and cell contacts. TES contains a PET domain in the NH(2)-terminus and three tandem LIM domains in the COOH-terminus. It has been hypothesized that interactions between two termini of TES might lead to a "closed" conformational state of the protein. Here, we provide evidence for different conformational states of TES. We confirmed that the NH(2)-terminus of TES can interact with its third LIM domain in the COOH-terminus by GST pull-down assays. In addition, antisera against the full-length or two truncations of TES were prepared to examine the relationship between the conformation and cellular distribution of the protein. We found that these antisera recognize different regions of TES and showed that TES is co-localised with the marker protein B23 in nucleolus, in addition to its localization in endoplasmic reticulum (ER). Furthermore, our co-immunoprecipitation (co-IP) analysis of TES and B23 demonstrated their co-existence in the same complex. Taken together, our results suggest that TES has different conformational states in different cellular compartments, and a "closed" conformational state of TES may be involved in nucleolar localization.

Unique spatial and cellular expression patterns of Hoxa5, Hoxb4 and Hoxb6 proteins in normal developing murine lung are modified in pulmonary hypoplasia

PubMed Central

Volpe, MaryAnn Vitoria; Wang, Karen Ting Wai; Nielsen, Heber Carl; Chinoy, Mala Romeshchandra

2009-01-01

Background Hox transcription factors modulate signaling pathways controlling organ morphogenesis and maintain cell fate and differentiation in adults. Retinoid signaling, key in regulating Hox expression, is altered in pulmonary hypoplasia. Information on pattern-specific expression of Hox proteins in normal lung development and in pulmonary hypoplasia is minimal. Our objective was to determine how pulmonary hypoplasia alters temporal, spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 proteins compared to normal lung development. Methods Temporal, spatial and cellular Hoxa5, Hoxb4 and Hoxb6 expression was studied in normal (untreated) and nitrofen-induced hypoplastic (NT-PH) lungs from gestational day 13.5, 16, 19 fetuses and neonates using western blot and immunohistochemistry. Results Modification of protein levels and spatial and cellular Hox expression patterns in NT-PH lungs was consistent with delayed lung development. Distinct protein isoforms were detected for each Hox protein. Expression levels of the Hoxa5 and Hoxb6 isoforms changed with development and further in NT-PH lungs. Compared to normal lungs, Gd19 and neonatal NT-PH lungs had decreased Hoxb6 and increased Hoxa5 and Hoxb4. Hoxa5 cellular localization changed from mesenchyme to epithelia earlier in normal lungs. Hoxb4 was expressed in mesenchyme and epithelial cells throughout development. Hoxb6 remained mainly in mesenchymal cells around distal airways. Conclusions Unique spatial and cellular expression of Hoxa5, Hoxb4 and Hoxb6 participates in branching morphogenesis and terminal sac formation. Altered Hox protein temporal and cellular balance of expression either contributes to pulmonary hypoplasia or functions as a compensatory mechanism attempting to correct abnormal lung development and maturation in this condition. PMID:18553509

Cellular degradation activity is maintained during aging in long-living queen bees.

PubMed

Hsu, Chin-Yuan; Qiu, Jiantai Timothy; Chan, Yu-Pei

2016-11-01

Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

Anderson localisation and optical-event horizons in rogue-soliton generation.

PubMed

Saleh, Mohammed F; Conti, Claudio; Biancalana, Fabio

2017-03-06

We unveil the relation between the linear Anderson localisation process and nonlinear modulation instability. Anderson localised modes are formed in certain temporal intervals due to the random background noise. Such localised modes seed the formation of solitary waves that will appear during the modulation instability process at those preferred intervals. Afterwards, optical-event horizon effects between dispersive waves and solitons produce an artificial collective acceleration that favours the collision of solitons, which could eventually lead to a rogue-soliton generation.

STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.

PubMed

Woroniuk, Anna; Porter, Andrew; White, Gavin; Newman, Daniel T; Diamantopoulou, Zoi; Waring, Thomas; Rooney, Claire; Strathdee, Douglas; Marston, Daniel J; Hahn, Klaus M; Sansom, Owen J; Zech, Tobias; Malliri, Angeliki

2018-05-29

The perinuclear actin cap is an important cytoskeletal structure that regulates nuclear morphology and re-orientation during front-rear polarisation. The mechanisms regulating the actin cap are currently poorly understood. Here, we demonstrate that STEF/TIAM2, a Rac1 selective guanine nucleotide exchange factor, localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB (NMMIIB), where it regulates perinuclear Rac1 activity. We show that STEF depletion reduces apical perinuclear actin cables (a phenotype rescued by targeting active Rac1 to the nuclear envelope), increases nuclear height and impairs nuclear re-orientation. STEF down-regulation also reduces perinuclear pMLC and decreases myosin-generated tension at the nuclear envelope, suggesting that STEF-mediated Rac1 activity regulates NMMIIB activity to promote stabilisation of the perinuclear actin cap. Finally, STEF depletion decreases nuclear stiffness and reduces expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

Beyond the redox imbalance: oxidative stress contributes to an impaired GLUT3 modulation in Huntington's disease

PubMed Central

Covarrubias-Pinto, Adriana; Moll, Pablo; Solís-Maldonado, Macarena; Acuña, Aníbal I.; Riveros, Andrea; Miró, María Paz; Papic, Eduardo; Beltrán, Felipe A.; Cepeda, Carlos; Concha, Ilona I.; Brauchi, Sebastián; Castro, Maite A.

2016-01-01

Failure in energy metabolism and oxidative damage are associated with Huntington’s disease (HD). Ascorbic acid released during synaptic activity inhibits use of neuronal glucose, favouring lactate uptake to sustain brain activity. Here, we observe a decreased expression of GLUT3 in STHdhQ111 cells (HD cells) and R6/2 mice (HD mice). Localisation of GLUT3 is decreased at the plasma membrane in HD cells affecting the modulation of glucose uptake by ascorbic acid. An ascorbic acid analogue without antioxidant activity is able to inhibit glucose uptake in HD cells. The impaired modulation of glucose uptake by ascorbic acid is directly related to ROS levels indicating that oxidative stress sequesters the ability of ascorbic acid to modulate glucose utilisation. Therefore, in HD, a decrease in GLUT3 localisation at the plasma membrane would contribute to an altered neuronal glucose uptake during resting periods while redox imbalance should contribute to metabolic failure during synaptic activity. PMID:26456058

Epidemiological Differences Between Localised and Non-Localised Low Back Pain

PubMed Central

Coggon, David; Ntani, Georgia; Walker-Bone, Karen; Palmer, Keith T; Felli, Vanda E; Harari, Raul; Barrero, Lope H; Felknor, Sarah A.; Gimeno, David; Cattrell, Anna; Vargas-Prada, Sergio; Bonzini, Matteo; Solidaki, Eleni; Merisalu, Eda; Habib, Rima R.; Sadeghian, Farideh; Kadir, M Masood; Warnakulasuriya, Sudath SP; Matsudaira, Ko; Nyantumbu, Busisiwe; Sim, Malcolm R; Harcombe, Helen; Cox, Ken; Sarquis, Leila M M; Marziale, Maria H; Harari, Florencia; Freire, Rocio; Harari, Natalia; Monroy, Magda V; Quintana, Leonardo A; Rojas, Marianela; Harris, E Clare; Serra, Consol; Martinez, J Miguel; Delclos, George; Benavides, Fernando G; Carugno, Michele; Ferrario, Marco M; Pesatori, Angela C; Chatzi, Leda; Bitsios, Panos; Kogevinas, Manolis; Oha, Kristel; Freimann, Tiina; Sadeghian, Ali; Peiris-John, Roshini J; Sathiakumar, Nalini; Wickremasinghe, A Rajitha; Yoshimura, Noriko; Kelsall, Helen L; Hoe, Victor C W; Urquhart, Donna M; Derrett, Sarah; McBride, David; Herbison, Peter; Gray, Andrew; Salazar Vega, Eduardo J.

2017-01-01

Study design Cross-sectional survey with longitudinal follow-up Objectives To test the hypothesis that pain which is localised to the low back differs epidemiologically from that which occurs simultaneously or close in time to pain at other anatomical sites Summary of background data Low back pain (LBP) often occurs in combination with other regional pain, with which it shares similar psychological and psychosocial risk factors. However, few previous epidemiological studies of LBP have distinguished pain that is confined to the low back from that which occurs as part of a wider distribution of pain. Methods We analysed data from a cohort study of musculoskeletal pain and associated disability in 47 occupational groups from 18 countries. Results Among 12,197 subjects at baseline, 609 (4.9%) reported localised LBP in the past month, and 3,820 (31.3%) non-localised LBP. Non-localised LBP was more frequently associated with sciatica in the past month (48.1% vs. 30.0% of cases), occurred on more days in the past month and past year, was more often disabling for everyday activities (64.1% vs. 47.3% of cases), and had more frequently led to medical consultation and sickness absence from work. It was also more often persistent when participants were followed up after a mean of 14 months (65.6% vs. 54.1% of cases). In adjusted Poisson regression analyses, non-localised LBP was differentially associated with female sex, older age, somatising tendency, poor mental health and report of time pressure at work,. There were also marked differences in the relative prevalence of localised and non-localised LBP by occupational group. Conclusions Future epidemiological studies should distinguish where possible between pain that is limited to the low back and LBP which occurs in association with pain at other anatomical locations. PMID:27820794

Development of a prototype of the tele-localisation system in radiotherapy using personal digital assistant via wireless communication.

PubMed

Wu, Vincent Wing-Cheung; Tang, Fuk-hay; Cheung, Wai-kwan; Chan, Kit-chi

2013-02-01

In localisation of radiotherapy treatment field, the oncologist is present at the simulator to approve treatment details produced by the therapist. Problems may arise if the oncologist is not available and the patient requires urgent treatment. The development of a tele-localisation system is a potential solution, where the oncologist uses a personal digital assistant (PDA) to localise the treatment field on the image sent from the simulator through wireless communication and returns the information to the therapist after his or her approval. Our team developed the first tele-localisation prototype, which consisted of a server workstation (simulator) for the administration of digital imaging and communication in medicine localisation images including viewing and communication with the PDA via a Wi-Fi network; a PDA (oncologist's site) installed with the custom-built programme that synchronises with the server workstation and performs treatment field editing. Trial tests on accuracy and speed of the prototype system were conducted on 30 subjects with the treatment regions covering the neck, skull, chest and pelvis. The average time required in performing the localisation using the PDA was less than 1.5 min, with the blocked field longer than the open field. The transmission speed of the four treatment regions was similar. The average physical distortion of the images was within 4.4% and the accuracy of field size indication was within 5.3%. Compared with the manual method, the tele-localisation system presented with an average deviation of 5.5%. The prototype system fulfilled the planned objectives of tele-localisation procedure with reasonable speed and accuracy. © 2012 The Authors. Journal of Medical Imaging and Radiation Oncology © 2012 The Royal Australian and New Zealand College of Radiologists.

Proteomics profiling of interactome dynamics by colocalisation analysis (COLA)† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6mb00701e Click here for additional data file. Click here for additional data file.

PubMed Central

Sailem, Heba Z.; Kümper, Sandra; Tape, Christopher J.; McCully, Ryan R.; Paul, Angela; Anjomani-Virmouni, Sara; Jørgensen, Claus; Poulogiannis, George; Marshall, Christopher J.

2017-01-01

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein–protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision. PMID:27824369

The therapeutic potential of plant-derived vaccines and antibodies.

PubMed

Rodgers, P B; Hamilton, W D; Adair, J R

1999-03-01

The production of recombinant proteins in plants is reviewed with a particular focus on plant-derived vaccines and antibodies for human healthcare. Issues relating to foreign gene expression, such as protein yield, localisation and glycosylation are also considered. Emphasis is placed on reporting progress with preclinical and clinical evaluation of plant-derived vaccines and antibodies. An assessment is made of the likely future direction of research and development in this area.

Resonant photoluminescence studies of carrier localisation in c-plane InGaN/GaN quantum well structures

NASA Astrophysics Data System (ADS)

Blenkhorn, W. E.; Schulz, S.; Tanner, D. S. P.; Oliver, R. A.; Kappers, M. J.; Humphreys, C. J.; Dawson, P.

2018-05-01

In this paper we report on changes in the form of the low temperature (12 K) photoluminescence spectra of an InGaN/GaN quantum well structure as a function of excitation photon energy. As the photon energy is progressively reduced we observe at a critical energy a change in the form of the spectra from one which is determined by the occupation of the complete distribution of hole localisation centres to one which is determined by the resonant excitation of specific localisation sites. This change is governed by an effective mobility edge whereby the photo-excited holes remain localised at their initial energy and are prevented from scattering to other localisation sites. This assignment is confirmed by the results of atomistic tight binding calculations which show that the wave function overlap of the lowest lying localised holes with other hole states is low compared with the overlap of higher lying hole states with other higher lying hole states.

Vectors for fluorescent protein tagging in Phytophthora: tools for functional genomics and cell biology.

PubMed

Ah-Fong, Audrey M V; Judelson, Howard S

2011-09-01

Fluorescent tagging has become the strategy of choice for examining the subcellular localisation of proteins. To develop a versatile community resource for this method in oomycetes, plasmids were constructed that allow the expression of either of four spectrally distinct proteins [cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and mCherry], alone or fused at their N- or C-termini, to sequences of interest. Equivalent sets of plasmids were made using neomycin or hygromycin phosphotransferases (nptII, hpt) as selectable markers, to facilitate double-labelling and aid work in diverse species. The fluorescent proteins and drug-resistance markers were fused to transcriptional regulatory sequences from the oomycete Bremia lactucae, which are known to function in diverse oomycetes, although the promoter in the fluorescence cassette (ham34) can be replaced easily by a promoter of interest. The function of each plasmid was confirmed in Phytophthora infestans. Moreover, fusion proteins were generated using targeting sequences for the endoplasmic reticulum, Golgi, mitochondria, nuclei, and peroxisomes. Studies of the distribution of the fusions in mycelia and sporangia provided insight into cellular organisation at different stages of development. This toolbox of vectors should advance studies of gene function and cell biology in Phytophthora and other oomycetes. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage

PubMed Central

Löfgren, Maria; Ekman, Stina; Svala, Emilia; Lindahl, Anders; Ley, Cecilia; Skiöldebrand, Eva

2014-01-01

Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell-like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor-like domain 7 (EGFL7), the stem cell-indicating factor Stro-1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin-1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro-1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro-1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell-like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage. PMID:25175365

Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

PubMed Central

2012-01-01

Background Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct. Methods In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR. Results In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla. Conclusions This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period. PMID:22958265

The Plasmodium berghei Ca2+/H+ Exchanger, PbCAX, Is Essential for Tolerance to Environmental Ca2+ during Sexual Development

PubMed Central

Guttery, David S.; Pittman, Jon K.; Frénal, Karine; Poulin, Benoit; McFarlane, Leon R.; Slavic, Ksenija; Wheatley, Sally P.; Soldati-Favre, Dominique; Krishna, Sanjeev; Tewari, Rita; Staines, Henry M.

2013-01-01

Ca2+ contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca2+ is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca2+ homeostatic control in apicomplexans uses a Ca2+/H+ exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from “round” form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca2+. Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca2+ homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission. PMID:23468629

Expression of membrane-associated proteins within single emulsion cell facsimiles.

PubMed

Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

2012-07-07

MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability

PubMed Central

Myers, Katie N.; Barone, Giancarlo; Ganesh, Anil; Staples, Christopher J.; Howard, Anna E.; Beveridge, Ryan D.; Maslen, Sarah; Skehel, J. Mark; Collis, Spencer J.

2016-01-01

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions. PMID:27739501

State-of-the-art on viral microRNAs in HPV infection and cancer development.

PubMed

Poltronieri, Palmiro; Sun, Binlian; Huang, Kai-Yao; Chang, Tzu-Hao; Lee, Tzong-Yi

2018-03-27

high-risk HPV subtypes are driving forces for human cancer development: HPV-16 and HPV-18 are responsible for most HPV-caused cancers. This review describes the present knowledge on HR-HPV genomes coding potential for viral miRNAs. HPV subtypes miRNA database, VIRmiRtar, has been constructed applying bioinformatics and a computational method, ViralMir, exploiting structural features, presence of hairpins, and validation by comparison with RNA sequencing datasets. Several miRNA candidates have been localised in the genomes of high-risk HPV subtypes. Among these, HPV-16 miR-1, miR-2 and miR-3. The database contains a list of host candidate gene targets that may be responsible for the oncogenesis in the various cellular environments. miRNA silencing therapies, based on specific cellular uptake of miRNA mimics and antagomiRs, directed towards HPV encoded miRNAs and/or microRNAs deregulated in the host cells, could be a valuable approach to support pharmaceutical interventions in the treatment of HPV dependent cancers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Time-resolved fluorescence microscopy to study biologically related applications using sol-gel derived and cellular media

NASA Astrophysics Data System (ADS)

Toury, Marion; Chandler, Lin; Allison, Archie; Campbell, David; McLoskey, David; Holmes-Smith, A. Sheila; Hungerford, Graham

2011-03-01

Fluorescence microscopy provides a non-invasive means for visualising dynamic protein interactions. As well as allowing the calculation of kinetic processes via the use of time-resolved fluorescence, localisation of the protein within cells or model systems can be monitored. These fluorescence lifetime images (FLIM) have become the preferred technique for elucidating protein dynamics due to the fact that the fluorescence lifetime is an absolute measure, in the main independent of fluorophore concentration and intensity fluctuations caused by factors such as photobleaching. In this work we demonstrate the use of a time-resolved fluorescence microscopy, employing a high repetition rate laser excitation source applied to study the influence of a metal surface on fluorescence tagged protein and to elucidate viscosity using the fluorescence lifetime probe DASPMI. These were studied in a cellular environment (yeast) and in a model system based on a sol-gel derived material, in which silver nanostructures were formed in situ using irradiation from a semiconductor laser in CW mode incorporated on a compact time-resolved fluorescence microscope (HORIBA Scientific DeltaDiode and DynaMyc).

Neurophysiological localisation of ulnar neuropathy at the elbow: Validation of diagnostic criteria developed by a taskforce of the Danish Society of clinical neurophysiology.

PubMed

Pugdahl, K; Beniczky, S; Wanscher, B; Johnsen, B; Qerama, E; Ballegaard, M; Benedek, K; Juhl, A; Ööpik, M; Selmar, P; Sønderborg, J; Terney, D; Fuglsang-Frederiksen, A

2017-11-01

This study validates consensus criteria for localisation of ulnar neuropathy at elbow (UNE) developed by a taskforce of the Danish Society of Clinical Neurophysiology and compares them to the existing criteria from the American Association of Neuromuscular and Electrodiagnostic Medicine (AANEM). The Danish criteria are based on combinations of conduction slowing in the segments of the elbow and forearm expressed in Z-scores, and difference between the segments in m/s. Examining fibres to several muscles and sensory fibres can increase the certainty of the localisation. Diagnostic accuracy for UNE was evaluated on 181 neurophysiological studies of the ulnar nerve from 171 peer-reviewed patients from a mixed patient-group. The diagnostic reference standard was the consensus diagnosis based on all available clinical, laboratory, and electrodiagnostic information reached by a group of experienced Danish neurophysiologists. The Danish criteria had high specificity (98.4%) and positive predictive value (PPV) (95.2%) and fair sensitivity (76.9%). Compared to the AANEM criteria, the Danish criteria had higher specificity (p<0.001) and lower sensitivity (p=0.02). The Danish consensus criteria for UNE are very specific and have high PPV. The Danish criteria for UNE are reliable and well suited for use in different centres as they are based on Z-scores. Copyright © 2017 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.

The Yeast PUF Protein Puf5 Has Pop2-Independent Roles in Response to DNA Replication Stress

PubMed Central

Traven, Ana; Lo, Tricia L.; Lithgow, Trevor; Heierhorst, Jörg

2010-01-01

PUFs are RNA binding proteins that promote mRNA deadenylation and decay and inhibit translation. Yeast Puf5 is the prototype for studying PUF-dependent gene repression. Puf5 binds to the Pop2 subunit of the Ccr4-Pop2-NOT mRNA deadenylase, recruiting the deadenylase and associated translational repressors to mRNAs. Here we used yeast genetics to show that Puf5 has additional roles in vivo that do not require Pop2. Deletion of PUF5 caused increased sensitivity to DNA replication stress in cells lacking Pop2, as well as in cells mutated for two activities recruited to mRNAs by the Puf5-Pop2 interaction, the deadenylase Ccr4 and the translational repressor Dhh1. A functional Puf5 RNA binding domain was required, and Puf5 cytoplasmic localisation was sufficient for resistance to replication stress, indicating posttranscriptional gene expression control is involved. In contrast to DNA replication stress, in response to the cell wall integrity pathway activator caffeine, PUF5 and POP2 acted in the same genetic pathway, indicating that functions of Puf5 in the caffeine response are mediated by Pop2-dependent gene repression. Our results support a model in which Puf5 uses multiple, Pop2-dependent and Pop2-independent mechanisms to control mRNA expression. The Pop2-independent roles for Puf5 could involve spatial control of gene expression, a proposition supported by our data indicating that the active form of Puf5 is localised to cytoplasmic foci. PMID:20498834

Electron localisation in static and time-dependent one-dimensional model systems

NASA Astrophysics Data System (ADS)

Durrant, T. R.; Hodgson, M. J. P.; Ramsden, J. D.; Godby, R. W.

2018-02-01

The most direct signature of electron localisation is the tendency of an electron in a many-body system to exclude other same-spin electrons from its vicinity. By applying this concept directly to the exact many-body wavefunction, we find that localisation can vary considerably between different ground-state systems, and can also be strongly disrupted, as a function of time, when a system is driven by an applied electric field. We use this measure to assess the well-known electron localisation function (ELF), both in its approximate single-particle form (often applied within density-functional theory) and its full many-particle form. The full ELF always gives an excellent description of localisation, but the approximate ELF fails in time-dependent situations, even when the exact Kohn-Sham orbitals are employed.

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.

2015-08-15

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of themore » P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.« less

Antenna array geometry optimization for a passive coherent localisation system

NASA Astrophysics Data System (ADS)

Knott, Peter; Kuschel, Heiner; O'Hagan, Daniel

2012-11-01

Passive Coherent Localisation (PCL), also known as Passive Radar, making use of RF sources of opportunity such as Radio or TV Broadcasting Stations, Cellular Phone Network Base Stations, etc. is an advancing technology for covert operation because no active radar transmitter is required. It is also an attractive addition to existing active radar stations because it has the potential to discover low-flying and low-observable targets. The CORA (Covert Radar) experimental passive radar system currently developed at Fraunhofer-FHR features a multi-channel digital radar receiver and a circular antenna array with separate elements for the VHF- and the UHF-range and is used to exploit alternatively Digital Audio (DAB) or Video Broadcasting (DVB-T) signals. For an extension of the system, a wideband antenna array is being designed for which a new discone antenna element has been developed covering the full DVB-T frequency range. The present paper describes the outline of the system and the numerical modelling and optimisation methods applied to solve the complex task of antenna array design: Electromagnetic full wave analysis is required for the parametric design of the antenna elements while combinatorial optimization methods are applied to find the best array positions and excitation coefficients for a regular omni-directional antenna performance. The different steps are combined in an iterative loop until the optimum array layout is found. Simulation and experimental results for the current system will be shown.

Progesterone signalling in broiler skeletal muscle is associated with divergent feed efficiency.

PubMed

Bottje, Walter; Kong, Byung-Whi; Reverter, Antonio; Waardenberg, Ashley J; Lassiter, Kentu; Hudson, Nicholas J

2017-02-24

We contrast the pectoralis muscle transcriptomes of broilers selected from within a single genetic line expressing divergent feed efficiency (FE) in an effort to improve our understanding of the mechanistic basis of FE. Application of a virtual muscle model to gene expression data pointed to a coordinated reduction in slow twitch muscle isoforms of the contractile apparatus (MYH15, TPM3, MYOZ2, TNNI1, MYL2, MYOM3, CSRP3, TNNT2), consistent with diminishment in associated slow machinery (myoglobin and phospholamban) in the high FE animals. These data are in line with the repeated transition from red slow to white fast muscle fibres observed in agricultural species selected on mass and FE. Surprisingly, we found that the expression of 699 genes encoding the broiler mitoproteome is modestly-but significantly-biased towards the high FE group, suggesting a slightly elevated mitochondrial content. This is contrary to expectation based on the slow muscle isoform data and theoretical physiological capacity arguments. Reassuringly, the extreme 40 most DE genes can successfully cluster the 12 individuals into the appropriate FE treatment group. Functional groups contained in this DE gene list include metabolic proteins (including opposing patterns of CA3 and CA4), mitochondrial proteins (CKMT1A), oxidative status (SEPP1, HIG2A) and cholesterol homeostasis (APOA1, INSIG1). We applied a differential network method (Regulatory Impact Factors) whose aim is to use patterns of differential co-expression to detect regulatory molecules transcriptionally rewired between the groups. This analysis clearly points to alterations in progesterone signalling (via the receptor PGR) as the major driver. We show the progesterone receptor localises to the mitochondria in a quail muscle cell line. Progesterone is sometimes used in the cattle industry in exogenous hormone mixes that lead to a ~20% increase in FE. Because the progesterone receptor can localise to avian mitochondria, our data continue to point to muscle mitochondrial metabolism as an important component of the phenotypic expression of variation in broiler FE.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells.

PubMed

Adigbli, D K; Wilson, D G G; Farooqui, N; Sousi, E; Risley, P; Taylor, I; Macrobert, A J; Loizidou, M

2007-08-20

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1-0.2 microg ml(-1)) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6-28%). Hypericin (0.1-0.2 microM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

PubMed Central

Adigbli, D K; Wilson, D G G; Farooqui, N; Sousi, E; Risley, P; Taylor, I; MacRobert, A J; Loizidou, M

2007-01-01

Multidrug resistance (MDR) is the major confounding factor in adjuvant solid tumour chemotherapy. Increasing intracellular amounts of chemotherapeutics to circumvent MDR may be achieved by a novel delivery method, photochemical internalisation (PCI). PCI consists of the co-administration of drug and photosensitiser; upon light activation the latter induces intracellular release of organelle-bound drug. We investigated whether co-administration of hypericin (photosensitiser) with mitoxantrone (MTZ, chemotherapeutic) plus illumination potentiates cytotoxicity in MDR cancer cells. We mapped the extent of intracellular co-localisation of drug/photosensitiser. We determined whether PCI altered drug-excreting efflux pump P-glycoprotein (Pgp) expression or function in MDR cells. Bladder and breast cancer cells and their Pgp-overexpressing MDR subclones (MGHU1, MGHU1/R, MCF-7, MCF-7/R) were given hypericin/MTZ combinations, with/without blue-light illumination. Pilot experiments determined appropriate sublethal doses for each. Viability was determined by the 3-[4,5-dimethylthiazolyl]-2,5-diphenyltetrazolium bromide assay. Intracellular localisation was mapped by confocal microscopy. Pgp expression was detected by immunofluorescence and Pgp function investigated by Rhodamine123 efflux on confocal microscopy. MTZ alone (0.1–0.2 μg ml−1) killed up to 89% of drug-sensitive cells; MDR cells exhibited less cytotoxicity (6–28%). Hypericin (0.1–0.2 μM) effects were similar for all cells; light illumination caused none or minimal toxicity. In combination, MTZ /hypericin plus illumination, potentiated MDR cell killing, vs hypericin or MTZ alone. (MGHU1/R: 38.65 and 36.63% increase, P<0.05; MCF-7/R: 80.2 and 46.1% increase, P<0.001). Illumination of combined MTZ/hypericin increased killing by 28.15% (P<0.05 MGHU1/R) compared to dark controls. Intracytoplasmic vesicular co-localisation of MTZ/hypericin was evident before illumination and at serial times post-illumination. MTZ was always found in sensitive cell nuclei, but not in dark resistant cell nuclei. In illuminated resistant cells there was some mobilisation of MTZ into the nucleus. Pgp expression remained unchanged, regardless of drug exposure. Pgp efflux was blocked by the Pgp inhibitor verapamil (positive control) but not impeded by hypericin. The increased killing of MDR cancer cells demonstrated is consistent with PCI. PCI is a promising technique for enhancing treatment efficacy. PMID:17667930

Evaluation of an automatic MR-based gold fiducial marker localisation method for MR-only prostate radiotherapy

NASA Astrophysics Data System (ADS)

Maspero, Matteo; van den Berg, Cornelis A. T.; Zijlstra, Frank; Sikkes, Gonda G.; de Boer, Hans C. J.; Meijer, Gert J.; Kerkmeijer, Linda G. W.; Viergever, Max A.; Lagendijk, Jan J. W.; Seevinck, Peter R.

2017-10-01

An MR-only radiotherapy planning (RTP) workflow would reduce the cost, radiation exposure and uncertainties introduced by CT-MRI registrations. In the case of prostate treatment, one of the remaining challenges currently holding back the implementation of an RTP workflow is the MR-based localisation of intraprostatic gold fiducial markers (FMs), which is crucial for accurate patient positioning. Currently, MR-based FM localisation is clinically performed manually. This is sub-optimal, as manual interaction increases the workload. Attempts to perform automatic FM detection often rely on being able to detect signal voids induced by the FMs in magnitude images. However, signal voids may not always be sufficiently specific, hampering accurate and robust automatic FM localisation. Here, we present an approach that aims at automatic MR-based FM localisation. This method is based on template matching using a library of simulated complex-valued templates, and exploiting the behaviour of the complex MR signal in the vicinity of the FM. Clinical evaluation was performed on seventeen prostate cancer patients undergoing external beam radiotherapy treatment. Automatic MR-based FM localisation was compared to manual MR-based and semi-automatic CT-based localisation (the current gold standard) in terms of detection rate and the spatial accuracy and precision of localisation. The proposed method correctly detected all three FMs in 15/17 patients. The spatial accuracy (mean) and precision (STD) were 0.9 mm and 0.5 mm respectively, which is below the voxel size of 1.1 × 1.1 × 1.2 mm3 and comparable to MR-based manual localisation. FM localisation failed (3/51 FMs) in the presence of bleeding or calcifications in the direct vicinity of the FM. The method was found to be spatially accurate and precise, which is essential for clinical use. To overcome any missed detection, we envision the use of the proposed method along with verification by an observer. This will result in a semi-automatic workflow facilitating the introduction of an MR-only workflow.

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Acute Inactivation of Primary Auditory Cortex Causes a Sound Localisation Deficit in Ferrets

PubMed Central

Wood, Katherine C.; Town, Stephen M.; Atilgan, Huriye; Jones, Gareth P.

2017-01-01

The objective of this study was to demonstrate the efficacy of acute inactivation of brain areas by cooling in the behaving ferret and to demonstrate that cooling auditory cortex produced a localisation deficit that was specific to auditory stimuli. The effect of cooling on neural activity was measured in anesthetized ferret cortex. The behavioural effect of cooling was determined in a benchmark sound localisation task in which inactivation of primary auditory cortex (A1) is known to impair performance. Cooling strongly suppressed the spontaneous and stimulus-evoked firing rates of cortical neurons when the cooling loop was held at temperatures below 10°C, and this suppression was reversed when the cortical temperature recovered. Cooling of ferret auditory cortex during behavioural testing impaired sound localisation performance, with unilateral cooling producing selective deficits in the hemifield contralateral to cooling, and bilateral cooling producing deficits on both sides of space. The deficit in sound localisation induced by inactivation of A1 was not caused by motivational or locomotor changes since inactivation of A1 did not affect localisation of visual stimuli in the same context. PMID:28099489

Probabilistic self-localisation on a qualitative map based on occlusions

NASA Astrophysics Data System (ADS)

Santos, Paulo E.; Martins, Murilo F.; Fenelon, Valquiria; Cozman, Fabio G.; Dee, Hannah M.

2016-09-01

Spatial knowledge plays an essential role in human reasoning, permitting tasks such as locating objects in the world (including oneself), reasoning about everyday actions and describing perceptual information. This is also the case in the field of mobile robotics, where one of the most basic (and essential) tasks is the autonomous determination of the pose of a robot with respect to a map, given its perception of the environment. This is the problem of robot self-localisation (or simply the localisation problem). This paper presents a probabilistic algorithm for robot self-localisation that is based on a topological map constructed from the observation of spatial occlusion. Distinct locations on the map are defined by means of a classical formalism for qualitative spatial reasoning, whose base definitions are closer to the human categorisation of space than traditional, numerical, localisation procedures. The approach herein proposed was systematically evaluated through experiments using a mobile robot equipped with a RGB-D sensor. The results obtained show that the localisation algorithm is successful in locating the robot in qualitatively distinct regions.

A new range-free localisation in wireless sensor networks using support vector machine

NASA Astrophysics Data System (ADS)

Wang, Zengfeng; Zhang, Hao; Lu, Tingting; Sun, Yujuan; Liu, Xing

2018-02-01

Location information of sensor nodes is of vital importance for most applications in wireless sensor networks (WSNs). This paper proposes a new range-free localisation algorithm using support vector machine (SVM) and polar coordinate system (PCS), LSVM-PCS. In LSVM-PCS, two sets of classes are first constructed based on sensor nodes' polar coordinates. Using the boundaries of the defined classes, the operation region of WSN field is partitioned into a finite number of polar grids. Each sensor node can be localised into one of the polar grids by executing two localisation algorithms that are developed on the basis of SVM classification. The centre of the resident polar grid is then estimated as the location of the sensor node. In addition, a two-hop mass-spring optimisation (THMSO) is also proposed to further improve the localisation accuracy of LSVM-PCS. In THMSO, both neighbourhood information and non-neighbourhood information are used to refine the sensor node location. The results obtained verify that the proposed algorithm provides a significant improvement over existing localisation methods.

Biomarkers in the Detection of Prostate Cancer in African Americans

DTIC Science & Technology

2012-09-01

176:1534–1542 19. Kim JW, Wieckowski E, Taylor DD, Reichert TE, Watkins S, Whiteside TL (2005) Fas ligand- positive membranous vesicles isolated from...Larregina AT, Shufesky WJ, Sullivan MMLG, Sullivan DBS, Papworth GD, ZahorchakAF, LogarAJ, Wang Z, Watkins SC, Falo, LD Jr, ThomsonAW (2004... Seth , Expression of the TMPRSS2:ERG fusion gene predicts cancer recurrence af- ter surgery for localised prostate cancer, British Journal of Cancer 97(12

Localised JAK/STAT Pathway Activation Is Required for Drosophila Wing Hinge Development

PubMed Central

Johnstone, Kirsty; Wells, Richard E.; Strutt, David; Zeidler, Martin P.

2013-01-01

Extensive morphogenetic remodelling takes place during metamorphosis from a larval to an adult insect body plan. These changes are particularly intricate in the generation of the dipteran wing hinge, a complex structure that is derived from an apparently simple region of the wing imaginal disc. Using the characterisation of original outstretched alleles of the unpaired locus as a starting point, we demonstrate the role of JAK/STAT pathway signalling in the process of wing hinge development. We show that differences in JAK/STAT signalling within the proximal most of three lateral folds present in the wing imaginal disc is required for fold morphology and the subsequent differentiation of the first and second auxiliary sclerites as well as the posterior notal wing process. Changes in these domains are consistent with the established fate map of the wing disc. We show that outstretched wing posture phenotypes arise from the loss of a region of Unpaired expression in the proximal wing fold and demonstrate that this results in a decrease in JAK/STAT pathway activity. Finally we show that reduction of JAK/STAT pathway activity within the proximal wing fold is sufficient to phenocopy the outstretched phenotype. Taken together, we suggest that localised Unpaired expression and hence JAK/STAT pathway activity, is required for the morphogenesis of the adult wing hinge, providing new insights into the link between signal transduction pathways, patterning and development. PMID:23741461

Driving mechanisms of passive and active transport across cellular membranes as the mechanisms of cell metabolism and development as well as the mechanisms of cellular distance reactions on hormonal expression and the immune response.

PubMed

Ponisovskiy, M R

2011-01-01

The article presents mechanisms of cell metabolism, cell development, cell activity, and maintenance of cellular stability. The literature is reviewed from the point of view of these concepts. The balance between anabolic and catabolic processes induces chemical potentials in the extracellular and intracellular media. The chemical potentials of these media are defined as the driving forces of both passive and active transport of substances across cellular membranes. The driving forces of substance transport across cellular membranes as in cellular metabolism and in immune responses and hormonal expressions are considered in the biochemical and biophysical models, reflecting the mechanisms for maintenance of stability of the internal medium and internal energy of an organism. The interactions of passive transport and active transport of substances across cellular walls promote cell proliferation, as well as the mechanism of cellular capacitors, promoting remote reactions across distance for hormonal expression and immune responses. The offered concept of cellular capacitors has given the possibility to explain the mechanism of remote responses of cells to new situations, resulting in the appearance of additional agents. The biophysical model develops an explanation of some cellular functions: cellular membrane action have been identified with capacitor action, based on the similarity of the structures and as well as on similarity of biophysical properties of electric data that confirm the action of the compound-specific interactions of cells within an organism, promoting hormonal expressions and immune responses to stabilize the thermodynamic system of an organism. Comparison of a cellular membrane action to a capacitor has given the possibility for the explanations of exocytosis and endocytosis mechanisms, internalization of the receptor-ligand complex, selection as a receptor reaction to a ligand by immune responses or hormonal effects, reflecting cellular distance reactions on the hormonal expressions, immune responses, and specificity of the mechanisms of immune reactions. Reviewing current research of cell activity, explanations are presented of mechanisms of apoptosis, autophagy, hormonal expression, and immune responses from the point of view of described cellular mechanisms. Thermodynamic laws are used to confirm the importance of the actions of these mechanisms for maintenance of stability of the internal medium and internal energy of an organism.

Strain localisation in mechanically layered rocks beneath detachment zones: insights from numerical modelling

NASA Astrophysics Data System (ADS)

Le Pourhiet, L.; Huet, B.; Labrousse, L.; Yao, K.; Agard, P.; Jolivet, L.

2013-04-01

We have designed a series of fully dynamic numerical simulations aimed at assessing how the orientation of mechanical layering in rocks controls the orientation of shear bands and the depth of penetration of strain in the footwall of detachment zones. Two parametric studies are presented. In the first one, the influence of stratification orientation on the occurrence and mode of strain localisation is tested by varying initial dip of inherited layering in the footwall with regard to the orientation of simple shear applied at the rigid boundary simulating a rigid hanging wall, all scaling and rheological parameter kept constant. It appears that when Mohr-Coulomb plasticity is being used, shear bands are found to localise only when the layering is being stretched. This corresponds to early deformational stages for inital layering dipping in the same direction as the shear is applied, and to later stages for intial layering dipping towards the opposite direction of shear. In all the cases, localisation of the strain after only γ=1 requires plastic yielding to be activated in the strong layer. The second parametric study shows that results are length-scale independent and that orientation of shear bands is not sensitive to the viscosity contrast or the strain rate. However, decreasing or increasing strain rate is shown to reduce the capacity of the shear zone to localise strain. In the later case, the strain pattern resembles a mylonitic band but the rheology is shown to be effectively linear. Based on the results, a conceptual model for strain localisation under detachment faults is presented. In the early stages, strain localisation occurs at slow rates by viscous shear instabilities but as the layered media is exhumed, the temperature drops and the strong layers start yielding plastically, forming shear bands and localising strain at the top of the shear zone. Once strain localisation has occured, the deformation in the shear band becomes extremely penetrative but the strength cannot drop since the shear zone has a finite thickness.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

PubMed

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-02-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

PubMed Central

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-01-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

Cellular and laminar expression of Dab-1 during the postnatal critical period in cat visual cortex and the effects of dark rearing.

PubMed

Kiser, Paul J; Liu, Zijing; Wilt, Steven D; Mower, George D

2011-04-06

This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5-week DR cats. By 10weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5- and 20-week-old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation. Copyright © 2011 Elsevier B.V. All rights reserved.

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

PubMed Central

Allen, Claire L.; Clare, Gerry; Stewart, Elizabeth A.; Branch, Matthew J.; McIntosh, Owen D.; Dadhwal, Megha; Dua, Harminder S.; Hopkinson, Andrew

2013-01-01

Purpose Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material. Methods AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays. Results Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM. Conclusions Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors. PMID:24205233

Nuclear localisation of 53BP1 is regulated by phosphorylation of the nuclear localisation signal.

PubMed

von Morgen, Patrick; Lidak, Tomas; Horejsi, Zuzana; Macurek, Libor

2018-06-01

Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin β and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined. Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import. Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair. © 2018 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Is the Pauli exclusion principle the origin of electron localisation?

NASA Astrophysics Data System (ADS)

Rincón, Luis; Torres, F. Javier; Almeida, Rafael

2018-03-01

In this work, we inquire into the origins of the electron localisation as obtained from the information content of the same-spin pair density, γσ, σ(r2∣r1). To this end, we consider systems of non-interacting and interacting identical Fermions contained in two simple 1D potential models: (1) an infinite potential well and (2) the Kronig-Penney periodic potential. The interparticle interaction is considered through the Hartree-Fock approximation as well as the configuration interaction expansion. Morover, the electron localisation is described through the Kullback-Leibler divergence between γσ, σ(r2∣r1) and its associated marginal probability. The results show that, as long as the adopted method properly includes the Pauli principle, the electronic localisation depends only modestly on the interparticle interaction. In view of the latter, one may conclude that the Pauli principle is the main responsible for the electron localisation.

TMV-Gate vectors: Gateway compatible tobacco mosaic virus based expression vectors for functional analysis of proteins

PubMed Central

Kagale, Sateesh; Uzuhashi, Shihomi; Wigness, Merek; Bender, Tricia; Yang, Wen; Borhan, M. Hossein; Rozwadowski, Kevin

2012-01-01

Plant viral expression vectors are advantageous for high-throughput functional characterization studies of genes due to their capability for rapid, high-level transient expression of proteins. We have constructed a series of tobacco mosaic virus (TMV) based vectors that are compatible with Gateway technology to enable rapid assembly of expression constructs and exploitation of ORFeome collections. In addition to the potential of producing recombinant protein at grams per kilogram FW of leaf tissue, these vectors facilitate either N- or C-terminal fusions to a broad series of epitope tag(s) and fluorescent proteins. We demonstrate the utility of these vectors in affinity purification, immunodetection and subcellular localisation studies. We also apply the vectors to characterize protein-protein interactions and demonstrate their utility in screening plant pathogen effectors. Given its broad utility in defining protein properties, this vector series will serve as a useful resource to expedite gene characterization efforts. PMID:23166857

Transient expression and cellular localization of recombinant proteins in cultured insect cells

USDA-ARS?s Scientific Manuscript database

Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling.

PubMed

Gu, Chunyan; Peng, Hailin; Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-08-22

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro . Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo . Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM.

Changes in cellular degradation activity in young and old worker honeybees (Apis mellifera).

PubMed

Hsu, Chin-Yuan; Chuang, Yu-Lung; Chan, Yu-Pei

2014-02-01

The trophocytes and fat cells of honeybees (Apis mellifera) have been used in cellular senescence studies, but the changes of cellular degradation activity with aging in workers are unknown. In this study, cellular degradation activity was evaluated in the trophocytes and fat cells of young and old workers reared in a field hive. The results showed the following: (1) 20S proteosome activity decreased with aging, whereas its expression increased with aging; (2) the expression of microtubule-associated protein 1 light chain 3-II (LC3-II) and the 70 kD heat shock cognate protein (Hsc70) decreased with aging; (3) the size and number of autophagic vacuoles decreased with aging; (4) p62/SQSTM1 and polyubiquitin aggregate expression decreased with aging; (5) lysosomal efficiency decreased with aging; and (6) molecular target of rapamycin (mTOR) expression increased with aging. These results indicate that young workers have higher levels of cellular degradation activity than old workers and that aging results in a decline in the cellular degradation activity in worker honeybees. Copyright © 2013 Elsevier Inc. All rights reserved.

FoxP1 marks medium spiny neurons from precursors to maturity and is required for their differentiation.

PubMed

Precious, S V; Kelly, C M; Reddington, A E; Vinh, N N; Stickland, R C; Pekarik, V; Scherf, C; Jeyasingham, R; Glasbey, J; Holeiter, M; Jones, L; Taylor, M V; Rosser, A E

2016-08-01

Identifying the steps involved in striatal development is important both for understanding the striatum in health and disease, and for generating protocols to differentiate striatal neurons for regenerative medicine. The most prominent neuronal subtype in the adult striatum is the medium spiny projection neuron (MSN), which constitutes more than 85% of all striatal neurons and classically expresses DARPP-32. Through a microarray study of genes expressed in the whole ganglionic eminence (WGE: the developing striatum) in the mouse, we identified the gene encoding the transcription factor Forkhead box protein P1 (FoxP1) as the most highly up-regulated gene, thus providing unbiased evidence for the association of FoxP1 with MSN development. We also describe the expression of FoxP1 in the human fetal brain over equivalent gestational stages. FoxP1 expression persisted through into adulthood in the mouse brain, where it co-localised with all striatal DARPP-32 positive projection neurons and a small population of DARPP-32 negative cells. There was no co-localisation of FoxP1 with any interneuron markers. FoxP1 was detectable in primary fetal striatal cells following dissection, culture, and transplantation into the adult lesioned striatum, demonstrating its utility as an MSN marker for transplantation studies. Furthermore, DARPP-32 expression was absent from FoxP1 knock-out mouse WGE differentiated in vitro, suggesting that FoxP1 is important for the development of DARPP-32-positive MSNs. In summary, we show that FoxP1 labels MSN precursors prior to the expression of DARPP-32 during normal development, and in addition suggest that FoxP1 labels a sub-population of MSNs that are not co-labelled by DARPP-32. We demonstrate the utility of FoxP1 to label MSNs in vitro and following neural transplantation, and show that FoxP1 is required for DARPP-32 positive MSN differentiation in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Biphasic zinc compartmentalisation in a human fungal pathogen.

PubMed

Crawford, Aaron C; Lehtovirta-Morley, Laura E; Alamir, Omran; Niemiec, Maria J; Alawfi, Bader; Alsarraf, Mohammad; Skrahina, Volha; Costa, Anna C B P; Anderson, Andrew; Yellagunda, Sujan; Ballou, Elizabeth R; Hube, Bernhard; Urban, Constantin F; Wilson, Duncan

2018-05-01

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Tracking intracellular uptake and localisation of alkyne tagged fatty acids using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Jamieson, Lauren E.; Greaves, Jennifer; McLellan, Jayde A.; Munro, Kevin R.; Tomkinson, Nicholas C. O.; Chamberlain, Luke H.; Faulds, Karen; Graham, Duncan

2018-05-01

Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.

Functionalized gold nanoparticles manifested as potent carriers for nucleolar targeting

NASA Astrophysics Data System (ADS)

Shahbazi, Reza; Ozcicek, Ilyas; Ozturk, Gurkan; Ulubayram, Kezban

2017-01-01

It is generally known that gold nanoparticles are localised in the cytoplasm and, if synthesised in small sizes or functionalized with specific proteins, they enter the cell nucleus. However, there is no report emphasising the importance of surface functionalization in their accumulation in the nucleolus. Here, for the first time in the literature, it is proposed that functionalization of gold nanoparticles with a thin layer of polyethyleneimine (PEI) spearheads them to the nucleolus of hard-to-transfect post-mitotic dorsal root ganglion neurones in a size-independent manner. As a potential for theranostic applications, it was found that functionalization with a thin layer of PEI affected the emission signal intensity of gold nanoparticles so that the cellular biodistribution of nanoparticles was visualised clearly under both confocal and two-photon microscopes.

Matter field Kähler metric in heterotic string theory from localisation

NASA Astrophysics Data System (ADS)

Blesneag, Ştefan; Buchbinder, Evgeny I.; Constantin, Andrei; Lukas, Andre; Palti, Eran

2018-04-01

We propose an analytic method to calculate the matter field Kähler metric in heterotic compactifications on smooth Calabi-Yau three-folds with Abelian internal gauge fields. The matter field Kähler metric determines the normalisations of the N = 1 chiral superfields, which enter the computation of the physical Yukawa couplings. We first derive the general formula for this Kähler metric by a dimensional reduction of the relevant supergravity theory and find that its T-moduli dependence can be determined in general. It turns out that, due to large internal gauge flux, the remaining integrals localise around certain points on the compactification manifold and can, hence, be calculated approximately without precise knowledge of the Ricci-flat Calabi-Yau metric. In a final step, we show how this local result can be expressed in terms of the global moduli of the Calabi-Yau manifold. The method is illustrated for the family of Calabi-Yau hypersurfaces embedded in P^1× P^3 and we obtain an explicit result for the matter field Kähler metric in this case.

Super-spreaders and the rate of transmission of the SARS virus

NASA Astrophysics Data System (ADS)

Small, Michael; Tse, C. K.; Walker, David M.

2006-03-01

We describe a stochastic small-world network model of transmission of the SARS virus. Unlike the standard Susceptible-Infected-Removed models of disease transmission, our model exhibits both geographically localised outbreaks and “super-spreaders”. Moreover, the combination of localised and long range links allows for more accurate modelling of partial isolation and various public health policies. From this model, we derive an expression for the probability of a widespread outbreak and a condition to ensure that the epidemic is controlled. Moreover, multiple simulations are used to make predictions of the likelihood of various eventual scenarios for fixed initial conditions. The main conclusions of this study are: (i) “super-spreaders” may occur even if the infectiousness of all infected individuals is constant; (ii) consistent with previous reports, extended exposure time beyond 3-5 days (i.e. significant nosocomial transmission) was the key factor in the severity of the SARS outbreak in Hong Kong; and, (iii) the spread of SARS can be effectively controlled by either limiting long range links (imposing a partial quarantine) or enforcing rapid hospitalisation and isolation of symptomatic individuals.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

PubMed Central

Mustapha, Nik M.; Tarr, Joanna M.; Kohner, Eva M.; Chibber, Rakesh

2010-01-01

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nε-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy. PMID:20652059

Acetylated sialic acid residues and blood group antigens localise within the epithelium in microvillous atrophy indicating internal accumulation of the glycocalyx

PubMed Central

Phillips, A D; Brown, A; Hicks, S; Schüller, S; Murch, S H; Walker-Smith, J A; Swallow, D M

2004-01-01

Background: Microvillous atrophy, a disorder of intractable diarrhoea in infancy, is characterised by the intestinal epithelial cell abnormalities of abnormal accumulation of periodic acid-Schiff (PAS) positive secretory granules within the apical cytoplasm and the presence of microvillous inclusions. The identity of the PAS positive material is not known, and the aim of this paper was to further investigate its composition. Methods: Formaldehyde fixed sections were stained with alcian blue/PAS to identify the acidic or neutral nature of the material, phenylhydrazine blocking was employed to stain specifically for sialic acid, and saponification determined the presence of sialic acid acetylation. The specificity of sialic acid staining was tested by digestion with mild sulphuric acid. Expression of blood group related antigens was tested immunochemically. Results: Alcian blue/PAS staining identified a closely apposed layer of acidic material on the otherwise neutral (PAS positive) brush border in controls. In microvillous atrophy, a triple layer was seen with an outer acidic layer, an unstained brush border region, and accumulation within the epithelium of a neutral glycosubstance that contained acetylated sialic acid. Blood group antigens were detected on the brush border, in mucus, and within goblet cells in controls. In microvillous atrophy they were additionally expressed within the apical cytoplasm of epithelial cells mirroring the PAS abnormality. Immuno electron microscopy localised expression to secretory granules. Conclusions: A neutral, blood group antigen positive, glycosubstance that contains acetylated sialic acid accumulates in the epithelium in microvillous atrophy. Previous studies have demonstrated that the direct and indirect constitutive pathways are intact in this disorder and it is speculated that the abnormal staining pattern reflects accumulation of glycocalyx related material. PMID:15542511

α-Synuclein staging in the amygdala of a Parkinson's disease model: cell types involved.

PubMed

Flores-Cuadrado, Alicia; Ubeda-Bañon, Isabel; Saiz-Sanchez, Daniel; de la Rosa-Prieto, Carlos; Martinez-Marcos, Alino

2015-01-01

Lewy bodies (ubiquitin and α-synuclein aggregates) can be detected in brain areas in a predictable sequence of six neuropathological stages in Parkinson's disease. Brainstem and olfactory structures are involved in stage 1, whereas the substantia nigra and amygdala are involved in stage 3, prior to cortical spreading. Amygdaloid pathology has been suggested to contribute to non-motor symptoms such as olfactory dysfunction and emotional impairment. This work analysed the distribution of α-synuclein at 16, 30, 43 and 56 weeks in the basolateral, central and cortical amygdaloid complexes of A53T transgenic mice. The expression of calbindin, calretinin and somatostatin was compared in control and transgenic animals. Co-localisation of these markers with α-synuclein was performed. Triple labeling of calbindin, somatostatin and α-synuclein was also investigated. Quantification was carried out using an optical dissector, ImageJ software and confocal microscopy. α-Synuclein-positive cells were mainly concentrated in the basolateral and cortical amygdaloid complexes with a non-significant increase over time from 16 to 30-43 weeks and a significant decrease thereafter. The expression of interneuron markers showed a significant decrease with aging in control animals. When comparing these markers between control and transgenic mice, calretinin was moderately decreased, but calbindin and somatostatin were highly reduced, particularly in the cortical amygdaloid complex. α-Synuclein mostly co-localised with calbindin and a number of these cells also co-expressed somatostatin. These data on α-synucleinopathy staging in the amygdala could help to explain non-motor symptoms as well as to understand the progression of Parkinson's disease in the brain. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Diagnostic accuracy of 3T magnetic resonance imaging in the preoperative localisation of parathyroid adenomas: comparison with ultrasound and 99mTc-sestamibi scans.

PubMed

Argirò, Renato; Diacinti, Daniele; Sacconi, Beatrice; Iannarelli, Angelo; Diacinti, Davide; Cipriani, Cristiana; Pisani, Daniela; Romagnoli, Elisabetta; Biffoni, Marco; Di Gioia, Cira; Pepe, Jessica; Bezzi, Mario; Letizia, Claudio; Minisola, Salvatore; Catalano, Carlo

2018-05-07

To evaluate the diagnostic performance of 3TMRI in comparison with ultrasound (US) and 99mTc-sestamibi scan for presurgical localisation of parathyroid adenomas (PTAs) in patients with primary hyperparathyroidism (PHPT). Fifty-seven patients affected by PHPT were prospectively enrolled and underwent US, 99mTc-sestamibi and 3TMRI. T2-weighted and post-contrast T1-weighted Iterative decomposition of water and fat with Echo Asymmetry and Least squares estimation (IDEAL) sequences were acquired. Diagnostic performance of US, 99mTc-sestamibi and MRI in localising PTAs to correct quadrant were compared according to surgical and pathological findings. According to surgical findings, US correctly localised 41/46 PTAs (sensitivity of 89.1%; specificity 97.5%; PPV 93.1% and NPV 95.6%); 99mTc-sestamibi correctly localised 38/46 PTAs (sensitivity 83.6%, specificity 98.3%, PPV 95% and NPV 93.7%). US and 99mTc-sestamibi combined had a sensitivity of 93.4% (43/46 PTAs), specificity of 98.3%, PPV 95% and NPV 98.3%. MRI correctly localised 45/46 PTAs (sensitivity 97.8%; specificity 97.5%; PPV 93.7% and NPV 99.2%). MRI was able to detect six adenomas missed by 99mTc-sestamibi and two adenomas missed by US. MRI and US were able to detect all enlarged parathyroid glands in patients with multiglandular disease. MRI identified six of seven ectopic adenomas. Our study demonstrated high diagnostic performance of 3T MRI in the preoperative PTAs quadrant localisation, as well as in patients with multiglandular disease and ectopic PTAs. MRI may be preferred to adequately select patient candidates for minimally invasive parathyroidectomy (MIP). • PTA(s) quadrant localisation by 3TMRI was more accurate than US+99mTc-sestamibi. • MRI identified all enlarged glands in multiglandular disease similarly to US. • MRI identified 6/7 ectopic PTAs similarly to 99mTc-sestamibi. • Presurgical PTA(s) localisation by 3TMRI select the optimal candidates for MIP.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

In an Ovine Model of Polycystic Ovary Syndrome (PCOS) Prenatal Androgens Suppress Female Fetal Renal Gluconeogenesis

PubMed Central

Connolly, Fiona; Rae, Michael T.; Späth, Katharina; Boswell, Lyndsey; McNeilly, Alan S.; Duncan, W. Colin

2015-01-01

Increased maternal androgen exposure during pregnancy programmes a polycystic ovary syndrome (PCOS)-like condition, with metabolic dysfunction, in adult female offspring. Other in utero exposures associated with the development of insulin resistance, such as intrauterine growth restriction and exposure to prenatal glucocorticoids, are associated with altered fetal gluconeogenesis. We therefore aimed to assess the effect of maternal androgenisation on the expression of PEPCK and G6PC in the ovine fetus. Pregnant Scottish Greyface sheep were treated with twice weekly testosterone propionate (TP; 100mg) or vehicle control from day 62 to day102 of gestation. At day 90 and day 112 fetal plasma and liver and kidney tissue was collected for analysis. PEPCK and G6PC expression were analysed by quantitative RT-PCR, immunohistochemistry and western blotting. PEPCK and G6PC were localised to fetal hepatocytes but maternal androgens had no effect on female or male fetuses. PEPCK and G6PC were also localised to the renal tubules and renal PEPCK (P<0.01) and G6PC (P = 0.057) were lower in females after prenatal androgenisation with no change in male fetuses. These tissue and sex specific observations could not be explained by alterations in fetal insulin or cortisol. The sexual dimorphism may be related to the increase in circulating estrogen (P<0.01) and testosterone (P<0.001) in females but not males. The tissue specific effects may be related to the increased expression of ESR1 (P<0.01) and AR (P<0.05) in the kidney when compared to the fetal liver. After discontinuation of maternal androgenisation female fetal kidney PEPCK expression normalised. These data further highlight the fetal and sexual dimorphic effects of maternal androgenisation, an antecedent to adult disease and the plasticity of fetal development. PMID:26148093

Acoustic emission source localization based on distance domain signal representation

NASA Astrophysics Data System (ADS)

Gawronski, M.; Grabowski, K.; Russek, P.; Staszewski, W. J.; Uhl, T.; Packo, P.

2016-04-01

Acoustic emission is a vital non-destructive testing technique and is widely used in industry for damage detection, localisation and characterization. The latter two aspects are particularly challenging, as AE data are typically noisy. What is more, elastic waves generated by an AE event, propagate through a structural path and are significantly distorted. This effect is particularly prominent for thin elastic plates. In these media the dispersion phenomenon results in severe localisation and characterization issues. Traditional Time Difference of Arrival methods for localisation techniques typically fail when signals are highly dispersive. Hence, algorithms capable of dispersion compensation are sought. This paper presents a method based on the Time - Distance Domain Transform for an accurate AE event localisation. The source localisation is found through a minimization problem. The proposed technique focuses on transforming the time signal to the distance domain response, which would be recorded at the source. Only, basic elastic material properties and plate thickness are used in the approach, avoiding arbitrary parameters tuning.

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling

PubMed Central

Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-01-01

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro. Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo. Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM. PMID:28915637

[Localised bronchi amyloidosis: a case report].

PubMed

Ayadi, Lobna; Khabir, Abdelmajid; Boudawara, Tahya; Makni, Saloua; Rekik, Wajdi Karim; Ayoub, Abdelkader; Jlidi, Rachid

2004-02-01

Localised pulmonary amyloïdosis is exceptional. Tracheobronchial symptoms are the most frequent. We report a case of a 68 year-old man complaining of cough and breathlessness. Bronchoscopy showed a submucosal infiltration with stenosis of left upper and lower bronchi. Multiples biopsy were performed and concluded to amyloïdosis of AL type. Our objective is to describe the anatomopathologic aspects of localised pulmonary amyloïdosis and to discuss its pathogeny.

Sensory Perception in the Human Research and Engineering Directorate: Thrust Areas and Recent Research 2011-2014

DTIC Science & Technology

2014-09-01

estimation in an open space. In: Glotin, editor. Soundscape semiotics: localisation and categorisation. Rijeka (Croatia): InTech; 2014. Available at...Glotin H, editor. Soundscape semiotics – localisation and categorisation. Rijeka (Croatia): InTech; 2014. Available at: http://www.intechopen.com...books/ soundscape -semiotics-localisation -and-categorisation/auditory-distance-estimation-in-an-open-space. The purpose of the study was to expand our

Inhibition of Macrophage CD36 Expression and Cellular Oxidized Low Density Lipoprotein (oxLDL) Accumulation by Tamoxifen: A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)γ-DEPENDENT MECHANISM.

PubMed

Yu, Miao; Jiang, Meixiu; Chen, Yuanli; Zhang, Shuang; Zhang, Wenwen; Yang, Xiaoxiao; Li, Xiaoju; Li, Yan; Duan, Shengzhong; Han, Jihong; Duan, Yajun

2016-08-12

Macrophage CD36 binds and internalizes oxidized low density lipoprotein (oxLDL) to facilitate foam cell formation. CD36 expression is activated by peroxisome proliferator-activated receptor γ (PPARγ). Tamoxifen, an anti-breast cancer medicine, has demonstrated pleiotropic functions including cardioprotection with unfully elucidated mechanisms. In this study, we determined that treatment of ApoE-deficient mice with tamoxifen reduced atherosclerosis, which was associated with decreased CD36 and PPARγ expression in lesion areas. At the cellular level, we observed that tamoxifen inhibited CD36 protein expression in human THP-1 monocytes, THP-1/PMA macrophages, and human blood monocyte-derived macrophages. Associated with decreased CD36 protein expression, tamoxifen reduced cellular oxLDL accumulation in a CD36-dependent manner. At the transcriptional level, tamoxifen decreased CD36 mRNA expression, promoter activity, and the binding of the PPARγ response element in CD36 promoter to PPARγ protein. Tamoxifen blocked ligand-induced PPARγ nuclear translocation and CD36 expression, but it increased PPARγ phosphorylation, which was due to that tamoxifen-activated ERK1/2. Furthermore, deficiency of PPARγ expression in macrophages abolished the inhibitory effect of tamoxifen on CD36 expression or cellular oxLDL accumulation both in vitro and in vivo Taken together, our study demonstrates that tamoxifen inhibits CD36 expression and cellular oxLDL accumulation by inactivating the PPARγ signaling pathway, and the inhibition of macrophage CD36 expression can be attributed to the anti-atherogenic properties of tamoxifen. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells

PubMed Central

Tawadros, T; Brown, M D; Hart, C A; Clarke, N W

2012-01-01

Background: High intake of omega-6 polyunsaturated fatty acids (PUFA) has been associated with clinical progression in prostate cancer (CaP). This study investigates the signalling mechanism by which the omega-6 PUFA arachidonic acid (AA) induces prostatic cellular migration to bone marrow stroma. Methods: Western blot analysis of the PC-3, PC3-GFP, DU 145 and LNCaP cells or their lipid raft (LR) components post AA stimulation was conducted in association with assays for adhesion and invasion through the bone marrow endothelial monolayers. Results: Arachidonic acid increased transendothelial migration of PC3-GFP cells (adhesion 37%±0.08, P=0.0124; transmigration 270%±0.145, P=0.0008). Akt, Src and focal adhesion kinase (FAK) pathways were induced by AA and integrally involved in transendothelial migration. LR were critical in AA uptake and induced Akt activity. Ephrin receptor A2 (EphA2), localised in LR, is expressed in DU 145 and PC-3 cells. Arachidonic acid induced a rapid increase of EphA2 Akt-dependent/ligand-independent activation, while knockdown of the EphrinA1 ligand decreased AA induced transendothelial migration, with an associated decrease in Src and FAK activity. Arachidonic acid activated Akt in EphA2− LNCaP cells but failed to induce BMEC transendothelial invasion. Conclusion: Arachidonic acid induced stimulation of EphA2 in vitro is associated fundamentally with CaP epithelial migration across the endothelial barrier. PMID:23037715

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Radioguided localisation of impalpable breast lesions using 99m-Technetium macroaggregated albumin: Lessons learnt during introduction of a new technique to guide preoperative localisation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Landman, Joanne; Kulawansa, Sagarika; McCarthy, Michael

2015-03-15

Preoperative wire-guided localisation (WGL) of impalpable breast lesions is widely used but can be technically difficult. Risks include wire migration, inaccurate placement, and inadequate surgical margins. Research shows that radioguided occult lesion localisation (ROLL) is quicker, easier, and can improve surgical and cosmetic outcomes. An audited introduction of ROLL was conducted to validate the technique as a feasible alternative to WGL. Fifty patients with single impalpable lesions and biopsy proven malignancy or indeterminate histology underwent WGL followed by intralesional radiopharmaceutical injection of 99m-Technetium macroaggregated albumin. Postprocedural mammography was performed to demonstrate wire position, and scintigraphy to evaluate radiopharmaceutical migration. Lymphoscintigraphymore » and intraoperative sentinel node biopsy were performed if indicated, followed by lesion localisation and excision using a gamma probe. Specimen imaging was performed, with immediate reexcision for visibly inadequate margins. Accurate localisation was achieved in 86% of patients with ROLL compared to 72% with WGL. All lesions were successfully removed, with clear margins in 71.8% of malignant lesions. Reexcision and intraoperative sentinel node localisation rates were equivalent to preaudit figures for WGL. ROLL was easy to perform and problems were infrequent. Inaccurate radiopharmaceutical placement necessitating WGL occurred in four patients. Minor radiopharmaceutical migration was common, but precluded using ROLL in only two cases. ROLL is effective, simple, inexpensive, and easily learnt; however, preoperative confirmation of correct radiopharmaceutical placement using mammography and the gamma probe is important to help ensure successful lesion removal. Insertion of a backup hookwire is recommended during the initial introduction of ROLL.« less

Fukunaga-Koontz feature transformation for statistical structural damage detection and hierarchical neuro-fuzzy damage localisation

NASA Astrophysics Data System (ADS)

Hoell, Simon; Omenzetter, Piotr

2017-07-01

Considering jointly damage sensitive features (DSFs) of signals recorded by multiple sensors, applying advanced transformations to these DSFs and assessing systematically their contribution to damage detectability and localisation can significantly enhance the performance of structural health monitoring systems. This philosophy is explored here for partial autocorrelation coefficients (PACCs) of acceleration responses. They are interrogated with the help of the linear discriminant analysis based on the Fukunaga-Koontz transformation using datasets of the healthy and selected reference damage states. Then, a simple but efficient fast forward selection procedure is applied to rank the DSF components with respect to statistical distance measures specialised for either damage detection or localisation. For the damage detection task, the optimal feature subsets are identified based on the statistical hypothesis testing. For damage localisation, a hierarchical neuro-fuzzy tool is developed that uses the DSF ranking to establish its own optimal architecture. The proposed approaches are evaluated experimentally on data from non-destructively simulated damage in a laboratory scale wind turbine blade. The results support our claim of being able to enhance damage detectability and localisation performance by transforming and optimally selecting DSFs. It is demonstrated that the optimally selected PACCs from multiple sensors or their Fukunaga-Koontz transformed versions can not only improve the detectability of damage via statistical hypothesis testing but also increase the accuracy of damage localisation when used as inputs into a hierarchical neuro-fuzzy network. Furthermore, the computational effort of employing these advanced soft computing models for damage localisation can be significantly reduced by using transformed DSFs.

PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases

PubMed Central

Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M

2006-01-01

Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016

bicoid RNA localization requires specific binding of an endosomal sorting complex

PubMed Central

Irion, Uwe; St Johnston, Daniel

2007-01-01

Summary paragraph: bicoid mRNA localises to the anterior of the Drosophila egg, where it is translated to form a morphogen gradient of Bicoid protein that patterns the head and thorax of the embryo. Although bicoid was the first identified localised cytoplasmic determinant1-4, little is known about how the mRNA is coupled to the microtubule-dependent transport pathway that targets it to the anterior, and it has been proposed that it is recognised by a complex of many redundant proteins, each of which binds to the localisation element in its 3'UTR with little or no specificity5. Indeed, the only known RNA-binding protein that co-localises with bicoid mRNA is Staufen, which binds non-specifically to dsRNA in vitro6, 7. Here we show that mutants in all subunits of the ESCRT-II complex (Vps22, Vps25 and Vps36) abolish the final Staufen-dependent step in bcd RNA localisation. ESCRT-II is a highly conserved component of the pathway that sorts ubiquitinated endosomal proteins into internal vesicles8, 9, and functions as a tumour-suppressor by removing activated receptors from the cytoplasm10, 11. However, the role of ESCRT-II in bicoid localisation appears to be independent of endosomal sorting, because mutations in ESCRT-I and III components have no effect of the targeting of bicoid mRNA. Instead, Vps36 functions by binding directly and specifically to stem-loop V of the bicoid 3'UTR through its N-terminal GLUE domain12, making it the first example of a sequence specific RNA-binding protein that recognises the bicoid localisation signal. Furthermore, Vps36 localises to the anterior of the oocyte in a bicoid mRNA-dependent manner, and is required for the subsequent recruitment of Staufen to the bicoid complex. This novel function of ESCRT-II as an RNA-binding complex is conserved in vertebrates, and may explain some of its roles that are independent of endosomal sorting. PMID:17268469

LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed.

PubMed

Boulard, C; Thévenin, J; Tranquet, O; Laporte, V; Lepiniec, L; Dubreucq, B

2018-05-01

The LAFL transcription factors LEC2, ABI3, FUS3 and LEC1 are master regulators of seed development. LEC2, ABI3 and FUS3 are closely related proteins that contain a B3-type DNA binding domain. We have previously shown that LEC1 (a NF-YB type protein) can increase LEC2 and ABI3 but not FUS3 activity. Interestingly, FUS3, LEC2 and ABI3 contain a B2 domain, the function of which remains elusive. We showed that LEC1 and LEC2 partially co-localised in the nucleus of developing embryos. By comparing protein sequences from various species, we identified within the B2 domains a set of highly conserved residues (i.e. TKxxARxxRxxAxxR). This domain directly interacts with LEC1 in yeast. Mutations of the conserved amino acids of the motif in the B2 domain abolished this interaction both in yeast and in moss protoplasts and did not alter the nuclear localisation of LEC2 in planta. Conversely, the mutations of key amino acids for the function of LEC1 in planta (D86K) prevented the interaction with LEC2. These results provide molecular evidences for the binding of LEC1 to B2-domain containing transcription factors, to form heteromers, involved in the control of gene expression. Copyright © 2018 Elsevier B.V. All rights reserved.

Acetylcholine and the alpha 7 nicotinic receptor: a potential therapeutic target for the treatment of periodontal disease?

PubMed Central

2013-01-01

Objectives The aim of this review is to examine the evidence for a functional cholinergic system operating within the periodontium and determine the evidence for its role in periodontal immunity. Introduction Acetylcholine can influence the immune system via the ‘cholinergic anti-inflammatory pathway’. This pathway is mediated by the vagus nerve which releases acetylcholine to interact with the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) on proximate immuno-regulatory cells. Activation of the α7nAChR on these cells leads to down-regulated expression of pro-inflammatory mediators and thus regulates localised inflammatory responses. The role of the vagus nerve in periodontal pathophysiology is currently unknown. However, non-neuronal cells can also release acetylcholine and express the α7nAChR; these include keratinocytes, fibroblasts, T cells, B cells and macrophages. Therefore, by both autocrine and paracrine methods non-neuronal acetylcholine can also be hypothesised to modulate the localised immune response. Methods A Pubmed database search was performed for studies providing evidence for a functional cholinergic system operating in the periodontium. In addition, literature on the role of the ‘cholinergic anti-inflammatory pathway’ in modulating the immune response was extrapolated to hypothesise that similar mechanisms of immune regulation occur within the periodontium. Conclusion The evidence suggests a functional nonneuronal ‘cholinergic anti-inflammatory pathway’ may operate in the periodontium and that this may be targeted therapeutically to treat periodontal disease. PMID:22777144

VvGONST-A and VvGONST-B are Golgi-localised GDP-sugar transporters in grapevine (Vitis vinifera L.).

PubMed

Utz, Daniella; Handford, Michael

2015-02-01

Plant nucleotide-sugar transporters (NSTs) are responsible for the import of nucleotide-sugar substrates into the Golgi lumen, for subsequent use in glycosylation reactions. NSTs are specific for either GDP- or UDP-sugars, and almost all transporters studied to date have been isolated from Arabidopsis thaliana L. In order to determine the conservation of the import mechanism in other higher plant species, here we report the identification and characterisation of VvGONST-A and VvGONST-B from grapevine (Vitis vinifera L. cv. Thompson Seedless), which are the orthologues of the GDP-sugar transporters GONST3 and GONST4 in Arabidopsis. Both grapevine NSTs possess the molecular features characteristic of GDP-sugar transporters, including a GDP-binding domain (GXL/VNK) towards the C-terminal. VvGONST-A and VvGONST-B expression is highest at berry setting and decreases throughout berry development and ripening. Moreover, we show using green fluorescent protein (GFP) tagged versions and brefeldin A treatments, that both are localised in the Golgi apparatus. Additionally, in vitro transport assays after expression of both NSTs in tobacco leaves indicate that VvGONST-A and VvGONST-B are capable of transporting GDP-mannose and GDP-glucose, respectively, but not a range of other UDP- and GDP-sugars. The possible functions of these NSTs in glucomannan synthesis and/or glycosylation of sphingolipids are discussed. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Biological processing of dinuclear ruthenium complexes in eukaryotic cells.

PubMed

Li, Xin; Heimann, Kirsten; Dinh, Xuyen Thi; Keene, F Richard; Collins, J Grant

2016-10-20

The biological processing - mechanism of cellular uptake, effects on the cytoplasmic and mitochondrial membranes, intracellular sites of localisation and induction of reactive oxygen species - of two dinuclear polypyridylruthenium(ii) complexes has been examined in three eukaryotic cells lines. Flow cytometry was used to determine the uptake of [{Ru(phen)2}2{μ-bb12}](4+) (Rubb12) and [Ru(phen)2(μ-bb7)Ru(tpy)Cl](3+) {Rubb7-Cl, where phen = 1,10-phenanthroline, tpy = 2,2':6',2''-terpyridine and bbn = bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane} in baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (HepG2) cell lines. The results demonstrated that the major uptake mechanism for Rubb12 and Rubb7-Cl was active transport, although with a significant contribution from carrier-assisted diffusion for Rubb12 and passive diffusion for Rubb7-Cl. Flow cytometry coupled with Annexin V/TO-PRO-3 double-staining was used to compare cell death by membrane damage or apoptosis. Rubb12 induced significant direct membrane damage, particularly with HepG2 cells, while Rubb7-Cl caused considerably less membrane damage but induced greater levels of apoptosis. Confocal microscopy, coupled with JC-1 assays, demonstrated that Rubb12 depolarises the mitochondrial membrane, whereas Rubb7-Cl had a much smaller affect. Cellular localisation experiments indicated that Rubb12 did not accumulate in the mitochondria, whereas significant mitochondrial accumulation was observed for Rubb7-Cl. The effect of Rubb12 and Rubb7-Cl on intracellular superoxide dismutase activity showed that the ruthenium complexes could induce cell death via a reactive oxygen species-mediated pathway. The results of this study demonstrate that Rubb12 predominantly kills eukaryotic cells by damaging the cytoplasmic membrane. As this dinuclear ruthenium complex has been previously shown to exhibit greater toxicity towards bacteria than eukaryotic cells, the results of the present study suggest that metal-based cationic oligomers can achieve selective toxicity against bacteria, despite exhibiting a non-specific membrane damage mechanism of action.

Rhenium tetrazolato complexes coordinated to thioalkyl-functionalised phenanthroline ligands: synthesis, photophysical characterisation, and incubation in live HeLa cells.

PubMed

Werrett, Melissa V; Wright, Phillip J; Simpson, Peter V; Raiteri, Paolo; Skelton, Brian W; Stagni, Stefano; Buckley, Alysia G; Rigby, Paul J; Massi, Massimiliano

2015-12-21

Three new complexes of formulation fac-[Re(CO)3(diim)L], where diim is either 1,10-phenanthroline or 1,10-phenanthroline functionalised at position 5 by a thioalkyl chain, and L is either a chloro or aryltetrazolato ancillary ligand, were synthesised and photophysically characterised. The complexes exhibit phosphorescent emission with maxima around 600 nm, originating from triplet metal-to-ligand charge transfer states with partially mixed ligand-to-ligand charge transfer character. The emission is relatively long-lived, within the 200-400 ns range, and with quantum yields of 2-4%. The complexes were trialed as cellular markers in live HeLa cells, along with two previously reported rhenium tetrazolato complexes bound to unsubstituted 1,10-phenanthroline. All five complexes exhibit good cellular uptake and non-specific perinuclear localisation. Upon excitation at 405 nm, the emission from the rhenium complexes could be clearly distinguished from autofluorescence, as demonstrated by spectral detection within the live cells. Four of the complexes did not appear to be toxic, however prolonged excitation could result in membrane blebbing. No major sign of photobleaching was detected upon multiple imaging on the same cell sample.

Vaccine induced differential expressions of miRNAs at cytolytic stage in chickens resistant or susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Gene expression regulation is critical for all cellular processes since dysregulation of it often results in elevated disease risk and compromised cellular immunity. MicroRNAs (miRNAs) directly regulate gene expression post-transcriptionally through base-pairing with regions in the 3’-untranslated s...

Cellular reprogramming dynamics follow a simple 1D reaction coordinate

NASA Astrophysics Data System (ADS)

Teja Pusuluri, Sai; Lang, Alex H.; Mehta, Pankaj; Castillo, Horacio E.

2018-01-01

Cellular reprogramming, the conversion of one cell type to another, induces global changes in gene expression involving thousands of genes, and understanding how cells globally alter their gene expression profile during reprogramming is an ongoing problem. Here we reanalyze time-course data on cellular reprogramming from differentiated cell types to induced pluripotent stem cells (iPSCs) and show that gene expression dynamics during reprogramming follow a simple 1D reaction coordinate. This reaction coordinate is independent of both the time it takes to reach the iPSC state as well as the details of the experimental protocol used. Using Monte-Carlo simulations, we show that such a reaction coordinate emerges from epigenetic landscape models where cellular reprogramming is viewed as a ‘barrier-crossing’ process between cell fates. Overall, our analysis and model suggest that gene expression dynamics during reprogramming follow a canonical trajectory consistent with the idea of an ‘optimal path’ in gene expression space for reprogramming.

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

PubMed

Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

2017-08-31

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

PubMed Central

Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

2017-01-01

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

Distinct physiological roles for the two L-asparaginase isozymes of Escherichia coli.

PubMed

Srikhanta, Yogitha N; Atack, John M; Beacham, Ifor R; Jennings, Michael P

2013-07-05

Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen. Copyright © 2013. Published by Elsevier Inc.

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

PubMed

Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

2015-12-01

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Assessing the Nature of the Distribution of Localised States in Bulk GaAsBi.

PubMed

Wilson, Tom; Hylton, Nicholas P; Harada, Yukihiro; Pearce, Phoebe; Alonso-Álvarez, Diego; Mellor, Alex; Richards, Robert D; David, John P R; Ekins-Daukes, Nicholas J

2018-04-24

A comprehensive assessment of the nature of the distribution of sub band-gap energy states in bulk GaAsBi is presented using power and temperature dependent photoluminescence spectroscopy. The observation of a characteristic red-blue-red shift in the peak luminescence energy indicates the presence of short-range alloy disorder in the material. A decrease in the carrier localisation energy demonstrates the strong excitation power dependence of localised state behaviour and is attributed to the filling of energy states furthest from the valence band edge. Analysis of the photoluminescence lineshape at low temperature presents strong evidence for a Gaussian distribution of localised states that extends from the valence band edge. Furthermore, a rate model is employed to understand the non-uniform thermal quenching of the photoluminescence and indicates the presence of two Gaussian-like distributions making up the density of localised states. These components are attributed to the presence of microscopic fluctuations in Bi content, due to short-range alloy disorder across the GaAsBi layer, and the formation of Bi related point defects, resulting from low temperature growth.

Assessment of localisation to auditory stimulation in post-comatose states: use the patient’s own name

PubMed Central

2013-01-01

Background At present, there is no consensus on how to clinically assess localisation to sound in patients recovering from coma. We here studied auditory localisation using the patient’s own name as compared to a meaningless sound (i.e., ringing bell). Methods Eighty-six post-comatose patients diagnosed with a vegetative state/unresponsive wakefulness syndrome or a minimally conscious state were prospectively included. Localisation of auditory stimulation (i.e., head or eyes orientation toward the sound) was assessed using the patient’s own name as compared to a ringing bell. Statistical analyses used binomial testing with bonferroni correction for multiple comparisons. Results 37 (43%) out of the 86 studied patients showed localisation to auditory stimulation. More patients (n=34, 40%) oriented the head or eyes to their own name as compared to sound (n=20, 23%; p<0.001). Conclusions When assessing auditory function in disorders of consciousness, using the patient’s own name is here shown to be more suitable to elicit a response as compared to neutral sound. PMID:23506054

[Knowledge and destiny or longevity and old age: the heritage of Homo sapiens].

PubMed

Goddio, A S

1994-12-01

Several theories have been proposed to explain ageing: limitation of the number of cell divisions or Hayflick's limit, the genetic theory, the action of free radicals, immune deficiency, etc. All of these theories share several points in common: their genetic determinism or repercussions which appear to be part of the heritage of complex organisms. Progress in genetics with chromosome decoding to localise genes and genetic manipulations or control of gene expression will probably allow an increased life expectancy, perhaps in the near future.

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues.

PubMed

McSorley, Theresa; Ort, Stephan; Hazra, Saugata; Lavie, Arnon; Konrad, Manfred

2008-03-05

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the k(cat) values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme.

N-terminal nesprin-2 variants regulate β-catenin signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa

2016-07-15

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragmentmore » of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.« less

Heat shock protein 70 in the rat nasal cavity: localisation and response to hyperthermia.

PubMed

Simpson, Sharon A; Alexander, David J; Reed, Celia J

2004-06-01

Heat shock proteins (HSPs) are a group of proteins that are rapidly induced in response to physiological stress, including hyperthermia and exposure to toxicants. Thus they may provide a useful index of toxicity in in vitro systems for screening for toxicity. We have recently developed a rat nasal explant system for investigating upper respiratory tract toxicity, and the aims of this study were to localise HSP70 within the rat nasal cavity and to characterise its response to hyperthermia. Constitutively, HSP70 was found to be predominantly localised to the sustentacular cells, basal cells and Bowman's glands of the olfactory epithelium (OE), with the most intense immunohistochemical staining at levels 3 and 4 of the posterior of the rat nasal cavity. Ethmoturbinates (ETs) and liver slices were exposed to heat shock (37 degrees and 43 degrees C, respectively) for 45 min and then returned to normal culture temperatures (31 degrees and 37 degrees C, respectively) for 24 h. In ETs, HSP72 was maximally induced 4-fold at 4 h after heat shock, and levels then returned to those of control tissue. ATP concentrations were markedly decreased up to 4 h after heat shock and then returned to control levels. In contrast, HSP72 levels in liver slices increased and ATP levels decreased steadily throughout the 24 h culture period. ETs were also able to withstand a 45-min heat shock at 43 degrees C, that is 12 degrees C above normal culture temperature. Incubation of ETs with cycloheximide prior to heat shock reduced the ability of the OE to recover from heat shock at 37 degrees C. Thus the OE of the rat nasal cavity expresses HSP72, and this protein appears to play an important role in the ability of the tissue to withstand hyperthermia.

Arc/Arg3.1 mRNA expression reveals a sub-cellular trace of prior sound exposure in adult primary auditory cortex

PubMed Central

Ivanova, Tamara; Matthews, Andrew; Gross, Christina; Mappus, Rudolph C.; Gollnick, Clare; Swanson, Andrew; Bassell, Gary J.; Liu, Robert C.

2011-01-01

Acquiring the behavioral significance of a sound has repeatedly been shown to correlate with long term changes in response properties of neurons in the adult primary auditory cortex. However, the molecular and cellular basis for such changes is still poorly understood. To address this, we have begun examining the auditory cortical expression of an activity-dependent effector immediate early gene (IEG) with documented roles in synaptic plasticity and memory consolidation in the hippocampus: Arc/Arg3.1. For initial characterization, we applied a repeated 10 minute (24 hour separation) sound exposure paradigm to determine the strength and consistency of sound-evoked Arc/Arg3.1 mRNA expression in the absence of explicit behavioral contingencies for the sound. We used 3D surface reconstruction methods in conjunction with fluorescent in-situ hybridization (FISH) to assess the layer-specific sub-cellular compartmental expression of Arc/Arg3.1 mRNA. We unexpectedly found that both the intranuclear and cytoplasmic patterns of expression depended on the prior history of sound stimulation. Specifically, the percentage of neurons with expression only in the cytoplasm increased for repeated versus singular sound exposure, while intranuclear expression decreased. In contrast, the total cellular expression did not differ, consistent with prior IEG studies of primary auditory cortex. Our results were specific for cortical layers 3–6, as there was virtually no sound driven Arc/Arg3.1 mRNA in layers 1–2 immediately after stimulation. Our results are consistent with the kinetics and/or detectability of cortical sub-cellular Arc/Arg3.1 mRNA expression being altered by the initial exposure to the sound, suggesting exposure-induced modifications in the cytoplasmic Arc/Arg3.1 mRNA pool. PMID:21334422

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

PubMed Central

Xue, Gondga; von Schubert, Conrad; Hermann, Pascal; Peyer, Martina; Maushagen, Regina; Schmuckli-Maurer, Jacqueline; Bütikofer, Peter; Langsley, Gordon; Dobbelaere, Dirk A.E.

2010-01-01

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite–host interactions during host cell division. PMID:20381541

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells.

PubMed

Fabrick, Jeffrey A; Hull, J Joe

2017-04-20

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.

PubMed

Hu, G; Lee, H; Price, S M; Shen, M M; Abate-Shen, C

2001-06-01

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

PubMed Central

2010-01-01

Background Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins. Results Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated. Conclusions The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface. PMID:20144183

Evidence of phase nucleation during olivine diffusion creep: a new perspective for mantle strain localisation

NASA Astrophysics Data System (ADS)

Précigout, Jacques; Stünitz, Holger

2017-04-01

Mantle strain localisation is of great importance for lithosphere dynamics, but the cause for this phenomenon remains very elusive, particularly in conditions of the strong and ductile uppermost mantle. In these latter, grain size reduction leading to diffusion creep in olivine is believed to be one of the best candidates to account for strain localisation. However, the mechanisms of grain size reduction in this regime are still poorly understood. Here we show the results of Griggs-type experiments that document grain size reduction and material weakening during wet olivine diffusion creep at 900 °C and 1.2 GPa. While occurring for both, mono-phase and two-phase aggregates, grain size reduction is coeval with strain localisation and local phase mixing in olivine-pyroxene aggregates. Based on evidence of fluid inclusions and cracks filled with a fine-grained phase mixture, we conclude that grain size reduces as a result of fluid-assisted nucleation. Cavitation induced by grain boundary sliding (creep cavitation) can be inferred, and may play a critical role for olivine grain size reduction. Amongst their implications for rock rheology in general, our findings highlight a key process for strain localisation in the ductile uppermost mantle. This study has been published under the reference: "Précigout, J., and Stünitz, H. (2016) Evidence of phase nucleation during olivine diffusion creep: a new perspective for mantle strain localisation. Earth and Planetary Science Letters 455: 94-105, doi:101016/j.epsl.2016.09.029".

Comparison of the time taken for localised breast surgery pre- and post-introduction of intra-operative digital specimen mammography.

PubMed

Carraro do Nascimento, Vinicius; Bourke, Anita G

2018-02-01

More than half of the patients with an impalpable malignant breast lesion have a mammographically detected and imaged-guided localisation, which can be technically challenging for the breast surgeon. Specimen imaging is used to confirm successful excision of the localised index lesion and has improved the operating list efficiency resulting in a higher number of excisions per surgical list. The aim of this study was to evaluate whether introducing IDSM (intra-operative digital specimen mammography) saved operation time for localised breast surgery. A single-centre retrospective review was undertaken to compare the operation time (from incision to wound closure) taken for excision of 114 consecutive image-guided localised impalpable breast lesions, performed using departmental specimen radiography (DSR), 6 months prior to the introduction of IDSM (Hologic, Trident ® ) in March 2013, with the theatre time taken for excision of 121 consecutive image-guided localised impalpable breast lesions in the 6 months following introduction of IDSM. There was no significant difference in mean surgical time, which were 47.8 (±27.3) minutes in the CSR group and 48.8 (±25.7) minutes in the IDSM group. We were expecting to confirm a reduction in theatre time with the introduction of IDSM. Surprisingly, no difference in operating times was demonstrated. Factors that influenced the impact of IDSM included the proximity of the imaging department to the operating theatre. © 2017 The Royal Australian and New Zealand College of Radiologists.

Multiparametric Determination of Radiation Risk

NASA Technical Reports Server (NTRS)

Richmond, Robert C.

2003-01-01

Predicting risk of human cancer following exposure to ionizing space radiation is challenging in part because of uncertainties of low-dose distribution amongst cells, of unknown potentially synergistic effects of microgravity upon cellular protein-expression, and of processing dose-related damage within cells to produce rare and late-appearing malignant transformation, degrade the confidence of cancer risk-estimates. The NASA- specific responsibility to estimate the risks of radiogenic cancer in a limited number of astronauts is not amenable to epidemiologic study, thereby increasing this challenge. Developing adequately sensitive cellular biodosimeters that simultaneously report 1) the quantity of absorbed close after exposure to ionizing radiation, 2) the quality of radiation delivering that dose, and 3) the risk of developing malignant transformation by the cells absorbing that dose could be useful for resolving these challenges. Use of a multiparametric cellular biodosimeter is suggested using analyses of gene-expression and protein-expression whereby large datasets of cellular response to radiation-induced damage are obtained and analyzed for expression-profiles correlated with established end points and molecular markers predictive for cancer-risk. Analytical techniques of genomics and proteomics may be used to establish dose-dependency of multiple gene- and protein- expressions resulting from radiation-induced cellular damage. Furthermore, gene- and protein-expression from cells in microgravity are known to be altered relative to cells grown on the ground at 1g. Therefore, hypotheses are proposed that 1) macromolecular expression caused by radiation-induced damage in cells in microgravity may be different than on the ground, and 2) different patterns of macromolecular expression in microgravity may alter human radiogenic cancer risk relative to radiation exposure on Earth. A new paradigm is accordingly suggested as a national database wherein genomic and proteomic datasets are registered and interrogated in order to provide statistically significant dose-dependent risk estimation of radiogenic cancer in astronauts.

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

PubMed

Aomatsu, Keiichi; Arao, Tokuzo; Abe, Kosuke; Kodama, Aya; Sugioka, Koji; Matsumoto, Kazuko; Kudo, Kanae; Kimura, Hideharu; Fujita, Yoshihiko; Hayashi, Hidetoshi; Nagai, Tomoyuki; Shimomura, Yoshikazu; Nishio, Kazuto

2012-02-16

The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

Impairment of CDKL5 nuclear localisation as a cause for severe infantile encephalopathy.

PubMed

Rosas-Vargas, H; Bahi-Buisson, N; Philippe, C; Nectoux, J; Girard, B; N'Guyen Morel, M A; Gitiaux, C; Lazaro, L; Odent, S; Jonveaux, P; Chelly, J; Bienvenu, T

2008-03-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome-like phenotype. To date, fewer than 20 different mutations have been reported. So far, no clear genotype-phenotype correlation has been established. We screened the entire coding region of CDKL5 in 151 affected girls with a clinically heterogeneous phenotype ranging from encephalopathy with epilepsy to atypical Rett syndrome by denaturing high liquid performance chromatography and direct sequencing, and we identified three novel missense mutations located in catalytic domain (p.Ala40Val, p.Arg65Gln, p.Leu220Pro). Segregation analysis showed that p.Arg65Gln was inherited from the healthy father, which rules out the involvement of CDKL5 in the aetiology of the phenotype in this patient. However, the de novo occurrence was shown for p.Ala40Val and p.Leu220Pro. The p.Ala40Val mutation was observed in two unrelated patients and represented the first recurrent mutation in the CDKL5 gene. For the two de novo mutations, we analysed the cellular localisation of the wild-type and CDKL5 mutants by transfection experiments. We showed that the two CDKL5 mutations cause mislocalisation of the mutant CDKL5 proteins in the cytoplasm. Interestingly these missense mutations that result in a mislocalisation of the CDKL5 protein are associated with severe developmental delay which was apparent within the first months of life characterised by early and generalised hypotonia, and autistic features, and as well as early infantile spasms.

A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.

PubMed

Diot, Alan; Hinks-Roberts, Alex; Lodge, Tiffany; Liao, Chunyan; Dombi, Eszter; Morten, Karl; Brady, Stefen; Fratter, Carl; Carver, Janet; Muir, Rebecca; Davis, Ryan; Green, Charlotte J; Johnston, Iain; Hilton-Jones, David; Sue, Carolyn; Mortiboys, Heather; Poulton, Joanna

2015-10-01

Mitophagy is a cellular mechanism for the recycling of mitochondrial fragments. This process is able to improve mitochondrial DNA (mtDNA) quality in heteroplasmic mtDNA disease, in which mutant mtDNA co-exists with normal mtDNA. In disorders where the load of mutant mtDNA determines disease severity it is likely to be an important determinant of disease progression. Measuring mitophagy is technically demanding. We used pharmacological modulators of autophagy to validate two techniques for quantifying mitophagy. First we used the IN Cell 1000 analyzer to quantify mitochondrial co-localisation with LC3-II positive autophagosomes. Unlike conventional fluorescence and electron microscopy, this high-throughput system is sufficiently sensitive to detect transient low frequency autophagosomes. Secondly, because mitophagy preferentially removes pathogenic heteroplasmic mtDNA mutants, we developed a heteroplasmy assay based on loss of m.3243A>G mtDNA, during culture conditions requiring oxidative metabolism ("energetic stress"). The effects of the pharmacological modulators on these two measures were consistent, confirming that the high throughput imaging output (autophagosomes co-localising with mitochondria) reflects mitochondrial quality control. To further validate these methods, we performed a more detailed study using metformin, the most commonly prescribed antidiabetic drug that is still sometimes used in Maternally Inherited Diabetes and Deafness (MIDD). This confirmed our initial findings and revealed that metformin inhibits mitophagy at clinically relevant concentrations, suggesting that it may have novel therapeutic uses. Copyright © 2015. Published by Elsevier Ltd.

New insights on the functional role of URG7 in the cellular response to ER stress.

PubMed

Armentano, Maria Francesca; Caterino, Marianna; Miglionico, Rocchina; Ostuni, Angela; Pace, Maria Carmela; Cozzolino, Flora; Monti, Maria; Milella, Luigi; Carmosino, Monica; Pucci, Piero; Bisaccia, Faustino

2018-04-28

Up-regulated Gene clone 7 (URG7) is an ER resident protein, whose expression is up-regulated in the presence of hepatitis B virus X antigen (HBxAg) during HBV infection. In virus-infected hepatocytes, URG7 shows an anti-apoptotic activity due to the PI3K/AKT signalling activation, does not seem to have tumorigenic properties, but it appears to promote the development and progression of fibrosis. However, the molecular mechanisms underlying URG7 activity remain largely unknown. To shed light on URG7 activity, we first analysed its interactome in HepG2 transfected cells: this analysis suggests that URG7 could have a role in affecting protein synthesis, folding and promoting proteins degradation. Moreover, keeping into account its subcellular localisation in the ER and that several viral infections give rise to ER stress, a panel of experiments was performed to evaluate a putative role of URG7 in ER stress. Our main results demonstrate that in ER-stressed cells URG7 is able to modulate the expression of Unfolded Protein Response (UPR) markers towards survival outcomes, up-regulating GRP78 protein and down-regulating the pro-apoptotic protein CHOP. Furthermore, URG7 reduces the ER stress by decreasing the amount of unfolded proteins, by increasing both the total protein ubiquitination and the AKT activation and reducing Caspase 3 activation. All together these data suggest that URG7 plays a pivotal role as a reliever of ER stress-induced apoptosis. This is the first characterisation of URG7 activity under ER stress conditions. The results presented here will help to hypothesise new strategies to counteract the antiapoptotic activity of URG7 in the context of the viral infection. © 2018 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Bidirectional nucleolar dysfunction in C9orf72 frontotemporal lobar degeneration.

PubMed

Mizielinska, Sarah; Ridler, Charlotte E; Balendra, Rubika; Thoeng, Annora; Woodling, Nathan S; Grässer, Friedrich A; Plagnol, Vincent; Lashley, Tammaryn; Partridge, Linda; Isaacs, Adrian M

2017-04-18

An intronic GGGGCC expansion in C9orf72 is the most common known cause of both frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat expansion leads to the generation of sense and antisense repeat RNA aggregates and dipeptide repeat (DPR) proteins, generated by repeat-associated non-ATG translation. The arginine-rich DPR proteins poly(glycine-arginine or GR) and poly(proline-arginine or PR) are potently neurotoxic and can localise to the nucleolus when expressed in cells, resulting in enlarged nucleoli with disrupted functionality. Furthermore, GGGGCC repeat RNA can bind nucleolar proteins in vitro. However, the relevance of nucleolar stress is unclear, as the arginine-rich DPR proteins do not localise to the nucleolus in C9orf72-associated FTLD/ALS (C9FTLD/ALS) patient brain. We measured nucleolar size in C9FTLD frontal cortex neurons using a three-dimensional, volumetric approach. Intriguingly, we found that C9FTLD brain exhibited bidirectional nucleolar stress. C9FTLD neuronal nucleoli were significantly smaller than control neuronal nucleoli. However, within C9FTLD brains, neurons containing poly(GR) inclusions had significantly larger nucleolar volumes than neurons without poly(GR) inclusions. In addition, expression of poly(GR) in adult Drosophila neurons led to significantly enlarged nucleoli. A small but significant increase in nucleolar volume was also observed in C9FTLD frontal cortex neurons containing GGGGCC repeat-containing RNA foci. These data show that nucleolar abnormalities are a consistent feature of C9FTLD brain, but that diverse pathomechanisms are at play, involving both DPR protein and repeat RNA toxicity.

Expression of renin–angiotensin system components in the early bovine embryo

PubMed Central

Pijacka, Wioletta; Hunter, Morag G; Broughton Pipkin, Fiona; Luck, Martin R

2012-01-01

The renin–angiotensin system (RAS), mainly associated with the regulation of blood pressure, has been recently investigated in female reproductive organs and the developing foetus. Angiotensin II (Ang II) influences oviductal gamete movements and foetal development, but there is no information about RAS in the early embryo. The aim of this study was to determine whether RAS components are present in the pre-implantation embryo, to determine how early they are expressed and to investigate their putative role at this stage of development. Bovine embryos produced in vitro were used for analysis of RAS transcripts (RT-PCR) and localisation of the receptors AGTR1 and AGTR2 (immunofluorescent labelling). We also investigated the effects of Ang II, Olmesartan (AGTR1 antagonist) and PD123319 (AGTR2 antagonist) on oocyte cleavage, embryo expansion and hatching. Pre-implanted embryos possessed AGTR1 and AGTR2 but not the other RAS components. Both receptors were present in the trophectoderm and in the inner cell mass of the blastocyst. AGTR1 was mainly localised in granular-like structures in the cytoplasm, suggesting its internalisation into clathrin-coated vesicles, and AGTR2 was found mainly in the nuclear membrane and in the mitotic spindle of dividing trophoblastic cells. Treating embryos with PD123319 increased the proportion of hatched embryos compared with the control. These results, the first on RAS in the early embryo, suggest that the pre-implanted embryo responds to Ang II from the mother rather than from the embryo itself. This may be a route by which the maternal RAS influences blastocyst hatching and early embryonic development. PMID:23781300

Molecular biology of myopia.

PubMed

Schaeffel, Frank; Simon, Perikles; Feldkaemper, Marita; Ohngemach, Sibylle; Williams, Robert W

2003-09-01

Experiments in animal models of myopia have emphasised the importance of visual input in emmetropisation but it is also evident that the development of human myopia is influenced to some degree by genetic factors. Molecular genetic approaches can help to identify both the genes involved in the control of ocular development and the potential targets for pharmacological intervention. This review covers a variety of techniques that are being used to study the molecular biology of myopia. In the first part, we describe techniques used to analyse visually induced changes in gene expression: Northern Blot, polymerase chain reaction (PCR) and real-time PCR to obtain semi-quantitative and quantitative measures of changes in transcription level of a known gene, differential display reverse transcription PCR (DD-RT-PCR) to search for new genes that are controlled by visual input, rapid amplification of 5' cDNA (5'-RACE) to extend the 5' end of sequences that are regulated by visual input, in situ hybridisation to localise the expression of a given gene in a tissue and oligonucleotide microarray assays to simultaneously test visually induced changes in thousands of transcripts in single experiments. In the second part, we describe techniques that are used to localise regions in the genome that contain genes that are involved in the control of eye growth and refractive errors in mice and humans. These include quantitative trait loci (QTL) mapping, exploiting experimental test crosses of mice and transmission disequilibrium tests (TDT) in humans to find chromosomal intervals that harbour genes involved in myopia development. We review several successful applications of this battery of techniques in myopia research.

Relevance of MET activation and genetic alterations of KRAS and E-cadherin for cetuximab sensitivity of gastric cancer cell lines.

PubMed

Heindl, Stefan; Eggenstein, Evelyn; Keller, Simone; Kneissl, Julia; Keller, Gisela; Mutze, Kathrin; Rauser, Sandra; Gasteiger, Georg; Drexler, Ingo; Hapfelmeier, Alexander; Höfler, Heinz; Luber, Birgit

2012-05-01

The therapeutic activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab in gastric cancer is currently being investigated. Reliable biomarkers for the identification of patients who are likely to benefit from the treatment are not available. The aim of the study was to examine the drug sensitivity of five gastric cancer cell lines towards cetuximab as a single agent and to establish predictive markers for chemosensitivity in this cell culture model. The effect of a combination of cetuximab with chemotherapy was compared between a sensitive and a nonsensitive cell line. EGFR expression, activation and localisation, the presence and subcellular localisation of the cell adhesion molecule E-cadherin as well as MET activation were examined by Western blot analysis, flow cytometry and immunofluorescence staining. Cells were treated with varying concentrations of cetuximab and cisplatin and 5-fluorouracil in tumour-relevant concentrations. The biological endpoint was cell viability, which was measured by XTT cell proliferation assay. Response to treatment was evaluated using statistical methods. We assessed the activity of cetuximab in five gastric cancer cell lines (AGS, KATOIII, MKN1, MKN28 and MKN45). The viability of two cell lines, MKN1 and MKN28, was significantly reduced by cetuximab treatment. High EGFR expression and low levels of receptor activation were associated with cetuximab responsiveness. MET activation as well as mutations of KRAS and CDH1 (gene encoding E-cadherin) was associated with cetuximab resistance. These data indicate that our examinations may be clinically relevant, and the candidate markers should therefore be tested in clinical studies.

Discriminating cellular heterogeneity using microwell-based RNA cytometry

PubMed Central

Dimov, Ivan K.; Lu, Rong; Lee, Eric P.; Seita, Jun; Sahoo, Debashis; Park, Seung-min; Weissman, Irving L.; Lee, Luke P.

2014-01-01

Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here, we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA-cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA-cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score. Moreover, we use Monte-Carlo simulation and RNA-cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We applied this system to characterize the RNA distributions of aging related genes in a highly purified mouse hematopoietic stem cell population. We identified genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during aging can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup. PMID:24667995

MagR Alone Is Insufficient to Confer Cellular Calcium Responses to Magnetic Stimulation

PubMed Central

Pang, Keliang; You, He; Chen, Yanbo; Chu, Pengcheng; Hu, Meiqin; Shen, Jianying; Guo, Wei; Xie, Can; Lu, Bai

2017-01-01

Magnetic manipulation of cell activity offers advantages over optical manipulation but an ideal tool remains elusive. The MagR protein was found through its interaction with cryptochrome (Cry) and the protein in solution appeared to respond to magnetic stimulation (MS). After we initiated an investigation on the specific role of MagR in cellular response to MS, a subsequent study claimed that MagR expression alone could achieve cellular activation by MS. Here we report that despite systematically testing different ways of measuring intracellular calcium and different MS protocols, it was not possible to detect any cellular or neuronal responses to MS in MagR-expressing HEK cells or primary neurons from the dorsal root ganglion and the hippocampus. By contrast, in neurons co-expressing MagR and channelrhodopin, optical but not MS increased calcium influx in hippocampal neurons. Our results indicate that MagR alone is not sufficient to confer cellular magnetic responses. PMID:28360843

Passive RFID Rotation Dimension Reduction via Aggregation

NASA Astrophysics Data System (ADS)

Matthews, Eric

Radio Frequency IDentification (RFID) has applications in object identification, position, and orientation tracking. RFID technology can be applied in hospitals for patient and equipment tracking, stores and warehouses for product tracking, robots for self-localisation, tracking hazardous materials, or locating any other desired object. Efficient and accurate algorithms that perform localisation are required to extract meaningful data beyond simple identification. A Received Signal Strength Indicator (RSSI) is the strength of a received radio frequency signal used to localise passive and active RFID tags. Many factors affect RSSI such as reflections, tag rotation in 3D space, and obstacles blocking line-of-sight. LANDMARC is a statistical method for estimating tag location based on a target tag's similarity to surrounding reference tags. LANDMARC does not take into account the rotation of the target tag. By either aggregating multiple reference tag positions at various rotations, or by determining a rotation value for a newly read tag, we can perform an expected value calculation based on a comparison to the k-most similar training samples via an algorithm called K-Nearest Neighbours (KNN) more accurately. By choosing the average as the aggregation function, we improve the relative accuracy of single-rotation LANDMARC localisation by 10%, and any-rotation localisation by 20%.

Immunogold staining procedure for the localisation of regulatory peptides.

PubMed

Varndell, I M; Tapia, F J; Probert, L; Buchan, A M; Gu, J; De Mey, J; Bloom, S R; Polak, J M

1982-01-01

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.

Non-Gaussian probabilistic MEG source localisation based on kernel density estimation☆

PubMed Central

Mohseni, Hamid R.; Kringelbach, Morten L.; Woolrich, Mark W.; Baker, Adam; Aziz, Tipu Z.; Probert-Smith, Penny

2014-01-01

There is strong evidence to suggest that data recorded from magnetoencephalography (MEG) follows a non-Gaussian distribution. However, existing standard methods for source localisation model the data using only second order statistics, and therefore use the inherent assumption of a Gaussian distribution. In this paper, we present a new general method for non-Gaussian source estimation of stationary signals for localising brain activity from MEG data. By providing a Bayesian formulation for MEG source localisation, we show that the source probability density function (pdf), which is not necessarily Gaussian, can be estimated using multivariate kernel density estimators. In the case of Gaussian data, the solution of the method is equivalent to that of widely used linearly constrained minimum variance (LCMV) beamformer. The method is also extended to handle data with highly correlated sources using the marginal distribution of the estimated joint distribution, which, in the case of Gaussian measurements, corresponds to the null-beamformer. The proposed non-Gaussian source localisation approach is shown to give better spatial estimates than the LCMV beamformer, both in simulations incorporating non-Gaussian signals, and in real MEG measurements of auditory and visual evoked responses, where the highly correlated sources are known to be difficult to estimate. PMID:24055702

Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells.

PubMed

Gillard, Ghislain; Shafaq-Zadah, Massiullah; Nicolle, Ophélie; Damaj, Raghida; Pécréaux, Jacques; Michaux, Grégoire

2015-05-01

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis. © 2015. Published by The Company of Biologists Ltd.

EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

PubMed

Cho, Min Chul; Cho, Sung Yong; Yoon, Cheol Yong; Lee, Seung Bae; Kwak, Cheol; Kim, Hyeon Hoe; Jeong, Hyeon

2015-01-01

To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells. Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively. In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation. Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Liwei; Zhao, Wenting; Zheng, Quanhui

2016-01-15

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. Themore » data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-β-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-β-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence. - Highlights: • The ribosomal L1 domain and lysine-rich region of CSIG were expressed. • They are critical for CSIG to regulate proliferation and senescence. • CSIG and its domains exhibit different subcellular distribution.« less

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors.

PubMed

Mulji, Alpa; Haslam, Carl; Brown, Fiona; Randle, Rebecca; Karamshi, Bhumika; Smith, Julia; Eagle, Robert; Munoz-Muriedas, Jordi; Taylor, Joanna; Sheikh, Arshad; Bridges, Angela; Gill, Kirsty; Jepras, Rob; Smee, Penny; Barker, Mike; Woodrow, Mike; Liddle, John; Thomas, Pamela; Jones, Emma; Gordon, Laurie; Tanner, Rob; Leveridge, Melanie; Hutchinson, Sue; Martin, Margaret; Brown, Murray; Kruidenier, Laurens; Katso, Roy

2012-01-01

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

PubMed Central

Clyde, Karen; Glaunsinger, Britt A.

2011-01-01

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection. PMID:21573023

Divergent RNA Localisation Patterns of Maternal Genes Regulating Embryonic Patterning in the Butterfly Pararge aegeria

PubMed Central

Carter, Jean-Michel; Gibbs, Melanie; Breuker, Casper J.

2015-01-01

The maternal effect genes responsible for patterning the embryo along the antero-posterior (AP) axis are broadly conserved in insects. The precise function of these maternal effect genes is the result of the localisation of their mRNA in the oocyte. The main developmental mechanisms involved have been elucidated in Drosophila melanogaster, but recent studies have shown that other insect orders often diverge in RNA localisation patterns. A recent study has shown that in the butterfly Pararge aegeria the distinction between blastodermal embryonic (i.e. germ band) and extra-embryonic tissue (i.e. serosa) is already specified in the oocyte during oogenesis in the ovariole, long before blastoderm cellularisation. To examine the extent by which a female butterfly specifies and patterns the AP axis within the region fated to be the germ band, and whether she specifies a germ plasm, we performed in situ hybridisation experiments on oocytes in P. aegeria ovarioles and on early embryos. RNA localisation of the following key maternal effect genes were investigated: caudal (cad), orthodenticle (otd), hunchback (hb) and four nanos (nos) paralogs, as well as TDRD7 a gene containing a key functional domain (OST-HTH/LOTUS) shared with oskar. TDRD7 was mainly confined to the follicle cells, whilst hb was exclusively zygotically transcribed. RNA of some of the nos paralogs, otd and cad revealed complex localisation patterns within the cortical region prefiguring the germ band (i.e. germ cortex). Rather interestingly, otd was localised within and outside the anterior of the germ cortex. Transcripts of nos-O formed a distinct granular ring in the middle of the germ cortex possibly prefiguring the region where germline stem cells form. These butterfly RNA localisation patterns are highly divergent with respect to other insects, highlighting the diverse ways in which different insect orders maternally regulate early embryogenesis of their offspring. PMID:26633019

A Genetic Approach to Promoter Recognition during Trans Induction of Viral Gene Expression

NASA Astrophysics Data System (ADS)

Coen, Donald M.; Weinheimer, Steven P.; McKnight, Steven L.

1986-10-01

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors.

Improvement of localised corrosion resistance of AISI 2205 Duplex Stainless Steel joints made by gas metal arc welding under electromagnetic interaction of low intensity

NASA Astrophysics Data System (ADS)

García-Rentería, M. A.; López-Morelos, V. H.; García-Hernández, R.; Dzib-Pérez, L.; García-Ochoa, E. M.; González-Sánchez, J.

2014-12-01

The resistance to localised corrosion of AISI 2205 duplex stainless steel plates joined by Gas Metal Arc Welding (GMAW) under the effect of electromagnetic interaction of low intensity (EMILI) was evaluated with sensitive electrochemical methods. Welds were made using two shielding gas mixtures: 98% Ar + 2% O2 (M1) and 97% Ar + 3% N2 (M2). Plates were welded under EMILI using the M1 gas with constant welding parameters. The modified microstructural evolution in the high temperature heat affected zone and at the fusion zone induced by application of EMILI during welding is associated with the increase of resistance to localised corrosion of the welded joints. Joints made by GMAW using the shielding gas M2 without the application of magnetic field presented high resistance to general corrosion but high susceptibility to undergo localised attack.

Differences in finger localisation performance of patients with finger agnosia.

PubMed

Anema, Helen A; Kessels, Roy P C; de Haan, Edward H F; Kappelle, L Jaap; Leijten, Frans S; van Zandvoort, Martine J E; Dijkerman, H Chris

2008-09-17

Several neuropsychological studies have suggested parallel processing of somatosensory input when localising a tactile stimulus on one's own by pointing towards it (body schema) and when localising this touched location by pointing to it on a map of a hand (body image). Usually these reports describe patients with impaired detection, but intact sensorimotor localisation. This study examined three patients with a lesion of the angular gyrus with intact somatosensory processing, but with selectively disturbed finger identification (finger agnosia). These patients performed normally when pointing towards the touched finger on their own hand but failed to indicate this finger on a drawing of a hand or to name it. Similar defects in the perception of other body parts were not observed. The findings provide converging evidence for the dissociation between body image and body schema and, more importantly, reveal for the first time that this distinction is also present in higher-order cognitive processes selectively for the fingers.

Flows and Stratification of an Enclosure Containing Both Localised and Vertically Distributed Sources of Buoyancy

NASA Astrophysics Data System (ADS)

Partridge, Jamie; Linden, Paul

2013-11-01

We examine the flows and stratification established in a naturally ventilated enclosure containing both a localised and vertically distributed source of buoyancy. The enclosure is ventilated through upper and lower openings which connect the space to an external ambient. Small scale laboratory experiments were carried out with water as the working medium and buoyancy being driven directly by temperature differences. A point source plume gave localised heating while the distributed source was driven by a controllable heater mat located in the side wall of the enclosure. The transient temperatures, as well as steady state temperature profiles, were recorded and are reported here. The temperature profiles inside the enclosure were found to be dependent on the effective opening area A*, a combination of the upper and lower openings, and the ratio of buoyancy fluxes from the distributed and localised source Ψ =Bw/Bp . Industrial CASE award with ARUP.

Scientific Benefit of Enlarging Gravitational Wave Detector Networks

NASA Astrophysics Data System (ADS)

Chu, Qi; Wen, Linqing; Blair, David

2012-06-01

Localising the sources of gravitational waves (GWs) in the sky is crucial to observing the electromagnetic counterparts of GW sources. The localisation capability is poor by a single GW detector yet can be improved by adding more detectors to the detector network. In this paper we review recent studies on scientific benefits of global detector networks and focus on their localisation capability. We employ Wen-Chen's formula to compare this merit of current and future detector networks for localising gravitational wave bursts. We find that the addition of a new detector located in Japan, or India, or Australia will increase angular resolution 3~5 fold with respect to current LIGO-Virgo network, and that the angular resolution improvement by adding a single detector in Australia is comparable to that achieved by adding detectors in both India and Japan. A six-site network achieves a 11-fold improvement in angular resolution compared with the existing three-site network.

Interdependency of formation and localisation of the Min complex controls symmetric plastid division.

PubMed

Maple, Jodi; Møller, Simon G

2007-10-01

Plastid division represents a fundamental biological process essential for plant development; however, the molecular basis of symmetric plastid division is unclear. AtMinE1 plays a pivotal role in selection of the plastid division site in concert with AtMinD1. AtMinE1 localises to discrete foci in chloroplasts and interacts with AtMinD1, which shows a similar localisation pattern. Here, we investigate the importance of Min protein complex formation during the chloroplast division process. Dissection of the assembly of the Min protein complex and determination of the interdependency of complex assembly and localisation in planta allow us to present a model of the molecular basis of selection of the division site in plastids. Moreover, functional analysis of AtMinE1 in bacteria demonstrates the level of functional conservation and divergence of the plastidic MinE proteins.

Kyste hydatique à localisation costo vertébrale

PubMed Central

Marouf, Rachid

2014-01-01

L'hydatidose est une affection parasitaire due à la contamination de l'homme par la forme larvaire de ténia échinococcus granulosus, la forme costo vertébrale est une localisation très rare qui représente 0,18 à 1,21% de l'ensemble des localisations hydatiques. Nous rapportons le cas d'une femme de 32 ans qui présente un kyste hydatique multi vésiculaire à localisation costovertébrale, traité par chirurgie radicale associée à un traitement médical anti parasitaire pour une durée de 6 mois, avec bonne évolution. L'atteinte costo-vertébrale par la maladie hydatique est rare et l’évolution est insidieuse. Malgré un traitement chirurgical radical, la fréquence des récidives rend le pronostic sombre. PMID:25922632

Absolute High-Precision Localisation of an Unmanned Ground Vehicle by Using Real-Time Aerial Video Imagery for Geo-referenced Orthophoto Registration

NASA Astrophysics Data System (ADS)

Kuhnert, Lars; Ax, Markus; Langer, Matthias; Nguyen van, Duong; Kuhnert, Klaus-Dieter

This paper describes an absolute localisation method for an unmanned ground vehicle (UGV) if GPS is unavailable for the vehicle. The basic idea is to combine an unmanned aerial vehicle (UAV) to the ground vehicle and use it as an external sensor platform to achieve an absolute localisation of the robotic team. Beside the discussion of the rather naive method directly using the GPS position of the aerial robot to deduce the ground robot's position the main focus of this paper lies on the indirect usage of the telemetry data of the aerial robot combined with live video images of an onboard camera to realise a registration of local video images with apriori registered orthophotos. This yields to a precise driftless absolute localisation of the unmanned ground vehicle. Experiments with our robotic team (AMOR and PSYCHE) successfully verify this approach.

bicoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring

PubMed Central

Trovisco, Vítor; Belaya, Katsiaryna; Nashchekin, Dmitry; Irion, Uwe; Sirinakis, George; Butler, Richard; Lee, Jack J; Gavis, Elizabeth R; St Johnston, Daniel

2016-01-01

bicoid mRNA localises to the Drosophila oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that bicoid mRNA particles undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore, bicoid mRNA localises normally in shot2A2, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus, bicoid mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that bicoid mRNA forms 110–120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring. DOI: http://dx.doi.org/10.7554/eLife.17537.001 PMID:27791980

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression

PubMed Central

QI, Xiaonan; LI, Huatao; CONG, Xia; WANG, Xin; JIANG, Zhongling; CAO, Rongfeng; TIAN, Wenru

2016-01-01

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 μg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation. PMID:27478062

Unbiased compound screening with a reporter gene assay highlights the role of p13 in the cardiac cellular stress response.

PubMed

Inoue, Naoki; Hirouchi, Taisei; Kasai, Atsushi; Higashi, Shintaro; Hiraki, Natsumi; Tanaka, Shota; Nakazawa, Takanobu; Nunomura, Kazuto; Lin, Bangzhong; Omori, Akiko; Hayata-Takano, Atsuko; Kim, Yoon-Jeong; Doi, Takefumi; Baba, Akemichi; Hashimoto, Hitoshi; Shintani, Norihito

2018-01-08

We recently showed that a 13-kDa protein (p13), the homolog protein of formation of mitochondrial complex V assembly factor 1 in yeast, acts as a potential protective factor in pancreatic islets under diabetes. Here, we aimed to identify known compounds regulating p13 mRNA expression to obtain therapeutic insight into the cellular stress response. A luciferase reporter system was developed using the putative promoter region of the human p13 gene. Overexpression of peroxisome proliferator-activated receptor gamma coactivator 1α, a master player regulating mitochondrial metabolism, increased both reporter activity and p13 expression. Following unbiased screening with 2320 known compounds in HeLa cells, 12 pharmacological agents (including 8 cardiotonics and 2 anthracyclines) that elicited >2-fold changes in p13 mRNA expression were identified. Among them, four cardiac glycosides decreased p13 expression and concomitantly elevated cellular oxidative stress. Additional database analyses showed highest p13 expression in heart, with typically decreased expression in cardiac disease. Accordingly, our results illustrate the usefulness of unbiased compound screening as a method for identifying novel functional roles of unfamiliar genes. Our findings also highlight the importance of p13 in the cellular stress response in heart. Copyright © 2017. Published by Elsevier Inc.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

PubMed

Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

2012-09-03

Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Selective coexpression of VEGF receptor 2 in EGFRvIII-positive glioblastoma cells prevents cellular senescence and contributes to their aggressive nature.

PubMed

Jones, Karra A; Gilder, Andrew S; Lam, Michael S; Du, Na; Banki, Michael A; Merati, Aran; Pizzo, Donald P; VandenBerg, Scott R; Gonias, Steven L

2016-05-01

In glioblastoma (GBM), the gene for epidermal growth factor receptor (EGFR) is frequently amplified. EGFR mutations also are common, including a truncation mutation that yields a constitutively active variant called EGFR variant (v)III. EGFRvIII-positive GBM progresses rapidly; however, the reason for this is not clear because the activity of EGFRvIII is attenuated compared with EGF-ligated wild-type EGFR. We hypothesized that EGFRvIII-expressing GBM cells selectively express other oncogenic receptors that support tumor progression. Mining of The Cancer Genome Atlas prompted us to test whether GBM cells in culture, which express EGFRvIII, selectively express vascular endothelial growth factor receptor (VEGFR)2. We also studied human GBM propagated as xenografts. We then applied multiple approaches to test the effects of VEGFR2 on GBM cell growth, apoptosis, and cellular senescence. In human GBM, EGFR overexpression and EGFRvIII positivity were associated with increased VEGFR2 expression. In GBM cells in culture, EGFRvIII-initiated cell signaling increased expression of VEGFR2, which prevented cellular senescence and promoted cell cycle progression. The VEGFR-selective tyrosine kinase inhibitor cediranib decreased tumor DNA synthesis, increased staining for senescence-associated β-galactosidase, reduced retinoblastoma phosphorylation, and increased p27(Kip1), all markers of cellular senescence. Similar results were obtained when VEGFR2 was silenced. VEGFR2 expression by GBM cells supports cell cycle progression and prevents cellular senescence. Coexpression of VEGFR2 by GBM cells in which EGFR signaling is activated may contribute to the aggressive nature of these cells. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A functional role of the extracellular domain of Crumbs in cell architecture and apicobasal polarity.

PubMed

Letizia, Annalisa; Ricardo, Sara; Moussian, Bernard; Martín, Nicolás; Llimargas, Marta

2013-05-15

Regulated cell shape changes in epithelial cells, which contribute to most organs and tissues, are at the basis of morphogenesis. Crumbs (Crb) is an essential apical determinant controlling epithelial apicobasal polarity. Here we provide evidence for a novel role of Crb apical localisation and stabilisation in controlling cell shape through apical domain organisation and adherens junction positioning. We find that Crb apical stabilisation requires the extracellular domain. In vivo results from Drosophila suggest that the extracellular domain assists Crb apical stabilisation by mediating Crb-Crb interactions at opposing cell membranes. We further confirm Crb-Crb extracellular interactions by showing that the extracellular domain of Crb is sufficient to promote cell aggregation in vitro. Furthermore, we report that Crb apical stabilisation mediated by the extracellular domain is also required for maintenance of Crb apicobasal polarity. Our results provide new insights into the mechanisms of apicobasal polarity and the cellular mechanisms of tissue architecture.

Mimicking phosphorylation of Ser-74 on human deoxycytidine kinase selectively increases catalytic activity for dC and dC analogues

PubMed Central

McSorley, Theresa; Ort, Stephan; Hazra, Saugata; Lavie, Arnon; Konrad, Manfred

2009-01-01

Intracellular phosphorylation of dCK on Ser-74 results in increased nucleoside kinase activity. We mimicked this phosphorylation by a Ser-74-Glu mutation in bacterially produced dCK and investigated kinetic parameters using various nucleoside substrates. The S74E mutation increases the kcat values 11-fold for dC, and 3-fold for the anti-cancer analogues dFdC and AraC. In contrast, the rate is decreased for the purine substrates. In HEK293 cells, we found that by comparing transiently transfected dCK(S74E)-GFP and wild-type dCK-GFP, mimicking the phosphorylation of Ser-74 has no effect on cellular localisation. We note that phosphorylation may represent a mechanism to enhance the catalytic activity of the relatively slow dCK enzyme. PMID:18258203

Chemical imaging of a Symbiodinium sp. cell using synchrotron infrared microspectroscopy: a feasibility study.

PubMed

Gordon, B R; Martin, D E; Bambery, K R; Motti, C A

2018-04-01

The symbiotic relationship between corals and Symbiodinium spp. is the key to the success and survival of coral reef ecosystems the world over. Nutrient exchange and chemical communication between the two partners provides the foundation of this key relationship, yet we are far from a complete understanding of these processes. This is due, in part, to the difficulties associated with studying an intracellular symbiosis at the small spatial scales required to elucidate metabolic interactions between the two partners. This feasibility study, which accompanied a more extensive investigation of fixed Symbiodinium cells (data unpublished), examines the potential of using synchrotron radiation infrared microspectroscopy (SR-IRM) for exploring metabolite localisation within a single Symbiodinium cell. In doing so, three chemically distinct subcellular regions of a single Symbiodinium cell were established and correlated to cellular function based on assignment of diagnostic chemical classes. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases.

PubMed

Jacobsen, Annette V; Murphy, James M

2017-06-15

Over the past decade, our understanding of the mechanisms by which pseudokinases, which comprise ∼10% of the human and mouse kinomes, mediate signal transduction has advanced rapidly with increasing structural, biochemical, cellular and genetic studies. Pseudokinases are the catalytically defective counterparts of conventional, active protein kinases and have been attributed functions as protein interaction domains acting variously as allosteric modulators of conventional protein kinases and other enzymes, as regulators of protein trafficking or localisation, as hubs to nucleate assembly of signalling complexes, and as transmembrane effectors of such functions. Here, by categorising mammalian pseudokinases based on their known functions, we illustrate the mechanistic diversity among these proteins, which can be viewed as a window into understanding the non-catalytic functions that can be exerted by conventional protein kinases. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Guine pig liver L-asparaginase. Separation, purification, and intracellular localisation of two distinct enzymatic activities.

PubMed

Rogez, J C; Plaquet, R; Biserte, G

1975-12-18

Two distinct L-asparaginase (EC 3.5.1.1) activities were detected in guinea pig liver: Asparaginase 1 and Asparaginase 2. Asparaginase 1 has been purified 272 fold from the crude homogenate; its molecular weight was evaluated by gel filtration to be about 150 000. The purified preparation was shown to be homogeneous by cellulose acetate strip and polyacrylamide disc-gel electrophoresis. Asparaginase 2 has been purified 63.5 fold from the crude homogenate. Its molecular weight was evaluated by gel filtration to be about 21 500. Cellulose acetate strip electrophoresis demonstrated two bands, one of which corresponded to Asparaginase 1 and the other to Asparaginase 2. Cellular fractionation in the ultracentrifuge, showed Asparaginase 1 to be present only in the cytosol fraction. Asparaginase 2 which was unstable at 105 000 X g seemed mostly localized in the mitochondria and secondarily in the cytoplasmic fraction.

Intermediates of Metabolism: From Bystanders to Signalling Molecules.

PubMed

Haas, Robert; Cucchi, Danilo; Smith, Joanne; Pucino, Valentina; Macdougall, Claire Elizabeth; Mauro, Claudio

2016-05-01

The integration of biochemistry into immune cell biology has contributed immensely to our understanding of immune cell function and the associated pathologies. So far, most studies have focused on the regulation of metabolic pathways during an immune response and their contribution to its success. More recently, novel signalling functions of metabolic intermediates are being discovered that might play important roles in the regulation of immunity. Here we describe the three long-known small metabolites lactate, acetyl-CoA, and succinate in the context of immunometabolic signalling. Functions of these ubiquitous molecules are largely dependent on their intra- and extracellular concentrations as well as their subcompartmental localisation. Importantly, the signalling functions of these metabolic intermediates extend beyond self-regulatory roles and include cell-to-cell communication and sensing of microenvironmental conditions to elicit stress responses and cellular adaptation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Towards the Physics of Calcium Signalling in Plants

PubMed Central

Vaz Martins, Teresa; Evans, Matthew J.; Woolfenden, Hugh C.; Morris, Richard J.

2013-01-01

Calcium is an abundant element with a wide variety of important roles within cells. Calcium ions are inter- and intra-cellular messengers that are involved in numerous signalling pathways. Fluctuating compartment-specific calcium ion concentrations can lead to localised and even plant-wide oscillations that can regulate downstream events. Understanding the mechanisms that give rise to these complex patterns that vary both in space and time can be challenging, even in cases for which individual components have been identified. Taking a systems biology approach, mathematical and computational techniques can be employed to produce models that recapitulate experimental observations and capture our current understanding of the system. Useful models make novel predictions that can be investigated and falsified experimentally. This review brings together recent work on the modelling of calcium signalling in plants, from the scale of ion channels through to plant-wide responses to external stimuli. Some in silico results that have informed later experiments are highlighted. PMID:27137393

Mycobacterium tuberculosis promotes genomic instability in macrophages.

PubMed

Castro-Garza, Jorge; Luévano-Martínez, Miriam Lorena; Villarreal-Treviño, Licet; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha Imelda; García-Vielma, Catalina; González-Hernández, Silvia; Cortés-Gutiérrez, Elva Irene

2018-03-01

Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Pirin Inhibits Cellular Senescence in Melanocytic Cells

PubMed Central

Licciulli, Silvia; Luise, Chiara; Scafetta, Gaia; Capra, Maria; Giardina, Giuseppina; Nuciforo, Paolo; Bosari, Silvano; Viale, Giuseppe; Mazzarol, Giovanni; Tonelli, Chiara; Lanfrancone, Luisa; Alcalay, Myriam

2011-01-01

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression. PMID:21514450

Revisiting the B-cell compartment in mouse and humans: more than one B-cell subset exists in the marginal zone and beyond.

PubMed

Garraud, Olivier; Borhis, Gwenoline; Badr, Gamal; Degrelle, Séverine; Pozzetto, Bruno; Cognasse, Fabrice; Richard, Yolande

2012-11-29

The immunological roles of B-cells are being revealed as increasingly complex by functions that are largely beyond their commitment to differentiate into plasma cells and produce antibodies, the key molecular protagonists of innate immunity, and also by their compartmentalisation, a more recently acknowledged property of this immune cell category. For decades, B-cells have been recognised by their expression of an immunoglobulin that serves the function of an antigen receptor, which mediates intracellular signalling assisted by companion molecules. As such, B-cells were considered simple in their functioning compared to the other major type of immune cell, the T-lymphocytes, which comprise conventional T-lymphocyte subsets with seminal roles in homeostasis and pathology, and non-conventional T-lymphocyte subsets for which increasing knowledge is accumulating. Since the discovery that the B-cell family included two distinct categories - the non-conventional, or extrafollicular, B1 cells, that have mainly been characterised in the mouse; and the conventional, or lymph node type, B2 cells - plus the detailed description of the main B-cell regulator, FcγRIIb, and the function of CD40(+) antigen presenting cells as committed/memory B-cells, progress in B-cell physiology has been slower than in other areas of immunology. Cellular and molecular tools have enabled the revival of innate immunity by allowing almost all aspects of cellular immunology to be re-visited. As such, B-cells were found to express "Pathogen Recognition Receptors" such as TLRs, and use them in concert with B-cell signalling during innate and adaptive immunity. An era of B-cell phenotypic and functional analysis thus began that encompassed the study of B-cell microanatomy principally in the lymph nodes, spleen and mucosae. The novel discovery of the differential localisation of B-cells with distinct phenotypes and functions revealed the compartmentalisation of B-cells. This review thus aims to describe novel findings regarding the B-cell compartments found in the mouse as a model organism, and in human physiology and pathology. It must be emphasised that some differences are noticeable between the mouse and human systems, thus increasing the complexity of B-cell compartmentalisation. Special attention will be given to the (lymph node and spleen) marginal zones, which represent major crossroads for B-cell types and functions and a challenge for understanding better the role of B-cell specificities in innate and adaptive immunology.

Microstructure of selective laser melted CM247LC nickel-based superalloy and its evolution through heat treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Divya, V.D., E-mail: dv272@cam.ac.uk; Muñoz-Moreno, R.; Messé, O.M.D.M.

2016-04-15

The selective laser melting of high temperature alloys is of great interest to the aerospace industry as it offers the prospect of producing more complex geometries than can be achieved with other manufacturing methods. In this study, the microstructure of the nickel-based superalloy, CM247LC, has been characterised following selective laser melting and after a post deposition heat treatment below the γ′ solvus temperature. In the as-deposited state, scanning electron microscopy with electron backscatter diffraction revealed a fine, cellular microstructure with preferential alignment of 〈001〉 along the build direction. A high dislocation density was seen at the periphery of the cells,more » indicating substantial localised deformation of the material. Fine primary MC carbides were also observed in the inter-cellular regions. High-resolution transmission electron microscopy identified the occurrence of very fine γ′ precipitates, approximately 5 nm in diameter, dispersed within the gamma phase. After heat treatment, the elongated cell colonies were observed to partially coalesce, accompanied by a decrease in dislocation density, producing columnar grains along the build direction. Cuboidal γ′ precipitates approximately 500 nm in diameter were observed to form in the recrystallised grains, accompanied by larger γ′ precipitates on the grain boundaries.« less

Transport of Gold Nanoparticles by Vascular Endothelium from Different Human Tissues

PubMed Central

Gromnicova, Radka; Kaya, Mehmet; Romero, Ignacio A.; Williams, Phil; Satchell, Simon; Sharrack, Basil; Male, David

2016-01-01

The selective entry of nanoparticles into target tissues is the key factor which determines their tissue distribution. Entry is primarily controlled by microvascular endothelial cells, which have tissue-specific properties. This study investigated the cellular properties involved in selective transport of gold nanoparticles (<5 nm) coated with PEG-amine/galactose in two different human vascular endothelia. Kidney endothelium (ciGENC) showed higher uptake of these nanoparticles than brain endothelium (hCMEC/D3), reflecting their biodistribution in vivo. Nanoparticle uptake and subcellular localisation was quantified by transmission electron microscopy. The rate of internalisation was approximately 4x higher in kidney endothelium than brain endothelium. Vesicular endocytosis was approximately 4x greater than cytosolic uptake in both cell types, and endocytosis was blocked by metabolic inhibition, whereas cytosolic uptake was energy-independent. The cellular basis for the different rates of internalisation was investigated. Morphologically, both endothelia had similar profiles of vesicles and cell volumes. However, the rate of endocytosis was higher in kidney endothelium. Moreover, the glycocalyces of the endothelia differed, as determined by lectin-binding, and partial removal of the glycocalyx reduced nanoparticle uptake by kidney endothelium, but not brain endothelium. This study identifies tissue-specific properties of vascular endothelium that affects their interaction with nanoparticles and rate of transport. PMID:27560685

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

PubMed

Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

2016-02-01

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

Antihypertensive methyldopa, labetalol, hydralazine, and clonidine reversed tumour necrosis factor-α inhibited endothelial nitric oxide synthase expression in endothelial-trophoblast cellular networks.

PubMed

Xu, Bei; Bobek, Gabriele; Makris, Angela; Hennessy, Annemarie

2017-03-01

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor-α (TNF-α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre-incubated with (or without) low dose TNF-α (0.5 ng/mL) or TNF-α plus soluble fms-like tyrosine kinase-1 (sFlt-1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR-8/SVneo cells were co-cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF-α on eNOS mRNA expression. After pre-incubating endothelial cells with TNF-α and sFlt-1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF-α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF-α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF-α. The anti-angiogenic molecule sFlt-1 may antagonise the potential benefit of these medications by interfering with the NOS pathway. © 2016 John Wiley & Sons Australia, Ltd.

Expression pattern and phosphorylation status of Smad2/3 in different subtypes of human first trimester trophoblast.

PubMed

Haider, S; Kunihs, V; Fiala, C; Pollheimer, J; Knöfler, M

2017-09-01

TGF-β superfamily members are thought to play a pivotal role in placental development and differentiation. However, their downstream effectors, the Smad transcription factors, have been poorly investigated in human trophoblasts. Expression and localisation of the canonical TGF-β targets Smad2/3 and their regulators (Smad4 and Smad7) were investigated in first trimester placenta and purified cytotrophoblast (CTB) subtypes using immunofluorescence, western blotting and qPCR. Canonical and non-canonical activation was analysed in nuclear/cytoplasmic extracts of trophoblast subtypes as well as in tissue sections using antibodies against Smad2/3, phosphorylated either at the C-terminus (pSmad2C/3C) or in their linker regions (pSmad2L/3L). Smad phosphorylation was also examined in differentiating extravillous trophoblasts (EVTs) in the absence or presence of decidual stromal cell (DSC)-conditioned medium. Smad2, Smad4 and Smad7 protein were uniformly expressed between 6th and 12th week placentae and the different isolated CTB subtypes. Activated pSmad2L was mainly detected in nuclei and cytoplasm of villous CTBs, whereas pSmad2C was absent from these cells. In contrast, pSmad2C could be detected in the cytoplasm of cell column trophoblasts and in the cytoplasm/nuclei of EVTs. Smad3 and its phosphorylated forms pSmad3C and pSmad3L specifically localised to EVT nuclei. During EVT differentiation autocrine activation of pSmad2C/3C and pSmad3L was observed. DSC-conditioned medium further increased Smad2/3 phosphorylation in EVTs. The lack of pSmad2C in villous CTBs suggests that other mitogens than TGF-β could promote Smad2 linker phosphorylation under homeostatic conditions. Whereas autocrine signalling activates Smad2/3 in differentiating EVTs, paracrine factors contribute to Smad phosphorylation in these cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

PubMed Central

Jeffery, Hannah C.; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E.; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K.; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R.; Adams, David H.; Klenerman, Paul; Oo, Ye H.

2016-01-01

Background & Aims Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. Methods The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Results Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Conclusions Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. PMID:26743076

Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1.

PubMed

Jeffery, Hannah C; van Wilgenburg, Bonnie; Kurioka, Ayako; Parekh, Krishan; Stirling, Kathryn; Roberts, Sheree; Dutton, Emma E; Hunter, Stuart; Geh, Daniel; Braitch, Manjit K; Rajanayagam, Jeremy; Iqbal, Tariq; Pinkney, Thomas; Brown, Rachel; Withers, David R; Adams, David H; Klenerman, Paul; Oo, Ye H

2016-05-01

Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored. The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1. Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17. Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Quantum State Diffusion

NASA Astrophysics Data System (ADS)

Percival, Ian

2005-10-01

1. Introduction; 2. Brownian motion and Itô calculus; 3. Open quantum systems; 4. Quantum state diffusion; 5. Localisation; 6. Numerical methods and examples; 7. Quantum foundations; 8. Primary state diffusion; 9. Classical dynamics of quantum localisation; 10. Semiclassical theory and linear dynamics.

Contralateral routing of signals disrupts monaural level and spectral cues to sound localisation on the horizontal plane.

PubMed

Pedley, Adam J; Kitterick, Pádraig T

2017-09-01

Contra-lateral routing of signals (CROS) devices re-route sound between the deaf and hearing ears of unilaterally-deaf individuals. This rerouting would be expected to disrupt access to monaural level cues that can support monaural localisation in the horizontal plane. However, such a detrimental effect has not been confirmed by clinical studies of CROS use. The present study aimed to exercise strict experimental control over the availability of monaural cues to localisation in the horizontal plane and the fitting of the CROS device to assess whether signal routing can impair the ability to locate sources of sound and, if so, whether CROS selectively disrupts monaural level or spectral cues to horizontal location, or both. Unilateral deafness and CROS device use were simulated in twelve normal hearing participants. Monaural recordings of broadband white noise presented from three spatial locations (-60°, 0°, and +60°) were made in the ear canal of a model listener using a probe microphone with and without a CROS device. The recordings were presented to participants via an insert earphone placed in their right ear. The recordings were processed to disrupt either monaural level or spectral cues to horizontal sound location by roving presentation level or the energy across adjacent frequency bands, respectively. Localisation ability was assessed using a three-alternative forced-choice spatial discrimination task. Participants localised above chance levels in all conditions. Spatial discrimination accuracy was poorer when participants only had access to monaural spectral cues compared to when monaural level cues were available. CROS use impaired localisation significantly regardless of whether level or spectral cues were available. For both cues, signal re-routing had a detrimental effect on the ability to localise sounds originating from the side of the deaf ear (-60°). CROS use also impaired the ability to use level cues to localise sounds originating from straight ahead (0°). The re-routing of sounds can restrict access to the monaural cues that provide a basis for determining sound location in the horizontal plane. Perhaps encouragingly, the results suggest that both monaural level and spectral cues may not be disrupted entirely by signal re-routing and that it may still be possible to reliably identify sounds originating on the hearing side. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 β: identification of miR-153 target genes with functions related to IA-2β in pancreas and brain.

PubMed

Mandemakers, W; Abuhatzira, L; Xu, H; Caromile, L A; Hébert, S S; Snellinx, A; Morais, V A; Matta, S; Cai, T; Notkins, A L; De Strooper, B

2013-07-01

We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2β. We also identified miR-153 target genes that correlated with IA-2β localisation and function. A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2β, quantitative PCR analysis of miR-153 and Ia-2β (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2β single knockout and Ia-2/Ia-2β double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2β, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2β in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2β knockout and Ia-2/Ia-2β double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2β expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. This study suggests the involvement of miR-153, IA-2β and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

Enhancement of reverse transfection efficiency by combining stimulated DNA surface desorption and electroporation

NASA Astrophysics Data System (ADS)

Creasey, Rhiannon; Hook, Andrew; Thissen, Helmut; Voelcker, Nicolas H.

2007-12-01

Transfection cell microarrays (TCMs) are a high-throughput, miniaturised cell-culture system utilising reverse transfection, in which cells are seeded onto a DNA array resulting in localised regions of transfected cells. TCMs are useful for the analysis of gene expression, and can be used to identify genes involved in many cellular processes. This is of significant interest in fields such as tissue engineering, diagnostic screening, and drug testing [1, 2]. Low transfection efficiency has so far limited the application and utility of this technique. Recently, the transfection efficiency of TCMs was improved by an application of a high voltage for a short period of time to the DNA array resulting in the electroporation of cells attached to the surface [3, 4]. Furthermore, application of a low voltage for a longer period of time to the DNA array was shown to improve the transfection efficiency by stimulating the desorption of attached DNA, increasing the concentration of DNA available for cellular uptake [5]. In the present study, the optimisation of the uptake of adsorbed DNA vectors by adherent cells, utilising a voltage bias without compromising cell viability was investigated. This was achieved by depositing negatively charged DNA plasmids onto a positively charged allylamine plasma polymer (ALAPP) layer deposited on highly doped p-type silicon wafers either using a pipettor or a microarray contact printer. Surface-dependant human embryonic kidney (HEK 293 line) cells were cultured onto the DNA vector loaded ALAPP spots and the plasmid transfection events were detected by fluorescence microscopy. Cell viability assays, including fluorescein diacetate (FDA) / Hoechst DNA labelling, were carried out to determine the number of live adherent cells before and after application of a voltage. A protocol was developed to screen for voltage biases and exposure times in order to optimise transfection efficiency and cell viability. Cross-contamination between the microarray spots carrying different DNA vectors was also investigated. By application of a voltage of 286 V/cm for 10 ms, transfection efficiency was doubled compared to using only transfection reagent, whilst maintaining a cell viability of 60-70% of the positive control.

Spatial reconstruction of single-cell gene expression

PubMed Central

Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

2015-01-01

Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

PubMed Central

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression.

PubMed

Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

2015-01-01

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Game of life on phyllosilicates: Gliders, oscillators and still life

NASA Astrophysics Data System (ADS)

Adamatzky, Andrew

2013-10-01

A phyllosilicate is a sheet of silicate tetrahedra bound by basal oxygens. A phyllosilicate automaton is a regular network of finite state machines - silicon nodes and oxygen nodes - which mimics structure of the phyllosilicate. A node takes states 0 and 1. Each node updates its state in discrete time depending on a sum of states of its three (silicon) or six (oxygen) neighbours. Phyllosilicate automata exhibit localisations attributed to Conway's Game of Life: gliders, oscillators, still lifes, and a glider gun. Configurations and behaviour of typical localisations, and interactions between the localisations are illustrated.

Distributed estimation of sensors position in underwater wireless sensor network

NASA Astrophysics Data System (ADS)

Zandi, Rahman; Kamarei, Mahmoud; Amiri, Hadi

2016-05-01

In this paper, a localisation method for determining the position of fixed sensor nodes in an underwater wireless sensor network (UWSN) is introduced. In this simple and range-free scheme, the node localisation is achieved by utilising an autonomous underwater vehicle (AUV) that transverses through the network deployment area, and that periodically emits a message block via four directional acoustic beams. A message block contains the actual known AUV position as well as a directional dependent marker that allows a node to identify the respective transmit beam. The beams form a fixed angle with the AUV body. If a node passively receives message blocks, it could calculate the arithmetic mean of the coordinates existing in each messages sequence, to find coordinates at two different time instants via two different successive beams. The node position can be derived from the two computed positions of the AUV. The major advantage of the proposed localisation algorithm is that it is silent, which leads to energy efficiency for sensor nodes. The proposed method does not require any synchronisation among the nodes owing to being silent. Simulation results, using MATLAB, demonstrated that the proposed method had better performance than other similar AUV-based localisation methods in terms of the rates of well-localised sensor nodes and positional root mean square error.

Gene expression profiling in the Cynomolgus macaque Macaca fascicularis shows variation within the normal birth range

PubMed Central

2011-01-01

Background Although an adverse early-life environment has been linked to an increased risk of developing the metabolic syndrome, the molecular mechanisms underlying altered disease susceptibility as well as their relevance to humans are largely unknown. Importantly, emerging evidence suggests that these effects operate within the normal range of birth weights and involve mechanisms of developmental palsticity rather than pathology. Method To explore this further, we utilised a non-human primate model Macaca fascicularis (Cynomolgus macaque) which shares with humans the same progressive history of the metabolic syndrome. Using microarray we compared tissues from neonates in the average birth weight (50-75th centile) to those of lower birth weight (5-25th centile) and studied the effect of different growth trajectories within the normal range on gene expression levels in the umbilical cord, neonatal liver and skeletal muscle. Results We identified 1973 genes which were differentially expressed in the three tissue types between average and low birth weight animals (P < 0.05). Gene ontology analysis identified that these genes were involved in metabolic processes including cellular lipid metabolism, cellular biosynthesis, cellular macromolecule synthesis, cellular nitrogen metabolism, cellular carbohydrate metabolism, cellular catabolism, nucleotide and nucleic acid metabolism, regulation of molecular functions, biological adhesion and development. Conclusion These differences in gene expression levels between animals in the upper and lower percentiles of the normal birth weight range may point towards early life metabolic adaptations that in later life result in differences in disease risk. PMID:21999700

Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

PubMed Central

Zhang, Wenwen; Yang, Xiaoxiao; Chen, Yuanli; Hu, Wenquan; Liu, Lipei; Zhang, Xiaomeng; Liu, Mengyang; Sun, Lei; Liu, Ying; Yu, Miao; Li, Xiaoju; Li, Luyuan; Zhu, Yan; Miao, Qing Robert; Han, Jihong; Duan, Yajun

2017-01-01

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression. PMID:29217477

Comprehensive Evaluation of the Contribution of X Chromosome Genes to Platinum Sensitivity

PubMed Central

Gamazon, Eric R.; Im, Hae Kyung; O’Donnell, Peter H.; Ziliak, Dana; Stark, Amy L.; Cox, Nancy J.; Dolan, M. Eileen; Huang, Rong Stephanie

2011-01-01

Utilizing a genome-wide gene expression dataset generated from Affymetrix GeneChip® Human Exon 1.0ST array, we comprehensively surveyed the role of 322 X chromosome gene expression traits on cellular sensitivity to cisplatin and carboplatin. We identified 31 and 17 X chromosome genes whose expression levels are significantly correlated (after multiple testing correction) with sensitivity to carboplatin and cisplatin, respectively, in the combined HapMap CEU and YRI populations (false discovery rate, FDR<0.05). Of those, 14 overlap for both cisplatin and carboplatin. Employing an independent gene expression quantification method, the Illumina Sentrix Human-6 Expression BeadChip, measured on the same HapMap cell lines, we found that 4 and 2 of these genes are significantly associated with carboplatin and cisplatin sensitivity respectively in both analyses. Two genes, CTPS2 and DLG3, were identified by both genome-wide gene expression analyses as correlated with cellular sensitivity to both platinating agents. The expression of DLG3 gene was also found to correlate with cellular sensitivity to platinating agents in NCI60 cancer cell lines. In addition, we evaluated the role of X chromosome gene expression to the observed differences in sensitivity to the platinums between CEU and YRI derived cell lines. Of the 34 distinct genes significantly correlated with either carboplatin or cisplatin sensitivity, 14 are differentially expressed (defined as p<0.05) between CEU and YRI. Thus, sex chromosome genes play a role in cellular sensitivity to platinating agents and differences in the expression level of these genes are an important source of variation that should be included in comprehensive pharmacogenomic studies. PMID:21252287

Congenital tuberculosis localised to the ear.

PubMed Central

Naranbhai, R C; Mathiassen, W; Malan, A F

1989-01-01

We report two infants who had localised congenital tuberculous otitis. In both cases the infants presented with an ear discharge and both mothers had been diagnosed as having miliary tuberculosis. Infection is thought to have occurred in utero or during birth. Images Figure PMID:2786383

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®)

PubMed Central

Kwon, Yong-Dae; Choi, Hyun-jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter

2014-01-01

PURPOSE The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. MATERIALS AND METHODS Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). RESULTS From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. CONCLUSION Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. CLINICAL RELEVANCE With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants. PMID:25352963

Fasting increases the phosphorylation of AMPK and expression of sirtuin1 in muscle of adult male northern elephant seals (Mirounga angustirostris).

PubMed

Lee, Debby; Martinez, Bridget; Crocker, Daniel E; Ortiz, Rudy M

2017-02-01

Fasting typically suppresses thyroid hormone (TH)-mediated cellular events and increases sirtuin 1 (SIRT1) activity. THs may regulate metabolism through nongenomic pathways and directly through activation of adenosine monophosphate-activated protein kinase (AMPK). Adult male elephant seals ( Mirounga angustirostris ) are active, hypermetabolic, and normothermic during their annual breeding fast, which is characterized by stable TH levels. However, the contribution of TH to maintenance of their fasting metabolism is unknown. To investigate the fasting effects on cellular TH-mediated events and its potential association with SIRT1 and AMPK, we quantified plasma TH levels, mRNA expressions of muscle SIRT1 and TH-associated genes as well as the phosphorylation of AMPK in adult, male northern elephant seals ( n  = 10/fasting period) over 8 weeks of fasting (early vs. late). Deiodinase type I (DI1) expression increased twofold with fasting duration suggesting that the potential for TH-mediated cellular signaling is increased. AMPK phosphorylation increased 61 ± 21% with fasting suggesting that cellular metabolism is increased. The mRNA expression of the TH transporter, monocarboxylate transporter 10 (MCT10), increased 2.4-fold and the TH receptor (THr β -1) decreased 30-fold suggesting that cellular uptake of T 4 is increased, but its subsequent cellular effects such as activation of AMPK are likely nongenomic. The up-regulation of SIRT1 mRNA expression (2.6-fold) likely contributes to the nongenomic activation of AMPK by TH, which may be necessary to maintain the expression of PGC-1 α These coordinated changes likely contribute to the up-regulation of mitochondrial metabolism to support the energetic demands associated with prolonged fasting in adult seals. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Transcriptional profiling of Epstein–Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection

PubMed Central

Zhang, Y; Ohyashiki, J H; Takaku, T; Shimizu, N; Ohyashiki, K

2006-01-01

Nasal NK/T-cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein–Barr virus (EBV). The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. To understand the pathogenesis of EBV-associated NK/T-cell lymphoproliferative disorders (LPD) and design new therapies, we employed a novel EBV DNA microarray to compare patterns of EBV expression in six cell lines established from EBV-associated NK/T-cell LPD. We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was expressed in SNK/T cells and the expression levels were preferentially high in cell lines from CAEBV infection. We also analyzsd the gene expression patterns of host cellular genes using a human oligonucleotide DNA microarray. We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression, which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD. PMID:16449999

Host defence peptides in human burns.

PubMed

Kaus, Aljoscha; Jacobsen, Frank; Sorkin, Michael; Rittig, Andrea; Voss, Bruno; Daigeler, Adrien; Sudhoff, Holger; Steinau, Hans-Ulrich; Steinstraesser, Lars

2008-02-01

The goal of this study was to analyse expression profiles of human epithelial host defence peptides in burned and unburned skin tissue, samples of which were obtained during debridements and snap-frozen in liquid nitrogen. Total RNA was isolated, and cDNA of epithelial host defence peptides and proteins (hCAP-18/LL-37, hBD1-hBD4, dermcidin, S100A7/psoriasin and RNAse7) was quantified by qRT-PCR. In situ hybridisation and immunohistochemical staining localised gene expression of hCAP-18/LL-37, hBD2 and hBD3 in histological sections. Most of the analysed host defence peptides and proteins showed higher mRNA levels in partial-thickness burns than in unburned tissue. In situ hybridisation revealed expression of hCAP-18/LL-37, hBD2 and hBD3 at the surface of burns that was independent of burn depth. However, the finding of higher host defence peptide gene expression rates does not correlate with the incidence of wound infection in burns. We hypothesise that the epithelial innate immune response in burns is complex.

La tuberculose extra-ganglionnaire primitive de la sphère ORL: à propos de 15 cas

PubMed Central

Touati, Mohamed Mliha; Darouassi, Youssef; Chihani, Mehdi; Lakouichmi, Mohammed; Tourabi, Khalid; Ammar, Haddou; Bouaity, Brahim

2014-01-01

Les localisations ORL extra ganglionnaires de la tuberculose sont rares. La symptomatologie clinique ainsi que les examens paracliniques sont souvent trompeurs,posant ainsi le problème de diagnostic différentiel avec la pathologie tumorale. Nous rapportons 15 cas de localisations extra ganglionnaires de tuberculose, colligés au service ORL et CCF de l'Hopital Militaire Avicenne de Marrakech colligés entre 2009 et 2013. L’âge moyen de nos patients est de 33 ans. L’étude topographique a montré 6 cas au niveau du cavum, un cas de miliaire tuberculeuse pharyngée, 4 cas laryngés; 2 localisations auriculaires; 1 parotidienne et 1 localisation sous maxillaire. Le diagnostic était anatomopathologiquedans tous les cas. Tous nos patients ont reçu un traitement antituberculeux avec une bonne évolution. Mots-clés: Tuberculose, amygdale, rhinopharynx, larynx, glandes salivaires,Oreille moyenne. PMID:25815100

Null stream analysis of Pulsar Timing Array data: localisation of resolvable gravitational wave sources

NASA Astrophysics Data System (ADS)

Goldstein, Janna; Veitch, John; Sesana, Alberto; Vecchio, Alberto

2018-04-01

Super-massive black hole binaries are expected to produce a gravitational wave (GW) signal in the nano-Hertz frequency band which may be detected by pulsar timing arrays (PTAs) in the coming years. The signal is composed of both stochastic and individually resolvable components. Here we develop a generic Bayesian method for the analysis of resolvable sources based on the construction of `null-streams' which cancel the part of the signal held in common for each pulsar (the Earth-term). For an array of N pulsars there are N - 2 independent null-streams that cancel the GW signal from a particular sky location. This method is applied to the localisation of quasi-circular binaries undergoing adiabatic inspiral. We carry out a systematic investigation of the scaling of the localisation accuracy with signal strength and number of pulsars in the PTA. Additionally, we find that source sky localisation with the International PTA data release one is vastly superior than what is achieved by its constituent regional PTAs.

Localised burst reconstruction from space-time PODs in a turbulent channel

NASA Astrophysics Data System (ADS)

Garcia-Gutierrez, Adrian; Jimenez, Javier

2017-11-01

The traditional proper orthogonal decomposition of the turbulent velocity fluctuations in a channel is extended to time under the assumption that the attractor is statistically stationary and can be treated as periodic for long-enough times. The objective is to extract space- and time-localised eddies that optimally represent the kinetic energy (and two-event correlation) of the flow. Using time-resolved data of a small-box simulation at Reτ = 1880 , minimal for y / h 0.25 , PODs are computed from the two-point spectral-density tensor Φ(kx ,kz , y ,y' , ω) . They are Fourier components in x, z and time, and depend on y and on the temporal frequency ω, or, equivalently, on the convection velocity c = ω /kx . Although the latter depends on y, a spatially and temporally localised `burst' can be synthesised by adding a range of PODs with specific phases. The results are localised bursts that are amplified and tilted, in a time-periodic version of Orr-like behaviour. Funded by the ERC COTURB project.

The lung in amyloidosis.

PubMed

Milani, Paolo; Basset, Marco; Russo, Francesca; Foli, Andrea; Palladini, Giovanni; Merlini, Giampaolo

2017-09-30

Amyloidosis is a disorder caused by misfolding of autologous protein and its extracellular deposition as fibrils, resulting in vital organ dysfunction and eventually death. Pulmonary amyloidosis may be localised or part of systemic amyloidosis.Pulmonary interstitial amyloidosis is symptomatic only if the amyloid deposits severely affect gas exchange alveolar structure, thus resulting in serious respiratory impairment. Localised parenchymal involvement may be present as nodular amyloidosis or as amyloid deposits associated with localised lymphomas. Finally, tracheobronchial amyloidosis, which is usually not associated with evident clonal proliferation, may result in airway stenosis.Because the treatment options for amyloidosis are dependent on the fibril protein type, the workup of all new cases should include accurate determination of the amyloid protein. Most cases are asymptomatic and need only a careful follow-up. Diffuse alveolar-septal amyloidosis is treated according to the underlying systemic amyloidosis. Nodular pulmonary amyloidosis is usually localised, conservative excision is usually curative and the long-term prognosis is excellent. Tracheobronchial amyloidosis is usually treated with bronchoscopic interventions or external beam radiation therapy. Copyright ©ERS 2017.

Analysis of lead toxicity in human cells.

PubMed

Gillis, Bruce S; Arbieva, Zarema; Gavin, Igor M

2012-07-27

Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels. The results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies.

Analysis of lead toxicity in human cells

PubMed Central

2012-01-01

Background Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. Results We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high zinc protoporphyrin blood levels. Conclusions The results of our study defined specific changes in gene and protein expression in response to lead challenges and determined the injurious effects of exposures to lead on a cellular level. This information can be used for documenting the health effects of exposures to lead which will facilitate identifying and monitoring efficacious treatments for lead-related maladies. PMID:22839698

Creatine biosynthesis and transport by the term human placenta.

PubMed

Ellery, Stacey J; Della Gatta, Paul A; Bruce, Clinton R; Kowalski, Greg M; Davies-Tuck, Miranda; Mockler, Joanne C; Murthi, Padma; Walker, David W; Snow, Rod J; Dickinson, Hayley

2017-04-01

Creatine is an amino acid derivative that is involved in preserving ATP homeostasis. Previous studies suggest an important role for the creatine kinase circuit for placental ATP turnover. Creatine is obtained from both the diet and endogenous synthesis, usually along the renal-hepatic axis. However, some tissues with a high-energy demand have an inherent capacity to synthesise creatine. In this study, we determined if the term human placenta has the enzymatic machinary to synthesise creatine. Eleven placentae were collected following elective term caesarean section. Samples from the 4 quadrants of each placenta were either fixed in formalin or frozen. qPCR was used to determine the mRNA expression of the creatine synthesising enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), and the creatine transporter (SLC6A8). Protein expression of AGAT and GAMT was quantified by Western blot, and observations of cell localisation of AGAT, GAMT and SLC6A8 made with immunohistochemistry. Synthesis of guanidinoacetate (GAA; creatine precursor) and creatine in placental homogenates was determined via GC-MS and HPLC, respectively. AGAT, GAMT and SLC6A8 mRNA and protein were detected in the human placenta. AGAT staining was identified in stromal and endothelial cells of the fetal capillaries. GAMT and SLC6A8 staining was localised to the syncytiotrophoblast of the fetal villi. Ex vivo, tissue homogenates produce both GAA (4.6 nmol mg protein -1 h -1 ) and creatine (52.8 nmol mg protein -1 h -1 ). The term human placenta has the capacity to synthesise creatine. These data present a new understanding of placental energy metabolism. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

PubMed

Heskes, Allison M; Sundram, Tamil C M; Boughton, Berin A; Jensen, Niels B; Hansen, Nikolaj L; Crocoll, Christoph; Cozzi, Federico; Rasmussen, Simon; Hamberger, Britta; Hamberger, Björn; Staerk, Dan; Møller, Birger L; Pateraki, Irini

2018-03-01

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species. © 2018 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

B Lymphocyte intestinal homing in inflammatory bowel disease

PubMed Central

2011-01-01

Background Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features. Methods The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression. Results The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004). Conclusion Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function. PMID:22208453

Expression of XNOA 36 in the mitochondrial cloud of Xenopus laevis oocytes.

PubMed

Vaccaro, M C; Wilding, M; Dale, B; Campanella, C; Carotenuto, R

2012-08-01

In Xenopus laevis oocytes a mitochondrial cloud (MC) is found between the nucleus and the plasma membrane at stages I-II of oogenesis. The MC contains RNAs that are transported to the future vegetal pole at stage II of oogenesis. In particular, germinal plasm mRNAs are found in the Message Transport Organiser (METRO) region, the MC region opposite to the nucleus. At stages II-III, a second pathway transports Vg1 and VegT mRNAs to the area where the MC content merges with the vegetal cortex. Microtubules become polarized at the sites of migration of Vg1 and VegT mRNAs through an unknown signalling mechanism. In early meiotic stages, the centrioles are almost completely lost with their remnants being dispersed into the cytoplasm and the MC, which may contain a MTOC to be used in the later localization pathway of the mRNAs. In mammals, XNOA 36 encodes a member of a highly conserved protein family and localises to the nucleolus or in the centromeres. In the Xenopus late stage I oocyte, XNOA 36 mRNA is transiently segregated in one half of the oocyte, anchored by a cytoskeletal network that contains spectrin. Here we found that XNOA 36 transcript also localises to the nucleoli and in the METRO region. XNOA 36 protein immunolocalization, using an antibody employed for the library immunoscreening that depicted XNOA 36 expression colonies, labels the migrating MC, the cytoplasm of stage I oocytes and in particular the vegetal cortex facing the MC. The possible role of XNOA 36 in mRNA anchoring to the vegetal cortex or in participating in early microtubule reorganization is discussed.

Label-free proteomic analysis of intestinal mucosa proteins in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

PubMed

Di, Guilan; Li, Hui; Zhang, Chao; Zhao, Yanjing; Zhou, Chuanjiang; Naeem, Sajid; Li, Li; Kong, Xianghui

2017-07-01

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 10 8  CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Arabidopsis thaliana GEX1 has dual functions in gametophyte development and early embryogenesis

USDA-ARS?s Scientific Manuscript database

GEX1 is a plasma membrane protein conserved among plant species, and was previously shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immedi...

RRM2 induces NF-{kappa}B-dependent MMP-9 activation and enhances cellular invasiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duxbury, Mark S.; Whang, Edward E.

2007-03-02

Ribonucleotide reductase is a dimeric enzyme that catalyzes conversion of ribonucleotide 5'-diphosphates to their 2'-deoxynucleotide forms, a rate-limiting step in the production of 2'-deoxyribonucleoside 5'-triphosphates required for DNA synthesis. The ribonucleotide reductase M2 subunit (RRM2) is a determinant of malignant cellular behavior in a range of human cancers. We examined the effect of RRM2 overexpression on pancreatic adenocarcinoma cellular invasiveness and nuclear factor-{kappa}B (NF-{kappa}B) transcription factor activity. RRM2 overexpression increases pancreatic adenocarcinoma cellular invasiveness and MMP-9 expression in a NF-{kappa}B-dependent manner. RNA interference (RNAi)-mediated silencing of RRM2 expression attenuates cellular invasiveness and NF-{kappa}B activity. NF-{kappa}B is a key mediator ofmore » the invasive phenotypic changes induced by RRM2 overexpression.« less

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation.

PubMed

Novakovic, Boris; Fournier, Thierry; Harris, Lynda K; James, Joanna; Roberts, Claire T; Yong, Hannah E J; Kalionis, Bill; Evain-Brion, Danièle; Ebeling, Peter R; Wallace, Euan M; Saffery, Richard; Murthi, Padma

2017-07-03

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Changes in myometrial expression of progesterone receptor membrane components 1 and 2 are associated with human parturition at term.

PubMed

Wang, Ray; Sheehan, Penelope M; Brennecke, Shaun P

2016-04-01

While the exact mechanism of human parturition remains unknown, functional progesterone withdrawal is believed to play a key regulatory role. Progesterone receptor membrane components 1 and 2 (PGRMC1, PGRMC2) are putative progesterone receptors and the aim of this project was to investigate their expression in human myometrium. Human term myometrium was obtained from the lower uterine segment incision in women undergoing elective (not-in-labour, NIL; n=11) and emergency Caesarean sections (in-labour, IL; n=10), following written consent. PGRMC1 and 2 expression was quantified using real-time reverse transcription polymerase chain reaction and western blot. Subcellular localisation was performed by immunohistochemistry and immunofluorescence. There was a significant decrease in PGRMC1 mRNA (P=0.0317) and protein expression (P=0.0151) in IL myometrium, compared with NIL myometrium. PGRMC2 mRNA expression (P=0.0151) was also decreased in IL myometrium, compared with NIL myometrium. Immunostaining studies confirmed the presence of PGRMC1 and 2 in smooth-muscle cells. Expression was perinuclear in NIL myometrium and more generalised and cytoplasmic in IL myometrium. The decrease in PGRMC1 expression and the translocation away from a perinuclear location for both PGRMC1 and 2 could contribute to a functional progesterone withdrawal that may ultimately initiate parturition.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu Ping; Jiang Xiaohong; Arcasoy, Murat O.

The role of erythropoietin receptor (EpoR) expression in tumor cells and the potential of EpoR-mediated signaling to contribute to cellular proliferation and invasiveness require further characterization. To determine whether EpoR expression and activation in tumor cells modulates intracellular signal transduction to promote cellular proliferation and migration, we employed a novel experimental model using human breast cancer cells engineered to stably express a constitutively active EpoR-R129C variant. EpoR-R129C expression resulted in increased cellular proliferation and migration of breast cancer cells and these effects were associated with significantly increased Epo-induced phosphorylation of ERK1/2, AKT and c-Jun-NH2-kinase (SAPK/JNK) proteins. Expression of the constitutivelymore » active EpoR-R129C receptor promoted the proliferation and migration of breast cancer cells via activation of ERK- and SAPK/JNK-dependent signaling pathways, respectively. These findings suggest that EpoR over-expression and activation in breast cancer cells has the potential to contribute to tumor progression by promoting the proliferation and invasiveness of the neoplastic cells.« less

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

PubMed Central

Mercer, Jacob L.; Argus, Joseph P.; Crabtree, Donna M.; Keenan, Melissa M.; Wilks, Moses Q.; Chi, Jen-Tsan Ashley; Bensinger, Steven J.

2015-01-01

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease. PMID:26075887

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

PubMed

Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

2014-09-15

Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM. © 2014. Published by The Company of Biologists Ltd.

AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann-Pick type C1 disease.

PubMed

Hughes, Michael P; Smith, Dave A; Morris, Lauren; Fletcher, Claire; Colaco, Alexandria; Huebecker, Mylene; Tordo, Julie; Palomar, Nuria; Massaro, Giulia; Henckaerts, Els; Waddington, Simon N; Platt, Frances M; Rahim, Ahad A

2018-06-05

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localised to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterised mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localises to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.

Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria.

PubMed

Miwa, Satomi; Czapiewski, Rafal; Wan, Tengfei; Bell, Amy; Hill, Kirsten N; von Zglinicki, Thomas; Saretzki, Gabriele

2016-10-22

Telomerase in its canonical function maintains telomeres in dividing cells. In addition, the telomerase protein TERT has non-telomeric functions such as shuttling to mitochondria resulting in a decreased oxidative stress, DNA damage and apoptosis. TERT protein persists in adult neurons and can co-localise to mitochondria under various stress conditions. We show here that TERT expression decreased in mouse brain during aging while release of reactive oxygen species (ROS) from the mitochondrial electron transport chain increased. Dietary restriction (DR) caused accumulation of TERT protein in mouse brain mitochondria correlating to decreased ROS release and improved learning and spatial short-term memory. Decreased mTOR signalling is a mediator of DR. Accordingly, feeding mice with rapamycin increased brain mitochondrial TERT and reduced ROS release. Importantly, the beneficial effects of rapamycin on mitochondrial function were absent in brains and fibroblasts from first generation TERT -/- mice, and when TERT shuttling was inhibited by the Src kinase inhibitor bosutinib. Taken together, our data suggests that the mTOR signalling pathway impinges on the mitochondrial localisation of TERT protein, which might in turn contribute to the protection of the brain by DR or rapamycin against age-associated mitochondrial ROS increase and cognitive decline.

Spatial reconstruction of single-cell gene expression data.

PubMed

Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

2015-05-01

Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

Localised sarcoptic mange in dogs: a retrospective study of 10 cases.

PubMed

Pin, D; Bensignor, E; Carlotti, D-N; Cadiergues, M C

2006-10-01

The authors report 10 cases of localised sarcoptic mange in dogs. In each case, lesions were localised to one precise area of the skin. Pruritus was present in nine cases and absent in one. Affected areas were the feet (one case), the face and/or the pinnae (six cases), the abdominal skin (one case), the flank (one case) and the lumbar area (one case). The types of lesions were erythema, papules, lichenification, scales, crusts and alopecia. Parasites were found in all cases except one, in which anti-immunoglobulin G Sarcoptes serology was positive. The acaricidal treatments given were lindane, ivermectin or selamectin and were all successful.

Selfish Spermatogonial Selection: Evidence from an Immunohistochemical Screen in Testes of Elderly Men

PubMed Central

Turner, Gareth D. H.; Dudka-Ruszkowska, Wioleta; Taylor, Stephen; Meyts, Ewa Rajpert-De; Goriely, Anne; Wilkie, Andrew O. M.

2012-01-01

The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia–caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively–represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection. PMID:22879958

The conserved Wdr8-hMsd1/SSX2IP complex localises to the centrosome and ensures proper spindle length and orientation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hori, Akiko; Morand, Agathe; Ikebe, Chiho

2015-12-04

The centrosome plays a pivotal role in a wide range of cellular processes and its dysfunction is causally linked to many human diseases including cancer and developmental and neurological disorders. This organelle contains more than one hundred components, and yet many of them remain uncharacterised. Here we identified a novel centrosome protein Wdr8, based upon the structural conservation of the fission yeast counterpart. We showed that Wdr8 constitutively localises to the centrosome and super resolution microscopy uncovered that this protein is enriched at the proximal end of the mother centriole. Furthermore, we identified hMsd1/SSX2IP, a conserved spindle anchoring protein, asmore » one of Wdr8 interactors by mass spectrometry. Wdr8 formed a complex and partially colocalised with hMsd1/SSX2IP. Intriguingly, knockdown of Wdr8 or hMsd1/SSX2IP displayed very similar mitotic defects, in which spindle microtubules became shortened and misoriented. Indeed, Wdr8 depletion resulted in the reduced recruitment of hMsd1/SSX2IP to the mitotic centrosome, though the converse is not true. Together, we propose that the conserved Wdr8-hMsd1/SSX2IP complex plays a critical role in controlling proper spindle length and orientation. - Highlights: • Human Wdr8 is a centrosomal protein enriched in the proximal end of the centriole. • Wdr8 and hMsd1/SSX2IP form a complex conserved in fungi. • Depletion of Wdr8 results in shorter, tilted spindle microtubules. • Depletion phenotypes of Wdr8 are very similar to those of hMsd1/SSX2IP knockdown.« less

A novel role for GSK3β as a modulator of Drosha microprocessor activity and MicroRNA biogenesis.

PubMed

Fletcher, Claire E; Godfrey, Jack D; Shibakawa, Akifumi; Bushell, Martin; Bevan, Charlotte L

2016-10-23

Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here, we identify the kinase GSK3β as an important modulator of miR biogenesis at Microprocessor level. Repression of GSK3β activity reduces Drosha activity toward pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisation of miR biogenesis proteins. Conversely, GSK3β activation increases Drosha activity and mature miR accumulation. GSK3β achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent upon presence of RNA. Indeed, GSK3β itself can immunoprecipitate pri-miRs, suggesting possible RNA-binding capacity. Kinase assays identify the mechanism for GSK3β-enhanced Drosha activity, which requires GSK3β nuclear localisation, as phosphorylation of Drosha at S 300 and/or S 302 ; confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature miRs in the presence of phospho-mimic Drosha. Functional implications of GSK3β-enhanced miR biogenesis are illustrated by increased levels of GSK3β-upregulated miR targets following GSK3β inhibition. These data, the first to link GSK3β with the miR cascade in humans, highlight a novel pro-biogenesis role for GSK3β in increasing miR biogenesis as a component of the Microprocessor complex with wide-ranging functional consequences. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The Future of Digital Working: Knowledge Migration and Learning

ERIC Educational Resources Information Center

Malcolm, Irene

2014-01-01

Against the backdrop of intensified migration linked to globalisation, this article considers the implications of knowledge migration for future digital workers. It draws empirically on a socio-material analysis of the international software localisation industry. Localisers' work requires linguistic, cultural and software engineering skills to…

IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena.

PubMed

Nakagawa, Hiroyuki; Miki, Hiroaki; Nozumi, Motohiro; Takenawa, Tadaomi; Miyamoto, Shigeaki; Wehland, Jürgen; Small, J Victor

2003-06-15

The insulin receptor tyrosine kinase substrate p53 (IRSp53) links Rac and WAVE2 and has been implicated in lamellipodia protrusion. Recently, however, IRSp53 has been reported to bind to both Cdc42 and Mena to induce filopodia. To shed independent light on IRSp53 function we determined the localisations and dynamics of IRSp53 and WAVE2 in B16 melanoma cells. In cells spread well on a laminin substrate, IRSp53 was localised by antibody labelling at the tips of both lamellipodia and filopodia. The same localisation was observed in living cells with IRSp53 tagged with enhanced green florescence protein (EGFP-IRSp53), but only during protrusion. From the transfection of deletion mutants the N-terminal region of IRSp53, which binds active Rac, was shown to be responsible for its localisation. Although IRSp53 has been reported to regulate filopodia formation with Mena, EGFP-IRSp53 showed the same localisation in MVD7 Ena/VASP (vasodilator stimulated phosphoprotein) family deficient cells. WAVE2 tagged with DsRed1 colocalised with EGFP-IRSp53 at the tips of protruding lamellipodia and filopodia and, in double-transfected cells, the IRSp53 signal in filopodia decreased before that of WAVE2 during retraction. These results suggest an alternative modulatory role for IRSp53 in the extension of both filopodia and lamellipodia, through WAVE2.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

PubMed Central

Watt, Stephen A.; Dayal, Jasbani H. S.; Wright, Sheila; Riddle, Megan; Pourreyron, Celine; McMillan, James R.; Kimble, Roy M.; Prisco, Marco; Gartner, Ulrike; Warbrick, Emma; McLean, W. H. Irwin; Leigh, Irene M.; McGrath, John A.; Salas-Alanis, Julio C.; Tolar, Jakub; South, Andrew P.

2015-01-01

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention. PMID:26380979

Heterochrony in the regulation of the developing marsupial limb.

PubMed

Chew, Keng Yih; Shaw, Geoffrey; Yu, Hongshi; Pask, Andrew J; Renfree, Marilyn B

2014-02-01

At birth, marsupial neonates have precociously developed forelimbs. The development of the tammar wallaby (Macropus eugenii) hindlimbs lags significantly behind that of the forelimbs. This differs from the grey short-tailed opossum, Monodelphis domestica, which has relatively similar fore- and hindlimbs at birth. This study examines the expression of the key patterning genes TBX4, TBX5, PITX1, FGF8, and SHH in developing limb buds in the tammar wallaby. All genes examined were highly conserved with orthologues from opossum and mouse. TBX4 expression appeared earlier in development than in the mouse, but later than in the opossum. SHH expression is restricted to the zone of polarising activity, while TBX5 (forelimb) and PITX1 (hindlimb) showed diffuse mRNA expression. FGF8 is specifically localised to the apical ectodermal ridge, which is more prominent than in the opossum. The most marked divergence in limb size in marsupials occurs in the kangaroos and wallabies. The faster development of the fore limb compared to that of the hind limb correlates with the early timing of the expression of the key patterning genes in these limbs. Copyright © 2013 Wiley Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-07-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

PubMed Central

Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

2015-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations ( healthy young UK men and Scandinavian diabetic patients ) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold ( P  < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P  < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

PubMed Central

Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

99mTc-EDDA/HYNIC-TOC and (18)F-FDG in thyroid cancer patients with negative (131)I whole-body scans.

PubMed

Gabriel, Michael; Froehlich, Franz; Decristoforo, Clemens; Ensinger, Christian; Donnemiller, Eveline; von Guggenberg, Elisabeth; Heute, Dirk; Moncayo, Roy

2004-03-01

Several studies have reported on the expression of somatostatin receptors in patients with differentiated thyroid cancer (DTC). The aim of this study was to evaluate the imaging abilities of a recently developed technetium-99m labelled somatostatin analogue, (99m)Tc-EDDA/HYNIC-TOC ((99m)Tc-TOC), in terms of precise localisation of disease. The study population comprised 54 patients (24 men, 30 women; age range 22-90 years) with histologically confirmed DTC who presented with recurrent or persistent disease as indicated by elevated Tg levels after initial treatment. All patients were negative on the iodine-131 post-therapy whole-body scans. Fluorine-18 fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) was performed in a subgroup of 36 patients. The study population consisted of two groups: Group A ( n=22) comprised patients with disease recurrence as shown by elevated Tg levels but without detectable pathology. In group B ( n=32), pre-existing lesions were known. Among the 54 cases, SSTR scintigraphy was true positive in 33 (61.1%), true negative in 4 (7.4%) and false negative in 17 (31.5%) cases, which resulted in a sensitivity of 66%. A total of 138 tumour foci were localised in 33 patients. The fraction of true positive (99m)Tc-TOC findings was positively correlated ( P<0.01) with elevated Tg levels (higher than 30 ng/ml). Despite two false positive findings, analysis on a lesion basis demonstrated better diagnostic efficacy with (18)F-FDG PET ( P<0.001); however, it also revealed substantial agreement between the imaging techniques [Cohen's kappa of 0.62 (0.47-0.78)]. In conclusion, scintigraphy with (99m)Tc-TOC might be a promising tool for treatment planning; it is easy to perform and showed sufficient accuracy for localisation diagnostics in thyroid cancer patients with recurrent or metastatic disease.

Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium.

PubMed Central

Chitambar, C R; Seligman, P A

1986-01-01

We have previously shown that human leukemic cells proliferate normally in serum-free media containing various transferrin forms, but the addition of transferrin-gallium leads to inhibition of cellular proliferation. Because gallium has therapeutic potential, the effects of transferrin-gallium on leukemic cell proliferation, transferrin receptor expression, and cellular iron utilization were studied. The cytotoxicity of gallium is considerably enhanced by its binding to transferrin and cytotoxicity can be reversed by transferrin-iron but not by other transferrin forms. Exposure to transferrin-gallium leads to a marked increase in cell surface transferrin binding sites, but despite this, cellular 59Fe incorporation is inappropriately low. Although shunting of transferrin-gallium to another cellular compartment has not been ruled out, other studies suggest that transferrin-gallium impairs intracellular release of 59Fe from transferrin by interfering with processes responsible for intracellular acidification. These studies, taken together, demonstrate that inhibition of cellular iron incorporation by transferrin-gallium is a prerequisite for inhibition of cellular proliferation. PMID:3465751

Regulation of cellular senescence by the essential caveolar component PTRF/Cavin-1

PubMed Central

Bai, Lin; Deng, Xiaoli; Li, Juanjuan; Wang, Miao; Li, Qian; An, Wei; A, Deli; Cong, Yu-Sheng

2011-01-01

Polymerase I and transcript release factor (PTRF, also known as Cavin-1) is an essential component in the biogenesis and function of caveolae. Here, we show that PTRF expression is increased in senescent human fibroblasts. Importantly, overexpression of PTRF induced features characteristic of cellular senescence, whereas reduced PTRF expression extended the cellular replicative lifespan. Interestingly, we found that PTRF localized primarily to the nuclei of young and quiescent WI-38 human fibroblasts, but translocated to the cytosol and plasma membrane during cellular senescence. Furthermore, electron microscopic analysis demonstrated an increased number of caveolar structures in senescent and PTRF-transfected WI-38 cells. Our data suggest that the role of PTRF in cellular senescence is dependent on its targeting to caveolae and its interaction with caveolin-1, which appeared to be regulated by the phosphorylation of PTRF. Taken together, our findings identify PTRF as a novel regulator of cellular senescence that acts through the p53/p21 and caveolar pathways. PMID:21445100

Murine Hyperglycemic Vasculopathy and Cardiomyopathy: Whole-Genome Gene Expression Analysis Predicts Cellular Targets and Regulatory Networks Influenced by Mannose Binding Lectin

PubMed Central

Zou, Chenhui; La Bonte, Laura R.; Pavlov, Vasile I.; Stahl, Gregory L.

2012-01-01

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL’s role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies. PMID:22375142

Moving with Modernisation and Civilisation: Taiwanese Nativist Education in the Early 1930s

ERIC Educational Resources Information Center

Huang, Hsuan-Yi

2017-01-01

This study examines localisation endeavours in contemporary Taiwan to explore the history of the Taiwanese localisation movement as a way of reimagining meanings of "Taiwaneseness" constructed under different historical circumstances. It focuses on the "xiangtu" (nativist) literature movement in colonial Taiwan in the early…

Chinese Localisation of Evergreen: An Open Source Integrated Library System

ERIC Educational Resources Information Center

Zou, Qing; Liu, Guoying

2009-01-01

Purpose: The purpose of this paper is to investigate various issues related to Chinese language localisation in Evergreen, an open source integrated library system (ILS). Design/methodology/approach: A Simplified Chinese version of Evergreen was implemented and tested and various issues such as encoding, indexing, searching, and sorting…

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion

PubMed Central

A.P., Sudheesh

2017-01-01

ABSTRACT Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3′-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP–PIPKIα-mediated 3′-end processing as a key anti-invasive mechanism in breast cancer. PMID:29203642

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

PubMed

A P, Sudheesh; Laishram, Rakesh S

2018-03-01

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors ( KISS1R , CDH1 , NME1 , CDH13 , FEZ1 , and WIF1 ) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-PIPKIα-mediated 3'-end processing as a key anti-invasive mechanism in breast cancer. Copyright © 2018 A.P. and Laishram.

Raman microscopy of bladder cancer cells expressing green fluorescent protein

NASA Astrophysics Data System (ADS)

Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

2016-11-01

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

PubMed Central

2013-01-01

Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts.

PubMed

Makpol, Suzana; Yeoh, Thong Wei; Ruslam, Farah Adilah Che; Arifin, Khaizurin Tajul; Yusof, Yasmin Anum Mohd

2013-08-16

Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.

Foliar Nutrient Distribution Patterns in Sympatric Maple Species Reflect Contrasting Sensitivity to Excess Manganese.

PubMed

Fernando, Denise R; Marshall, Alan T; Lynch, Jonathan P

2016-01-01

Sugar maple and red maple are closely-related co-occurring tree species significant to the North American forest biome. Plant abiotic stress effects including nutritional imbalance and manganese (Mn) toxicity are well documented within this system, and are implicated in enhanced susceptibility to biotic stresses such as insect attack. Both tree species are known to overaccumulate foliar manganese (Mn) when growing on unbuffered acidified soils, however, sugar maple is Mn-sensitive, while red maple is not. Currently there is no knowledge about the cellular sequestration of Mn and other nutrients in these two species. Here, electron-probe x-ray microanalysis was employed to examine cellular and sub-cellular deposition of excessively accumulated foliar Mn and other mineral nutrients in vivo. For both species, excess foliar Mn was deposited in symplastic cellular compartments. There were striking between-species differences in Mn, magnesium (Mg), sulphur (S) and calcium (Ca) distribution patterns. Unusually, Mn was highly co-localised with Mg in mesophyll cells of red maple only. The known sensitivity of sugar maple to excess Mn is likely linked to Mg deficiency in the leaf mesophyll. There was strong evidence that Mn toxicity in sugar maple is primarily a symplastic process. For each species, leaf-surface damage due to biotic stress including insect herbivory was compared between sites with acidified and non-acidified soils. Although it was greatest overall in red maple, there was no difference in biotic stress damage to red maple leaves between acidified and non-acidified soils. Sugar maple trees on buffered non-acidified soil were less damaged by biotic stress compared to those on unbuffered acidified soil, where they are also affected by Mn toxicity abiotic stress. This study concluded that foliar nutrient distribution in symplastic compartments is a determinant of Mn sensitivity, and that Mn stress hinders plant resistance to biotic stress.

Foliar Nutrient Distribution Patterns in Sympatric Maple Species Reflect Contrasting Sensitivity to Excess Manganese

PubMed Central

Fernando, Denise R.; Marshall, Alan T.; Lynch, Jonathan P.

2016-01-01

Sugar maple and red maple are closely-related co-occurring tree species significant to the North American forest biome. Plant abiotic stress effects including nutritional imbalance and manganese (Mn) toxicity are well documented within this system, and are implicated in enhanced susceptibility to biotic stresses such as insect attack. Both tree species are known to overaccumulate foliar manganese (Mn) when growing on unbuffered acidified soils, however, sugar maple is Mn-sensitive, while red maple is not. Currently there is no knowledge about the cellular sequestration of Mn and other nutrients in these two species. Here, electron-probe x-ray microanalysis was employed to examine cellular and sub-cellular deposition of excessively accumulated foliar Mn and other mineral nutrients in vivo. For both species, excess foliar Mn was deposited in symplastic cellular compartments. There were striking between-species differences in Mn, magnesium (Mg), sulphur (S) and calcium (Ca) distribution patterns. Unusually, Mn was highly co-localised with Mg in mesophyll cells of red maple only. The known sensitivity of sugar maple to excess Mn is likely linked to Mg deficiency in the leaf mesophyll. There was strong evidence that Mn toxicity in sugar maple is primarily a symplastic process. For each species, leaf-surface damage due to biotic stress including insect herbivory was compared between sites with acidified and non-acidified soils. Although it was greatest overall in red maple, there was no difference in biotic stress damage to red maple leaves between acidified and non-acidified soils. Sugar maple trees on buffered non-acidified soil were less damaged by biotic stress compared to those on unbuffered acidified soil, where they are also affected by Mn toxicity abiotic stress. This study concluded that foliar nutrient distribution in symplastic compartments is a determinant of Mn sensitivity, and that Mn stress hinders plant resistance to biotic stress. PMID:27391424

Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains.

PubMed

Tummala, Seshu B; Junne, Stefan G; Paredes, Carlos J; Papoutsakis, Eleftherios T

2003-12-30

Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms. Copyright 2003; Wiley Periodicals, Inc.

Digital sorting of complex tissues for cell type-specific gene expression profiles.

PubMed

Zhong, Yi; Wan, Ying-Wooi; Pang, Kaifang; Chow, Lionel M L; Liu, Zhandong

2013-03-07

Cellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations. Here, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies. The results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Hongxing, E-mail: Hongxing.Zhao@igp.uu.se; Chen, Maoshan; Lind, Sara Bergström

The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phasemore » and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction. - Highlights: • The expression of 398 lncRNAs showed a distinct temporal pattern during Ad2 infection. • 80% of the deregulated lncRNAs were up-regulated during the late phase of infection. • The deregulated lncRNAs potentiallyinteract with 33 cellular RNA binding proteins. • These RBPs are involved in RNA splicing, nuclear export and translation. • Adenovirus exploits the cellular lncRNA network to optimize its replication.« less

DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

Minimal hardware Bluetooth tracking for long-term at-home elder supervision.

PubMed

Kelly, Damian; McLoone, Sean; Farrell, Ronan

2010-01-01

The ability to automatically detect the location of an elder within their own home is a significant enabler of remote elder supervision and interaction applications. This location information is typically generated via a myriad of sensors throughout the home environment. Even with high sensor redundancy, there are still situations where traditional elder monitoring systems are unable to resolve the location of the elder. This work develops a minimal infrastructure radio-frequency localisation system for long-term elder location tracking. An RFID room-labelling technique is employed and with it, the localisation system developed in this work is shown to exhibit superior performance to more traditional localisation systems in realistic long-term deployments.

[Extragenital endometriosis: Parietal, thoracic, diaphragmatic and nervous lesions. CNGOF-HAS Endometriosis Guidelines].

PubMed

Merlot, B; Ploteau, S; Abergel, A; Rubob, C; Hocke, C; Canis, M; Fritel, X; Roman, H; Collinet, P

2018-03-01

According to some studies, extragenital endometriosis represents 5% of the localisations. Its prevalence seems to be underestimated. The extra pelvic localisation can make the diagnosis more difficult. Nevertheless, the recurrent and catamenial symptomatology can evoke this pathology. Surgery seems to be the unique efficient treatment for parietal lesions. Pain linked to nervous lesions (peripheric and sacral roots) seems to be underestimated and difficult to diagnose because of various localisations. Neurolysis seems to have encouraging results. Diaphragmatic lesions are often discovered either incidentally during laparoscopy, or by pulmonary symptomatology as recurrent catamenial pneumothorax or cyclic thoracic pain. Surgical treatment seems as well to be efficient. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

PubMed Central

Majumder, Mrinmoyee; House, Reniqua; Palanisamy, Nallasivam; Qie, Shuo; Day, Terrence A.; Neskey, David; Diehl, J. Alan

2016-01-01

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells. PMID:27606879

Proteomic analysis of the response of α-ketoglutarate-producer Yarrowia lipolytica WSH-Z06 to environmental pH stimuli.

PubMed

Guo, Hongwei; Wan, Hui; Chen, Hongwen; Fang, Fang; Liu, Song; Zhou, Jingwen

2016-10-01

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

Cellular Factors Shape 3D Genome Landscape

Cancer.gov

Researchers, using novel large-scale imaging technology, have mapped the spatial location of individual genes in the nucleus of human cells and identified 50 cellular factors required for the proper 3D positioning of genes. These spatial locations play important roles in gene expression, DNA repair, genome stability, and other cellular activities.

Role of BAG3 in cancer progression: A therapeutic opportunity.

PubMed

De Marco, Margot; Basile, Anna; Iorio, Vittoria; Festa, Michelina; Falco, Antonia; Ranieri, Bianca; Pascale, Maria; Sala, Gianluca; Remondelli, Paolo; Capunzo, Mario; Firpo, Matthew A; Pezzilli, Raffaele; Marzullo, Liberato; Cavallo, Pierpaolo; De Laurenzi, Vincenzo; Turco, Maria Caterina; Rosati, Alessandra

2018-06-01

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours. Copyright © 2017 Elsevier Ltd. All rights reserved.

Visualisation of the mechanosensitive channel of large conductance in bacteria using confocal microscopy.

PubMed

Norman, Christel; Liu, Zhen-Wei; Rigby, Paul; Raso, Albert; Petrov, Yevgeniy; Martinac, Boris

2005-07-01

The mechanosensitive channel of large conductance (MscL) plays an important role in the survival of bacterial cells to hypo-osmotic shock. This channel has been extensively studied and its sequence, structure and electrophysiological characteristics are well known. Here we present a method to visualise MscL in living bacteria using confocal microscopy. By creating a gene fusion between mscl and the gene encoding the green fluorescent protein (GFP) we were able to express the fusion protein MscL-GFP in bacteria. We show that MscL-GFP is present in the cytoplasmic membrane and forms functional channels. These channels have the same characteristics as wild-type MscL, except that they require more pressure to open. This method could prove an interesting, non-invasive, tool to study the localisation and the regulation of expression of MscL in bacteria.

Nationalisation, Localisation and Globalisation in Finnish Higher Education

ERIC Educational Resources Information Center

Valimaa, Jussi

2004-01-01

This article analyses and discusses the interplay between the social processes of nationalisation, localisation and globalisation in a single European nation state. The view of nationalisation put forward draws on a national case study based on historical and sociological research findings. The second part of the article presents a case study of…

Collaborations between Foreign-Invested Enterprises and China's VET Schools: Making the System Work amid Localised Skill Shortages

ERIC Educational Resources Information Center

Li, Yiqiong; Sheldon, Peter

2014-01-01

This article examines collaborative initiatives individual foreign-invested enterprises (FIEs) develop with China's vocational education and training (VET) schools amid localised shortages of skilled workers. It thus focuses on employer initiatives in responding to VET system weaknesses rather than, as is common, those weaknesses. Using Suzhou…

Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors.

PubMed

Roderick, H L; Campbell, A K; Llewellyn, D H

1997-03-24

The multi-functional protein calreticulin (CRT) is normally found within the lumen of the endoplasmic reticulum (ER). However, some of its proposed functions require it to be located within the nucleus, where its presence is contentious. We have investigated this in live COS7, HeLa and LM(TK-) cells using green fluorescent protein (GFP)-fusion proteins. GFP-CRT, and GFP, with an ER signal peptide and a KDEL sequence (ER-GFP), were localised to the ER. In addition, GFP-CRT was located in the nucleus of all the cell types at low levels. The higher levels of nuclear fluorescence in LM(TK-) and HeLa cells suggested that glucocorticoid receptors might enhance nuclear localisation of calreticulin. Dexamethasone treatment of LM(TK-) cells doubled the amount of nuclear GFP-CRT, but did not affect the localisation of a GFP-CRT fusion in which the glucocorticoid receptor-binding N-domain of calreticulin had been deleted. Thus, despite ER targeting and retention signals, calreticulin is also located within the nucleus where its presence increases due to its interaction with glucocorticoid receptors.

Phosphorylation of Tat-interactive protein 60 kDa by protein kinase C epsilon is important for its subcellular localisation.

PubMed

Sapountzi, Vasileia; Logan, Ian R; Nelson, Glyn; Cook, Susan; Robson, Craig N

2008-01-01

Tat-interactive protein 60 kDa is a nuclear acetyltransferase that both coactivates and corepresses transcription factors and has a definitive function in the DNA damage response. Here, we provide evidence that Tat-interactive protein 60 kDa is phosphorylated by protein kinase C epsilon. In vitro, protein kinase C epsilon phosphorylates Tat-interactive protein 60 kDa on at least two sites within the acetyltransferase domain. In whole cells, activation of protein kinase C increases the levels of phosphorylated Tat-interactive protein 60 kDa and the interaction of Tat-interactive protein 60 kDa with protein kinase C epsilon. A phosphomimetic mutant Tat-interactive protein 60 kDa has distinct subcellular localisation compared to the wild-type protein in whole cells. Taken together, these findings suggest that the protein kinase C epsilon phosphorylation sites on Tat-interactive protein 60 kDa are important for its subcellular localisation. Regulation of the subcellular localisation of Tat-interactive protein 60 kDa via phosphorylation provides a novel means of controlling Tat-interactive protein 60 kDa function.

Counterintuitive electron localisation from density-functional theory with polarisable solvent models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dale, Stephen G., E-mail: sdale@ucmerced.edu; Johnson, Erin R., E-mail: erin.johnson@dal.ca

2015-11-14

Exploration of the solvated electron phenomena using density-functional theory (DFT) generally results in prediction of a localised electron within an induced solvent cavity. However, it is well known that DFT favours highly delocalised charges, rendering the localisation of a solvated electron unexpected. We explore the origins of this counterintuitive behaviour using a model Kevan-structure system. When a polarisable-continuum solvent model is included, it forces electron localisation by introducing a strong energetic bias that favours integer charges. This results in the formation of a large energetic barrier for charge-hopping and can cause the self-consistent field to become trapped in local minimamore » thus converging to stable solutions that are higher in energy than the ground electronic state. Finally, since the bias towards integer charges is caused by the polarisable continuum, these findings will also apply to other classical polarisation corrections, as in combined quantum mechanics and molecular mechanics (QM/MM) methods. The implications for systems beyond the solvated electron, including cationic DNA bases, are discussed.« less

The geometry of propagating rifts

NASA Astrophysics Data System (ADS)

McKenzie, Dan

1986-03-01

The kinematics of two different processes are investigated, both of which have been described as rift propagation. Courtillot uses this term to describe the change from distributed to localised extension which occurs during the early development of an ocean basin. The term localisation is instead used here to describe this process, to distinguish it from Hey's type of propagation. Localisation generally leads to rotation of the direction of magnetisation. To Hey propagation means the extension of a rift into the undeformed plate beyond a transform fault. Detail surveys of the Galapagos rift have shown that the propagating and failing rifts are not connected by a single transform fault, but by a zone which is undergoing shear. The principal deformation is simple shear, and the kinematics of this deformation are investigated in some detail. The strike of most of the lineations observed in the area can be produced by such deformation. The mode of extension on the propagating rift appears to be localised for some periods but to be distributed for others. Neither simple kinematic arguments nor stretching of the lithosphere with conservation of crust can account for the observed variations in water depth.

Reliability of horizontal and vertical tube shift techniques in the localisation of supernumerary teeth.

PubMed

Mallineni, S K; Anthonappa, R P; King, N M

2016-12-01

To assess the reliability of the vertical tube shift technique (VTST) and horizontal tube shift technique (HTST) for the localisation of unerupted supernumerary teeth (ST) in the anterior region of the maxilla. A convenience sample of 83 patients who attended a major teaching hospital because of unerupted ST was selected. Only non-syndromic patients with ST and who had complete clinical and radiographic and surgical records were included in the study. Ten examiners independently rated the paired set of radiographs for each technique. Chi-square test, paired t test and kappa statistics were employed to assess the intra- and inter-examiner reliability. Paired sets of 1660 radiographs (830 pairs for each technique) were available for the analysis. The overall sensitivity for VTST and HTST was 80.6 and 72.1% respectively, with slight inter-examiner and good intra-examiner reliability. Statistically significant differences were evident between the two localisation techniques (p < 0.05). Localisation of unerupted ST using VTST was more successful than HTST in the anterior region of the maxilla.

Mechanisms for localising calcineurin and CaMKII in dendritic spines.

PubMed

Penny, Christopher J; Gold, Matthew G

2018-05-27

Calcineurin and calmodulin-dependent protein kinase II (CaMKII) are both highly abundant in neurons, and both are activated by calmodulin at similar Ca 2+ concentrations in the test tube. However, they fulfill opposite functions in dendritic spines, with CaMKII activity driving long-term synaptic potentiation following large influxes of Ca 2+ through NMDA-type glutamate receptors (NMDARs), and calcineurin responding to smaller influxes of Ca 2+ through the same receptors to induce long-term depression. In this review, we explore the notion that precise dynamic localisation of the two enzymes at different sites within dendritic spines is fundamental to this behavior. We describe the structural basis of calcineurin and CaMKII localisation by their interaction with proteins including AKAP79, densin-180, α-actinin, and NMDARs. We then consider how interactions with these proteins likely position calcineurin and CaMKII at different distances from Ca 2+ microdomains emanating from the mouths of NMDARs in order to drive the divergent responses. We also highlight shortcomings in our current understanding of synaptic localisation of these two important signalling enzymes. Copyright © 2017. Published by Elsevier Inc.

The regulatory software of cellular metabolism.

PubMed

Segrè, Daniel

2004-06-01

Understanding the regulation of metabolic pathways in the cell is like unraveling the 'software' that is running on the 'hardware' of the metabolic network. Transcriptional regulation of enzymes is an important component of this software. A recent systematic analysis of metabolic gene-expression data in Saccharomyces cerevisiae reveals a complex modular organization of co-expressed genes, which could increase our ability to understand and engineer cellular metabolic functions.

Differential tolerance of two Gammarus pulex populations transplanted from different metallogenic regions to a polymetal gradient.

PubMed

Khan, Farhan R; Irving, Jennifer R; Bury, Nicolas R; Hogstrand, Christer

2011-03-01

The River Hayle, Cornwall, UK exhibits pronounced Cu and Zn concentration gradients which were used to compare the metal handling abilities of two populations of Gammarus pulex (Crustacea: Amphipoda). One population was native to the Hayle region (Drym) and presumably has been historically impacted by elevated Cu and Zn levels, whilst naïve gammarids were collected from the River Cray, Kent, UK. Both populations were subject to a 32 day in situ exposure at four R. Hayle sites (Drym, Godolphin, Relubbus and St. Erth). Mortality (LT50), Cu and Zn accumulation and sub-cellular distribution, and oxidative stress (malondialdehyde production) increased with the expected Cu and Zn bioavailabilities at the four sites (i.e. Godolphin>Relubbus>St. Erth>Drym). The naïve population experienced greater metal induced effects in terms of Cu and Zn accumulation, oxidative stress responses and lower LT50s. Analysis of Cu and Zn sub-cellular distribution, however, revealed no significant differences in metal handling. In both populations each metal was localised predominantly to the sub-cellular fraction containing metal bound to metallothionein-like proteins (MTLP) or that holding both metal-rich granules (MRG) and exoskeleton, MTLP and MRG binding being indicative of metal detoxification. However, a greater capacity for detoxified metal storage is not a mechanism implicated in the perceived tolerance of the historically impacted gammarids. Instead our results suggest that the historically impacted population was adapted for lower uptake of Cu and Zn leading to lower bioaccumulation, stress response and ultimately mortality. These results demonstrate not only the usefulness of the in situ methodology, but also that differences in population exposure history can cause significant differences in metal responses during exposure at higher concentrations. Copyright © 2011 Elsevier B.V. All rights reserved.

Partitioning of glutamine synthesised by the isolated perfused human placenta between the maternal and fetal circulations☆

PubMed Central

Day, P.E.L.; Cleal, J.K.; Lofthouse, E.M.; Goss, V.; Koster, G.; Postle, A.; Jackson, J.M.; Hanson, M.A.; Jackson, A.A.; Lewis, R.M.

2013-01-01

Introduction Placental glutamine synthesis has been demonstrated in animals and is thought to increase the availability of this metabolically important amino acid to the fetus. Glutamine is of fundamental importance for cellular replication, cellular function and inter-organ nitrogen transfer. The objective of this study was to investigate the role of glutamate/glutamine metabolism by the isolated perfused human placenta in the provision of glutamine to the fetus. Methods Glutamate metabolism was investigated in the isolated dually perfused human placental cotyledon. U–13C-glutamate was used to investigate the movement of carbon and 15N-leucine to study movement of amino-nitrogen. Labelled amino acids were perfused via maternal or fetal arteries at defined flow rates. The enrichment and concentration of amino acids in the maternal and fetal veins were measured following 5 h of perfusion. Results Glutamate taken up from the maternal and fetal circulations was primarily converted into glutamine the majority of which was released into the maternal circulation. The glutamine transporter SNAT5 was localised to the maternal-facing membrane of the syncytiotrophoblast. Enrichment of 13C or 15N glutamine in placental tissue was lower than in either the maternal or fetal circulation, suggesting metabolic compartmentalisation within the syncytiotrophoblast. Discussion Placental glutamine synthesis may help ensure the placenta's ability to supply this amino acid to the fetus does not become limiting to fetal growth. Glutamine synthesis may also influence placental transport of other amino acids, metabolism, nitrogen flux and cellular regulation. Conclusions Placental glutamine synthesis may therefore be a central mechanism in ensuring that the human fetus receives adequate nutrition and is able to maintain growth. PMID:24183194

Novel technology of molecular radio-guidance for lymph node dissection in recurrent prostate cancer by PSMA-ligands.

PubMed

Rauscher, Isabel; Horn, Thomas; Eiber, Matthias; Gschwend, Jürgen E; Maurer, Tobias

2018-04-01

Recently, prostate-specific membrane antigen-radioguided surgery (PSMA-RGS) has been introduced as a promising new and individual treatment concept in patients with localised recurrent prostate cancer (PC). In the following, we want to review our experience with PSMA-RGS in patients with localised biochemical recurrent PC. A non-systematic review of the literature was carried out with focus on technical and logistical aspects of PSMA-RGS. Furthermore, published data on intraoperative detection of metastatic lesions compared to preoperative PSMA-PET and postoperative histopathology, postoperative complications as well as oncological follow-up data are summarized. Finally, relevant aspects on prerequisites for PSMA-RGS, patient selection, and the potential benefit of additional salvage radiotherapy or potential future applications of robotic PSMA-RGS with drop-in γ-probes are discussed. First results show that PSMA-RGS is very sensitive and specific in tracking suspicious lesions intraoperatively. Prerequisite for patient selection and localisation of tumour recurrence is a positive Ga-HBED-CC PSMA positron-emission tomography (PET) scan with preferably only singular soft tissue or lymph node recurrence after primary treatment. Furthermore, PSMA-RGS has the potential to positively influence oncological outcome. PSMA-RGS seems to be of high value in patients with localised PC recurrence for exact localisation and resection of oftentimes small metastatic lesions using intraoperative and ex vivo γ-probe measurements. However, patient identification on the basis of Ga-HBED-CC-PSMA PET imaging as well as clinical parameters is crucial to obtain satisfactory results.

Proteomic profiling of the human T-cell nucleolus.

PubMed

Jarboui, Mohamed Ali; Wynne, Kieran; Elia, Giuliano; Hall, William W; Gautier, Virginie W

2011-12-01

The nucleolus, site of ribosome biogenesis, is a dynamic subnuclear organelle involved in diverse cellular functions. The size, number and organisation of nucleoli are cell-specific and while it remains to be established, the nucleolar protein composition would be expected to reflect lineage-specific transcriptional regulation of rDNA genes and have cell-type functional components. Here, we describe the first characterisation of the human T-cell nucleolar proteome. Using the Jurkat T-cell line and a reproducible organellar proteomic approach, we identified 872 nucleolar proteins. In addition to ribosome biogenesis and RNA processing networks, network modeling and topological analysis of nucleolar proteome revealed distinct macromolecular complexes known to orchestrate chromatin structure and to contribute to the regulation of gene expression, replication, recombination and repair, and chromosome segregation. Furthermore, among our dataset, we identified proteins known to functionally participate in T-cell biology, including RUNX1, ILF3, ILF2, STAT3, LSH, TCF-1, SATB1, CTCF, HMGB3, BCLAF1, FX4L1, ZAP70, TIAM1, RAC2, THEMIS, LCP1, RPL22, TOPK, RETN, IFI-16, MCT-1, ISG15, and 14-3-3τ, which support cell-specific composition of the Jurkat nucleolus. Subsequently, the nucleolar localisation of RUNX1, ILF3, STAT3, ZAP70 and RAC2 was further validated by Western Blot analysis and immunofluorescence microscopy. Overall, our T-cell nucleolar proteome dataset not only further expands the existing repertoire of the human nucleolar proteome but support a cell type-specific composition of the nucleolus in T cell and highlights the potential roles of the nucleoli in lymphocyte biology. Copyright © 2011 Elsevier Ltd. All rights reserved.

The concept of the CCN protein family revisited: a centralized coordination network.

PubMed

Perbal, Bernard

2018-03-01

The wide array of biological properties attributed to the CCN family of proteins (Perbal in Lancet 363(9402):62-64, 2004) led me to reconsider the possible relationship and roles that these proteins may play as a team, instead of acting on their own as individual regulators in various signaling pathways. The dynamic model which I present in this review stems from the contribution of the biological properties that we established for CCN3, one of the three founding members of the CCN family, which was identified by our group as the first CCN protein showing growth inhibitory properties (1992), expressed mainly in quiescent cells (1996), and showing anti-tumor activities in several cellular models both ex vivo and in vivo. At the present time CCN3 is the only member of the family that has been reported to negatively act on the progression of the cell cycle. The unique dual localisation of CCN3 in the nucleus and outside cells, either at the membrane or in the extracellular matrix, that I first established in 1999, and that now appears to be shared by several other CCN proteins, is a unique essential feature which can no longer be ignored. Based on the structural and functional properties of CCN3, shared by most of the CCN family members, I propose an « all in one » concept in which CCN proteins are team members with specific functions that are aimed at the same goal. This model accounts both for the functional specificity of the various CCN proteins, their sequential and opposite or complementary effects in various biological context, and for the biological consequences of their physical interaction and biological cross-regulation.

Differential retention of alpha-vitamin E is correlated with its transporter gene expression and growth inhibition efficacy in prostate cancer cells.

PubMed

Ni, Jing; Pang, See-Too; Yeh, Shuyuan

2007-04-01

Epidemiological studies showed Vit E has protective effects against prostate cancer (PCa). Interestingly, different prostate cancer cells have different sensitivity to alpha-Vit E or VES treatment. The goal of this study is to determine whether cellular Vit E bioavailability and its transport proteins are important contributing factors. alpha-Vit E and its ester form, VES, were used to treat prostate cancer LNCaP, PC3, and DU145 cells, and their growth rates were determined by MTT assay. Cellular levels of Vit E were quantified using HPLC as the index of bioavailability. The expression levels of Vit E transport proteins were determined by real-time PCR. Among these PCa cells, only LNCaP cells were sensitive to 20 microM alpha-Vit E treatment, while both LNCaP and PC3 cells were sensitive to 20 microM VES treatment. Coordinately, cellular levels of alpha-Vit E and VES positively correlated to their inhibitory effects. Further study found expression levels of Vit E transport proteins, including tocopherol associated protein (TAP), scavenger receptor class B type I (SR-BI), alpha-tocopherol transfer protein (TTP), and ATP binding cassette transporter A1 (ABCA1), were different in various PCa cells, which may contribute to cellular Vit E bioavailability. This notion is further supported by the findings that overexpression or knockdown of TTP could coordinately alter cellular alpha-Vit E levels in PCa cells. Antiproliferative efficacy of alpha-Vit E is correlated with its cellular bioavailability in PCa cells. Modulating the expression of the efflux or influx transporters could sensitize the growth inhibition efficacy of Vit E in prostate cancer cells.

CD133+CD54+CD44+ circulating tumor cells as a biomarker of treatment selection and liver metastasis in patients with colorectal cancer

PubMed Central

Wang, Cun; Huang, Qiaorong; Meng, Wentong; Yu, Yongyang; Yang, Lie; Peng, Zhihai; Hu, Jiankun; Li, Yuan; Mo, Xianming; Zhou, Zongguang

2016-01-01

Introduction Liver is the most common site of distant metastasis in colorectal cancer (CRC). Early diagnosis and appropriate treatment selection decides overall prognosis of patients. However, current diagnostic measures were basically imaging but not functional. Circulating tumor cells (CTCs) known as hold the key to understand the biology of metastatic mechanism provide a novel and auxiliary diagnostic strategy for CRC with liver metastasis (CRC-LM). Results The expression of CD133+ and CD133+CD54+CD44+ cellular subpopulations were higher in the peripheral blood of CRC-LM patients when compared with those without metastasis (P<0.001). Multivariate analysis proved the association between the expression of CD133+CD44+CD54+ cellular subpopulation and the existence of CRC-LM (P<0.001). The combination of abdominal CT/MRI, CEA and the CD133+CD44+CD54+ cellular subpopulation showed increased detection and discrimination rate for liver metastasis, with a sensitivity of 88.2% and a specificity of 92.4%. Meanwhile, it also show accurate predictive value for liver metastasis (OR=2.898, 95% C.I.1.374–6.110). Materials and Method Flow cytometry and multivariate analysis was performed to detect the expression of cancer initiating cells the correlation between cellular subpopulations and liver metastasis in patients with CRC. The receiver operating characteristic curves combined with the area under the curve were generated to compare the predictive ability of the cellular subpopulation for liver metastasis with current CT and MRI images. Conclusions The identification, expression and application of CTC subpopulations will provide an ideal cellular predictive marker for CRC liver metastasis and a potential marker for further investigation. PMID:27764803

CREG1 enhances p16INK4a-induced cellular senescence

PubMed Central

Moolmuang, Benchamart

2011-01-01

Cellular senescence is an irreversible growth arrest that is activated in normal cells upon shortening of telomere and other cellular stresses. Bypassing cellular senescence is a necessary step for cells to become immortal during oncogenic transformation. During the spontaneous immortalization of Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes 1) expression was decreased during immortalization and increased in senescence. Moreover, we found that repression of CREG1 expression occurs via an epigenetic mechanism, promoter DNA methylation. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, cancers commonly seen in Li-Fraumeni Syndrome. In addition, we found that p16INK4a is also downregulated in immortal cells and that coexpression of CREG1 and p16INK4a, an inhibitor of CDK4/6 and Rb phosphorylation, has a greater effect than either CREG1 and p16INK4a alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS fibroblasts, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16INK4a inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity, thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, this is the first evidence to demonstrate that CREG1 enhances p16INK4a-induced senescence by transcriptional repression of cell cycle-regulated genes. PMID:21263217

Modelling of deformation around magmatic intrusions with application to gold-related structures in the Yilgarn Craton, Western Australia

NASA Astrophysics Data System (ADS)

Zhang, Y.; Karrech, A.; Schaubs, P. M.; Regenauer-Lieb, K.; Poulet, T.; Cleverley, J. S.

2012-03-01

This study simulates rock deformation around high temperature granite intrusions and explores how gold bearing shear zones near intrusions were developed in the Yilgarn, using a new continuum damage mechanics algorithm that considers the temperature and time dependent elastic-visco-plastic constitutive behaviour of crustal materials. The results demonstrate that strain rates have the most significant effects on structural patterns for both extensional and compressional cases. Smaller strain rates promote the formation of narrow high-strain shear zones and strong strain localisation along the flank or shoulder areas of the intrusion and cold granite dome. Wider diffuse shear zones are developed under higher strain rates due to strain hardening. The cooling of the intrusion to background temperatures occurred over a much shorter time interval when compared to the duration of deformation and shear zones development. Strong strain localisation near the intrusion and shear zone development in the crust occurred under both extensional and compressional conditions. There is always clear strain localisation around the shoulders of the intrusion and the flanks of the "cold" granitic dome in early deformation stages. In the models containing a pre-existing fault, strain localisation near the intrusion became asymmetric with much stronger localisation and the development of a damage zone at the shoulder adjacent to the reactivated fault. At higher deformation stages, the models produced a range of structural patterns including graben and half graben basin (extension), "pop-up" wedge structures (compression), tilted fault blocks and switch of shear movement from reverse to normal on shear zones. The model explains in part why a number of gold deposits (e.g. Wallaby and Paddington deposits) in the Yilgarn were formed near the flank of granite-cored domes and deep "tapping" faults, and shows that the new modelling approach is capable of realistically simulating high strain localisation and shear zone development.

Feasibility of imaging epileptic seizure onset with EIT and depth electrodes.

PubMed

Witkowska-Wrobel, Anna; Aristovich, Kirill; Faulkner, Mayo; Avery, James; Holder, David

2018-06-01

Imaging ictal and interictal activity with Electrical Impedance Tomography (EIT) using intracranial electrode mats has been demonstrated in animal models of epilepsy. In human epilepsy subjects undergoing presurgical evaluation, depth electrodes are often preferred. The purpose of this work was to evaluate the feasibility of using EIT to localise epileptogenic areas with intracranial electrodes in humans. The accuracy of localisation of the ictal onset zone was evaluated in computer simulations using 9M element FEM models derived from three subjects. 5 mm radius perturbations imitating a single seizure onset event were placed in several locations forming two groups: under depth electrode coverage and in the contralateral hemisphere. Simulations were made for impedance changes of 1% expected for neuronal depolarisation over milliseconds and 10% for cell swelling over seconds. Reconstructions were compared with EEG source modelling for a radially orientated dipole with respect to the closest EEG recording contact. The best accuracy of EIT was obtained using all depth and 32 scalp electrodes, greater than the equivalent accuracy with EEG inverse source modelling. The localisation error was 5.2 ± 1.8, 4.3 ± 0 and 46.2 ± 25.8 mm for perturbations within the volume enclosed by depth electrodes and 29.6 ± 38.7, 26.1 ± 36.2, 54.0 ± 26.2 mm for those without (EIT 1%, 10% change, EEG source modelling, n = 15 in 3 subjects, p < 0.01). As EIT was insensitive to source dipole orientation, all 15 perturbations within the volume enclosed by depth electrodes were localised, whereas the standard clinical method of visual inspection of EEG voltages, only localised 8 out of 15 cases. This suggests that adding EIT to SEEG measurements could be beneficial in localising the onset of seizures. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The surgical manegement of metastases to humerus-clinical evaluation.

PubMed

Chrobok, Adam; Spindel, Jerzy; Miszczyk, Leszek; Koczy, Bogdan; Pilecki, Bogdan; Jarosz, Adam; Mrozek, Tomasz

2003-06-30

Background. The humerus is a common localisation of cancer metastases. The restoration of anatomical order and tumor resection within humerus is important for patients quality of everyday life and for their pain relief. The surgical treatment is one of the most important part of the whole oncological ways of tratment.
The study objective was a clinical assesment of tumor resection and reconstruction within humerus according to matastasis localisation and the choice of surgical technique.
Material and methods. In the years 1999-2002 19 patients underwent surgery due to pathological fracture or/and cancer metastasis within humerus. The shaft localisation of the tumor was found in 8 cases and in 11 patients the metastatic foci were found in proximnal diaphysis. In patients with proximal diaphysis localisation of the tumor the partial resection with subsequent joint exchange procedure was made. The humeral shaft metastatic cancer changes were treated by the segmental resection with subsequent surgical cement filling or auto/allogenical bone grafting combined with intramedullary nail or AO/ASIF plate stabilisation. The average follow-up period was 8,5 months.
Results. In patients after resection with shoulder joint alloplasty according to the Enneking test a very good result was found in 7 and very good in 4 cases.
In patients with femoral shaft metastatic tumor locaslisation, 6 good and 2 fair results were found. In 1 patient after 2 months rehabilitation a reoperation was needed due to the mechanical destabilisation. The best results were found in patients after intramedullary nailing.An acute soft tissue inflamation requiering surgical treatment was found in 1 patient after shoulder joint replacement.
Conclusions. After clinical analysis of the material we highly recomend the partial humeral bone resection with subsequent shoulder alloplasty in patients with proximal humeral diaphysis metastatic localisation. In cases with shaft localisation a better results were found after intramedullary nailing compared to common plate technique.

Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

PubMed

Meissner, Barbara; Rogalski, Teresa; Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G

2011-01-01

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.

Cellular Transcription Factors Induced in Trigeminal Ganglia during Dexamethasone-Induced Reactivation from Latency Stimulate Bovine Herpesvirus 1 Productive Infection and Certain Viral Promoters

PubMed Central

Workman, Aspen; Eudy, James; Smith, Lynette; Frizzo da Silva, Leticia; Sinani, Devis; Bricker, Halie; Cook, Emily; Doster, Alan

2012-01-01

Bovine herpesvirus 1 (BHV-1), an alphaherpesvirinae subfamily member, establishes latency in sensory neurons. Elevated corticosteroid levels, due to stress, reproducibly triggers reactivation from latency in the field. A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves consistently induces reactivation from latency. Lytic cycle viral gene expression is detected in sensory neurons within 6 h after DEX treatment of latently infected calves. These observations suggested that DEX stimulated expression of cellular genes leads to lytic cycle viral gene expression and productive infection. In this study, a commercially available assay—Bovine Gene Chip—was used to compare cellular gene expression in the trigeminal ganglia (TG) of calves latently infected with BHV-1 versus DEX-treated animals. Relative to TG prepared from latently infected calves, 11 cellular genes were induced more than 10-fold 3 h after DEX treatment. Pentraxin three, a regulator of innate immunity and neurodegeneration, was stimulated 35- to 63-fold after 3 or 6 h of DEX treatment. Two transcription factors, promyelocytic leukemia zinc finger (PLZF) and Slug were induced more than 15-fold 3 h after DEX treatment. PLZF or Slug stimulated productive infection 20- or 5-fold, respectively, and Slug stimulated the late glycoprotein C promoter more than 10-fold. Additional DEX-induced transcription factors also stimulated productive infection and certain viral promoters. These studies suggest that DEX-inducible cellular transcription factors and/or signaling pathways stimulate lytic cycle viral gene expression, which subsequently leads to successful reactivation from latency in a small subset of latently infected neurons. PMID:22190728

Development of a discriminatory biocompatibility testing model for non-precious dental casting alloys.

PubMed

McGinley, Emma Louise; Fleming, Garry J P; Moran, Gary P

2011-12-01

To develop an enhanced, reproducible and discriminatory biocompatibility testing model for non-precious dental casting alloys, prepared to a clinically relevant surface finishing condition, using TR146 oral keratinocyte cells. Comparative biocompatibility was determined following direct and indirect exposure of TR146 cells to two nickel-chromium (Ni-Cr) and a cobalt-chromium (Co-Cr) alloy-discs. The surface roughness of the discs was determined using a contact stylus profilometer and the elemental ion release by inductively coupled plasma mass spectrometry (ICP-MS). Subsequent biocompatibility analysis included cell morphology, cell density measurements with Trypan blue exclusion assay, inflammatory cytokine expression with ELISAs, cellular metabolic activity using XTT and cellular toxicity using lactate dehydrogenase (LDH) release assay. TR146 cell morphology was altered following direct and indirect exposure to the Ni-Cr alloys but not the Co-Cr alloy. Significant reductions (all P<0.001) in viable cell density measurements, cellular metabolic activity, significant increases inflammatory cytokine expression and cellular toxicity were observed when TR146 cells were exposed to the Ni-Cr alloys. Significant decreases in cell density measurements, cellular metabolic activity, significant increases inflammatory cytokine expression and cellular toxicity for the Ni-Cr d.Sign(®)15 alloy compared with d.Sign(®)10 alloy were identifiable (all P<0.001). Cellular toxicity was attributed to nickel ion release levels in solution detected by ICP-MS analysis. Nickel ions from the Ni-Cr alloys permeated the epithelial cells and activated a proinflammatory response, namely IL-1a, IL-8 and PGE2 expression. Further evidence of nickel ioninduced cell death was supported by the decreased biocompatibility of the highest nickel ion releasing alloy (d.Sign(®)15 compared with d.Sign(®)10) and the increased biocompatibility of the Co-Cr (d.Sign(®)30) alloy where nickel ions were absent. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms

NASA Technical Reports Server (NTRS)

Baldwin, Kenneth M.; Haddad, Fadia

2002-01-01

The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

Interaction with caveolin-1 modulates vascular ATP-sensitive potassium (KATP) channel activity

PubMed Central

Davies, Lowri M; Purves, Gregor I; Barrett-Jolley, Richard; Dart, Caroline

2010-01-01

ATP-sensitive potassium channels (KATP channels) of arterial smooth muscle are important regulators of arterial tone, and hence blood flow, in response to vasoactive transmitters. Recent biochemical and electron microscopic evidence suggests that these channels localise to small vesicular invaginations of the plasma membrane, known as caveolae, and interact with the caveolae-associated protein, caveolin. Here we report that interaction with caveolin functionally regulates the activity of the vascular subtype of KATP channel, Kir6.1/SUR2B. Pinacidil-evoked recombinant whole-cell Kir6.1/SUR2B currents recorded in HEK293 cells stably expressing caveolin-1 (69.6 ± 8.3 pA pF−1, n= 8) were found to be significantly smaller than currents recorded in caveolin-null cells (179.7 ± 35.9 pA pF−1, n= 6; P < 0.05) indicating that interaction with caveolin may inhibit channel activity. Inclusion in the pipette-filling solution of a peptide corresponding to the scaffolding domain of caveolin-1 had a similar inhibitory effect on whole-cell Kir6.1/SUR2B currents as co-expression with full-length caveolin-1, while a scrambled version of the same peptide had no effect. Interestingly, intracellular dialysis of vascular smooth muscle cells with the caveolin-1 scaffolding domain peptide (SDP) also caused inhibition of pinacidil-evoked native whole-cell KATP currents, indicating that a significant proportion of vascular KATP channels are susceptible to block by exogenously applied SDP. In cell-attached recordings of Kir6.1/SUR2B single channel activity, the presence of caveolin-1 significantly reduced channel open probability (from 0.05 ± 0.01 to 0.005 ± 0.001; P < 0.05) and the amount of time spent in a relatively long-lived open state. These changes in kinetic behaviour can be explained by a caveolin-induced shift in the channel's sensitivity to its physiological regulator MgADP. Our findings thus suggest that interaction with caveolin-1 suppresses vascular-type KATP channel activity. Since caveolin expression is regulated by cellular free cholesterol and plasma levels of low-density lipoprotein (LDL), this interaction may have implications in both the physiological and pathophysiological control of vascular function. PMID:20624795

Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

PubMed Central

Dougherty, W G; Semler, B L

1993-01-01

Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

Is the etiology of eosinophilic esophagitis in adults a response to allergy or reflux injury? Study of cellular proliferation markers.

PubMed

Lewis, C J; Lamb, C A; Kanakala, V; Pritchard, S; Armstrong, G R; Attwood, S E A

2009-01-01

Recent research suggests that allergy may be the key factor in the etiology of eosinophilic esophagitis (EE); however, historically, the condition was hypothesized as related to reflux injury to the esophageal mucosa. We studied this hypothesis by comparing markers of inflammation and cellular proliferation in EE and reflux esophagitis. Lower esophageal biopsies of adult patients with EE (n = 10), reflux esophagitis (n = 8), and normal controls (n = 13) were assessed quantitatively for the expression of the cyclooxygenase-2 (COX-2) enzyme, cellular proliferation, and oncogenic resistance to apoptosis using monoclonal antibodies for COX-2, Ki-67, and Bcl-2, respectively. Normal esophageal epithelium demonstrated weak diffuse uptake of COX-2 stain in the basal layer. No COX-2 expression was demonstrated in the EE group, significantly less than the control and reflux groups (P < 0.01 and P < 0.001, respectively). Cellular proliferation measured by Ki-67 expression was higher in EE and reflux compared with control (P < 0.001 and P < 0.01). Ki-67 expression, and thus degree of hyperplasia, appeared greater in EE than reflux, but was not statistically significant (P = 0.228). The degree of apoptosis was similar in all study groups. EE and reflux esophagitis are proliferative conditions expressing Ki-67 in higher concentrations than control. Mucosal proliferation in reflux esophagitis is COX-2 dependent. This novel research in EE has demonstrated downregulation of COX-2 expression compared with reflux esophagitis and control. We hypothesize that the allergy-related cytokine IL-13 known to inhibit COX-2 expression and found in high concentrations in EE as responsible for this. The pathogenesis of EE is likely dependent on allergy rather than reflux injury to the esophagus.

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

PubMed

Kilaparty, Surya P; Agarwal, Rakhee; Singh, Pooja; Kannan, Krishnaswamy; Ali, Nawab

2016-07-01

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

Globalisation and Localisation in Music Education in Hong Kong and Taiwan

ERIC Educational Resources Information Center

Ho, Wai-Chung

2013-01-01

The purpose of the study is to analyse and discuss the influences of globalisation and localisation on music education in Hong Kong and Taiwan. It argues that the reform of music education concerns changes to the contents of the curriculum that envisage the cultural and political developments that arise from processes of globalisation and…

The management of anterior tooth wear using gold palatal veneers in canine guidance.

PubMed

Eliyas, S; Martin, N

2013-03-01

Localised anterior tooth wear can be managed using minimally invasive techniques with conservation of tooth structure and preservation of pulp vitality. This article describes and illustrates with two clinical cases, the management of localised tooth wear, with the restoration of canine guidance by a combination of gold palatal veneers and direct composite restorations.

Application of the Localisation Platform Crowdin in Translator Education

ERIC Educational Resources Information Center

Kudla, Dominik

2017-01-01

This article is an attempt to briefly describe the potential of the online localisation platform Crowdin for the education of the future translators at universities and in private courses or workshops. This description is provided on the basis of the information gathered through the user experience of the author and uses the example of the Khan…

Robust weak anti-localisation effect in strongly textured nanocrystalline Bi2Se3 samples

NASA Astrophysics Data System (ADS)

Pereira, V. M. M.; Henriques, M. S. C.; Paixão, J. A.

2018-05-01

Topological insulators are a quantum state of matter that has recently created a great interest among the scientific community, with Bi2Se3 being one of the most extensively studied materials. Here, we demonstrate that polycrystalline nanostructured samples of Bi2Se3 preserve the existence of topological surface states, where electrons cannot be localised. The nanosheet crystals were synthesised by a microwave-assisted method and their structure, composition and morphology thoroughly characterised. The transport properties of a textured polycrystalline sample with strong preferred orientation along the c-axis were measured, showing the presence of the weak anti-localisation effect and Shubnikov-de Haas oscillations. These features are robust against the presence of non-magnetic impurities and structural defects.

Immunobiological Aspects of erbB Receptors in Breast Cancer

DTIC Science & Technology

2000-08-01

receptor . The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were...determine Cells were washed twice with cold phosphate-buffered saline which cellular substrates couple with the receptor complex. (PBS) and lysed with 1...turnover, receptor proteins suggests that these substrates are properly lo- and cellular transformation in NEN757 cells (Qian et al., cated for

Macromolecular Expression and Function: A New Paradigm for NASA Risk Assessment

NASA Technical Reports Server (NTRS)

Richmond, Robert

2003-01-01

Predicting risks in humans of either acute effects such as bone loss or muscle wasting, or late effects such as cancer, is challenging. To an approximation, this is because uncertainties of exposure to stress factors or toxic agents and the uniformity of processing subsequent damage at the cellular level within a complex set of biological variables degrade the confidence of predicting pathologic outcome. A cellular biodosimeter that simultaneously reports 1) the type of damage due to that exposure, 2) the quantity of damage incurred by that exposure, and 3) the dataset used to assess risk of developing pathologic outcome caused by that exposure would therefore be useful for predicting ultimate risks faced by an individual, such as an astronaut. It is suggested that such a biodosimeter can be based upon analyses of gene-expression and protein expression whereby large datasets of cellular response to damage are obtained and analyzed for expression-profiles correlated with established end points and molecular markers predictive for risks being assessed. The usefulness of multiparametric cellular biodosimeters could be realized by quantitatively profiling these datasets using techniques of bioinformatics. Such an approach contributes to the foundation of molecular epidemiology as a new scientific discipline, and represents a new paradigm of risk assessment.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells.

PubMed

Udono, Miyako; Fujii, Kaoru; Harada, Gakuro; Tsuzuki, Yumi; Kadooka, Keishi; Zhang, Pingbo; Fujii, Hiroshi; Amano, Maho; Nishimura, Shin-Ichiro; Tashiro, Kosuke; Kuhara, Satoru; Katakura, Yoshinori

2015-11-27

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells

PubMed Central

Udono, Miyako; Fujii, Kaoru; Harada, Gakuro; Tsuzuki, Yumi; Kadooka, Keishi; Zhang, Pingbo; Fujii, Hiroshi; Amano, Maho; Nishimura, Shin-Ichiro; Tashiro, Kosuke; Kuhara, Satoru; Katakura, Yoshinori

2015-01-01

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program. PMID:26611489

Identifying the molecular basis of functions in the transcriptome of the social amoeba Dictyostelium discoideum.

PubMed

Whitney, T J; Gardner, D G; Mott, M L; Brandon, M

2010-03-09

The unusual life cycle of Dictyostelium discoideum, in which an extra-cellular stressor such as starvation induces the development of a multicellular fruiting body consisting of stalk cells and spores from a culture of identical amoebae, provides an excellent model for investigating the molecular control of differentiation and the transition from single- to multi-cellular life, a key transition in development. We utilized serial analysis of gene expression (SAGE), a molecular method that is unbiased by dependence on previously identified genes, to obtain a transcriptome from a high-density culture of amoebae, in order to examine the transition to multi-cellular development. The SAGE method provides relative expression levels, which allows us to rank order the expressed genes. We found that a large number of ribosomal proteins were expressed at high levels, while various components of the proteosome were expressed at low levels. The only identifiable transmembrane signaling system components expressed in amoebae are related to quorum sensing, and their expression levels were relatively low. The most highly expressed gene in the amoeba transcriptome, dutA untranslated RNA, is a molecule with unknown function that may serve as an inhibitor of translation. These results suggest that high-density amoebae have not initiated development, and they also suggest a mechanism by which the transition into the development program is controlled.

Cellular expansion and gene expression in the developing grape (Vitis vinifera L.).

PubMed

Schlosser, J; Olsson, N; Weis, M; Reid, K; Peng, F; Lund, S; Bowen, P

2008-01-01

Expression profiles of genes involved in cell wall metabolism and water transport were compared with changes in grape (Vitis vinifera L.) berry growth, basic chemical composition, and the shape, size, and wall thickness of cells within tissues of the berry pericarp. Expression of cell wall-modifying and aquaporin genes in berry pericarp tissues generally followed a bimodal expression profile with high levels of expression coinciding with the two periods of rapid berry growth, stages I and III, and low levels of expression corresponding to the slow-growth period, stage II. Cellular expansion was observed throughout all tissues during stage I, and only mesocarp cellular expansion was observed during stage III. Expansion of only exocarp cells was evident during transition between stages II and III. Cell wall-modifying and aquaporin gene expression profiles followed similar trends in exocarp and mesocarp tissues throughout berry development, with the exception of the up-regulation of pectin methylesterase, pectate lyase, two aquaporin genes (AQ1 and AQ2), and two expansin genes (EXP3 and EXPL) during stage II, which was delayed in the exocarp tissue compared with mesocarp tissue. Exocarp endo-(1-->3)-beta-glucanase and expansin-like gene expression was concurrent with increases in epidermal and hypodermal cell wall thickness. These results indicate a potential role of the grape berry skin in modulating grape berry growth.

Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

USGS Publications Warehouse

Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

2011-01-01

The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

Genome-wide localization and expression profiling establish Sp2 as a sequence-specific transcription factor regulating vitally important genes

PubMed Central

Terrados, Gloria; Finkernagel, Florian; Stielow, Bastian; Sadic, Dennis; Neubert, Juliane; Herdt, Olga; Krause, Michael; Scharfe, Maren; Jarek, Michael; Suske, Guntram

2012-01-01

The transcription factor Sp2 is essential for early mouse development and for proliferation of mouse embryonic fibroblasts in culture. Yet its mechanisms of action and its target genes are largely unknown. In this study, we have combined RNA interference, in vitro DNA binding, chromatin immunoprecipitation sequencing and global gene-expression profiling to investigate the role of Sp2 for cellular functions, to define target sites and to identify genes regulated by Sp2. We show that Sp2 is important for cellular proliferation that it binds to GC-boxes and occupies proximal promoters of genes essential for vital cellular processes including gene expression, replication, metabolism and signalling. Moreover, we identified important key target genes and cellular pathways that are directly regulated by Sp2. Most significantly, Sp2 binds and activates numerous sequence-specific transcription factor and co-activator genes, and represses the whole battery of cholesterol synthesis genes. Our results establish Sp2 as a sequence-specific regulator of vitally important genes. PMID:22684502

Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide

PubMed Central

Fomenko, Dmitri E.; Koc, Ahmet; Agisheva, Natalia; Jacobsen, Michael; Kaya, Alaattin; Malinouski, Mikalai; Rutherford, Julian C.; Siu, Kam-Leung; Jin, Dong-Yan; Winge, Dennis R.; Gladyshev, Vadim N.

2011-01-01

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H2O2 response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to activate and repress gene expression in response to H2O2. Further studies involving redox transcription factors suggested that thiol peroxidases are major regulators of global gene expression in response to H2O2. The data suggest that thiol peroxidases sense and transfer oxidative signals to the signaling proteins and regulate transcription, whereas a direct interaction between H2O2 and other cellular proteins plays a secondary role. PMID:21282621

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation.

PubMed

Ali, Mehboob; Heyob, Kathryn; Jacob, Naduparambil K; Rogers, Lynette K

2016-09-01

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASP(pS157), but no changes in cofilin1/VASP(pS239) in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASP(pS157) or cofilin1/VASP(pS239) suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220-31. ©2016 AACR. ©2016 American Association for Cancer Research.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and is Modified by DHA Supplementation

PubMed Central

Ali, Mehboob; Heyob, Kathryn; Jacob, Naduparambil K.; Rogers, Lynette K.

2016-01-01

Profilin 1, cofilin 1, and vasodialator stimulated phosphoprotein (VASP) are actin binding proteins (ABP) which regulate actin remodelling and facilitate cancer cell metastases. MiR~17–92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. Additionally, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1 and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by western blot and confocal analysis. MiR~17–92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASPpS157 but no changes in cofilin1/VASPpS239 in the human malignant tissues compared to normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASPpS157 or cofilin1/VASPpS239 suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR~17–92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR~17–92, and DHA as a novel therapeutic. PMID:27496138

UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins.

PubMed

Pacini, Laura; Ceraolo, Maria Grazia; Venuti, Assunta; Melita, Giusi; Hasan, Uzma A; Accardi, Rosita; Tommasino, Massimo

2017-10-01

Several lines of evidence indicate that cutaneous human papillomavirus (HPV) types belonging to the beta genus of the HPV phylogenetic tree synergize with UV radiation in the development of skin cancer. Accordingly, the E6 and E7 oncoproteins from some beta HPV types are able to deregulate pathways related to immune response and cellular transformation. Toll-like receptor 9 (TLR9), in addition to playing a role in innate immunity, has been shown to be involved in the cellular stress response. Using primary human keratinocytes as experimental models, we have shown that UV irradiation (and other cellular stresses) activates TLR9 expression. This event is closely linked to p53 activation. Silencing the expression of p53 or deleting its encoding gene affected the activation of TLR9 expression after UV irradiation. Using various strategies, we have also shown that the transcription factors p53 and c-Jun are recruited onto a specific region of the TLR9 promoter after UV irradiation. Importantly, the E6 and E7 oncoproteins from beta HPV38, by inducing the accumulation of the p53 antagonist ΔNp73α, prevent the UV-mediated recruitment of these transcription factors onto the TLR9 promoter, with subsequent impairment of TLR9 gene expression. This study provides new insight into the mechanism that mediates TLR9 upregulation in response to cellular stresses. In addition, we show that HPV38 E6 and E7 are able to interfere with this mechanism, providing another explanation for the possible cooperation of beta HPV types with UV radiation in skin carcinogenesis. IMPORTANCE Beta HPV types have been suggested to act as cofactors in UV-induced skin carcinogenesis by altering several cellular mechanisms activated by UV radiation. We show that the expression of TLR9, a sensor of damage-associated molecular patterns produced during cellular stress, is activated by UV radiation in primary human keratinocytes (PHKs). Two transcription factors known to be activated by UV radiation, p53 and c-Jun, play key roles in UV-activated TLR9 expression. The E6 and E7 oncoproteins from beta HPV38 strongly inhibit UV-activated TLR9 expression by preventing the recruitment of p53 and c-Jun to the TLR9 promoter. Our findings provide additional support for the role that beta HPV types play in skin carcinogenesis by preventing activation of specific pathways upon exposure of PHKs to UV radiation. Copyright © 2017 American Society for Microbiology.

An online database for plant image analysis software tools.

PubMed

Lobet, Guillaume; Draye, Xavier; Périlleux, Claire

2013-10-09

Recent years have seen an increase in methods for plant phenotyping using image analyses. These methods require new software solutions for data extraction and treatment. These solutions are instrumental in supporting various research pipelines, ranging from the localisation of cellular compounds to the quantification of tree canopies. However, due to the variety of existing tools and the lack of central repository, it is challenging for researchers to identify the software that is best suited for their research. We present an online, manually curated, database referencing more than 90 plant image analysis software solutions. The website, plant-image-analysis.org, presents each software in a uniform and concise manner enabling users to identify the available solutions for their experimental needs. The website also enables user feedback, evaluations and new software submissions. The plant-image-analysis.org database provides an overview of existing plant image analysis software. The aim of such a toolbox is to help users to find solutions, and to provide developers a way to exchange and communicate about their work.

Gastric form of alpha chain disease.

PubMed Central

Coulbois, J; Galian, P; Galian, A; Couteaux, B; Danon, F; Rambaud, J

1986-01-01

A case of alpha chain disease, involving the stomach only, is reported in an Algerian man suffering from epigastric pains. Upper digestive tract fibreoptic endoscopy showed two antral ulcers and an ulcerative gastritis pattern, which promptly disappeared with cimetidine treatment. Antral biopsies at a distance from the ulcers, but not of the ulcer crater itself, disclosed a dense infiltration of antral lamina propria by mature or sometimes atypical plasma cells. On transmural surgical antral biopsy, the infiltrate spread to the superficial part of the submucosa. No other localisation of the disease was found in spite of multiple biopsies obtained by endoscopy, with a peroral capsule and during staging laparotomy. The alpha chain disease protein was absent from serum and urine, but found in the gastric juice and in the cytoplasma of the cellular infiltrate (alpha 1 subclass). A complete clinical, endoscopic, histological and immunological remission was observed after a six months' course of oral tetracycline. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:3087826

Fluorescent probes for the simultaneous detection of multiple analytes in biology.

PubMed

Kolanowski, Jacek L; Liu, Fei; New, Elizabeth J

2018-01-02

Many of the key questions facing cellular biology concern the location and concentration of chemical species, from signalling molecules to metabolites to exogenous toxins. Fluorescent sensors (probes) have revolutionised the understanding of biological systems through their exquisite sensitivity to specific analytes. Probe design has focussed on selective sensors for individual analytes, but many of the most pertinent biological questions are related to the interaction of more than one chemical species. While it is possible to simultaneously use multiple sensors for such applications, data interpretation will be confounded by the fact that sensors will have different uptake, localisation and metabolism profiles. An alternative solution is to instead use a single probe that responds to two analytes, termed a dual-responsive probe. Recent progress in this field has yielded exciting probes, some of which have demonstrated biological application. Here we review work that has been carried out to date, and suggest future research directions that will harness the considerable potential of dual-responsive fluorescent probes.

A physico-genetic module for the polarisation of auxin efflux carriers PIN-FORMED (PIN)

NASA Astrophysics Data System (ADS)

Hernández-Hernández, Valeria; Barrio, Rafael A.; Benítez, Mariana; Nakayama, Naomi; Romero-Arias, José Roberto; Villarreal, Carlos

2018-05-01

Intracellular polarisation of auxin efflux carriers is crucial for understanding how auxin gradients form in plants. The polarisation dynamics of auxin efflux carriers PIN-FORMED (PIN) depends on both biomechanical forces as well as chemical, molecular and genetic factors. Biomechanical forces have shown to affect the localisation of PIN transporters to the plasma membrane. We propose a physico-genetic module of PIN polarisation that integrates biomechanical, molecular, and cellular processes as well as their non-linear interactions. The module was implemented as a discrete Boolean model and then approximated to a continuous dynamic system, in order to explore the relative contribution of the factors mediating PIN polarisation at the scale of single cell. Our models recovered qualitative behaviours that have been experimentally observed and enable us to predict that, in the context of PIN polarisation, the effects of the mechanical forces can predominate over the activity of molecular factors such as the GTPase ROP6 and the ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN RIC1.

Mycobacterium tuberculosis promotes genomic instability in macrophages

PubMed Central

Castro-Garza, Jorge; Luévano-Martínez, Miriam Lorena; Villarreal-Treviño, Licet; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha Imelda; García-Vielma, Catalina; González-Hernández, Silvia; Cortés-Gutiérrez, Elva Irene

2018-01-01

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection. PMID:29412354

Phylogenetic relationships, stage-specific expression and localisation of a unique family of inactive cysteine proteases in Sarcoptes scabiei.

PubMed

Fernando, Deepani D; Reynolds, Simone L; Zakrzewski, Martha; Mofiz, Ehtesham; Papenfuss, Anthony T; Holt, Deborah; Fischer, Katja

2018-05-16

Scabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites. We postulated that the SMIPP-Cs have evolved as an adaptation to the parasitic lifestyle of the scabies mite. To formulate testable hypotheses for their functions and to propose possible strategies for translational research we investigated whether the SMIPP-Cs are common to all scabies mite varieties and where within the mite body as well as when throughout the parasitic life-cycle they are expressed. SMIPP-C sequences from human, pig and dog mites were analysed bioinformatically and the phylogenetic relationships between the SMIPP-C multi-copy gene families of human, pig and dog mites were established. Results suggest that amplification of the SMIPP-C genes occurred in a common ancestor and individual genes evolved independently in the different mite varieties. Recombinant human mite SMIPP-C proteins were produced and used for murine polyclonal antibody production. Immunohistology on skin sections from human patients localised the SMIPP-Cs in the mite gut and in mite faeces within in the epidermal skin burrows. SMIPP-C transcription into mRNA in different life stages was assessed in human and pig mites by reverse transcription followed by droplet digital PCR (ddPCR). High transcription levels of SMIPP-C genes were detected in the adult female life stage in comparison to all other life stages. The fact that the SMIPP-Cs are unique to three Sarcoptes varieties, present in all burrowing life stages and highly expressed in the digestive system of the infective adult female life stage may highlight an essential role in parasitism. As they are excreted from the gut in scybala they presumably are able to interact or interfere with host proteins present in the epidermis.

Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15

PubMed Central

Mahendrasingam, Shanthini; Fettiplace, Robert; Alagramam, Kumar N.; Cross, Ellen

2017-01-01

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15. PMID:29069081

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport

PubMed Central

Sanders, Anna A. W. M.; Li, Chunmei; Kennedy, Julie; Cai, Jerry; Scheidel, Noemie; Kennedy, Breandán N.; Morin, Ryan D.; Leroux, Michel R.; Blacque, Oliver E.

2016-01-01

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base. PMID:27930654

Whole-Organism Developmental Expression Profiling Identifies RAB-28 as a Novel Ciliary GTPase Associated with the BBSome and Intraflagellar Transport.

PubMed

Jensen, Victor L; Carter, Stephen; Sanders, Anna A W M; Li, Chunmei; Kennedy, Julie; Timbers, Tiffany A; Cai, Jerry; Scheidel, Noemie; Kennedy, Breandán N; Morin, Ryan D; Leroux, Michel R; Blacque, Oliver E

2016-12-01

Primary cilia are specialised sensory and developmental signalling devices extending from the surface of most eukaryotic cells. Defects in these organelles cause inherited human disorders (ciliopathies) such as retinitis pigmentosa and Bardet-Biedl syndrome (BBS), frequently affecting many physiological and developmental processes across multiple organs. Cilium formation, maintenance and function depend on intracellular transport systems such as intraflagellar transport (IFT), which is driven by kinesin-2 and IFT-dynein motors and regulated by the Bardet-Biedl syndrome (BBS) cargo-adaptor protein complex, or BBSome. To identify new cilium-associated genes, we employed the nematode C. elegans, where ciliogenesis occurs within a short timespan during late embryogenesis when most sensory neurons differentiate. Using whole-organism RNA-Seq libraries, we discovered a signature expression profile highly enriched for transcripts of known ciliary proteins, including FAM-161 (FAM161A orthologue), CCDC-104 (CCDC104), and RPI-1 (RP1/RP1L1), which we confirm are cilium-localised in worms. From a list of 185 candidate ciliary genes, we uncover orthologues of human MAP9, YAP, CCDC149, and RAB28 as conserved cilium-associated components. Further analyses of C. elegans RAB-28, recently associated with autosomal-recessive cone-rod dystrophy, reveal that this small GTPase is exclusively expressed in ciliated neurons where it dynamically associates with IFT trains. Whereas inactive GDP-bound RAB-28 displays no IFT movement and diffuse localisation, GTP-bound (activated) RAB-28 concentrates at the periciliary membrane in a BBSome-dependent manner and undergoes bidirectional IFT. Functional analyses reveal that whilst cilium structure, sensory function and IFT are seemingly normal in a rab-28 null allele, overexpression of predicted GDP or GTP locked variants of RAB-28 perturbs cilium and sensory pore morphogenesis and function. Collectively, our findings present a new approach for identifying ciliary proteins, and unveil RAB28, a GTPase most closely related to the BBS protein RABL4/IFT27, as an IFT-associated cargo with BBSome-dependent cell autonomous and non-autonomous functions at the ciliary base.

Chronically sun-damaged melanomas express low levels of nuclear glutathione-S-transferase-π: an epidemiological and clinicopathological study in Italy.

PubMed

Campione, Elena; Medda, Emanuela; Paternò, Evelin J; Diluvio, Laura; Ricozzi, Ilaria; Carboni, Isabella; Costanza, Gaetana; Rossi, Piero; Rapanotti, Cristina; Di Stefani, Alessandro; Chimenti, Sergio; Bianchi, Luca; Orlandi, Augusto

2015-01-01

The detoxifying enzyme glutathione-s-transferase pi (GST-π) is present in keratinocytes and melanocytes and exerts a protective role against tumour progression. Melanomas close to melanocytic naevus remnants occur less frequently on sun-exposed areas, whereas solar dermal elastosis, hallmark of chronic sun-damage, characterise melanomas on sun-exposed skin. We evaluated the expression of GST-π in 113 melanomas associated to melanocytic naevus remnants or to solar dermal elastosis, classified according to clinical characteristics, history of sun exposure, histological subtypes and AJCC staging. Chronically sun-damaged melanomas, identified by moderate-severe solar dermal elastosis, showed a lower nuclear GST-π expression and a higher thickness than those related to melanocytic naevus remnants (p < 0.03). Multivariate logistic regression analysis demonstrated that male gender and chronic sun-exposure are independent risk factors significantly associated to melanomas localised on the trunk (OR = 3.36, 95% CI: 1.31-8.65; OR = 5.97, 95% CI: 1.71-20.87). If confirmed on a larger series, lower expression of nuclear GST-π in melanoma cells could represent a possible marker of chronically sun-damaged melanoma pathogenesis.

The pathophysiological role of PEDF in bone diseases.

PubMed

Broadhead, M L; Akiyama, T; Choong, P F M; Dass, C R

2010-04-01

First discovered in 1991 as a factor secreted by retinal pigment epithelial cells, the potency of pigment epithelium derived factor (PEDF) as an anti-angiogenic has led to examination of its role in active bone growth, repair and remodelling. In the musculoskeletal system, PEDF expression occurs particularly at sites of active bone formation. Expression has been noted in osteoblasts and to a lesser degree osteoclasts, the major classes of bone cells. In fact, PEDF is capable of inducing differentiation of precursor cells into mature osteoblasts. Expression and localisation are closely linked with that of vascular endothelial growth factor (VEGF). Studies at the epiphyseal plate have revealed that PEDF expression plays a key role in endochondral ossification, and beyond this may account for the epiphyseal plate's innate ability to resist neoplastic cell invasion. Collagen-1, the major protein in bone, is avidly bound by PEDF, implicating an important role played by this protein on PEDF function, possibly through MMP-2 and -9 activity. Surprisingly, the role of PEDF has not been evaluated more widely in bone disorders, so the challenge ahead lies in a more diverse evaluation of PEDF in various osteologic pathologies including osteoarthritis and fracture healing.

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma

PubMed Central

Stronach, Euan A.; Cunnea, Paula; Turner, Christina; Guney, Tankut; Aiyappa, Radhika; Jeyapalan, Senthuran; de Sousa, Camila H.; Browne, Alacoque; Magdy, Nesreen; Studd, James B.; Sriraksa, Ruethairat; Gabra, Hani; El-Bahrawy, Mona

2015-01-01

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease. PMID:26267317

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

PubMed

Stronach, Euan A; Cunnea, Paula; Turner, Christina; Guney, Tankut; Aiyappa, Radhika; Jeyapalan, Senthuran; de Sousa, Camila H; Browne, Alacoque; Magdy, Nesreen; Studd, James B; Sriraksa, Ruethairat; Gabra, Hani; El-Bahrawy, Mona

2015-10-13

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

Inhibitory effects of OK-432 (Picibanil) on cellular proliferation and adhesive capacity of breast carcinoma cells.

PubMed

Horii, Yoshio; Iino, Yuichi; Maemura, Michio; Horiguchi, Jun; Morishita, Yasuo

2005-02-01

We investigated the potent inhibitory effects of OK-432 (Picibanil) on both cellular adhesion and cell proliferation of estrogen-dependent (MCF-7) or estrogen-independent (MDA-MB-231) breast carcinoma cells. Cellular proliferation of both MCF-7 and MDA-MB-231 cells was markedly inhibited in a dose-dependent manner, when the carcinoma cells were exposed to OK-432. Cell attachment assay demonstrated that incubation with OK-432 for 24 h reduced integrin-mediated cellular adhesion of both cell types. However, fluorescence activated cell sorter (FACS) analysis revealed that incubation with OK-432 for 24 h did not decrease the cell surface expressions of any integrins. These results suggest that the binding avidity of integrins is reduced by OK-432 without alteration of the integrin expression. We conclude that OK-432 inhibits integrin-mediated cellular adhesion as well as cell proliferation of breast carcinoma cells regardless of estrogen-dependence, and that these actions of OK-432 contribute to prevention or inhibition of breast carcinoma invasion and metastasis.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung.

PubMed

Groneberg, D A; Eynott, P R; Döring, F; Dinh, Q Thai; Oates, T; Barnes, P J; Chung, K F; Daniel, H; Fischer, A

2002-01-01

Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the beta-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative D-Ala-L-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung

PubMed Central

Groneberg, D; Eynott, P; Doring, F; Thai, D; Oates, T; Barnes, P; Chung, K; Daniel, H; Fischer, A

2002-01-01

Background: Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Methods: Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the ß-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. Results: PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative D-Ala-L-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. Conclusions: These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics. PMID:11809991

FOXA1 and SOX9 Expression in the Developing Urogenital Sinus of the Tammar Wallaby (Macropus eugenii).

PubMed

Gamat, Melissa; Chew, Keng Yih; Shaw, Geoffrey; Renfree, Marilyn B

2015-01-01

The mammalian prostate is a compact structure in humans but multi-lobed in mice. In humans and mice, FOXA1 and SOX9 play pivotal roles in prostate morphogenesis, but few other species have been examined. We examined FOXA1 and SOX9 in the marsupial tammar wallaby, Macropus eugenii, which has a segmented prostate more similar to human than to mouse. In males, prostatic budding in the urogenital epithelium (UGE) was initiated by day 24 postpartum (pp), but in the female the UGE remained smooth and had begun forming the marsupial vaginal structures. FOXA1 was upregulated in the male urogenital sinus (UGS) by day 51 pp, whilst in the female UGS FOXA1 remained basal. FOXA1 was localised in the UGE in both sexes between day 20 and 80 pp. SOX9 was upregulated in the male UGS at day 21-30 pp and remained high until day 51-60 pp. SOX9 protein was localised in the distal tips of prostatic buds which were highly proliferative. The persistent upregulation of the transcription factors SOX9 and FOXA1 after the initial peak and fall of androgen levels suggest that in the tammar, as in other mammals, these factors are required to sustain prostate differentiation, development and proliferation as androgen levels return to basal levels. © 2015 S. Karger AG, Basel.

Yellow Fever Virus Modulates the Expression of Key Proteins Related to the microRNA Pathway in the Human Hepatocarcinoma Cell Line HepG2.

PubMed

Holanda, Gustavo Moraes; Casseb, Samir Mansour Moraes; Mello, Karla Fabiane Lopes; Vasconcelos, Pedro Fernando Costa; Cruz, Ana Cecília Ribeiro

2017-06-01

Yellow fever is a zoonotic disease caused by the yellow fever virus (YFV) and transmitted by mosquitoes of the family Culicidae. It is well known that cellular and viral microRNAs (miRNAs) are involved in modulation of viral and cellular gene expression, as well as immune response, and are considered by the scientific community as possible targets for an effective therapy against viral infections. This regulation may be involved in different levels of infection and clinical symptomatology. We used viral titration techniques, viral kinetics from 24 to 96 hours postinfection (hpi), and analyzed the expression of key proteins related to the miRNA pathway by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of Dicer was different when compared over the course of infection by the distinct YFV genotypes. Drosha expression was similar during infection by YFV genotype 1 or 2, with a decrease in their expression over time and a slight increase in 96 hpi. Ago1, Ago2, and Ago4 showed different levels of expression between the viral genotypes: for YFV genotype 1 infection, Ago1 presented a positive expression, while for YFV genotype 2, it showed a negative expression, when compared with negative controls. We conclude that YFV infection modulates the proteins involved in miRNA biogenesis, which can regulate both viral replication and cellular immune response.

Small RNA Profiling of Influenza A Virus-Infected Cells Identifies miR-449b as a Regulator of Histone Deacetylase 1 and Interferon Beta

PubMed Central

Buggele, William A.; Krause, Katherine E.; Horvath, Curt M.

2013-01-01

The mammalian antiviral response relies on the alteration of cellular gene expression, to induce the production of antiviral effectors and regulate their activities. Recent research has indicated that virus infections can induce the accumulation of cellular microRNA (miRNA) species that influence the stability of host mRNAs and their protein products. To determine the potential for miRNA regulation of cellular responses to influenza A virus infection, small RNA profiling was carried out using next generation sequencing. Comparison of miRNA expression profiles in uninfected human A549 cells to cells infected with influenza A virus strains A/Udorn/72 and A/WSN/33, revealed virus-induced changes in miRNA abundance. Gene expression analysis identified mRNA targets for a cohort of highly inducible miRNAs linked to diverse cellular functions. Experiments demonstrate that the histone deacetylase, HDAC1, can be regulated by influenza-inducible miR-449b, resulting in altered mRNA and protein levels. Expression of miR-449b enhances virus and poly(I:C) activation of the IFNβ promoter, a process known to be negatively regulated by HDAC1. These findings demonstrate miRNA induction by influenza A virus infection and elucidate an example of miRNA control of antiviral gene expression in human cells, defining a role for miR-449b in regulation of HDAC1 and antiviral cytokine signaling. PMID:24086750

A meta-analysis to evaluate the cellular processes regulated by the interactome of endogenous and over-expressed estrogen receptor alpha.

PubMed

Simões, Joana; Amado, Francisco M; Vitorino, Rui; Helguero, Luisa A

2015-01-01

The nature of the proteins complexes that regulate ERα subcellular localization and activity is still an open question in breast cancer biology. Identification of such complexes will help understand development of endocrine resistance in ER+ breast cancer. Mass spectrometry (MS) has allowed comprehensive analysis of the ERα interactome. We have compared six published works analyzing the ERα interactome of MCF-7 and HeLa cells in order to identify a shared or different pathway-related fingerprint. Overall, 806 ERα interacting proteins were identified. The cellular processes were differentially represented according to the ERα purification methodology, indicating that the methodologies used are complementary. While in MCF-7 cells, the interactome of endogenous and over-expressed ERα essentially represents the same biological processes and cellular components, the proteins identified were not over-lapping; thus, suggesting that the biological response may differ as the regulatory/participating proteins in these complexes are different. Interestingly, biological processes uniquely associated to ERα over-expressed in HeLa cell line included L-serine biosynthetic process, cellular amino acid biosynthetic process and cell redox homeostasis. In summary, all the approaches analyzed in this meta-analysis are valid and complementary; in particular, for those cases where the processes occur at low frequency with normal ERα levels, and can be identified when the receptor is over-expressed. However special effort should be put into validating these findings in cells expressing physiological ERα levels.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

PubMed Central

Warenius, H M; Jones, M; Gorman, T; McLeish, R; Seabra, L; Barraclough, R; Rudland, P

2000-01-01

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign PMID:10993658

The atomic structure of polar and non-polar InGaN quantum wells and the green gap problem.

PubMed

Humphreys, C J; Griffiths, J T; Tang, F; Oehler, F; Findlay, S D; Zheng, C; Etheridge, J; Martin, T L; Bagot, P A J; Moody, M P; Sutherland, D; Dawson, P; Schulz, S; Zhang, S; Fu, W Y; Zhu, T; Kappers, M J; Oliver, R A

2017-05-01

We have used high resolution transmission electron microscopy (HRTEM), aberration-corrected quantitative scanning transmission electron microscopy (Q-STEM), atom probe tomography (APT) and X-ray diffraction (XRD) to study the atomic structure of (0001) polar and (11-20) non-polar InGaN quantum wells (QWs). This paper provides an overview of the results. Polar (0001) InGaN in QWs is a random alloy, with In replacing Ga randomly. The InGaN QWs have atomic height interface steps, resulting in QW width fluctuations. The electrons are localised at the top QW interface by the built-in electric field and the well-width fluctuations, with a localisation energy of typically 20meV. The holes are localised near the bottom QW interface, by indium fluctuations in the random alloy, with a localisation energy of typically 60meV. On the other hand, the non-polar (11-20) InGaN QWs contain nanometre-scale indium-rich clusters which we suggest localise the carriers and produce longer wavelength (lower energy) emission than from random alloy non-polar InGaN QWs of the same average composition. The reason for the indium-rich clusters in non-polar (11-20) InGaN QWs is not yet clear, but may be connected to the lower QW growth temperature for the (11-20) InGaN QWs compared to the (0001) polar InGaN QWs. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Analysis of Mitochondrial haemoglobin in Parkinson's disease brain.

PubMed

Shephard, Freya; Greville-Heygate, Oliver; Liddell, Susan; Emes, Richard; Chakrabarti, Lisa

2016-07-01

Mitochondrial dysfunction is an early feature of neurodegeneration. We have shown there are mitochondrial haemoglobin changes with age and neurodegeneration. We hypothesised that altered physiological processes are associated with recruitment and localisation of haemoglobin to these organelles. To confirm a dynamic localisation of haemoglobin we exposed Drosophila melanogaster to cyclical hypoxia with recovery. With a single cycle of hypoxia and recovery we found a relative accumulation of haemoglobin in the mitochondria compared with the cytosol. An additional cycle of hypoxia and recovery led to a significant increase of mitochondrial haemoglobin (p<0.05). We quantified ratios of human mitochondrial haemoglobin in 30 Parkinson's and matched control human post-mortem brains. Relative mitochondrial/cytosolic quantities of haemoglobin were obtained for the cortical region, substantia nigra and cerebellum. In age matched post-mortem brain mitochondrial haemoglobin ratios change, decreasing with disease duration in female cerebellum samples (n=7). The change is less discernible in male cerebellum (n=18). In cerebellar mitochondria, haemoglobin localisation in males with long disease duration shifts from the intermembrane space to the outer membrane of the organelle. These new data illustrate dynamic localisation of mitochondrial haemoglobin within the cell. Mitochondrial haemoglobin should be considered in the context of gender differences characterised in Parkinson's disease. It has been postulated that cerebellar circuitry may be activated to play a protective role in individuals with Parkinson's. The changing localisation of intracellular haemoglobin in response to hypoxia presents a novel pathway to delineate the role of the cerebellum in Parkinson's disease. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

The cost-effectiveness of active surveillance compared to watchful waiting and radical prostatectomy for low risk localised prostate cancer.

PubMed

Lao, Chunhuan; Edlin, Richard; Rouse, Paul; Brown, Charis; Holmes, Michael; Gilling, Peter; Lawrenson, Ross

2017-08-08

Radical prostatectomy is the most common treatment for localised prostate cancer in New Zealand. Active surveillance was introduced to prevent overtreatment and reduce costs while preserving the option of radical prostatectomy. This study aims to evaluate the cost-effectiveness of active surveillance compared to watchful waiting and radical prostatectomy. Markov models were constructed to estimate the life-time cost-effectiveness of active surveillance compared to watchful waiting and radical prostatectomy for low risk localised prostate cancer patients aged 45-70 years, using national datasets in New Zealand and published studies including the SPCG-4 study. This study was from the perspective of the Ministry of Health in New Zealand. Radical prostatectomy is less costly than active surveillance in men aged 45-55 years with low risk localised prostate cancer, but more costly for men aged 60-70 years. Scenario analyses demonstrated significant uncertainty as to the most cost-effective option in all age groups because of the unavailability of good quality of life data for men under active surveillance. Uncertainties around the likelihood of having radical prostatectomy when managed with active surveillance also affect the cost-effectiveness of active surveillance against radical prostatectomy. Active surveillance is less likely to be cost-effective compared to radical prostatectomy for younger men diagnosed with low risk localised prostate cancer. The cost-effectiveness of active surveillance compared to radical prostatectomy is critically dependent on the 'trigger' for radical prostatectomy and the quality of life in men on active surveillance. Research on the latter would be beneficial.

MHC II-β chain gene expression studies define the regional organization of the thymus in the developing bony fish Dicentrarchus labrax (L.).

PubMed

Picchietti, S; Abelli, L; Guerra, L; Randelli, E; Proietti Serafini, F; Belardinelli, M C; Buonocore, F; Bernini, C; Fausto, A M; Scapigliati, G

2015-02-01

MHC II-β chain gene transcripts were quantified by real-time PCR and localised by in situ hybridization in the developing thymus of the teleost Dicentrarchus labrax, regarding the specialization of the thymic compartments. MHC II-β expression significantly rose when the first lymphoid colonization of the thymus occurred, thereafter increased further when the organ progressively developed cortex and medulla regions. The evolving patterns of MHC II-β expression provided anatomical insights into some mechanisms of thymocyte selection. Among the stromal cells transcribing MHC II-β, scattered cortical epithelial cells appeared likely involved in the positive selection, while those abundant in the cortico-medullary border and medulla in the negative selection. These latter most represent dendritic cells, based on typical localization and phenotype. These findings provide further proofs that efficient mechanisms leading to maturation of naïve T cells are operative in teleosts, strongly reminiscent of the models conserved in more evolved gnathostomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

The expression of ADAM12 (meltrin alpha) in human giant cell tumours of bone.

PubMed

Tian, B L; Wen, J M; Zhang, M; Xie, D; Xu, R B; Luo, C J

2002-12-01

To examine the expression of ADAM12 (meltrin alpha), a member of the disintegrin and metalloprotease (ADAM) family, in human giant cell tumours of the bone, skeletal muscle tissue from human embryos, and human adult skeletal muscle tissue. ADAM12 mRNA was detected by reverse transcription polymerase chain reaction and in situ hybridisation. ADAM12 mRNA was detected in 14 of the 20 giant cell tumours of bone and in three of the six tumour cell cultures. The expression of ADAM12 in cells cultured from the tumour was linked to the presence of multinucleated giant cells. ADAM12 mRNA could not be detected in the five adult skeletal muscle tissue samples, although it was found in the two embryonic skeletal muscle tissue samples. ADAM12 mRNA was localised to the cytoplasm of multinucleated giant cells and some mononuclear stromal cells. These results indicate that multinucleated giant cells are formed by the cell fusion of mononuclear stromal cells in giant cell tumours of bone and that ADAM12 is involved in the cell fusion process.

Effects of pulpotomy using mineral trioxide aggregate on prostaglandin transporter and receptors in rat molars.

PubMed

Ohkura, Naoto; Edanami, Naoki; Takeuchi, Ryosuke; Tohma, Aiko; Ohkura, Mariko; Yoshiba, Nagako; Yoshiba, Kunihiko; Ida-Yonemochi, Hiroko; Ohshima, Hayato; Okiji, Takashi; Noiri, Yuichiro

2017-07-31

Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E 2 (PGE 2 ) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE 2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

Exploring the Function of Online Narratives to Develop Critical Thinking and Localisation of Knowledge in an International Science Program

ERIC Educational Resources Information Center

Hicks, Marianne; Tham, Melissa; Brookes, Rowan

2017-01-01

e-learning practitioners have long recognised the benefits of using online training to achieve knowledge transfer, less is understood about facilitating the sharing of values, attitudes, critical thinking, and localisation using online platforms. In this article an online learning platform in the context of an international scientific program was…

Nonlinear Light Dynamics in Multi-Core Structures

DTIC Science & Technology

2017-02-27

be generated in continuous- discrete optical media such as multi-core optical fiber or waveguide arrays; localisation dynamics in a continuous... discrete nonlinear system. Detailed theoretical analysis is presented of the existence and stability of the discrete -continuous light bullets using a very...and pulse compression using wave collapse (self-focusing) energy localisation dynamics in a continuous- discrete nonlinear system, as implemented in a