RESUMEN
As the quest toward novel materials proceeds, improved characterization technologies are needed. In particular, the atomic thickness in graphene and other 2D materials renders some conventional technologies obsolete. Characterization technologies at wafer level are needed with enough sensitivity to detect strain in order to inform fabrication. In this work, NEXAFS spectroscopy was combined with simulations to predict lattice parameters of graphene grown on copper and further transferred to a variety of substrates. The strains associated with the predicted lattice parameters are in agreement with experimental findings. The approach presented here holds promise to effectively measure strain in graphene and other 2D systems at wafer levels to inform manufacturing environments.
RESUMEN
The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.
Asunto(s)
Ligamentos Colaterales/metabolismo , Proteoglicanos/metabolismo , Animales , Bovinos , Ligamentos Colaterales/patología , Técnicas de Cultivo , Decorina , Proteínas de la Matriz Extracelular , Líquido Intracelular/metabolismo , Marcaje Isotópico , Peso Molecular , Radioisótopos de AzufreRESUMEN
The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The beta subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase alpha-subunit (PHY; EC 1.14.11.2)- and beta-subunit (PDI; EC 5.3.4.1)-encoding genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.
Asunto(s)
Caenorhabditis elegans/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Colágeno/metabolismo , Expresión Génica , Genes de Helminto , Hidroxiprolina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genética , Homología de Secuencia de Aminoácido , EsqueletoRESUMEN
Vertebrate genes with sequence similarity to the Drosophila homeobox gene, sine oculis (so), constitute the SIX family. There is notable expression of members of this family in anterior neural structures, and several SIX genes have been shown to play roles in vertebrate and insect development, or have been implicated in maintenance of the differentiated state of tissues. Mutations in three of these genes in man (SIX5, SIX6 and SIX3) are associated with severe phenotypes, and therefore, the cloning of other human genes from this family is of interest. We have cloned and characterised the gene that encodes human SIX2, elucidated its gene structure and conducted expression studies in a range of tissues. SIX2 is widely expressed in the late first-trimester fetus, but has a limited range of expression sites in the adult. The expression pattern of SIX2 and its localisation to chromosome 2p15-p16 will be of use in assessing its candidacy in human developmental disorders.
Asunto(s)
Genes/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , Cromosomas Humanos Par 2/genética , Clonación Molecular , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Células Híbridas , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
Cyclophilin 3 (CYP-3) is one of the most abundantly expressed cyclophilin isoforms in the free living nematode Caenorhabditis elegans. The detailed post-embryonic expression pattern of the cyp-3 transcript is unusual, peaking during early larval development. The spatial expression pattern was examined via reporter gene analysis demonstrating that the cyp-3 transcript is exclusively expressed in the single anterior excretory cell. Recombinant cyclophilin 3 has been purified, crystallized and solved to a resolution of 1.8 A. The peptidyl-prolyl isomerase activity of CYP-3 has been characterized against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 2.4 x 10(6) M(-1) s(-1). The immunosuppressive drug cyclosporin A binds and inhibits CYP-3 with an IC(50) value of 16 nM, comparable with the range of values found for human cyclophilin A. The x-ray structure shows that the overall fold and active site geometry is similar to other cyclophilin structures. There are however a number of distinctive features, and we use this structure and amino acid sequence alignment analysis to identify a subgroup of "divergent-loop cyclophilins". This subgroup has a number of uniquely conserved features: an additional loop between residues 48 and 54 (KSGKPLH); two cysteine residues (Cys(40) and Cys(168)) that are in close proximity but remain in the unoxidized form, and two other conserved residues, His(54) and Glu(83). We suggest that these features are functionally important for the role played by this class of cyclophilins during cellular responses to stress caused by changes in the redox environment or by up-regulation of cellular activity. This study represents a detailed biological, biochemical, and structural characterization of a single cyclophilin isoform in the model organism Caenorhabditis elegans.
Asunto(s)
Caenorhabditis elegans/enzimología , Isomerasa de Peptidilprolil/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Ciclosporina/farmacocinética , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil/efectos de los fármacos , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Isoformas de Proteínas , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de TiempoAsunto(s)
Caenorhabditis elegans/genética , Isomerasa de Peptidilprolil/genética , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Clonación Molecular/métodos , Vectores Genéticos , Estadios del Ciclo de Vida , Isomerasa de Peptidilprolil/biosíntesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesisRESUMEN
A divergent multi-domain cyclophilin from the filarial nematodes Brugia malayi, Onchocerca volvulus and Dirofilaria immitis has a highly conserved orthologue in the free-living nematodes Caenorhabditis elegans and C. briggsae. Cyclophilins are the receptors for the immunosuppressive and anti-parasitic agent cyclosporin A and additionally these ubiquitously expressed proteins have protein folding capabilities, and exhibit proline isomerase activity. These divergent nematode cyclophilins (CYP-4 isoforms) are three domain proteins, which share 63-88% identity and have highly conserved differences present in their functionally important cyclosporin A binding and proline isomerase domains. This unusual class of nematode cyclophilins has been studied in the model nematode C. elegans, revealing a unique temporal and spatial expression pattern. The cyp-4 transcript is most abundantly expressed in the early larval stages and is expressed exclusively in the body-wall striated muscle cells. An important functional role was established for this divergent enzyme, as specific double-stranded RNA interference experiments resulted in progeny with a phenotypically lumpy appearance. This morphological defect was predominantly expressed in the early larval stages and is consistent with an effect on body-wall muscle cell development. This study has established that this highly conserved family of nematode cyclophilins has a tissue-specific, functional role in early larval development and supports the use of C. elegans as a model for the study of orthologues in the experimentally less amenable parasitic nematodes.
Asunto(s)
Nematodos/enzimología , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Secuencia Conservada , ADN Complementario , Regulación del Desarrollo de la Expresión Génica , Genes de Helminto , Genes Reporteros , Humanos , Isoenzimas , Larva/enzimología , Datos de Secuencia Molecular , Desarrollo de Músculos , Nematodos/genética , Nematodos/crecimiento & desarrollo , Isomerasa de Peptidilprolil/química , ARN Bicatenario/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Bovine collateral ligament synthesized a 35S-labeled large proteoglycan species which eluted with a Kav of approximately 0.27 on Sepharose CL-2B and contained only chondroitin sulfate chains with a molecular mass of approximately 32 kDa. Fluorography of the 35S-labeled core proteins derived from the large ligament proteoglycan revealed a broad range of molecular masses above approximately 200 kDa, which was of comparable size to the four major endogenous core protein bands derived from this proteoglycan detected with 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides. The core proteins derived from the large ligament proteoglycan exhibited immunoreactivity of 12/21/1-C-6, an antibody specific for a peptide epitope common to both the G1 and G2 domains of aggrecan. Four major core protein bands with molecular masses greater than approximately 200 kDa derived from the large ligament proteoglycan, were detected using the antibodies raised against versican from bovine aorta or human fibroblasts. Compared with aggrecan, the 35S-labeled large ligament proteoglycan was distributed over a broader range of buoyant densities in an associative caesium chloride density gradient. This polydispersity may be indicative of differences in the degree of glycosylation as well as heterogeneity in the size of the large ligament proteoglycan core proteins. The 35S-labeled large ligament proteoglycan also demonstrated the ability to form complexes with an aggrecan aggregate preparation, the majority of which could not be dissociated by the presence of HA10-50. These findings indicate that the large chondrotin sulfate proteoglycan synthesized by bovine collateral ligament may be a versican-like proteoglycan which exhibited the potential to form like protein-stabilized complexes.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/química , Ligamentos Colaterales/química , Proteínas de la Matriz Extracelular , Agrecanos , Animales , Bovinos , Centrifugación por Gradiente de Densidad , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Disacáridos/inmunología , Técnicas In Vitro , Lectinas Tipo C , Masculino , Peso Molecular , Oligosacáridos/farmacología , Unión Proteica , Proteoglicanos/metabolismoRESUMEN
This paper investigates the kinetics and mechanism of loss of the two major proteoglycan species from cultures of bovine collateral ligament. Following incubation of ligament with [35S]sulfate after 6 days in culture, the rate of loss of the predominant proteoglycan species, decorin, from the matrix was shown to be much slower (t1/2 approximately 18 days) than that of the large chondroitin sulfate proteoglycan (t1/2 approximately 1.4 days). Analysis of 35S-labeled proteoglycans released into the medium between Days 11 and 15 of the culture period on a column of Sepharose CL-4B revealed that these macromolecules constituted mainly decorin of similar hydrodynamic size to that present in the matrix. Furthermore, analysis of core proteins using gel electrophoresis followed by fluorography or immunodetection with LF-94, an antibody directed against the amino-terminal region of decorin, indicated that the core proteins of decorin released into the medium and those remaining in the matrix of ligament cultures had a similar molecular mass (approximately 49 kDa). Analysis of both the 35S-labeled and endogenous macromolecules using 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides, revealed that three core proteins with molecular masses greater than approximately 200 kDa were present in the matrix. Four additional core proteins (range approximately 80-200 kDa) derived from the large proteoglycan were detected in the medium of ligament cultures. These findings indicate that, unlike decorin, the loss of the large chondroitin sulfate proteoglycan from the matrix of ligament cultures involved proteolytic cleavage of its core protein. No difference in the hydrodynamic size of the 35S-labeled glycosaminoglycan chains derived from either proteoglycan species remaining in the matrix or released into the medium of ligament cultures was observed.