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1.
Nanoscale ; 10(2): 690-704, 2018 Jan 03.
Article in English | MEDLINE | ID: mdl-29242877

ABSTRACT

Multicore superparamagnetic nanoparticles have been proposed as ideal tools for some biomedical applications because of their high magnetic moment per particle, high specific surface area and long term colloidal stability. Through controlled aggregation and packing of magnetic cores it is possible to obtain not only single-core but also multicore and hollow spheres with internal voids. In this work, we compare toxicological properties of single and multicore nanoparticles. Both types of particles showed moderate in vitro toxicity (MTT assay) tested in Hep G2 (human hepatocellular carcinoma) and Caco-2 (human colorectal adenocarcinoma) cells. The influence of surface chemistry in their biological behavior was also studied after functionalization with O,O'-bis(2-aminoethyl) PEG (2000 Da). For the first time, these nanoparticles were evaluated in a Xenopus laevis model studying their whole organism toxicity and their impact upon iron metabolism. The degree of activation of the metabolic pathway depends on the size and surface charge of the nanoparticles which determine their uptake. The results also highlight the potential of Xenopus laevis model bridging the gap between in vitro cell-based assays and rodent models for toxicity assessment to develop effective nanoparticles for biomedical applications.


Subject(s)
Magnetite Nanoparticles , Xenopus laevis/metabolism , Animals , Biotransformation , Caco-2 Cells , Embryo, Nonmammalian , Hep G2 Cells , Humans , Iron/metabolism , Particle Size , Toxicity Tests
2.
Development ; 128(11): 2063-73, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11493528

ABSTRACT

Wnt signalling functions in many tissues and during different stages of animal development to produce very specific responses. In early Xenopus embryos there is a dramatic change in response to Wnt signalling within only a few hours of development. Wnt signalling in very early embryos leads to a dorsalising response, which establishes the endogenous dorsal axis. Only a few hours later in development, almost the opposite happens: Xwnt-8 functions to pattern the embryonic mesoderm by promoting ventral and lateral mesoderm. The specificity of the response could conceivably be carried out by differential use of different signal transduction pathways, many of which have recently been described. We have found, however, that this dramatic shift in response to Wnt signalling in early Xenopus is not brought about by differential use of distinct signal transduction pathways. In fact beta-catenin, a downstream component of the canonical Wnt signal transduction pathway, functions not only in the early dorsalising response but also in the later ventrolateral-promoting response. Interaction of beta-catenin with the XTcf-3 transcription factor is required for the early dorsalising activity. In contrast, our experiments suggest that late Wnt signalling in the ventrolateral mesoderm does not require a similar dependency of beta-catenin function on XTcf-3. Our results highlight the potential versatility of the canonical Wnt pathway to interact with tissue-specific factors downstream of beta-catenin, in order to achieve tissue-specific effects.


Subject(s)
Blastocyst/metabolism , Cytoskeletal Proteins/metabolism , HMGB Proteins , Proto-Oncogene Proteins/metabolism , Signal Transduction , Trans-Activators , Transcription Factors/metabolism , Zebrafish Proteins , Animals , Animals, Genetically Modified , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Glycogen Synthase Kinase 3 , Lithium , Mesoderm , Proto-Oncogene Proteins/genetics , TCF Transcription Factors , Transcription Factor 3 , Transcription Factor 7-Like 1 Protein , Transcription Factors/genetics , Wnt Proteins , Xenopus Proteins , Xenopus laevis/embryology , beta Catenin
3.
Gene ; 262(1-2): 291-9, 2001 Jan 10.
Article in English | MEDLINE | ID: mdl-11179695

ABSTRACT

Paxillin is a focal adhesion scaffolding protein, which has been proposed to play a role in focal adhesion dynamics. We have isolated a cDNA clone of the Drosophila homologue of paxillin. Comparison of the Drosophila paxillin sequence with those of vertebrate paxillins shows strong conservation of the LIM domains and LD repeats. Using the Drosophila genomic sequence we have identified two partial curated transcripts and deduced the structure of the paxillin gene. No homologues of other members of the paxillin family such as HIC-5 or leupaxin are to be found in the Drosophila genome. Surprisingly paxillin mRNA is expressed in a restricted pattern during embryogenesis. In particular it is strongly expressed in cells and tissues undergoing cell shape changes or cell migration. Many of the sites of expression are also known to be sites of integrin function or FAK expression. The data support a role for paxillin as an adapter and/or signaling protein during developmental processes involving integrin-mediated adhesion.


Subject(s)
Cell Adhesion Molecules/genetics , Cytoskeletal Proteins/genetics , Drosophila/genetics , Gene Expression Regulation, Developmental , Phosphoproteins/genetics , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion Molecules/metabolism , Cell Movement/genetics , Cell Size/genetics , Cloning, Molecular , Cytoskeletal Proteins/metabolism , Drosophila/embryology , Drosophila Proteins , Embryo, Nonmammalian , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Mice , Paxillin , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Repetitive Sequences, Amino Acid
4.
Curr Biol ; 10(14): 849-52, 2000 Jul 13.
Article in English | MEDLINE | ID: mdl-10899005

ABSTRACT

The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.


Subject(s)
Xenopus laevis/embryology , Xenopus laevis/genetics , Animals , Animals, Genetically Modified , Cytoskeletal Proteins , Gene Expression Regulation, Developmental , Genes, Reporter , Green Fluorescent Proteins , HSP70 Heat-Shock Proteins/genetics , Luminescent Proteins/genetics , Phenotype , Promoter Regions, Genetic , Proteins/genetics , Recombinant Fusion Proteins/genetics , Signal Transduction , Wnt Proteins , Zebrafish Proteins
5.
J Cell Biol ; 147(2): 417-34, 1999 Oct 18.
Article in English | MEDLINE | ID: mdl-10525545

ABSTRACT

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the alpha6beta4 integrin and have shown that the cytoplasmic domain of the beta4 subunit associates with an NH(2)-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with alpha6beta4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that beta4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the beta4 cytoplasmic domain. Mapping of the beta4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that beta4 can compete out the plectin ABD fragment from its association with F-actin. The ability of beta4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament-anchoring hemidesmosomes when beta4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.


Subject(s)
Actins/metabolism , Antigens, Surface/metabolism , Desmosomes/metabolism , Integrins/metabolism , Intermediate Filament Proteins/metabolism , Intermediate Filaments/metabolism , Keratinocytes/metabolism , Cell Line, Transformed , Desmosomes/ultrastructure , Humans , Immunohistochemistry , Integrin alpha6beta4 , Keratinocytes/ultrastructure , Plectin , Protein Binding , Transfection
6.
Mech Dev ; 86(1-2): 203-7, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10446283

ABSTRACT

A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain.


Subject(s)
Brain/embryology , Gene Expression Regulation, Developmental , Heart/embryology , Receptors, Cell Surface/genetics , Receptors, G-Protein-Coupled , Xenopus Proteins , Xenopus/embryology , Amino Acid Sequence , Animals , Embryo, Nonmammalian , Frizzled Receptors , Gastrula , Humans , Mice , Molecular Sequence Data , Receptors, Cell Surface/metabolism , Sequence Homology, Amino Acid , Xenopus/genetics
7.
Genomics ; 21(3): 510-6, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7959727

ABSTRACT

Mouse cDNA clones coding for a desmocollin and a desmoglein, desmosomal cadherins that are putative adhesion molecules of the desmosome type of cell-cell junction characteristically found in epithelial tissues, have been isolated and sequenced. From sequence comparisons with the known human and bovine desmosomal cadherins, these clones represent a mouse Dsc3 and Dsg1. By interspecific backcross analysis, these genes were found to be closely linked in the proximal region of mouse chromosome 18, a region having conserved synteny with human chromosome 18. From these results, and recently reported linkage of DSG1 and DSG2 on human chromosome 18 at 18q12.1 in a deletion panel of somatic cell hybrids, all the desmosomal cadherins genes so far examined are clustered on chromosome 18 in human and mouse, which may have implications for gene expression. We further show that the human DSC3 gene, previously reported to be located on chromosome 9, also maps to human chromosome 18.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 18 , Cytoskeletal Proteins/genetics , Genetic Linkage , Membrane Glycoproteins/genetics , Mice/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Adhesion Molecules/genetics , Cloning, Molecular , DNA Primers , Desmocollins , Desmoglein 1 , Desmoglein 2 , Desmogleins , Desmoplakins , Hominidae/genetics , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid
9.
Genomics ; 10(3): 640-5, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1889810

ABSTRACT

We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).


Subject(s)
Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 6 , Chromosomes, Human, Pair 7 , Chromosomes, Human, Pair 9 , Cytoskeletal Proteins/genetics , Desmosomes , Animals , Base Sequence , Chromosome Mapping , Desmocollins , Desmogleins , Desmoplakins , Genes , Humans , Hybrid Cells , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Rats , gamma Catenin
10.
J Biol Chem ; 266(16): 10438-45, 1991 Jun 05.
Article in English | MEDLINE | ID: mdl-2037591

ABSTRACT

We have cloned the human genes coding for desmosomal glycoproteins DGII and DGIII, found in desmosomal cell junctions, and sequencing shows that they are related to the cadherin family of cell adhesion molecules. Thus a new super family of cadherin-like molecules exists which also includes the other major desmosomal glycoprotein, DGI (Wheeler, G. N., Parker, A. E., Thomas, C. L., Ataliotis, P., Poynter, D., Arnemann, J., Rutman, A. J., Pidsley, S. C., Watt, F. M., Rees, D. A., Buxton, R. S., and Magee, A. I. (1991) Proc. Natl. Acad. Sci. U.S.A., in press). DGIII differs from DGII by the addition of a 46-base pair exon containing an in-frame stop codon resulting in mature protein molecular weights of 84,633 for DGII and 78,447 for DGIII. The unique carboxyl-terminal region of DGII contains a potential serine phosphorylation site explaining why only DGII is phosphorylated on serine. The cadherin cell adhesion recognition sequence (His-Ala-Val) is replaced by Phe-Ala-Thr, suggesting that DGII/III may be adhesive molecules using a different mechanism.


Subject(s)
Cadherins/genetics , Desmosomes/metabolism , Glycoproteins/genetics , RNA Splicing , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cadherins/metabolism , Cloning, Molecular , DNA/genetics , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Female , Glycoproteins/metabolism , Humans , Male , Mice , Molecular Sequence Data , Phosphorylation , Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Nucleic Acid
11.
Proc Natl Acad Sci U S A ; 88(11): 4796-800, 1991 Jun 01.
Article in English | MEDLINE | ID: mdl-1711210

ABSTRACT

Among the variety of specialized intercellular junctions, those of the adherens type have the most obvious association with cytoskeletal elements. This may be with the actin microfilament system as in the zonula adherens or with intermediate filaments as in the macula adherens, or desmosome. In the former case, it is clear that transmembrane glycoproteins of the cadherin family are important adhesive components of the molecular assembly. We now show for desmosomes that a major glycoprotein component (desmosomal glycoprotein DGI) has extensive homology with the cadherins, defining an extended family, but also has unique features in its cytoplasmic domain that are likely to be relevant to the association with intermediate rather than actin filaments. A novel 282-residue extension contains repeats of approximately 29 amino acid residues predicted to have an antiparallel beta-sheet structure, followed by a glycine-rich sequence. As in the cadherins, the extracellular domain contains possible Ca2(+)-binding sequences and a potential protease processing site. The cell adhesion recognition region (His-Ala-Val) of the cadherins is modified to Arg-Ala-Leu.


Subject(s)
Cadherins/genetics , Cytoskeletal Proteins/genetics , Desmosomes/physiology , Epidermis/physiology , Keratinocytes/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cattle , Cytoskeletal Proteins/isolation & purification , Desmoplakins , Gene Library , Humans , Molecular Sequence Data , Poly A/genetics , Poly A/isolation & purification , RNA/genetics , RNA/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , Sequence Homology, Nucleic Acid
12.
Biochem J ; 270(2): 463-8, 1990 Sep 01.
Article in English | MEDLINE | ID: mdl-2119173

ABSTRACT

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.


Subject(s)
Galactosidases , Immune Sera , Laminin/analysis , Placenta/analysis , Recombinant Fusion Proteins/immunology , Yolk Sac/analysis , beta-Galactosidase , Amino Acid Sequence , Animals , Antibody Specificity , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Glycosylation , Humans , Laminin/immunology , Mice , Molecular Sequence Data , Molecular Weight , Rats , Sequence Homology, Nucleic Acid
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