RESUMO
Dental caries is a cariogenic bacteria-mediated, fermentable carbohydrate-driven dynamic disease. The new ecological hypothesis for dentin caries suggests that an alteration in the microbial community and the presence of specific metabolic pathway genes contribute to the initiation and progression of caries. This study aimed to determine the structural and functional characteristics of a microbial community of human deep-dentin carious lesions. Sixteen deep-dentin carious lesions were obtained from the first permanent molars of 8 patients aged 9 to 18 y. Shotgun metagenomic sequencing was used to measure the microbial composition and abundance at the phylum, class, order, family, genus, and species levels. Functional analysis of the DNA sequencing data set was also performed and compared among different layers of the lesions using DIAMOND software against the Kyoto Encyclopedia of Genes and Genomes database. This study found that in the deep-dentin carious lesions, Actinobacteria (35.8%) and Firmicutes (31.2%) were the most prevalent phyla, followed by Bacteroidetes (13.6%), Proteobacteria (3.6%), and Fusobacteria (2.5%). The microbial composition varied among the individuals, but there were no significant differences in the distribution of the relative microbial abundance between the superficial layers and the deep layers. Although 14.5% of the top 10 taxa were identified as Lactobacillus at the genus level, only 25% of the deep-dentin carious samples showed Lactobacillus as the most abundant genus. Other abundant taxa included Actinomyces (10.5%), Olsenella (9.4%), Prevotella (8.8%), Propionibacterium (7.2%), Streptococcus (3.9%), Selenomonas (3.7%), Corynebacterium (1.9%), Leptotrichia (1.4%), and Parascardovia (1.1%). The most abundant pathway identified in the KEGG database was the metabolic pathway containing 101,427 annotated genes, which consisted of 51.4% of all annotated genes. The carbohydrate metabolism pathway, amino acid metabolism, and membrane transport were the functional traits of the level 2 pathways. These findings suggest that the potent interaction within the microbial communities in deep-dentin carious lesions may play a fundamental role in caries etiology.
Assuntos
Cárie Dentária , Microbiota , Adolescente , Criança , Dentina , Humanos , Metagenoma , Microbiota/genética , StreptococcusRESUMO
Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program.
Assuntos
Diagnóstico Bucal/instrumentação , Dispositivos Lab-On-A-Chip , Sistemas Automatizados de Assistência Junto ao Leito , Desenho de Equipamento , Líquido do Sulco Gengival/química , Humanos , Imunoensaio/instrumentação , Microfluídica/instrumentação , Técnicas de Diagnóstico Molecular/instrumentação , Saliva/química , Fatores de TempoRESUMO
In this review, the authors survey the large number of antibacterial and antiviral proteins present in human saliva. Of interest, most of these antibacterial proteins display antiviral activity, typically against specific viral pathogens. The review focuses on one protein that interacts with both bacteria and viruses-gp340, originally referred to as salivary agglutinin. In the oral cavity, soluble gp340 binds to and aggregates a variety of bacteria, and this is thought to increase bacterial clearance from the mouth. However, when bound to the tooth surface, gp340 promotes bacterial adherence. In the oral cavity, most gp340 is found soluble in saliva and can function as a specific inhibitor of infectivity of HIV-1 and influenza A. In contrast, in the female reproductive track, most gp340 is bound to the cell surface, where it can promote HIV-1 infection.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Antivirais , Infecções por HIV/metabolismo , Receptores de Superfície Celular/fisiologia , Proteínas e Peptídeos Salivares/fisiologia , Antivirais/metabolismo , Aderência Bacteriana , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , HIV-1/metabolismo , Humanos , Vírus da Influenza A/metabolismo , Modelos Moleculares , Orthomyxoviridae , Ligação Proteica , Conformação Proteica , Receptores de Superfície Celular/química , Proteínas Supressoras de TumorRESUMO
The amelogenin proteins regulate enamel mineral formation in the developing tooth. The human AMELX gene, which encodes the amelogenin proteins, is located within an intron of the Arhgap 6 gene. ARHGAP 6 encodes a Rho GAP, which regulates activity of Rho A, a small G protein involved in intracellular signal transduction. Mice were generated in which the entire ARHGAP 6 gene was deleted by Cre-mediated recombination, which also removed the nested Amel X gene. Enamel from these mice appeared chalky white, and the molars showed excessive wear. The enamel layer was hypoplastic and non-prismatic, whereas other dental tissues had normal morphology. This phenotype is similar to that reported for Amel X null mice, which have a short deletion that removed the region surrounding the translation initiation site, and resembles some forms of X-linked amelogenesis imperfecta in humans. Analysis of the enamel from the Arhgap 6/Amel X-deleted mice verifies that the Amel X gene is nested within the murine Arhgap 6 gene and shows that removal of the entire Amel X gene leads to a phenotype similar to the earlier Amel X null mouse results, in which no amelogenin protein was detected. However, an unusual layer of aprismatic enamel covers the enamel surface, which may be related to the 1.1-Mb deletion, which included Arhgap 6 in these mice.
Assuntos
Proteínas do Esmalte Dentário/genética , Esmalte Dentário/patologia , Proteínas Ativadoras de GTPase/genética , Deleção de Genes , Camundongos Transgênicos/genética , Amelogênese Imperfeita/genética , Amelogenina , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dente/metabolismo , Dente/ultraestruturaRESUMO
The development of up-converting phosphor reporter particles has added a powerful tool to modern detection technologies. Carefully constructed phosphor reporters have core-shell structures with surface functional groups suitable for standard bio-conjugations. These reporters are chemically stable, possess the unique property of infrared up-conversion, and are readily detected. In contrast to conventional fluorescent reporters, up-converting phosphor particles do not bleach and allow permanent excitation with simultaneous signal integration. A large anti-Stokes shift (up to 500 nm) separates discrete emission peaks from the infrared excitation source. Along with the unmatched contrast in biological specimens due to the absence of autofluorescence upon infrared excitation, up-converting phosphor technology (UPT) has unique properties for highly-sensitive particle-based assays. The production and characteristics of UPT reporter particles as well as their application in various bioassays is reviewed.
Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Raios Infravermelhos , Medições Luminescentes , Nanotecnologia/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Espectrofotometria Infravermelho/métodos , Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Cerâmica , Metais Terras Raras , Sondas Moleculares , Nanotecnologia/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Espectrofotometria Infravermelho/instrumentaçãoRESUMO
Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.
Assuntos
Amelogênese/genética , Proteínas do Esmalte Dentário/genética , Amelogenina , Sequência de Aminoácidos , Animais , Cruzamento , Bovinos , Esmalte Dentário/metabolismo , Esmalte Dentário/ultraestrutura , Hipoplasia do Esmalte Dentário/genética , Hipoplasia do Esmalte Dentário/ultraestrutura , Proteínas do Esmalte Dentário/deficiência , Proteínas do Esmalte Dentário/metabolismo , Feminino , Vetores Genéticos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Dente Molar/química , Dente Molar/ultraestrutura , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
The amelogenin proteins are the most abundant organic components of developing dental enamel. Their importance for the proper mineralization of enamel is evident from the association between previously identified mutations in the X-chromosomal gene that encodes them and the enamel defect amelogenesis imperfecta. In this investigation, an adult male presenting with a severe hypoplastic enamel phenotype was found to have a single base deletion at the codon for amino acid 110 of the X-chromosomal 175-amino acid amelogenin protein. The proband's mother, who also has affected enamel, carries the identical deletion on one of her X-chromosomes, while the father has both normal enamel and DNA sequence. This frameshift mutation deletes part of the coding region for the repetitive portion of amelogenin as well as the hydrophilic tail, replacing them with a 47-amino acid segment containing nine cysteine residues. While greater than 60% of the protein is predicted to be intact, the severity of this phenotype illustrates the importance of the C-terminal region of the amelogenin protein for the formation of enamel with normal thickness.
Assuntos
Amelogênese Imperfeita/genética , Proteínas do Esmalte Dentário/genética , Cromossomo X/genética , Adulto , Amelogenina , Sequência de Aminoácidos , Análise Mutacional de DNA , Feminino , Mutação da Fase de Leitura , Ligação Genética , Genótipo , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Aberrações dos Cromossomos SexuaisRESUMO
Working with cultured dermal fibroblasts derived from control individuals and patients with systemic sclerosis (SSc), we have examined the effects of protein kinase C-delta (PKC-delta) on type I collagen biosynthesis and steady-state levels of COL1A1 and COL3A1 mRNAs. Rottlerin, a specific inhibitor of PKC-delta, exerted a powerful, dose-dependent inhibition of type I and type III collagen gene expression in normal and SSc cells. Optimal rottlerin concentrations caused a 70-90% inhibition of type I collagen production, a >80% reduction in COL1A1 mRNA, and a >70% reduction in COL3A1 mRNA in both cell types. In vitro nuclear transcription assays and transient transfections with COL1A1 promoter deletion constructs demonstrated that rottlerin profoundly reduced COL1A1 transcription and that this effect required a 129-bp promoter region encompassing nucleotides -804 to -675. This COL1A1 segment imparted rottlerin sensitivity to a heterologous promoter. Cotransfections of COL1A1 promoter constructs with a dominant-negative PKC-delta expression plasmid showed that suppression of this kinase silenced COL1A1 promoter activity. The results indicate that PKC-delta participates in the upregulation of collagen gene transcription in SSc and suggest that treatment with PKC-delta inhibitors could suppress fibrosis in this disease.
Assuntos
Colágeno/biossíntese , Colágeno/metabolismo , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Isoenzimas/fisiologia , Proteína Quinase C/fisiologia , Escleroderma Sistêmico/enzimologia , Escleroderma Sistêmico/metabolismo , Acetofenonas/metabolismo , Benzopiranos/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Clonagem Molecular , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Colágeno Tipo III/metabolismo , Relação Dose-Resposta a Droga , Genes Dominantes , Humanos , Microscopia de Fluorescência , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteína Quinase C-delta , RNA Mensageiro/metabolismo , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.
Assuntos
Odontogênese/fisiologia , Transdução de Sinais/fisiologia , Transativadores/genética , Fator de Crescimento Transformador beta , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Bovinos , Comunicação Celular/fisiologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Proteínas de Membrana/genética , Mesoderma/citologia , RNA Mensageiro/análise , Germe de Dente/química , Germe de Dente/embriologia , Transativadores/análiseRESUMO
It is possible that many of the fibrogenic effects of transforming growth factor-beta (TGF-beta) are mediated by connective tissue growth factor (CTGF). In the present work, we show that TGF-beta1 produces a 5- to 6-fold increase in CTGF expression by cultured human lung fibroblasts that is due mainly to increased transcription. The half-life of CTGF mRNA is 1.96 h, consistent with its role as a cytokine. In addition to requiring Smad activity, based upon the effects of specific inhibitors, the TGF-beta intracellular signaling pathway requires the activity of a phosphatidylcholine-specific phospholipase C, a protein kinase C, and one or more tyrosine kinases. It is also likely that the pathway requires a member of the Ras superfamily of small GTPases, but not trimeric G proteins. Pharmacologic inhibition of TGF-beta stimulation of CTGF expression may be an effective therapeutic approach to a variety of undesirable fibrotic reactions.
Assuntos
Fibroblastos/metabolismo , Substâncias de Crescimento/biossíntese , Proteínas Imediatamente Precoces/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Pulmão/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Células Cultivadas , Toxina da Cólera/farmacologia , Fator de Crescimento do Tecido Conjuntivo , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Substâncias de Crescimento/genética , Humanos , Proteínas Imediatamente Precoces/genética , Pulmão/citologia , Pulmão/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Fator de Crescimento Transformador beta1 , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologia , Proteínas ras/metabolismoRESUMO
Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine to adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N terminal to the proteinase cleavage site in amelogenin for enamel matrix metalloproteinase-20 (MMP-20), also known as enamelysin. MMP-20 effects the release of tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate MMP-20 hydrolyzes the putative mutated amelogenin cleavage site. The proteolytic site was modeled as a substrate by two synthetic peptides, P1 (SYGYEPMGGWLHHQ) and M1 (SYGYETMGGWLHHQ), selected from residue 36-49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the oligopeptides and the truncated peptides were separated by reversed phase HPLC and identified by mass spectrometry. The results demonstrate that both peptides are cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. However, the apparent first order rate of digestion of the mutation containing peptide by rMMP-20 was approximately 25 times slower than that of the non-mutated peptide. This study suggests that the reduced rate of TRAP formation due to a single amino acid substitution may alter enamel formation and consequently result in amelogenesis imperfecta.
Assuntos
Amelogênese Imperfeita/metabolismo , Proteínas do Esmalte Dentário/metabolismo , Metaloproteinases da Matriz/metabolismo , Cromossomo X , Amelogenina , Aminoácidos , Humanos , Hidrólise , Espectrometria de Massas/métodos , Metaloproteinase 20 da Matriz , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.
Assuntos
Núcleo Celular/metabolismo , Proteínas da Matriz Extracelular , Matriz Extracelular/metabolismo , Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Transformador beta , Bexiga Urinária/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Músculo Liso/citologia , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bexiga Urinária/citologiaRESUMO
OBJECTIVE: To examine the effects of specific inhibition of geranylgeranyl transferase I on the expression of types I and III collagen genes in normal and systemic sclerosis (SSc) dermal fibroblasts in vitro. METHODS: Fibroblasts from 2 normal subjects and 4 SSc patients were incubated with 2-10 microM of GGTI-298, a specific geranylgeranyl transferase inhibitor. Type I collagen and fibronectin production were determined by enzyme-linked immunosorbent assay. Steady-state messenger RNA (mRNA) levels for alpha1(I), alpha2(I), and alpha1(III) collagens and fibronectin were assessed by Northern hybridization, and the transcription of the alpha1(I) collagen gene was examined by transient transfections with a reporter construct containing -5.3 kb of the gene. RESULTS: GGTI-298 caused a dose-dependent inhibition of type I collagen production and a reduction in the steady-state levels of alpha1(I), alpha2(I), and alpha1(III) mRNA in normal and SSc cells. A 60-70% inhibition of type I collagen production and a 70-80% reduction in the mRNA levels for alpha1(I), alpha2(I), and alpha1(III) were observed at 10 microM GGTI-298. In contrast, the expression of fibronectin, cyclooxygenase 1, and GAPDH was not affected. The effects on alpha1(I) collagen mRNA resulted from a profound reduction in transcription of the alpha1(I) collagen gene promoter. GGTI-298 did not affect cellular viability or morphology. CONCLUSION: These results demonstrate that specific inhibition of geranylgeranyl prenylation causes a potent and selective inhibition of expression of the genes encoding types I and III collagens, without affecting cellular viability. The findings indicate that inhibition of geranylgeranyl prenylation should be further studied as a potential therapeutic approach for SSc and other fibrosing diseases.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Colágeno/metabolismo , Escleroderma Sistêmico/enzimologia , Pele/enzimologia , Actinas/efeitos dos fármacos , Actinas/genética , Actinas/metabolismo , Benzamidas/farmacologia , Linhagem Celular , Linhagem Celular Transformada , Colágeno/efeitos dos fármacos , Colágeno/genética , Ciclo-Oxigenase 1 , Inibidores Enzimáticos/farmacologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibronectinas/metabolismo , Humanos , Isoenzimas/metabolismo , Proteínas de Membrana , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/metabolismo , Escleroderma Sistêmico/patologia , Pele/patologia , Transcrição Gênica/efeitos dos fármacosRESUMO
During limb development, chondrocytes located at the epiphyseal tip of long bone models give rise to articular tissue, whereas the more numerous chondrocytes in the shaft undergo maturation, hypertrophy, and mineralization and are replaced by bone cells. It is not understood how chondrocytes follow these alternative pathways to distinct fates and functions. In this study we describe the cloning of C-1-1, a novel variant of the ets transcription factor ch-ERG. C-1-1 lacks a short 27-amino acid segment located approximately 80 amino acids upstream of the ets DNA binding domain. We found that in chick embryo long bone anlagen, C-1-1 expression characterizes developing articular chondrocytes, whereas ch-ERG expression is particularly prominent in prehypertrophic chondrocytes in the growth plate. To analyze the function of C-1-1 and ch-ERG, viral vectors were used to constitutively express each factor in developing chick leg buds and cultured chondrocytes. We found that virally driven expression of C-1-1 maintained chondrocytes in a stable and immature phenotype, blocked their maturation into hypertrophic cells, and prevented the replacement of cartilage with bone. It also induced synthesis of tenascin-C, an extracellular matrix protein that is a unique product of developing articular chondrocytes. In contrast, virally driven expression of ch-ERG significantly stimulated chondrocyte maturation in culture, as indicated by increases in alkaline phosphatase activity and deposition of a mineralized matrix; however, it had modest effects in vivo. The data show that C-1-1 and ch-ERG have diverse biological properties and distinct expression patterns during skeletogenesis, and are part of molecular mechanisms by which limb chondrocytes follow alternative developmental pathways. C-1-1 is the first transcription factor identified to date that appears to be instrumental in the genesis and function of epiphyseal articular chondrocytes.
Assuntos
Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Diferenciação Celular/genética , Condrócitos/enzimologia , Proteínas de Ligação a DNA , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Transativadores , Fatores de Transcrição/genética , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Animais , Sequência de Bases , Calcificação Fisiológica/genética , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Clonagem Molecular , Expressão Gênica , Hibridização In Situ , Técnicas In Vitro , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Botões de Extremidades/enzimologia , Proteínas Oncogênicas/genética , Especificidade de Órgãos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tenascina/biossíntese , Tenascina/genética , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG , TransfecçãoRESUMO
Alignment of tropoelastin molecules during the process of elastogenesis is thought to require fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, fibrillin-1 and fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of fibrillin-2 and fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.
Assuntos
Proteínas dos Microfilamentos/química , Fragmentos de Peptídeos/química , Tropoelastina/química , Sequência de Aminoácidos , Anticorpos , Sítios de Ligação , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Elasticidade , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Glicosilação , Humanos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tropoelastina/metabolismoRESUMO
The cytokine transforming growth factor-beta (TGF-beta) has multiple effects on a variety of cell types, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. In the present work, we demonstrate that TGF-beta1 produces a fourfold increase in transcription of the fibronectin gene in cultured human fetal lung fibroblasts with only a small increase in mRNA stability resulting in a significant increase in fibronectin mRNA steady state level. A corresponding increase in production of fibronectin protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that geranylgeranylated, but not farnesylated or acylated protein(s), protein kinase C-delta, phosphatidylcholine-specific phospholipse C, tyrosine kinase activity, and stress-activated protein kinase p38 are required for this TGF-beta1 effect. Trimeric G proteins and mitogen-activated protein kinases erk1 and erk2 do not appear to be involved. While these results emphasize the complexities involved in the control of extracellular matrix synthesis by TGF-beta, they also identify reaction sites that may be amenable to pharmacologic modulation. Such modulation could be of great advantage in the treatment of a wide variety of undesirable fibrotic reactions.
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Isoenzimas/metabolismo , Pulmão/metabolismo , Proteína Quinase C/metabolismo , Transcrição Gênica/fisiologia , Fator de Crescimento Transformador beta/farmacologia , Fosfolipases Tipo C/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular , Cicloeximida/farmacologia , Fibroblastos/metabolismo , Genisteína/farmacologia , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Norbornanos , Inibidores de Fosfodiesterase/farmacologia , Proteína Quinase C-delta , Prenilação de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiocarbamatos , Tionas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Condrócitos/fisiologia , Proteínas da Matriz Extracelular , Osteogênese/fisiologia , Ácido 1-Carboxiglutâmico/biossíntese , Ácido 1-Carboxiglutâmico/genética , Ácido 1-Carboxiglutâmico/metabolismo , Animais , Osso e Ossos/metabolismo , Calcificação Fisiológica/fisiologia , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/embriologia , Microscopia Eletrônica de Varredura , Minerais/metabolismo , Osteogênese/efeitos dos fármacos , Vitamina K/biossíntese , Vitamina K/genética , Vitamina K/metabolismo , Varfarina/farmacologia , Proteína de Matriz GlaRESUMO
Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato , Glicoproteínas de Membrana/metabolismo , Tropoelastina/metabolismo , Animais , Aorta/metabolismo , Moléculas de Adesão Celular , Relação Dose-Resposta a Droga , Heparitina Sulfato/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Ligação Proteica , Proteoglicanas/metabolismo , Proteínas Recombinantes/metabolismo , Albumina Sérica/metabolismo , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
The TGF-betas are multipotent in their biological activity, modulating cell growth and differentiation as well as extracellular matrix deposition and degradation. Most of these activities involve modulation of gene transcription. However, TGF-beta1 has been shown previously to substantially increase the expression of elastin by stabilization of tropoelastin mRNA through a signaling pathway which involves a phosphatidylcholine-specific phospholipase and a protein kinase C. The present results, through the use of specific inhibitors of geranylgeranyl transferase I, farnesyl transferase, and acyl transferase, demonstrate that geranylgeranylated and acylated, but not farnesyslated protein(s) is required for this TGF-beta1 effect. In addition, the general tyrosine kinase inhibitor genistein completely blocked this TGF-beta1 effect. The results suggest that the TGF-beta1 signaling pathway requires not only receptor ser/thr kinase activity, but also tyrosine kinase and small GTPase activities.
Assuntos
Aciltransferases/metabolismo , Alquil e Aril Transferases/metabolismo , Elastina/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica , Fator de Crescimento Transformador beta/farmacologia , Tropoelastina/genética , Benzamidas/farmacologia , Linhagem Celular , Toxina da Cólera/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos , GTP Fosfo-Hidrolases/metabolismo , Humanos , Pulmão , Metionina/análogos & derivados , Metionina/farmacologia , Proteína Quinase C/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Prenilação de Proteína/fisiologia , Transcrição Gênica/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Human submandibular saliva reduces human immunodeficiency virus type 1 (HIV-1) infection in vitro. To define the mechanism of inhibition, virus was incubated with saliva or medium, velocity sucrose gradient centrifugation was performed, and fractions were analyzed for p24 and gp120. The results show that after incubation with saliva, the envelope glycoprotein was displaced from both a laboratory-adapted and a low-passage clinical HIV-1 isolate. To identify the salivary protein(s) responsible, submandibular saliva was fractionated by anion- exchange chromatography. Protein fractions containing anti-HIV activity were assayed for their ability to strip gp120 from virus. The partially purified active fractions contained two high-molecular-weight sialyated glycoproteins identified as salivary agglutinin and mucin, as well as several lower-molecular-weight proteins. It thus appears that specific salivary proteins interact with HIV-1 to strip gp120 from the virus with a resultant decrease in infectivity.