Search PubMed⌕ Search

Biomedical subjects

C M Kielty

Publications and source records attributed to C M Kielty.

At least 55 records · Page 3Linked to original sources
PubMed1995

The calcium binding properties and molecular organization of epidermal growth factor-like domains in human fibrillin-1.

Human fibrillin-1 is a 350-kDa glycoprotein found in 10-nm connective tissue microfibrils. Mutations in the gene encoding this protein cause the Marfan syndrome, a disease characterized by cardiovascular, ocular, and skeletal abnormalities. Fibrillin-1 has a modular structure that includes 47 epidermal growth factor-like (EGF-like) domains, 43 of which contain a consensus sequence associated with calcium binding. A mutation causing an Asn-2144 --> Ser amino acid change in one of the potential calcium binding residues has been described in a patient with the Marfan syndrome. We have chemically synthesized a wild-type EGF-like domain (residues 2126-2165 of human fibrillin-1) and a mutant EGF-like domain containing the Asn-2144 --> Ser amino acid change and measured calcium binding to each using 1H-NMR spectroscopy. The wild-type domain binds calcium with a similar affinity to isolated EGF-like domains from coagulation factors IX and X; however, the mutant domain exhibits > 5-fold reduction in affinity. Rotary shadowing of fibrillin-containing microfibrils, isolated from dermal fibroblast cultures obtained from the Marfan patient, shows that the mutation does not prevent assembly of fibrillin into microfibrils but does alter the appearance of the interbead region. We have modeled a region of fibrillin-1 (residues 2126-2331) encompassing five calcium binding EGF-like domains, using data derived from the recently determined crystal structure of a calcium binding EGF-like domain from human factor IX. Our model suggests that these fibrillin-1 EGF-like domains adopt a helical arrangement stabilized by calcium and that defective calcium binding to a single EGF-like domain results in distortion of the helix. We propose a mechanism for the interaction of contiguous arrays of calcium binding EGF-like domains within the microfibril.

Amino Acid Sequence↗
PubMed1995

Fibrillin-containing microfibrils: structure and function in health and disease.

Fibrillin-containing microfibrils are a unique class of connective tissue macromolecules whose critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the recent linkage of their principal structural component fibrillin to Marfan syndrome, a heritable disorder with pleiotrophic connective tissue manifestations. The complexity of the structure: function relationships of these macromolecules was highlighted by the recent elucidation of the primary structure of fibrillin and characterisation of fibrillin mutations in Marfan patients. This review examines current understanding of the expression and assembly of fibrillin and describes new approaches which are now being applied to elucidate the many outstanding structural, organisational and functional aspects of the fibrillin-containing microfibrils.

Connective Tissue↗
PubMed1995

Marfan syndrome: fibrillin expression and microfibrillar abnormalities in a family with predominant ocular defects.

We have found abnormal fibrillin microfibrils isolated from tissues and cell cultures from two cousins with Marfan syndrome whose major clinical abnormality is bilateral ectopia lentis, but who also have skeletal involvement but no cardiovascular defects. Ultrastructural analysis of ciliary zonules showed the presence of abundant loose microfibril bundles which in many places appeared disorganised. Microfibrils isolated from ciliary zonules and vitreous were highly fragmented when examined by rotary shadowing electron microscopy. Investigation of microfibrils elaborated by patient dermal fibroblasts showed remarkable variations in periodicity and packing. The synthesis and secretion of fibrillin by these cells was confirmed electrophoretically with the identification of metabolically labelled immunoprecipitated fibrillin (M(r) 300,000) in medium and cell layer compartments. These data show that fibrillin expression is normal but that assembled microfibrils are manifestly abnormal both morphologically and functionally. The occurrence of microfibrils with variable periodicities and susceptibility to fragmentation suggests that structural weakness is probably the primary cause of lens dislocation in these patients.

Actin Cytoskeleton↗
PubMed1995

Cysteine-to-arginine point mutation in a 'hybrid' eight-cysteine domain of FBN1: consequences for fibrillin aggregation and microfibril assembly.

Mutations in the FBN1 gene encoding the microfibrillar glycoprotein fibrillin cause Marfan syndrome, a relatively common autosomal dominant connective tissue disease. Causative FBN1 mutations appear to be dispersed throughout the coding frame, and to date no predictable genotype: phenotype correlations have emerged. We have identified a point mutation within an eight-cysteine 'hybrid' motif of the fibrillin polypeptide which results in the substitution of an arginine for a cysteine, in a patient severely affected in the cardiovascular, skeletal and ocular systems. We have utilised cell cultures from various tissues of this patient to investigate the effects of this mutation on fibrillin expression and deposition, and the consequences in terms of microfibril assembly and organisation. We have established that there is no difference in the expression of normal and mutant alleles, and fibrillin synthesis, secretion and deposition are also normal. However, the rate of fibrillin aggregation is reduced and microfibrillar assemblies are both remarkably scarce and morphologically abnormal. These data clearly demonstrate that the mutated allele interferes with normal assembly, and strongly implicate this particular region of the fibrillin-1 molecule in stabilising microfibrillar assemblies.

Actin Cytoskeleton↗
PubMed1995

Expression of type VI collagen mRNAs in human endometrium during the menstrual cycle and first trimester of pregnancy.

The pattern of type VI collagen deposition in human endometrium throughout the menstrual cycle and in decidua of the first trimester of pregnancy was studied. Immunohistochemical analysis demonstrated a dense microfibrillar network of collagen VI in the stroma of preimplantation endometrium which was reduced during the peri-implantation period and no longer detected in first trimester decidua. However, type VI collagen was consistently present within blood vessel walls in both endometrium and decidua. Using in situ hybridization, mRNAs encoding alpha 1(VI), alpha 2(VI) and alpha 3(VI) chains within endometrial stromal and vascular cells were identified. All three mRNA species are abundant in the villous mesenchyme of the first trimester placenta. The detection of collagen VI mRNA species in the endometrium throughout the menstrual cycle suggests that the apparent decrease in abundance of extracellular immunoreactive fibrils may be a consequence of translational control, matrix redistribution or turnover. In contrast, in the first trimester of pregnancy, collagen VI protein was mainly absent from the decidual stroma and amounts of mRNA were very low, indicating a significant reduction in production. Loss of stromal type VI collagen contributes to the remodelling of the maternal extracellular matrix of pregnancy.

Animals↗
PubMed1994

Analyses of truncated fibrillin caused by a 366 bp deletion in the FBN1 gene resulting in Marfan syndrome.

We studied fibrillin synthesis in cultured fibroblasts from 11 members of a three-generation family with Marfan syndrome, caused by a large in-frame deletion in FBN1 (the fibrillin gene) leading to a loss of 366 bases in the corresponding fibrillin mRNA. Metabolic labelling with [35S]Met/Cys and SDS/PAGE allowed unequivocal identification of normal and truncated fibrillin in all cell strains harbouring the deletion. In culture medium, fibrillin and its truncated counterpart were predominant, whereas their respective larger precursors were found only in traces. This proportion, however, was markedly shifted towards the normal and truncated precursors by EGTA and reversed by the addition of calcium, which confirmed the existence of profibrillin and its probably calcium-dependent conversion into fibrillin. Tunicamycin caused increased electrophoretic mobility of normal and truncated molecules without changing their apparent size differences. Intracellularly, only profibrillin was found; in the mutant cells truncated and normal profibrillin molecules were present in similar amounts and both populations were secreted and deposited simultaneously into the extracellular matrix; there, however, truncated profibrillin only became easily detectable after treatment of cells with dextran sulphate, which increased the amount of extractable profibrillin. Immunofluorescence microscopy in patients' cultures identified fibrillin-containing microfibrils which appeared to be moderately reduced both in amount and diameter. Ultrastructural analysis by rotary-shadowing and immunogold electron microscopy demonstrated the presence of numerous beaded domains reacting with fibrillin antibodies, but no intact fibrillin microfibrils in patient's cell-layer extracts, in contrast with the extensive microfibrils elaborated by control cultures. Our findings suggest, that in the patients' cell cultures all microfibrils contained the truncated fibrillin molecules.

Actin Cytoskeleton↗
PubMed1994

Catabolism of intact fibrillin microfibrils by neutrophil elastase, chymotrypsin and trypsin.

We present ultrastructural and biochemical evidence for the turnover of intact fibrillin microfibrils by the serine proteinases, neutrophil elastase, chymotrypsin and trypsin. Rotary shadowing electron microscopy revealed that serine proteinase treatment of intact microfibrils isolated from foetal bovine skin resulted in extensive degradation. Microfibrils were destroyed by neutrophil elastase and effectively disrupted by chymotrypsin and trypsin, with no morphologically identifiable arrays remaining. Evidence of defined fibrillin degradation products was obtained by Western blotting of these enzyme-treated fibrillin assemblies. Fibrillin immunoprecipitated from dermal fibroblast culture medium was also comprehensively degraded by these enzymes. These observations demonstrate that serine proteinases are potent effectors for the physiological and pathological catabolism of microfibrils, and suggest a key role in elastic fibre degradation.

Animals↗
PubMed1994

Fibrillin secretion and microfibril assembly by Marfan dermal fibroblasts.

The Marfan syndrome has been linked to the FBN1 gene encoding the microfibrillar glycoprotein fibrillin. To date, there have been no descriptions of microfibrillar abnormalities characteristic of this connective tissue disorder, although biochemical analyses have highlighted apparent abnormalities in fibrillin synthesis, secretion and processing. We have conducted a biochemical and ultrastructural investigation of fibrillin expression and assembly by a panel of dermal fibroblast lines from patients with Marfan syndrome and related diseases. The study has highlighted marked differences between cells in terms of secretion and aggregation of newly-synthesised fibrillin. In addition, electron microscopic visualization of fibrillin assemblies has clearly demonstrated for the first time the plethora of microfibrillar abnormalities that underlie this heterogeneous disorder. These data emphasize the molecular complexity that is a feature of the diverse clinical phenotypes exhibited by Marfan patients.

Autoradiography↗
PubMed1994

Abnormal fibrillin assembly by dermal fibroblasts from two patients with Marfan syndrome.

The microfibrillar glycoprotein fibrillin is linked to the Marfan syndrome, an autosomal dominant connective tissue disorder. In this study, fibrillin synthesis, deposition and assembly has been investigated in Marfan dermal fibroblast lines from two unrelated patients for whom distinct mutations in the fibrillin gene FBN1 have been identified. In patient NB, a point mutation has occurred which causes an amino acid substitution and the other patient (GK) has a deletion in one allele. The two cell lines were broadly comparable with respect to de novo fibrillin synthesis and its distribution between medium and cell layer compartments. Electrophoresis of fibrillin immunoprecipitates confirmed the presence of fibrillin in medium and cell layers. GK cells secreted an additional higher relative molecular mass fibrillin-immunoreactive component. The time-course of fibrillin secretion was similar for the two lines, but differences in fibrillin aggregation were apparent. Rotary shadowing electron microscopy of extracted cell layers demonstrated the presence of abundant and extensive microfibrils in NB cell layers. These were abnormal in their gross morphology in comparison to microfibrils isolated from control cultures. No periodic microfibrillar structures were isolated from GK cell layers. These studies underline the need to classify fibrillin defects in terms of biochemical and ultrastructural criteria. Examination of the effects of individual mutations on microfibril organization will be particularly informative in elucidating the relationship between microfibril dysfunction and the complex clinical manifestations of Marfan patients.

Cell Line↗
PubMed1993

The role of calcium in the organization of fibrillin microfibrils.

The microfibrillar glycoprotein fibrillin has a multidomain structure which contains forty-three epidermal growth factor-like motifs with calcium-binding consensus sequences. We have utilized intact microfibrils isolated from human dermal fibroblast cultures to investigate the putative influence of bound calcium on microfibrillar organization and integrity. Incubation with EDTA or EGTA rapidly resulted in gross disruption of microfibril morphology. The treatment induced disorganization of the interbead domains although the regular beaded arrangement was always apparent. These changes were readily reversible on replacing calcium, indicating that the treatment had not compromised microfibrillar integrity. The data localize calcium binding EGF-like repeats to the interbead domains and indicate that lateral packing of fibrillin monomers is calcium-dependent. This arrangement suggests how mutations in epidermal growth factor-like domains of fibrillin might cause the disruption in microfibril organization and interactions which underlies the clinical symptoms of some Marfan syndrome patients.

Adult↗
PubMed1993

Catabolism of intact type VI collagen microfibrils: susceptibility to degradation by serine proteinases.

We present the first direct biochemical evidence for the turnover of intact type VI collagen microfibrils. Matrix-degrading enzymes of the serine proteinase class, including rat mast cell chymases I and II, human mast cell tryptase, neutrophil elastase, cathepsin G and trypsin, were able to catabolize intact type VI collagen microfibrils isolated from foetal bovine skin and metabolically labelled intact type VI collagen immunoprecipitated from fibroblast culture medium. By contrast, intact type VI collagen was not degraded by the human matrix metalloproteinases, MMP-1, MMP-2, MMP-3 and MMP-9. These data have important implications for the stability of type VI collagen in connective tissues and highlight the potential role of serine proteinases both in normal type VI collagen turnover and in inflammatory conditions characterized by matrix degradation.

Animals↗
PubMed1993

An immunohistochemical study examining the role of collagen type VI in the rodent periodontal ligament.

The distribution of collagen types I and VI has been examined in the periodontal ligaments of rat incisor and molar teeth using cryosections and immunohistochemical staining procedures. The stain for collagen type I was uniform in all the ligaments examined. Uniform staining for collagen type VI was evident only in the ligament of the fully erupted molars, and the stain was absent from the mid-zone of the ligaments of the erupting molars and incisors. The staining pattern of the collagen type VI antibodies is consistent with previous reports which have suggested that the removal of collagen type VI precedes the degradation of major banded collagen fibrils in remodelling connective tissues. The removal of collagen type VI from the mid-zone of the incisor ligament may precede the remodelling events which facilitate tooth eruption.

Animals↗
PubMed1993

Microfibrillar elements in the synovial joint: presence of type VI collagen and fibrillin-containing microfibrils.

OBJECTIVES: The aims were to isolate and positively identify the microfibrillar elements which have been observed in the synovial lining. In addition, synovial fluid was examined for these elements to improve the understanding of the role of these structures in health and disease. METHODS: Bacterial collagenase digestion of bovine synovial linings and human and bovine synovial fluids was used to release intact, non-denatured microfibrillar elements. The microfibrils were isolated by Sepharose CL-2B chromatography and viewed by rotary shadowing. They were characterised by immunogold labelling with specific antibodies. RESULTS: Intact type VI collagen microfibrils and fibrillin-containing microfibrils were isolated and positively identified in the synovial lining from bovine ankle joints by immunogold labelling. Type VI collagen microfibrils were also present in the synovial fluid. CONCLUSIONS: The role of the microfibrillar elements in vivo is not fully understood, but their distribution in the synovial lining suggests they have an important role in the mechanical and physical properties of this tissue. The presence of type VI collagen microfibrils in synovial fluid poses the intriguing possibility that it may represent a product of microfibril turnover and a potential early marker for rheumatoid arthritis. Alternatively, type VI collagen may be specifically secreted into the synovial fluid to interact with hyaluronan and form part of the structure of synovial fluid.

Actin Cytoskeleton↗
PubMed1993

Localization of von Willebrand factor binding domains to endothelial extracellular matrix and to type VI collagen.

We have recently shown that von Willebrand factor (vWF) binds to endothelial and fibroblastic extracellular matrixes (ECM) in a dose-dependent, specific, and saturable way. To localize the domain on the vWF subunit responsible for this interaction, purified proteolytic fragments of vWF were compared for their ability to inhibit 125I-vWF binding to ECM. A tryptic dimeric fragment of 116 kD (T116), extending from amino acid (aa) residues 449 to 728, produced a significant inhibition of 125I-vWF binding to the ECM. In contrast, P34 (aa 1-272), SpI (aa 911-1,365), and SpII (aa 1,366-2,050) had no significant effect on 125I-vWF binding to the ECM. Using an immunofluorescence technique, we identified type VI collagen and heparan sulfate in the endothelial ECM. 125I-vWF was found to bind specifically to purified type VI collagen. Unlabeled vWF and SpIII were able to completely inhibit 125I-vWF binding to type VI collagen. T116 and SpI appeared as competitors of this interaction, whereas P34 and SpII were not. Our data suggest that vWF binds to the endothelial ECM through the T116 fragment and that T116 and SpI each contain a binding site for type VI collagen. Heparin is known to be a vWF ligand, but did not appear as a competitor of vWF binding to the ECM, nor did heparan sulfate.

Collagen↗
PubMed1993

Synthesis and assembly of fibrillin by fibroblasts and smooth muscle cells.

The expression and assembly of the microfibrillar glycoprotein fibrillin has been investigated in cultures of nuchal ligament fibroblasts, skin fibroblasts and vascular smooth muscle cells. The level of fibrillin expression varied with the cell type and growth conditions. Higher levels of synthesis were recorded in quiescent post-confluent cells than in actively dividing subconfluent cultures. Nuchal ligament fibroblasts consistently synthesized the highest levels of fibrillin. Growth of cells in the presence of ascorbate resulted in an increased proportion of newly synthesized fibrillin retained within cell layers. Fibrillin was immunoprecipitated from medium and cell layer extracts in the form of monomers and high-M(r) disulphide-bonded aggregates. Rotary shadowing electron microscopy of cell layer extracts and collagen gels provided direct evidence for the assembly of extensive intact microfibrils by smooth muscle cells and fibroblast cultures. Gel filtration chromatography of medium and cell layer extracts, in combination with immunoprecipitation of column fractions, provided a means of analysing the size distribution and assembly of newly synthesized fibrillin. This cell culture approach provides an opportunity to evaluate normal and aberrant synthesis and assembly of fibrillin in a wide range of cell types.

Actin Cytoskeleton↗