RESUMEN
Prolyl 3-hydroxylation is a rare collagen type I post translational modification in fibrillar collagens. The primary 3Hyp substrate sites in type I collagen are targeted by an endoplasmic reticulum (ER) complex composed by cartilage associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and prolyl cis/trans isomerase B, whose mutations cause recessive forms of osteogenesis imperfecta with impaired levels of α1(I)3Hyp986. The absence of collagen type I 3Hyp in wild type zebrafish provides the unique opportunity to clarify the role of the complex in vertebrate. Zebrafish knock outs for crtap and p3h1 were generated by CRISPR/Cas9. Mutant fish have the typical OI patients' reduced size, body disproportion and altered mineralization. Vertebral body fusions, deformities and fractures are accompanied to reduced size, thickness and bone volume. Intracellularly, collagen type I is overmodified, and partially retained causing enlarged ER cisternae. In the extracellular matrix the abnormal collagen type I assembles in disorganized fibers characterized by altered diameter. The data support the defective chaperone role of the 3-hydroxylation complex as the primary cause of the skeletal phenotype.
Asunto(s)
Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de la Matriz Extracelular/genética , Osteogénesis Imperfecta/genética , Prolil Hidroxilasas/genética , Animales , Sistemas CRISPR-Cas , Ciclofilinas/genética , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Hidroxilación , Osteogénesis Imperfecta/metabolismo , Fenotipo , Prolil Hidroxilasas/química , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
OBJECTIVE: We previously demonstrated the ability of matrilin-3 to modulate the gene expression profile of primary human chondrocytes (PHCs) toward a state favoring cartilage catabolism. The structure within matrilin-3 responsible for the induction of these catabolic genes is unknown. Here, we investigated the potential of matrilin-3 (MATN3) and truncated matrilin-3 proteins, in both monomeric and oligomeric form, to stimulate interleukin (IL)-6 release in PHCs. METHODS: We expressed full-length matrilin-3 oligomers, matrilin-3 von Willebrand factor A (VWA) domain oligomers, matrilin-3 four epidermal growth factor (EGF) domain oligomers, matrilin-3 monomers without oligomerization domains, matrilin-3 VWA domain monomers, and matrilin-3 4EGF monomers. We then incubated PHCs in the absence or presence of full-length matrilin-3 or one of the truncated matrilin-3 proteins and finally determined the release of IL-6 in cell-culture supernatants. RESULTS: The addition of full-length matrilin-3 oligomers, matrilin-3 VWA domain oligomers, and, less pronounced, matrilin-3 monomers without oligomerization domains, and matrilin-3 4EGF-oligomers to the cell-culture medium led to a significant induction of IL-6 in PHCs. DISCUSSION: Based on recombinant expression of different matrilin-3 domains in both monomeric and oligomeric form, this work demonstrated that the VWA1 domain of matrilin-3 is primarily responsible for the induction of IL-6 release and that the oligomerization of the VWA1 domain markedly promotes its activity.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Proteínas de la Matriz Extracelular/genética , Interleucina-6/metabolismo , Proteínas Matrilinas/farmacología , Anciano , Proteína Receptora de AMP Cíclico , Humanos , Proteínas Matrilinas/genética , Persona de Mediana EdadRESUMEN
We have isolated and characterized the gene encoding mouse matrilin-4 (Matn4), an extracellular matrix protein present in a broad spectrum of tissues. The gene spanned 16 kb, consisted of 12 exons, and localized to chromosome 2. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, did not follow the GT-AG rule and belonged to the subgroup of introns having AT-AC at the ends. Matn4 contained two exons in the 5' UTR that could be alternatively spliced. We localized a major and a minor transcription start site to two different untranslated exons: exon 0a and exon 0b. Matn4 divergently overlapped 5' with the gene encoding RBP-L (for recombining binding protein suppressor of hairless-like; Rbpsuhl), a transcription factor with homology to RBP-JK. Exon 1 of Rbpsuhl was located in the second intron of Matn4, whereas exon 0a, the first exon of Matn4, was located in the second intron of Rbpsuhl. The second exons of the respective genes overlapped in an antisense orientation. We mapped the major transcription start of Rbpsuhl to a position approximately 150 nt upstream of the splice acceptor site of the first intron, leading to the synthesis of a truncated variant of RBP-L probably missing the amino-terminal 121 amino acid residues. We analyzed the expression of the different Matn4 and Rbpsuhl transcripts by quantitative RT-PCR; this showed the highest expression for both genes in lung and brain. In situ hybridization of brain sections showed a partially overlapping expression pattern for the two genes.
Asunto(s)
Regiones no Traducidas 5'/genética , Proteínas de Unión al ADN/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Genes Sobrepuestos/genética , Factores de Transcripción/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Mapeo Cromosómico/métodos , Proteínas de Unión al ADN/biosíntesis , Orden Génico/genética , Biblioteca Genómica , Proteínas Matrilinas , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Edición de ARN/genética , ARN Mensajero/genéticaRESUMEN
Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapter proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purified using affinity tags, and subjected to biochemical characterization. The largest oligomeric form of recombinantly expressed full-length matrilin-4 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electrophoresis, and mass spectrometry. Proteolytically processed matrilin-4 species were also detected. The cleavage occurs in the short linker region between the second von Willebrand factor A-like domain and the coiled-coil domain leading to the release of large fragments and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts similar degradation products could be detected, indicating that a related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos. Affinity-purified antibodies detect a broad expression in dense and loose connective tissue, bone, cartilage, central and peripheral nervous systems and in association with basement membranes. In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Membrana Basal/metabolismo , Línea Celular , Dimerización , Embrión de Mamíferos , Proteínas de la Matriz Extracelular/química , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Sustancias Macromoleculares , Proteínas Matrilinas , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Subunidades de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Cráneo/metabolismo , Transcripción Genética , Transfección , Factor de von Willebrand/químicaRESUMEN
Matrilin-3 is a recently identified member of the superfamily of proteins containing von Willebrand factor A-like domains and is able to form hetero-oligomers with matrilin-1 (cartilage matrix protein) via a C-terminal coiled-coil domain. Full-length matrilin-3 and a fragment lacking the assembly domain were expressed in 293-EBNA cells, purified, and subjected to biochemical characterization. Recombinantly expressed full-length matrilin-3 occurs as monomers, dimers, trimers, and tetramers, as detected by electron microscopy and SDS-polyacrylamide gel electrophoresis, whereas matrilin-3, purified from fetal calf cartilage, forms homotetramers as well as hetero-oligomers of variable stoichiometry with matrilin-1. In the matrix formed by cultured chondrosarcoma cells, matrilin-3 is found in a filamentous, collagen-dependent network connecting cells and in a collagen-independent pericellular network. Affinity-purified antibodies detect matrilin-3 expression in a variety of mouse cartilaginous tissues, such as sternum, articular, and epiphyseal cartilage, and in the cartilage anlage of developing bones. It is found both inside the lacunae and in the interterritorial matrix of the resting, proliferating, hypertrophic, and calcified cartilage zones, whereas the expression is lower in the superficial articular cartilage. In trachea and in costal cartilage of adult mice, an expression was seen in the perichondrium. Furthermore, matrilin-3 is found in bone, and its expression is, therefore, not restricted to chondroblasts and chondrocytes.
Asunto(s)
Desarrollo Óseo , Proteínas de la Matriz Extracelular/metabolismo , Animales , Animales Recién Nacidos , Bovinos , Condrocitos/metabolismo , Colágeno/metabolismo , Tejido Conectivo/crecimiento & desarrollo , Electroforesis en Gel Bidimensional , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Espectrometría de Masas , Proteínas Matrilinas , Ratones , Microscopía Electrónica , Conformación Proteica , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestructuraRESUMEN
The gene for murine matrilin-3, an extracellular matrix protein present in cartilage, was isolated and further characterized. The gene spans 23.4 kb and comprises 8 exons; with one exception, this reflects the modular structure of the protein. The major and a minor transcription start site were determined by RNase protection assays to positions approximately 72 nt and 87 nt upstream of the ATG codon, respectively. The promoter contains a TATA-like box 32 bp upstream of the main transcription start as well as several potential binding sites for eukaryotic transcription factors. As in all known matrilin genes, the last intron, separating the exons coding for the coiled-coil domain, does not follow the GT-AG rule and belongs to the subgroup of introns having AT-AC at the ends that are spliced by the U12-type spliceosome. The mouse matrilin-3 gene does not contain hidden exon sequences coding for the second vWFA-like domain present in all other matrilins. The intron that could possibly contain such sequences instead shows 75% repetitive sequences, indicating an evolutionary process that has led to the loss of sequences coding for vWFA2. Single-strand conformation polymorphism analysis was used to map the Matn3 gene to the proximal end of Chr 12, linked to the genes Synd1, Apob, Dntb, and Kif3c.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Exones , Intrones , Proteínas Matrilinas , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas , Mapeo RestrictivoRESUMEN
The composition and the content of gangliosides changes during physiological growth and differentiation as well as in neoplastic cell transformation. In order to determine if ganglioside profiles correlate with brain tumour malignancy, the ganglioside distribution was determined in 31 gliomas of astrocytic origin and in non-tumour tissue by a recently developed microbore high performance liquid chromatography (HPLC) method. Glioma malignancy was graded according to the grading system proposed by the World Health Organization (WHO) in 1993. In general, an increase of GD3 and a decrease of normal brain gangliosides correlated with a higher grade of malignancy. Pilocytic astrocytomas Grade I had a distinctive ganglioside profile, histologically as well as biochemically. Although they are low-grade gliomas, the pilocytic astrocytomas exhibited a GD3 content comparable to anaplastic gliomas and could only be biochemically distinguished from other tumour grades by relatively high type "b" ganglioside levels. Thus, ganglioside composition not only reflects anaplasia but can also be used to indicate biological characteristics of tumours of different histogenetic origin.
Asunto(s)
Neoplasias Encefálicas/química , Cromatografía Líquida de Alta Presión , Gangliósidos/análisis , Glioma/química , Astrocitoma/química , Astrocitoma/patología , Neoplasias Encefálicas/patología , Transformación Celular Neoplásica/química , Transformación Celular Neoplásica/patología , Glioblastoma/química , Glioblastoma/patología , Glioma/patología , HumanosRESUMEN
We have recently cloned a cDNA for mouse matrilin-4. By sequence comparison we identified the 12 kb long human matrilin-4 gene as a part of a high-throughput genomic sequence (HS453C12) in the databases. Additionally we found a human matrilin-4 expressed sequence tag (H54037) in the database that had been mapped to chromosome 20q13.1-2. The gene contains 10 exons and, like the matrilin-1 gene, the human matrilin-4 gene contains an AT-AC intron between the two exons encoding the coiled-coil domain. The cDNA sequence of human matrilin-4 was determined by sequencing of RT-PCR products obtained from mRNA of the human embryonic kidney cell line HEK 293. At the amino acid level it showed an overall sequence identity to the mature mouse matrilin-4 of 91% with a maximum of 97% in the second vWFA-like module. Alternative splicing leads to three different mRNAs. They all encode the putative signal peptide, the two vWFA-like domains and the potential coiled-coil alpha-helical oligomerisation domain but differ in that either one, two or three EGF-like domains are retained in the mature mRNA. Due to a G to A mutation at the splice donor site of intron C, the third exon encodes an untranslated pseudo-exon specifying the first EGF-like domain when compared to mouse matrilin-4.
Asunto(s)
Empalme Alternativo , Cromosomas Humanos Par 20 , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario , Bases de Datos como Asunto , Exones , Proteínas de la Matriz Extracelular/biosíntesis , Variación Genética , Humanos , Intrones , Sustancias Macromoleculares , Proteínas Matrilinas , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Mouse cDNA encoding for matrilin-4 was cloned and the primary structure of this fourth member of the matrilin family was deduced from the nucleotide sequence. The protein precursor of 624 amino acids consists of a putative signal peptide, two vWFA-like domains linked by four epidermal growth factor-like modules and a potential coiled-coil alpha-helical oligomerization domain at the C-terminus. The predicted Mr of the mature protein is 66 442. Expression in lung, brain, sternum, kidney and heart was detected by Northern blot analysis of mouse mRNA. Additionally an alternatively spliced mRNA lacking the sequence coding for the first vWFA domain was found in 7 weeks old mice leading to a protein precursor of 434 amino acids and a predicted Mr of the mature protein of 45468.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Bases de Datos Factuales , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Expresión Génica , Proteínas Matrilinas , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Alineación de Secuencia , Lugares Marcados de Secuencia , Distribución Tisular , Factor de von Willebrand/metabolismoRESUMEN
A mouse cDNA encoding for matrilin-3, the third member of the novel matrilin family of extracellular matrix proteins, was cloned. The protein precursor of 481 amino acids consists of a putative signal peptide, a short positively charged sequence, a single vWFA-like domain followed by four epidermal growth factor-like modules and a potential coiled-coil alpha-helical oligomerization domain at the C-terminus. It is the smallest member of the matrilin family with a predicted Mr of the mature protein of 48 902. The primary structure of a C-terminal portion of 310 amino acids of the human matrilin-3 was determined and showed a sequence identity to the mouse matrilin-3 of 84.8%. Northern blot hybridization of mouse matrilin-3 mRNA showed a 2.9 kb mRNA expressed in sternum, femur and trachea and indicates a cartilage-specific expression.
Asunto(s)
Proteínas de la Matriz Extracelular/química , Glicoproteínas/química , Factor de von Willebrand/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Proteína de la Matriz Oligomérica del Cartílago , Proteínas de la Matriz Extracelular/genética , Femenino , Glicoproteínas/genética , Humanos , Pulmón/química , Proteínas Matrilinas , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Alineación de Secuencia , Factor de von Willebrand/genéticaRESUMEN
A highly sensitive analytical method was developed that allows the separation of ganglioside mixtures and quantification of individual non-derivatized gangliosides in the concentration range between 2 pmol and 1 nmol. Gangliosides were separated with a gradient of acetonitrile/phosphate buffer on a 1 mm diameter microbore HPLC column packed with Spherisorb-NH2. They eluted according to their number of sialic acid residues with increasing phosphate and decreasing acetonitrile concentrations. The separation of different gangliosides with equal sialic acid content is also described. The column effluent was monitored at the maximum of absorption at 197 nm. The sensitivity is higher than resorcinol staining of fractionated gangliosides by thin layer chromatography, previously the standard method for ganglioside analysis. The separated gangliosides can be analyzed by further methods. The HPLC method described here has been applied to the analysis of serum and oligodendroglioma specimens.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Gangliósidos/análisis , Oligodendroglioma/química , Cromatografía Líquida de Alta Presión/instrumentación , Femenino , Gangliósidos/sangre , Gangliósidos/clasificación , Humanos , Sensibilidad y Especificidad , Espectrofotometría UltravioletaRESUMEN
Eight mollicellins (depsidones) were assayed for mutagenicity and antibacterial activity in Salmonella/microsome tests involving histidine reversion and forward mutation to 8-azaguanine resistance. Two of them, mollicellins C and E, which contain a 3-methylbutenoic acid moiety, were mutagenic and bactericidal for Salmonella typhimurium in the absence of microsomes. Mollicellins D and F, each containing a chlorine atom, were bactericidal but not mutagenic. The mutagenic activity was completely abolished and the antibiotic activity was greatly reduced by coincubation with rat liver microsomes.
Asunto(s)
Antiinfecciosos , Ascomicetos/metabolismo , Chaetomium/metabolismo , Mutágenos , Micotoxinas/farmacología , Salmonella typhimurium/efectos de los fármacos , Animales , Antibacterianos , Azaguanina/farmacología , Fenómenos Químicos , Química , Farmacorresistencia Microbiana , Histidina/metabolismo , Microsomas Hepáticos/metabolismo , Mutación , Micotoxinas/biosíntesis , Ratas , Salmonella typhimurium/genéticaRESUMEN
Production of luteoskyrin, a hepatotoxin synthesized by Penicillium islandicum Sopp., was studied with various fermentation methods. Best results were obtained in static fermentations on glutinous rice at 30 degrees C. The isolated yield of pure luteoskyrin was approximately 400 mg per kg of rice. Also produced were skyrin, islandicin, iridoskyrin, rubroskyrin, chrysophanol, mannitol, and erythritol. A new metabolite, which we call pibasterol, was also isolated.
Asunto(s)
Naftoquinonas/biosíntesis , Penicillium/metabolismo , Pigmentos Biológicos/biosíntesis , Antraquinonas/aislamiento & purificación , Fenómenos Químicos , Química , Medios de Cultivo , Grano Comestible , Fermentación , Pigmentos Biológicos/aislamiento & purificación , EsterolesRESUMEN
The production of the new mycotoxin malformin C by a solid substrate fermentation is described. Malformin C is highly toxic (mean lethal dose = 0.9 mg/kg) and exerts antibacterial activity against a variety of gram-positive and gram-negative organisms.
Asunto(s)
Aspergillus niger/metabolismo , Aspergillus/metabolismo , Micotoxinas/biosíntesis , Bacterias/efectos de los fármacos , Grano Comestible/microbiología , Fermentación , Micotoxinas/farmacología , Especificidad de la EspecieRESUMEN
Production of cyclopiazonic acid by Aspergillus flavus is reported for the first time. A procedure for its production by agitated solid substrate fermentation on red wheat is described along with the isolation procedure and physical and chemical properties of this indole derivative. The compound has been found to exert antibacterial activity.
Asunto(s)
Aspergillus flavus/metabolismo , Micotoxinas/biosíntesis , Bacillus megaterium/efectos de los fármacos , Compuestos Heterocíclicos/biosíntesis , Compuestos Heterocíclicos/aislamiento & purificación , Micotoxinas/aislamiento & purificaciónRESUMEN
This report describes an improved procedure for production of cytochalasin E and tremorgens by solid substrate, agitated fermentation of Aspergillus clavatus on pearled barley.