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C M Kielty

Publications and source records attributed to C M Kielty.

71 records · Page 4Linked to original sources
PubMed1993

Microfibrillar assemblies of foetal bovine skin. Developmental expression and relative abundance of type VI collagen and fibrillin.

Intact type VI collagen microfibrils and fibrillin-containing microfibrils were isolated from foetal bovine skin and investigated immunochemically and ultrastructurally. Substantial variations were detected in the abundance and macromolecular assembly of these structures at progressive stages of gestation. Microfibrils of collagen VI were increasingly abundant in skin through foetal development from late first trimester to term. The pattern of expression of fibrillin-containing microfibrils in foetal skin differed from that of collagen VI. Fibrillin-containing microfibrils were particularly sparse in first trimester skin, and present only as short assemblies. However, by early second trimester there had been a sharp increase in the abundance and length of these fibrillin-containing microfibrils. These observations are consistent with early second trimester being a key phase of fibrillin assembly. In the third trimester, fibrillin-containing microfibrils were frequently isolated in association with amorphous material. This information on the pattern of expression and assembly of collagen VI microfibrils and fibrillin-containing microfibrils in foetal skin implies temporally and functionally distinct contributions of these two components to the establishment of the fibrous dermal matrix.

Animals↗
PubMed1992

Microfibrillar components in dental pulp: presence of both type VI collagen- and fibrillin-containing microfibrils.

Microfibrillar elements were isolated from developing and formed bovine dental pulp by a procedure involving bacterial collagenase tissue digestion and chromatography on Sepharose CL-2B. Two microfibrillar assemblies could be demonstrated. Type VI collagen microfibrils with a characteristic periodicity of about 100 nm appeared as long, thin, flexible filaments. In a number of cases these structures aggregated by lateral association. Microfibrils of 10-14 nm dia were identified as containing fibrillin on the basis of their distinctive, periodic, beaded morphology. In addition to long, single strands there were instances of chains coalescing to give amorphous aggregates. No differences in the type of microfibrillar assemblies were evident between developing and formed pulp, although fibrillin-containing microfibrils were more abundant in formed pulp.

Actin Cytoskeleton↗
PubMed1992

Type VI collagen microfibrils: evidence for a structural association with hyaluronan.

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.

Animals↗
PubMed1992

Attachment of human vascular smooth muscles cells to intact microfibrillar assemblies of collagen VI and fibrillin.

Human vascular smooth muscle cells have been used to assess the implied role of connective tissue microfibrils as cellular ligands. Preparations of intact high-M(r) microfibrillar assemblies of collagen VI and of fibrillin, respectively, were isolated from foetal bovine skin and used as ligands in cell attachment and spreading assays. Intact collagen VI microfibrils were capable of mediating cell attachment and partial spreading. Cell attachment assays using ligands composed of defined collagen VI fragments generated by pepsin or bacterial collagenase digestions demonstrated that both the triple-helical and non-collagenous domains of collagen VI had cell adhesion activity, although at reduced levels relative to intact microfibrils. Fibronectin was identified as a modulator of intact collagen VI microfibril-mediated cell attachment. These observations are indicative of complex multiple interactions between collagen VI microfibrils and smooth muscle cells. Purified fibrillin-containing microfibrils were also shown to support smooth muscle cell adhesion. Both pepsin-resistant and pepsin-sensitive domains of fibrillin exhibited some cell attachment activity, but at reduced levels relative to the intact fibrillin microfibrils. These data provide the first direct evidence of a physiological role for intact microfibrillar assemblies in cell-matrix interactions, and the involvement of integrin cell surface receptors containing the beta 1 subunit.

Animals↗
PubMed1991

Isolation and ultrastructural analysis of microfibrillar structures from foetal bovine elastic tissues. Relative abundance and supramolecular architecture of type VI collagen assemblies and fibrillin.

Extensive intact assemblies of matrix macromolecules have been solubilized from foetal calf skin, nuchal ligament and aorta by a new procedure that includes bacterial collagenase digestion under non-reducing, non-denaturing conditions and gel filtration chromatography. Type VI collagen was identified as the major microfibrillar element of these tissues by SDS-PAGE analysis and Western blotting. Rotary shadowing electron microscopy of these preparations revealed by far the most abundant and extensive arrays of intact collagen VI microfibrils isolated to date. The distinct microfibrillar species, fibrillin, which was identified on the basis of its periodicity and morphology, was also solubilized in abundance by this protocol. Analysis of these complex polymers has generated new information on their supramolecular architecture and relative abundance in these tissues. The protocol also demonstrates that the release of intact collagen VI microfibrils from these tissues is largely dependent on the removal of the major collagen fibrils.

Animals↗
PubMed1990

Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells.

The chain composition and relative abundance of type VI collagen synthesized by cells cultured from foetal bovine nuchal ligament and skin were compared with those of the type VI collagen present in these foetal tissues. Immunoprecipitation of intact collagen VI from medium and cell layers of nuchal ligament fibroblasts and skin fibroblasts at confluence revealed collagen type VI molecules with a chain composition consistent with an [alpha 1(VI)alpha 2(VI)alpha 3(VI)] monomeric assembly. Maintenance of cells in a post-confluent quiescent state promoted a marked phenotypic change in these ratios, with increased concentrations of assemblies composed of equimolar ratios of alpha 1(VI) and alpha 2(VI) chains detected in the medium of these cultures. Analysis of steady-state concentrations of mRNA for alpha 1(VI) and alpha 2(VI) chains revealed these species to be present in increased abundance at post-confluence in all the cultures, but no corresponding increase was observed in the alpha 3(VI) mRNA. In order to assess the physiological significance of these observations, the chain composition of the collagen VI content of the corresponding foetal tissues was assessed by Western blotting after extraction in guanidinium isothiocyanate under reducing conditions. Extracts of nuchal ligament revealed a collagen VI chain composition consistent with a heterotrimeric chain assembly. In contrast, the skin extracts revealed an abundance of alpha 1(VI) and alpha 2(VI) chains with only traces of the alpha 3(VI) chain detected. Increased equimolar concentrations of the alpha 1(VI)-chain and alpha 2(VI)-chain mRNAs in skin again reflected the increased concentrations of these polypeptide chains. Type VI collagen was present in greater abundance both in the nuchal ligament and in the corresponding nuchal-ligament fibroblast cultures. The results indicate that the chain composition of type VI collagen is subject to modulation at the level of transcription as a result of variations in the proliferative state of the cells, and demonstrate that different isoforms of collagen VI occur in foetal development.

Animals↗
PubMed1985

Type X collagen, a product of hypertrophic chondrocytes.

The synthesis of collagen types IX and X by explants of chick-embryo cartilages was investigated. When sternal cartilage labelled for 24h with [3H]proline was extracted with 4M-guanidinium chloride, up to 20% of the 3H-labelled collagen laid down in the tissue could be accounted for by the low-Mr collagenous polypeptides (H and J chains) of type IX collagen; but no type X collagen could be detected. Explants of tibiotarsal and femoral cartilages were found to synthesize type IX collagen mainly in zones 1 and 2 of chondrocyte proliferation and elongation, whereas type X collagen was shown to be a product of the hypertrophic chondrocytes in zone 3. Pulse-chase experiments with tibiotarsal (zone-3) explants demonstrated a time-dependent conversion of type X procollagen into a smaller species whose polypeptides were of Mr 49 000. The processed chains [alpha 1(X) chains] were shown by peptide mapping techniques to share a common identity with the pro alpha 1(X) chains of Mr 59 000. No evidence for processing of type IX collagen was obtained in analogous pulse-chase experiments with sternal tissue. When chondrocytes from tibiotarsal cartilage (zone 3) were cultured on plastic under standard conditions for 4-10 weeks they released large amounts of type X procollagen into the medium. However, 2M-MgCl2 extracts of the cell layer were found to contain mainly the processed collagen comprising alpha 1(X) chains. The native type X procollagen purified from culture medium was shown by rotary shadowing to occur as a short rod-like molecule 148 nm in length with a terminal globular extension, whereas the processed species comprising alpha 1(X) chains of Mr 49 000 was detected by electron microscopy as the linear 148 nm segment.

Animals↗
PubMed1984

Embryonic chick cartilage collagens. Differences in the low-Mr species present in sternal cartilage and tibiotarsal articular cartilage.

The collagenous polypeptides present in embryonic chick sternal and tibiotarsal cartilages have been solubilised by digestion with pepsin and separated by salt fractionation. Type II collagen, 1 alpha 2 alpha 3 alpha collagen, and two polypeptides (apparent molecular mass 150 and 42 kDa), which were reducible to a number of smaller peptides, were extracted from both tissues. However, also present in the peptic digests of tibiotarsal cartilages was a major non-reducible highly-soluble polypeptide of 45 kDa. This short-chain collagen is apparently identical to the pepsinized product of G collagen (Mr 59 000), a major low-Mr procollagen-like species previously detected in chick chondrocyte cultures.

Amino Acids↗
PubMed1983

Identification and partial characterization of three low-molecular-weight collagenous polypeptides synthesized by chondrocytes cultured within collagen gels in the absence and in the presence of fibronectin.

Culture of chick-embryo sternal-cartilage chondrocytes within three-dimensional collagen gels promotes the synthesis of three low-molecular-weight collagenous polypeptides. The proportions of these novel collagens synthesized and released into the medium are markedly influenced by the presence or the absence of fibronectin in the serum supplement. Chondrocytes cultured on plastic dishes appear to synthesize only small amounts of these low-molecular-weight species. The three species (designated G, H and J) were characterized with respect to the proportion of [14C]proline incorporated into each polypeptide occurring as hydroxy[14C]proline and with respect to their susceptibilities to bacterial collagenase. On the basis of their electrophoretic mobilities under reducing conditions, the G, H and J polypeptides were calculated to have Mr 59 000, 69 000 and 84 000 respectively. Chymotrypsin digestion converted the G collagen into a species containing polypeptides of Mr 45 000, whereas the H and J polypeptides yielded a single band of Mr 53 000. The H and J polypeptides were found to occur as disulphide-linked aggregates, as was the chymotrypsin-digestion product. Peptide 'mapping' has shown that G, H and J polypeptides show no common identity and are distinct from the known interstitial collagens. Native G collagen was digested by human collagenase to discrete products, whereas H and J chains were not cleaved under identical conditions.

Animals↗
PubMed1982

Regulation of expression of liver-specific enzymes. II. Activation and chromosomal localization of soluble glutamate-pyruvate transaminase.

1. The expression of soluble GPT (E.C. 2. 6. 1. 2) was analysed in seventeen independent rat hepatoma/human somatic cell hybrids and in forty-one subclones derived from two of these hybrids. 2. As judged by electrophoretic mobility, twelve hybrid clones expressed rat GPT activity only and three expressed strong rat and weak human GPT activity but no heteromeric isozymes. In the remaining two hybrids, only human GPT was demonstrable. 3. The segregation of GPT and marker enzymes in the primary hybrid cells and the subclones suggests that the human structural GPT locus is on chromosome 8. 4. The re-expression of rat GPT in segregating subclones derived from two primary hybrids which had extinguished this function, could not be correlated with presence or absence of any particular human chromosome(s).

Alanine Transaminase↗
PubMed1982

Regulation of expression of liver-specific enzymes. III. Further analysis of a series of rat hepatoma X human somatic cell hybrids.

1. The expression of twelve liver-specific enzymes was analysed in twenty-one independent rat hepatoma X human somatic cell hybrids, and in some cases up to forty-one subclones were also tested. 2. Seventeen hybrids continued to express most of the rat liver-specific enzymes and in some cases human isozymes of glutamate-pyruvate transaminase, alpha-glycerophosphate dehydrogenase, guanine deaminase, alcohol dehydrogenase and pyruvate kinase were clearly identified. 3. Analysis of the segregation of the human liver-specific enzymes in these hybrids led to the assignment of human GPT to chromosome 8 (previously reported, Kielty, Povey & Hopkinson, 1982) and suggests the assignment of human GPD1 to chromosome 12. 4. The expression of the various liver-specific enzymes in these hybrids appeared to be controlled by independent regulatory mechanisms. 5. Four unusual reverse segregant hybrids were also analysed, and in these no liver-specific enzyme activity was demonstrable.

Alcohol Oxidoreductases↗
PubMed1981

Regulation of expression of liver-specific enzymes. I. Detection in mammalian tissues and cultured cells.

Nineteen enzymes showing highest activity in liver were examined in human and rodent tissues and cultured cells using starch-gel electrophoresis. The rat hepatoma line Faza 967 strongly expressed 13 of these enzymes. A series of somatic cell hybrids, constructed between Faza and cells of non-hepatic origin derived from man or from Chinese hamster, were examined for expression of these enzymes. Some of the human/rat hybrids continued to produce rat liver-specific enzymes, and the human forms of the enzymes glutamate-pyruvate transaminase, alpha-glycerophosphate dehydrogenase, alcohol dehydrogenase and pyruvate kinase L were reexpressed in a few cases.

Aldehyde Dehydrogenase↗
PubMedDate not supplied

Three-dimensional reconstructions of extracellular matrix polymers using automated electron tomography.

The extracellular matrix is an intricate network of macromolecules which provides support for cells and a framework for tissues. The detailed structure and organisation of most matrix polymers is poorly understood. These polymers have a complex ultrastructure, and it has proved a major challenge both to define their structural organisation and to relate this to their biological function. However, new approaches using automated electron tomography are beginning to reveal important insights into the molecular assembly and structural organisation of two of the most abundant polymer systems in the extracellular matrix. We have generated three-dimensional reconstructions of collagen fibrils from bovine cornea and fibrillin microfibrils from ciliary zonules. Analysis of these data has provided new insights into the organisation and function of these large macromolecular assemblies.

Animals↗
PubMedDate not supplied

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues.

We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.

Actin Cytoskeleton↗