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Ubiquitously
Terms modified by Ubiquitously Selected AbstractsDelayed embryonic development and impaired cell growth and survival in Actg1 null mice,CYTOSKELETON, Issue 9 2010Tina M. Bunnell Abstract Actins are among the most highly expressed proteins in eukaryotes and play a central role in nearly all aspects of cell biology. While the intricate process of development undoubtedly requires a properly regulated actin cytoskeleton, little is known about the contributions of different actin isoforms during embryogenesis. Of the six actin isoforms, only the two cytoplasmic actins, ,cyto - and ,cyto -actin, are ubiquitously expressed. We found that ,cyto -actin null (Actg1,/,) mice were fully viable during embryonic development, but most died within 48 h of birth due to respiratory failure and cannibalization by the parents. While no morphogenetic defects were identified, Actg1,/, mice exhibited stunted growth during embryonic and postnatal development as well as delayed cardiac outflow tract formation that resolved by birth. Using primary mouse embryonic fibroblasts, we confirm that ,cyto -actin is not required for cell migration. The Actg1,/, cells, however, exhibited growth impairment and reduced cell viability, defects which perhaps contribute to the stunted growth and developmental delays observed in Actg1,/, embryos. Since the total amount of actin protein was maintained in Actg1,/, cells, our data suggests a distinct requirement for ,cyto -actin in cell growth and survival. © 2010 Wiley-Liss, Inc. [source] Profilin-1 overexpression restores adherens junctions in MDA-MB-231 breast cancer cells in R-cadherin-dependent mannerCYTOSKELETON, Issue 12 2009Li Zou Abstract Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, is downregulated in several different types of adenocarcinoma and elicits tumor-suppressive effect on breast cancer cell lines. MDA-MB-231 (MDA-231), a breast cancer cell line that displays all the characteristics of post-epithelial-to-mesenchymal transition and does not form cell,cell adhesion, can be reverted to an epithelioid phenotype by Pfn1 overexpression. This morphological transition is caused by restoration of adherens junctions (AJ) requiring Pfn1's interaction with actin. Pfn1 overexpression increases the expression level of R-cadherin (a type of cadherin that is endogenously expressed in the parental cell line) and restores AJ in MDA-231 cells in R-cadherin-dependent manner. These findings highlight important role of Pfn1 in the regulation of epithelial cell,cell adhesion. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Rab6 family proteins interact with the dynein light chain protein DYNLRB1CYTOSKELETON, Issue 3 2008Bas Wanschers Abstract The small GTPase Rab6 is a key regulator in the retrograde transfer from endosomes via the Golgi to the ER. Three isoforms of Rab6 have been identified, the ubiquitously expressed Rab6A and Rab6A,, and the brain specific Rab6B. Recent studies have shown that Rab6A, is the major isoform regulating this retrograde transport. Cytoplasmic dynein is the main motor protein complex for this transport. Dynein consists of two heavy chains, two intermediate chains, four light intermediatechains and several light chains, called roadblock/LC7 proteins or DYNLRB proteins. In mammalian cells two light chain isoforms have been identified, DYNLRB1 and DYNLRB2. We here show with yeast-two-hybrid, co-immunoprecipitation and pull down studies that DYNLRB1 specifically interacts with all three Rab6 isoforms and co-localises at the Golgi. This is the first example of a direct interaction between Rab6 isoforms and the dynein complex. Pull down experiments showed further preferred association of DYNLRB1 with GTP-bound Rab6A and interestingly GDP-bound Rab6A, and Rab6B. In addition DYNLRB1 was found in the Golgi apparatus where it co-localises with EYFP-Rab6 isoforms. DYNLRB is a putative modulator of the intrinsic GTPase activity of GTP-binding proteins. In vitro we were not able to reproduce this effect on Rab6 GTPase activity. Cell Motil. Cytoskeleton 2008. © 2007 Wiley-Liss, Inc. [source] Caveolin-1 polarization in migrating endothelial cells is directed by substrate topology not chemoattractant gradientCYTOSKELETON, Issue 11 2006Virginie Santilman Abstract Polarization is a hallmark of migrating cells, and an asymmetric distribution of proteins is essential to the migration process. Caveolin-1 is highly polarized in migrating endothelial cells (EC). Several studies have shown caveolin-1 accumulation in the front of migrating EC while others report its accumulation in the EC rear. In this paper we address these conflicting results on polarized localization of caveolin-1. We find evidence for the hypothesis that different modes of locomotion lead to differences in protein polarization. In particular, we show that caveolin-1 is primarily localized in the rear of cells migrating on a planar substrate, but in the front of cells traversing a three-dimensional pore. We also show that a chemoattractant, present either as a gradient or ubiquitously in the medium, does not alter caveolin-1 localization in cells in either mode of locomotion. Thus we conclude that substrate topology, and not the presence of a chemoattractant, directs the polarization of caveolin-1 in motile ECs. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory systemDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2010Jun Takatoh Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure-specific monoclonal antibodies (HepSS-1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS-1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin-A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events. [source] Molecular cloning and sequence analysis of an ascidian egg ,-N-acetylhexosaminidase with a potential role in fertilizationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2003Ryo Koyanagi ,-N-Acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm,egg coat binding. In ascidians and mammals, ,-hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of ,-hexosaminidase. In the present study, P. mammillata,-hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known ,-hexosaminidases; however, P. mammillata,-hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata,-hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of ,-hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg ,-hexosaminidase forms are specific for the oocyte, test cells and follicle cells. [source] Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vesselsDEVELOPMENTAL DYNAMICS, Issue 7 2008Emma J. Gordon Abstract Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels. Developmental Dynamics 237:1901,1909, 2008. © 2008 Wiley-Liss, Inc. [source] Differential expression of Na,K-ATPase , and , subunit genes in the developing zebrafish inner earDEVELOPMENTAL DYNAMICS, Issue 3 2003Brian Blasiole Abstract We have used whole-mount in situ hybridization to analyze Na,K-ATPase , and , subunit gene expression in the developing zebrafish ear. Four ,1-like (,1a.1, ,1a.2, ,1a.4, and ,1a.5) and two , (,1a and ,2b) subunit genes are expressed in ear beginning at mid-somitogenesis. Each gene exhibits a distinct spatial and temporal expression pattern. The ,1a.1 gene was ubiquitously expressed in the otic epithelium from mid-somitogenesis to 24 hr postfertilization (hpf). Expression of this gene was gradually reduced and by 48 hpf, ,1a.1 transcripts were no longer detectable in the ear. The ,1a.2 and ,1a.5 genes were expressed in regions that correspond to the anterior macula, lateral crista, and semicircular canal projections up to 48 hpf. At later stages, expression of these genes was limited to cells in the dorsolateral septum and semicircular canal projections. ,1a.4 and ,1a transcripts were ubiquitously expressed during ear development and were present in most otic tissues at 5 days postfertilization (dpf). Expression of the ,2b gene, on the other hand, was restricted to subsets of cells that form sensory epithelia. These results strongly suggest different functional roles for individual Na,K-ATPase genes in zebrafish ear development. Na,K-ATPase genes are likely to represent useful markers for the analysis of zebrafish otogenesis. Development Dynamics, 2003. © 2003 Wiley-Liss, Inc. [source] Coordinated and conserved expression of alphoid repeat and alphoid repeat-tagged coding sequencesDEVELOPMENTAL DYNAMICS, Issue 1 2003Yin-Xiong Li Abstract We have found an alpha-like simple-sequence DNA repeat that is differentially expressed during early embryogenesis in both chick and zebrafish. Before and during the primitive streak stage, transcripts of the alphoid repeat sequence were ubiquitously expressed throughout zebrafish and chick embryos. After headfold formation, expression was limited to the cardiac neural crest, the head, and the heart. Two types of alphoid repeat sequence transcripts were identified: alphoid repeat RNA and alphoid repeat-tagged mRNA (ES,T). Several of the ES,Ts were identified by (1) searching expressed sequence tag databases, (2) arbitrary rapid amplification of cDNA ends (RACE), and (3) screening embryonic cDNA libraries. The alphoid element was located in the 3, untranslated region of one ES,T that was obtained by RACE. The ES,T sequences encoded a variety of different types of proteins, but all were expressed within tissues that were positive for the alphoid repeat RNA. The presence of two types of coordinately expressed alphoid-like repeat transcripts in maternal RNA with subsequent restriction to the head and heart, and the conservation of these features in disparate vertebrate embryos, suggest that the alphoid repeat sequence may serve as a control element in the gene regulation network. Developmental Dynamics 228:72,81, 2003. © 2003 Wiley-Liss, Inc. [source] In humans the adiponectin receptor R2 is expressed predominantly in adipose tissue and linked to the adipose tissue expression of MMIF-1DIABETES OBESITY & METABOLISM, Issue 4 2010K. Kos In this study, the regional adipose tissue-adiponectin (AT-ADN) and adiponectin receptor (R1 and R2) expression and their relation with metabolic parameters, circulating and AT-derived cytokine expressions were compared. Paired subcutaneous adipose tissue (SCAT) and visceral adipose tissue (VAT) were taken from 18 lean and 39 obese humans, AT-mRNA expression of adipokines analysed by RT-PCR and corresponding serum levels by enzyme-linked immunosorbent assay (ELISA). R1 and R2 adipocyte expression was compared with 17 other human tissues. ADN-gene expression was lower in VAT than SCAT [mean (SD) 1.54 (1.1) vs. 2.84 (0.87); p < 0.001], and lower in obese subjects (VAT : p = 0.01;SCAT : p < 0.001). SCAT-ADN correlated positively with serum ADN (r = 0.33;p = 0.036) but not VAT-ADN. AT expressions of ADN and macrophage migration inhibiting factor (MMIF), IL18 and cluster of differentiation factor 14 (CD14) in both depots showed inverse correlations. R1 and R2 were expressed ubiquitously and R2 highest in SCAT, and this is much higher (×100) than R1 (×100). R expression was similar in lean and obese subjects and unrelated to the metabolic syndrome, however, receptors correlated with VAT-MMIF (R 1: r = 0.4;p = 0.008;R 2: r = 0.35,p = 0.02) and SCAT-MMIF expression (R 2: r = 0.43;p = 0.004). Unlike ADN, its receptors are expressed in many human tissues. Human R2 expression is not highest in the liver but in AT where it is associated with MMIF expression. The adiponectin-dependent insulin-sensitizing action of thiazolidinediones is thus probably to differ amongst species with weaker effects on the human liver. [source] Sphingosine-1-phosphate and FTY720 as anti-atherosclerotic lipid compoundsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2007M. Tölle Abstract All stages of atherosclerosis have been identified as a chronic vascular inflammatory disease. In the last few years there is increasing evidence that endogenous lysophospholipids such as sphingosine-1-phosphate (S1P) have potent anti-inflammatory properties. The S1P analogue FTY720 that has been developed as a potent, orally active, immunosuppressant in the field of transplantation and autoimmune disease has interesting effects on inflammatory processes in the arterial vessel wall. S1P targets five specific S1P receptors (S1P1,5), which are ubiquitously expressed. S1P1,3 receptor expression is identified in arterial vessels. S1P and FTY720 show potent silencing effects on some vascular proinflammatory mechanisms in endothelial and vascular smooth muscle cells. In addition, the interaction of monocytes with the vessel wall is inhibited. As shown recently, FTY720 can effectively reduce the progression of atherosclerosis in apolipoprotein E-deficient mice having a high-cholesterol diet. It is not entirely clear which S1P receptor subtype is mainly involved in this process. However, it is currently speculated that the S1P3 and probably the S1P1 is involved in the anti-atherosclerotic effects of FTY720. This review summarizes the current knowledge about S1P- and FTY720-effects on mechanisms of vascular inflammatory disease. In addition S1P receptor subtypes are identified which might be interesting for molecular drug targeting. [source] PU.1 is required for transcriptional activation of the Stat6 response element in the Ig, promoterEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2003Marko Pesu Abstract Signal transducer and activator of transcription 6 (Stat6) has a crucial role in regulation of IL-4-induced gene responses. Stat6-binding sites are present in the promoters of both ubiquitously and cell-type-specifically expressed genes. The promoter regions of IL-4-inducible genes contain cis -acting elements for several transcription factors that act in concert with Stat6 and are also likely to modulate lineage-specific gene expression. We have observed that the Stat6 response element from the B-cell-specific Ig, promoter is readily activated upon IL-4 stimulation in B cells but not in non-hematopoietic cells. A minimal low-affinity PU.1-core-binding sequence (5,-AGAA-3,) was identified within the Stat6 DNA-binding site in the Ig, promoter. Ectopic expression of the myeloid- and B-cell-specific transcription factor PU.1 restored the IL-4-inducibility of the Ig,-Stat6 response element in HepG2 cells, and the induction required an intact PU.1-binding sequence. Both the transactivation and the DNA-binding domains of PU.1 were required for induction of Stat6-mediated transcription. The co-operation between PU.1 and Stat6 in transactivation of the Ig, gene represents a molecular mechanism for the fine-tuning of cell-type-restricted expression of IL-4-induced gene responses. [source] Codon 129 polymorphism and the E200K mutation do not affect the cellular prion protein isoform composition in the cerebrospinal fluid from patients with Creutzfeldt,Jakob diseaseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Matthias Schmitz Abstract The cellular prion protein (PrPc) is a multifunctional, highly conserved and ubiquitously expressed protein. It undergoes a number of modifications during its post-translational processing, resulting in different PrPc glycoforms and truncated PrPc fragments. Limited data are available in humans on the expression and cleavage of PrPc. In this study we investigated the PrPc isoform composition in the cerebrospinal fluid from patients with different human prion diseases. The first group of patients was affected by sporadic Creutzfeldt,Jakob disease exhibiting different PrP codon 129 genotypes. The second group contained patients with a genetic form of Creutzfeldt,Jakob disease (E200K). The third group consisted of patients with fatal familial insomnia and the last group comprised cases with the Gerstmann,Sträussler,Scheinker syndrome. We examined whether the PrP codon 129 polymorphism in sporadic Creutzfeldt,Jakob disease as well as the type of prion disease in human patients has an impact on the glycosylation and processing of PrPc. Immunoblotting analyses using different monoclonal PrPc antibodies directed against various epitopes of PrPc revealed, for all examined groups of patients, a consistent predominance of the glycosylated PrPc isoforms as compared with the unglycosylated form. In addition, the antibody SAF70 recognized a variety of PrPc fragments with sizes of 21, 18, 13 and 12 kDa. Our findings indicate that the polymorphisms at PrP codon 129, the E200K mutation at codon 200 or the examined types of human transmissible spongiform encephalopathies do not exert a measurable effect on the glycosylation and processing of PrPc in human prion diseases. [source] Requirement of the tumour suppressor APC for the clustering of PSD-95 and AMPA receptors in hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Atsushi Shimomura Abstract Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC,PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors; however, the clustering of an N -methyl- d -aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses. [source] Multi-directional differentiation of doublecortin- and NG2-immunopositive progenitor cells in the adult rat neocortex in vivoEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Yasuhisa Tamura Abstract In the adult mammalian brain, multipotent stem or progenitor cells involved in reproduction of neurons and glial cells have been well investigated only in very restricted regions; the subventricular zone of the lateral ventricle and the dentate gyrus in the hippocampal formation. In the neocortex, a series of in vitro studies has suggested the possible existence of neural progenitor cells possessing neurogenic and/or gliogenic potential in adult mammals. However, the cellular properties of the cortical progenitor cells in vivo have not been fully elucidated. Using 5,-bromodeoxyuridine labeling and immunohistochemical analysis of cell differentiation markers, we found that a subpopulation of NG2-immunopositive cells co-expressing doublecortin (DCX), an immature neuron marker, ubiquitously reside in the adult rat neocortex. Furthermore, these cells are the major population of proliferating cells in the region. The DCX(+)/NG2(+) cells reproduced the same daughter cells, or differentiated into DCX(+)/NG2(,) (approximately 1%) or DCX(,)/NG2(+) (approximately 10%) cells within 2 weeks after cell division. The DCX(+)/NG2(,) cells were also immunopositive for TUC-4, a neuronal linage marker, suggesting that these cells were committed to neuronal cell differentiation, whereas the DCX(,)/NG2(+) cells showed faint immunoreactivity for glutathione S-transferase (GST)-pi, an oligodendrocyte lineage marker, in the cytoplasm, suggesting glial cell lineage, and thereafter the cells differentiated into NG2(,)/GST-pi(+) mature oligodendrocytes after a further 2 weeks. These findings indicate that DCX(+)/NG2(+) cells ubiquitously exist as ,multipotent progenitor cells' in the neocortex of adult rats. [source] From meiosis to postmeiotic events: Uncovering the molecular roles of the meiosis-specific recombinase Dmc1FEBS JOURNAL, Issue 3 2010Wataru Kagawa In meiosis, the accurate segregation of maternal and paternal chromosomes is accomplished by homologous recombination. A central player in meiotic recombination is the Dmc1 recombinase, a member of the RecA/Rad51 recombinase superfamily, which is widely conserved from viruses to humans. Dmc1 is a meiosis-specific protein that functions with the ubiquitously expressed homolog, the Rad51 recombinase, which is essential for both mitotic and meiotic recombination. Since its discovery, it has been speculated that Dmc1 is important for unique aspects of meiotic recombination. Understanding the distinctive properties of Dmc1, namely, the features that distinguish it from Rad51, will further clarify the mechanisms of meiotic recombination. Recent structural, biochemical, and genetic findings are now revealing the molecular mechanisms of Dmc1-mediated homologous recombination and its regulation by various recombination mediators. [source] UXT interacts with the transcriptional repressor protein EVI1 and suppresses cell transformationFEBS JOURNAL, Issue 15 2007Roger McGilvray The EVI1 transcriptional repressor is critical to the normal development of a variety of tissues and participates in the progression of acute myeloid leukaemias. The repressor domain (Rp) was used to screen an adult human kidney yeast two-hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated. Enforced expression of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity. The Rp-binding site for UXT was determined and non-UXT-binding Evi1 mutants (Evi1,706,707) were developed which retain the ability to bind the corepressor mCtBP2. Evi1,706,707 transforms Rat1 fibroblasts, showing that the interaction is not essential for Evi1-mediated cell transformation. However, Evi1,706,707 produces an increased proportion of large colonies relative to wild-type, showing that endogenous UXT has an inhibitory effect on Evi1 biological activity. Exogenous UXT still suppresses Evi1,706,707-mediated cell transformation, indicating that it inhibits cell proliferation and/or survival by both Evi1-dependent and Evi1-independent mechanisms. These observations are consistent with the growth-suppressive function attributed to UXT in human prostate cancer. Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1. [source] Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosiscFEBS JOURNAL, Issue 16 2005Melissa C. Miles Multiple ankyrin repeat motif-containing proteins play an important role in protein,protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s). [source] Identification of a 250 kDa putative microtubule-associated protein as bovine ferritinFEBS JOURNAL, Issue 3 2005Evidence for a ferritin, microtubule interaction We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072,1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances. [source] Enhanced expression of Mcm proteins in cancer cells derived from uterine cervixFEBS JOURNAL, Issue 6 2003Yukio Ishimi Minichromosome maintenance proteins (Mcm) 2,7 play essential roles in eukaryotic DNA replication. Several reports have indicated the usefulness of Mcm proteins as markers of cancer cells in histopathological diagnosis. However, their mode of expression and pathophysiological significance in cancer cells remain to be clarified. We compared the level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells and normal human fibroblast WI-38 cells. All the proteins examined were detected in HeLa and GM cells at 6,10 times the level found in WI-38 cells on average. This increase was observed both in total cellular proteins and in the chromatin-bound fraction. Consistently, Mcm2 mRNA was enriched in HeLa cells to approximately four times the level in WI-38 cells, and the synthesis of Mcm4, 6 and 7 proteins was accelerated in HeLa cells. Immunohistochemical studies of surgical materials from human uterine cervix showed that Mcm3 and 4 are ubiquitously expressed in cancer cells. Further, the positive rate and level of Mcm3 and 4 expression appeared to be higher in cancer cells than in normal proliferating cells of the uterine cervix and dysplastic cells, suggesting that they can be useful markers to distinguish these cells. [source] Physical characterization of plakophilin 1 reconstituted with and without zincFEBS JOURNAL, Issue 14 2000Ilse Hofmann Plakophilin 1 (PKP1) belongs to the arm -repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The , helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 ± 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20,w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 × 10,7 cm2·s,1. The molecular mass of 2.44 × 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation. [source] Cloning of the guanylate kinase homologues AGK-1 and AGK-2 from Arabidopsis thaliana and characterization of AGK-1FEBS JOURNAL, Issue 2 2000Vinod Kumar Guanylate kinase is an essential enzyme for nucleotide metabolism, phosphorylating GMP to GDP or dGMP to dGDP. The low molecular mass cytosolic forms of guanylate kinase are implicated primarily in the regulation of the supply of guanine nucleotides to cell signalling pathways. The high molecular mass and membrane-associated forms of guanylate kinase homologues, notably found in neuronal tissues, are assigned roles in cell junction organization and transmembrane regulation. Here, we describe the first plant guanylate kinase-encoding genes, AGK1 and AGK2, from Arabidopsis thaliana. The nucleotide sequences of their genomic and cDNA clones predict proteins that carry N-terminal and C-terminal extensions of the guanylate kinase-like domain. The amino acid sequences of this domain share 46,52% identity with guanylate kinases from yeast, Escherichia coli, human, mouse and Caenorhabditis elegans. Arabidopsis guanylate kinases (AGKs) exhibit a high degree of conservation of active site residues and sequence motifs in common with other nucleoside monophosphate kinases, which suggests overall structural similarity of the plant proteins. Although bacterially expressed AGK-1 is enzymatically much less active than yeast guanylate kinase, its kinase domain is shown to complement yeast GUK1 recessive lethal mutations. AGKs are expressed ubiquitously in plant tissues with highest transcriptional activity detected in roots. The identification of AGKs provides new perspectives for understanding the role of guanylate kinases in plant cell signalling pathways. [source] Involvement of the MP1 scaffold protein in ERK signaling regulation during Drosophila wing developmentGENES TO CELLS, Issue 11 2008Emmanuèle Mouchel-Vielh Mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved transduction pathways involved in many cellular processes. Kinase modules are associated with scaffold proteins that regulate signaling by providing critical spatial and temporal specificities. Some of these scaffold proteins have been shown to be conserved, both in sequence and function. In mouse, the scaffold MP1 (MEK Partner 1) forms a signaling complex with MEK1 and ERK1. In this work, we focus on Drosophila MP1 (dMP1). We show that dMP1 is expressed ubiquitously during embryonic and larval development. By in vitro and in vivo experiments, we show that dMP1 is located in the cytoplasm and the nuclei, and that it interacts with MEK and ERK. Genetic studies with transgenic Drosophila lines allowing either dMP1 over-expression or dMP1 down-regulation by RNA interference highlight dMP1 function in the control of cell differentiation during development of the Drosophila wing. [source] Regulated expression and dynamic changes in subnuclear localization of mammalian Rad18 under normal and genotoxic conditionsGENES TO CELLS, Issue 8 2005Sadaharu Masuyama Rad18 plays a crucial role in postreplication repair in both lower eukaryotes and higher eukaryotes. However, regulation of the Rad18 expression in higher eukaryotes is largely unknown. We found that the RAD18 transcript is expressed ubiquitously in various tissues and very highly in the testis in mammals. Although human RAD18 (hRAD18) transcription levels fluctuate during the cell cycle, being maximal in the late S and minimal in the early G1, the protein levels remain constant throughout the cell cycle. Following UV-irradiation, hRAD18 transcription levels decrease significantly, but Rad18 protein levels change little. The protein levels are maintained at least in part by enhanced translation rates. hRad18 localizes in the nucleus in two forms: a diffused form and a condensed form forming nuclear dots. These nuclear dots disperse rapidly in the nucleoplasm after treatments with various genotoxic agents, resulting in an enhancement of the intranuclear Rad18 concentration of the diffused form. No de novo protein synthesis is required for this process. These results suggest that in higher eukaryotes, the maintenance and dynamic translocation of Rad18 protein is important for postreplication repair. [source] PRDX4, a member of the peroxiredoxin family, is fused to AML1 (RUNX1) in an acute myeloid leukemia patient with a t(X;21)(p22;q22)GENES, CHROMOSOMES AND CANCER, Issue 4 2004Yanming Zhang The AML1 gene (also known as RUNX1) at 21q22 codes for core binding factor (CBF) ,, which forms a heterodimer with CBF , that acts as a transcriptional activating factor. CBF is a critical regulator in the generation and differentiation of definitive hematopoietic stem cells and is frequently disrupted in leukemia through chromosome translocations. We cloned a novel AML1 partner gene, PRDX4, in an X;21 translocation in a 74-year-old male patient diagnosed with acute myeloid leukemia,M2. Chromosome analysis detected a t(X;21)(p22;q22) as the sole abnormality in bone marrow samples. The involvement of AML1 was confirmed by fluorescence in situ hybridization studies. Using 3, RACE-PCR, we cloned a fusion between exon 5 of AML1 and exon 2 of PRDX4. RT-PCR confirmed the fusion and detected another fusion between exon 6 of AML1 and exon 2 of PRDX4, indicating alternative splicing of exon 6 of AML1 in the fusion transcripts. PRDX4 is one of six peroxiredoxin-family genes that are highly conserved in eukaryotes and prokaryotes and are ubiquitously expressed. Peroxiredoxin genes exhibit thioredoxin-dependent peroxidase activity and have been implicated in a number of other cellular functions such as cell proliferation and differentiation. PRDX4 plays a regulatory role in the activation of the transcription factor NF-,B and is significantly down-regulated in acute promyelocytic leukemia. This is the first example of antioxidant enzyme involvement in a chromosome translocation in leukemia. © 2004 Wiley-Liss, Inc. [source] Generation of cortactin floxed mice and cellular analysis of motility in fibroblastsGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 9 2009Shinji Tanaka Abstract Cortactin is an F-actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttnflox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttnflox/flox embryos depleted cortactin within days, without disturbing F-actin distribution and localization of multiple actin-binding proteins. Cre-mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttnflox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F-actin organization and cell migration in Cttn null-MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin-dependent cell motility. genesis 47:638,646, 2009. © 2009 Wiley-Liss, Inc. [source] Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis,GENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 6 2007Aurora Badaloni Abstract Improved and modular tools are needed for the neuroanatomical dissection of CNS axonal tracts, and to study the cell-intrinsic and cell-extrinsic cues that govern their assembly and plasticity. Here we describe a general purpose transgenic tracer that can be used to visualize axonal tracts and synaptic terminals in any region of the embryonic neural tube or postnatal CNS, on any wild type or mutant genetic background. The construct permits CRE-inducible expression of a dicistronic axonal marker encoding two surface reporter proteins: a farnesylated GFP and the human Placental Alkaline Phosphatase (PLAP). Both proteins localize alongside the neuronal surface, permitting the concomitant detection of cell body, neurites, and presynaptic and postsynaptic sites in the same neuron. This provides a CRE-inducible dual system for imaging neural circuits in vivo, and to study their assembly and remodeling in cultured neurons, neural stem cells, and tissue explants derived from the reporter line. Unlike existing lines, this reporter does not encode a ubiquitously expressed, floxable LacZ gene, permitting the simultaneous analysis of beta galactosidase activity in mutant lines. genesis 45:405,412, 2007. © 2007 Wiley-Liss, Inc. [source] N-myc downstream-regulated gene 1 expression in injured sciatic nervesGLIA, Issue 4 2004Kazuho Hirata Abstract N-myc downstream-regulated gene 1 (NDRG1)/RTP/Drg1/Cap43/rit42/TDD5/Ndr1 is expressed ubiquitously and has been proposed to play a role in growth arrest and cell differentiation. A recent study showed that mutation of this gene is responsible for hereditary motor and sensory neuropathy-Lom. However, the role of this gene in the peripheral nervous system is not fully understood. In our study, rabbit polyclonal antibodies were raised against this gene product and were used to examine changes in its expression over the time course of Wallerian degeneration and ensuing regeneration after crush injury of mouse sciatic nerves. Fluorescent immunohistochemistry showed that NDRG1 was expressed over the intact nerve fibers. Double labeling with a Schwann cell (SC) marker, S-100 protein (S-100), revealed that NDRG1 was localized in the cytoplasm of S-100-positive Schwann cells (SCs). NDRG1 expression was maintained in the early stage of myelin degradation but was then markedly depleted at the end stage of myelin degradation when frequent occurrence of BrdU-labeled SCs was observed (at 7,9 days). The depletion of NDRG1 at this time point was also confirmed by Western blotting analysis. NDRG1 expression finally recovered at the stage of remyelination, with immunoreactivity stronger than that in intact nerves. These findings suggest that NDRG1 may play an important role in the terminal differentiation of SCs during nerve regeneration. © 2004 Wiley-Liss, Inc. [source] Nucleophosmin (NPM1) mutations in adult and childhood acute myeloid leukaemia: towards definition of a new leukaemia entity,HEMATOLOGICAL ONCOLOGY, Issue 4 2009Rachel Rau Abstract Nucleophosmin (NPM) is a ubiquitously expressed chaperone protein that shuttles rapidly between the nucleus and cytoplasm, but predominantly resides in the nucleolus. It plays key roles in ribosome biogenesis, centrosome duplication, genomic stability, cell cycle progression and apoptosis. Somatic mutations in exon 12 of the NPM gene (NPM1) are the most frequent genetic abnormality in adult acute myeloid leukaemia (AML), found in approximately 35% of all cases and up to 60% of patients with normal karyotype (NK) AML. In children, NPM1 mutations are far less frequent, occurring in 8,10% of all AML cases, and in approximately 25% of those with a NK. NPM1 mutations lead to aberrant localization of the NPM protein into the cytoplasm, thus the designation, NPMc+ AML. NPMc+ AML is seen predominantly in patients with a NK and is essentially mutually exclusive of recurrent chromosomal translocations. Patients with NPM1 mutations are twice as likely as those who lack an NPM1 mutation to also have a FMS-like tyrosine kinase (FLT3) internal tandem duplication (ITD) mutation. NPMc+ AML is also characterized by a unique gene expression signature and microRNA signature. NPMc+ AML has important prognostic significance, as NPMc+ AML, in the absence of a coexisting FLT3-ITD mutation, is associated with a favourable outcome. NPM1 mutations have also shown great stability during disease evolution, and therefore represent a possible marker for minimal residual disease detection. Given its distinctive biologic and clinical features and its clear clinical relevance, NPMc+ AML is included as a provisional entity in the 2008 WHO classifications. There is still much to be learned about this genetic alteration, including its exact role in leukaemogenesis, how it interacts with other mutations and why it confers a more favourable prognosis. Further, it represents a potential therapeutic target warranting research aimed at identifying novel small molecules with activity in NPMc+ AML. Copyright © 2009 John Wiley & Sons, Ltd. [source] Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor,induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase,HEPATOLOGY, Issue 6 2009Erwin Daniel Brenndörfer The hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability, viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication, on the one hand, and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. The study describes a novel mechanism by which HCV influences the activation of the epithelial growth factor receptor/Akt pathway through a nonstructural (NS)3/4A-dependent down-regulation of the ubiquitously expressed tyrosine phosphatase T cell protein tyrosine phosphatase (TC-PTP). NS3/4A is demonstrated to cleave TC-PTP protease-dependently in vitro at two cleavage sites. The in vivo relevance of this finding is supported by the fact that down-regulation of TC-PTP protein expression could also be demonstrated in HCV-infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. Conclusion: This down-regulation of TC-PTP results in an enhancement of epithelial growth factor (EGF)-induced signal transduction and increases basal activity of Akt, which is demonstrated to be essential for the maintenance of sufficient viral replication. Hence, therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects, further diminishing viral replication. (HEPATOLOGY 2009;49:1810,1820.) [source] | ||||||||||||||||||||||||||||||||||||||||