RESUMEN
We have cloned and sequenced the cDNAs for quail cornea keratan sulfate proteoglycan core proteins, keratocan and mimecan. The deduced quail keratocan protein contains a single conservative amino acid difference from the chick sequence, whereas quail mimecan protein contains a 58 amino acid-long avian-unique sequence that shares no homology with mammalian mimecan. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that keratocan and lumican are expressed at highest levels in cornea, whereas mimecan mRNA is expressed at a much lower level. Keratocan is expressed only in quail cornea, whereas mimecan is expressed in many different tissues as four transcripts of different sizes. Both lumican and mimecan are expressed at lowest levels in brain, liver and sternum.
Asunto(s)
Córnea/embriología , Proteínas del Ojo/genética , Glicoproteínas/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Córnea/metabolismo , ADN Complementario , Proteínas del Ojo/metabolismo , Glicoproteínas/metabolismo , Datos de Secuencia Molecular , Proteoglicanos/metabolismo , Codorniz , Distribución TisularRESUMEN
We have cloned and sequenced the cDNAs for quail cornea proteoglycan core proteins, decorin and lumican. Comparison of deduced amino acid sequences shows that two of five amino acid differences in the mature protein between quail and chick decorin, and two of three for lumican, are non-conservative. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that decorin and lumican are most highly expressed in cornea, and that both are also highly expressed at approximately equal levels in most other tissues. Decorin is highly expressed in sclera and sternum, whereas lumican is expressed in these tissues, as well as in liver, at very low levels. Both decorin and lumican are expressed at lowest levels in brain.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Córnea/embriología , Proteínas del Ojo/genética , Sulfato de Queratano/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Córnea/metabolismo , ADN Complementario , Decorina , Proteínas de la Matriz Extracelular , Expresión Génica , Lumican , Datos de Secuencia Molecular , CodornizRESUMEN
The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence. The expression of each of the three corneal KSPGs in the developing and mature chick cornea was investigated by competitive PCR and immuno-biochemical analysis of corneal extracts. Competitive PCR was used to determine the message levels for chick lumican, keratocan and osteoglycin in embryonic day 9, 12, 15, 18 and adult corneas. Results showed that lumican mRNA fluctuated during development but remained at a relatively high level while keratocan and osteoglycin message levels declined steadily from day 9 to adult. Additionally, lumican mRNA was present at higher levels, during all stages of corneal development, than keratocan and at much higher levels than osteoglycin. Antibodies shown to be specific for each KSPG were used to characterize proteoglycans isolated from embryonic and adult chick corneas. KSPGs from embryonic corneas eluted 1-2 fractions earlier on Q-Sepharose than KSPG from adult corneas. Additionally, Western blot analysis showed that embryonic KSPGs were more keratanase-resistant, endo-beta-galactosidase sensitive than adult KSPGs. The results of this study indicate an alteration in sulfation or the fine structure of the glycosaminoglycan chains occurs during corneal maturation for the 3 KSPGs.
Asunto(s)
Pollos/crecimiento & desarrollo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/crecimiento & desarrollo , Sulfato de Queratano/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Embrión de Pollo , Pollos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/genética , Córnea/metabolismo , ADN Complementario/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Sulfato de Queratano/genética , Lumican , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteoglicanos/metabolismoRESUMEN
The cornea contains, as a major element, a transparent stroma produced and maintained by keratocytes (fibroblasts). Through molecular biology studies using cultured human corneal fibroblasts, a cDNA that was shown to be novel was isolated and sequenced. This novel gene product, named SH3-domain binding protein 4 (SH3BP4), contains a 5.6-kb message that is present in normal human corneal fibroblasts and all tissues examined, with higher levels in pancreas, placenta, heart, and kidney. SH3BP4 was localized by FISH analysis to human chromosome 2q37.1-q37.2 near the telomere. The deduced sequence for SH3BP4 was found to contain a 963-amino-acid open reading frame that has homology to a 479-amino-acid protein in GenBank called EH-binding protein. Although the entire sequence of EH-binding protein aligns with SH3BP4, the alignment is not complete or contiguous. Therefore, SH3BP4 has an additional 73 amino acids at the N-terminus and an additional 411 amino acids near the C-terminus that are not present in EH-binding protein. Consensus sequence domains identified in SH3BP4 include a SH3 domain, three N-P-F motifs, a P-X-X-P motif noted for binding to SH3 domains, a bipartite nuclear targeting signal, and a tyrosine phosphorylation site. SH3BP4 homologies and consensus sequence sites indicate that it may be involved in a newly identified cascade of proteins involved in endocytosis, intracellular sorting, and the cell cycle.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Córnea/citología , ADN Complementario/genética , Fibroblastos/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Humanos Par 2/genética , Clonación Molecular , Fibroblastos/citología , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Mapeo Físico de Cromosoma , Homología de Secuencia de Aminoácido , Dominios Homologos src/genéticaRESUMEN
Corneal proteoglycans have chondroitin/dermatan and keratan sulfate (KS) chains and belong to the leucine-rich proteoglycan gene family. Corneal KS is N-linked to Asn of an NX(S/T) site through a complex oligosaccharide linkage region. Only some sites receive KS, whereas others remain in a high mannose form. To determine whether the attachment of KS was biased toward specific sites, we isolated trypsin-digested KS-containing fragments of chick corneal proteoglycans and sequenced the peptides. Results showed that all of the peptides sequenced aligned to the deduced amino acid sequence of either chick lumican or chick keratocan at the first, third, and fourth potential N-linked sites. Sites 1 and 4 in lumican and keratocan are in a homologous location. By analogy with the structure of ribonuclease inhibitor (a Leu-rich repeat containing protein), the KS chains would extend outward on the outer face of a horseshoe-like structure. The amino acid sequences surrounding the potential N-linked sites were also compared. Sites receiving KS tend to have a higher occurrence of aromatic residues, in particular Phe, located within 3 amino acids of NX(S/T). These conserved Phe residues may have a role in the conversion of high mannose N-linked oligosaccharides to polylactosamine and/or keratan sulfate.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/química , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/metabolismo , Sulfato de Queratano/química , Sulfato de Queratano/metabolismo , Oligosacáridos/química , Proteoglicanos/química , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , Proteoglicanos Tipo Condroitín Sulfato/genética , Clonación de Organismos , ADN Complementario , Glicosilación , Sulfato de Queratano/genética , Lumican , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Proteoglicanos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Mapeo Cromosómico , Córnea/metabolismo , Sulfato de Queratano/genética , Proteoglicanos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Lumican , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8 causes an increase in the percentage of cells lacking focal adhesions. Most of the IL-8-stimulated cells not only exhibit a lack of focal adhesions but also have a migratory phenotype that includes a protrusive leading edge and trailing tail. In addition, IL-8 was found to promote primary fibroblast chemotaxis in modified Boyden chambers as well as chemokinesis on serum-coated coverslips. Human primary fibroblasts were also found to specifically bind to IL-8 with high affinity. We have previously shown that a lack of focal structures in primary fibroblasts can be used as an index of chemokinetic locomotion and have fully characterized this system using newborn rat heart conditioned medium. The main stimulus in heart conditioned medium that is responsible for the lack of focal adhesions in the majority of cells can be immunoprecipitated using a polyclonal antibody against recombinant human IL-8. Additionally, video microscopy assays using heart conditioned medium depleted with the IL-8 antibody show an increase in the percentage of stationary cells, a consequent decrease in the percentage of migrating cells, and a twofold increase in the mitotic rate. Interleukin-1 alpha and tumor necrosis factor-alpha, which are early inflammatory cytokines, have been previously shown to stimulate IL-8 production in macrophages, fibroblasts, endothelial and epithelial cells. Our findings indicate that these two cytokines also cause an increase in the percentage of fibroblasts without focal adhesions. Additionally, this increase in cells lacking focal structures can be largely attributed to the production and subsequent autocrine action of a factor immunoprecipitated with an IL-8 antibody. Conversely, GRO-alpha, which has a high homology with IL-8, does not cause a similar increase in the percentage of cells lacking focal adhesions, but was not antagonistic to the effects of IL-8.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Interleucina-8/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Encía , Humanos , Interleucina-1/farmacología , Interleucina-8/metabolismo , Cinética , Microscopía por Video , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Fibroblast migration is an integral component of biological processes such as wound healing and embryogenesis. Previous experiments examining fibroblast locomotion from tissue explants have shown that migrating fibroblasts lack, or contain only transient, focal adhesions (focal contacts). Focal adhesions are specialized regions of tight cell-matrix interaction, assembled by a complex process of transmembrane signalling. Although the explant model has been used for studying several aspects of fibroblast locomotion, it is limited by the lack of control over migration, and only a small percentage of the cells actually locomoting. Therefore, we have developed an in vitro model for cultured fibroblast strains where the presence or absence of focal adhesions can be manipulated, and in the latter case 70% of these cells become locomotory. The stimulus used to decrease the percentage of cells containing focal adhesions, and hence enhance locomotion, was newborn rat heart-conditioned medium (HCM). Addition of HCM to rat embryo fibroblasts induced both chemokinesis and chemotaxis. Cells disassembled focal adhesions on a variety of extracellular matrix substrates after approximately 6 h of stimulation with HCM; conversely, removal of HCM promoted reformation of focal adhesions within 12-24 h. HCM-stimulated fibroblasts which lacked focal adhesions concomitantly lacked F-actin stress fibers and focal concentrations of vinculin and talin. Therefore, fibroblast migration can be readily controlled in an on-off manner through conditioned medium, which influences the absence or presence of focal adhesions.
Asunto(s)
Fibroblastos/citología , Células 3T3/citología , Células 3T3/efectos de los fármacos , Animales , Animales Recién Nacidos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Citoesqueleto/ultraestructura , Embrión de Mamíferos/citología , Matriz Extracelular/ultraestructura , Fibroblastos/efectos de los fármacos , Encía/citología , Corazón/fisiología , Humanos , Ratones , Miocardio/citología , Ratas , Transducción de Señal , Vinculina/metabolismoRESUMEN
Cell-mediated cytotoxicity involves a localized secretory process in which lytic agents stored in specialized granules of the effector cells are released upon contact with the appropriate target cell membrane and cause membrane damage. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibits cytotoxicity of natural killer cells, cytotoxic T-lymphocytes and lymphokine-activated killer cells. This inhibition is due to an effect of CCCP on the cytolytic cells, rather than on their targets, and is reversible. Treatment with CCCP does not inhibit the formation of effector-target conjugates, but seems to affect the programming of the effector cells for lysis. CCCP only inhibits lysis if added during a certain period of the lytic cycle: it has an effect only if added before, or within 5 minutes of the initiation of killing by a pulse of Ca++. Effector cells treated with CCCP retain their characteristic beta-glucuronidase-positive granules, but in the presence of the drug, these are no longer oriented to face the contact area with the target cell membrane.