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D R Keene

Publications and source records attributed to D R Keene.

77 records · Page 5Linked to original sources
PubMed1986

The tissue form of type VII collagen is an antiparallel dimer.

We recently reported the partial characterization of a new human collagen termed Type VII. This molecule is distinctive among the collagen family in that it contains three identical subunit alpha chains within a triple helical domain 424 nm in length. The molecule contains three identical alpha chains which are genetically distinct from other known collagens. Previous studies indicate that a portion of the limited pepsin-solubilized molecules appears to exist as antiparallel dimers associated by disulfide bonds. In this report, we demonstrate that the major tissue form of Type VII collagen is a dimer, associated by disulfide bonds through a 60-nm overlap of the aminoterminal triple helical ends. Intermolecular disulfide bonds occur only within this overlap region. Interchain disulfide bonds exist in the carboxyl terminal 7% of the molecule and may exist within the overlap region as well. Disulfide bond-stabilized aggregates larger than dimers are not seen.

Amino Acids↗
PubMed1986

Type VII collagen is a major structural component of anchoring fibrils.

Anchoring fibrils are specialized fibrous structures found in the subbasal lamina underlying epithelia of several external tissues. Based upon their sensitivity to collagenase and the similarity in banding pattern to artificially created segment-long spacing crystallites (SLS) of collagens, several authors have suggested that anchoring fibrils are lateral aggregates of collagenous macromolecules. We recently reported the similarity in length and banding pattern of anchoring fibrils to type VII collagen SLS crystallites. We now report the construction and characterization of a murine monoclonal antibody specific for type VII collagen. The epitope identified by this antibody has been mapped to the carboxyl terminus of the major helical domain of this molecule. The presence of type VII collagen as detected by indirect immunofluorescence in a variety of tissues corresponds exactly with ultrastructural observations of anchoring fibrils. Ultrastructural immunolocalization of type VII collagen using a 5-nm colloidal gold-conjugated second antibody demonstrates metal deposition upon anchoring fibrils at both ends of these structures, as predicted by the location of the epitope on type VII collagen. Type VII collagen is synthesized by primary cultures of amniotic epithelial cells. It is also produced by KB cells (an epidermoid carcinoma cell line) and WISH (a transformed amniotic cell line).

Animals↗
PubMed1986

Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils.

A new connective tissue protein, which we call fibrillin, has been isolated from the medium of human fibroblast cell cultures. Electrophoresis of the disulfide bond-reduced protein gave a single band with an estimated molecular mass of 350,000 D. This 350-kD protein appeared to possess intrachain disulfide bonds. It could be stained with periodic acid-Schiff reagent, and after metabolic labeling, it contained [3H]glucosamine. It could not be labeled with [35S]sulfate. It was resistant to digestion by bacterial collagenase. Using mAbs specific for fibrillin, we demonstrated its widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule. Electron microscopic immunolocalization with colloidal gold conjugates specified its location to a class of extracellular structural elements described as microfibrils. These microfibrils possessed a characteristic appearance and averaged 10 nm in diameter. Microfibrils around the amorphous cores of the elastic fiber system as well as bundles of microfibrils without elastin cores were labeled equally well with antibody. Immunolocalization suggested that fibrillin is arrayed periodically along the individual microfibril and that individual microfibrils may be aligned within bundles. The periodicity of the epitope appeared to match the interstitial collagen band periodicity. In contrast, type VI collagen, which has been proposed as a possible microfibrillar component, was immunolocalized with a specific mAb to small diameter microfilaments that interweave among the large, banded collagen fibers; it was not associated with the system of microfibrils identified by the presence of fibrillin.

Actin Cytoskeleton↗