Jerome Gross

The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation☆

Active synthesis of hyaluronate has been demonstrated in the early “dedifferentiated” blastema of the regenerating newt limb. Incorporation of isotopic precursors in vivo into hyaluronate is maximal at approximately 10 days of regeneration, whereas incorporation into chondroitin sulfate is maximal at 25–35 days. At 10 days, incorporation into hyaluronate is greatest in the most distal portion of the stump, grading off in the distal to proximal direction. Concomitant with a sharp decrease in incorporation into hyaluronate after 10–15 days of regeneration, hyaluronidase activity becomes measurable in the blastema and persists at a significant level until 40 days of regeneration, i.e., during the period of maximal chondroitin sulfate synthesis and cartilage differentiation. Turnover experiments indicated that hyaluronate was degraded rapidly at 25 days but not at 10 days of regeneration. Incorporation into hyaluronate was considerably greater than into chondroitin sulfate at all time points tested between 3 and 48 hours after administration of isotope to animals with 10-day regenerates. The reverse was true in animals with 25-day regenerates. It is concluded that hyaluronate is associated with blastema formation, and its possible role in the regulation of mesenchymal cell behavior is discussed.

Collagenolytic activity during mammalian wound repair

Abstract Collagenolytic activity has been demonstrated in cultures of tissues from cutaneous mammalian wounds cultured on substrate gels of reconstituted collagen. As much as 90–100% of the insoluble collagen substrate was broken down at neutral pH, and at least 60% of the product was dialyzable. In wound edge cultures containing both epithelium and mesenchyme, collagenolysis was initially localized around the former. Variable collagenase activity was produced by granulation tissue alone, but none by dermis or by unwounded loose connective tissue. Unwounded skin revealed weaker collagenolytic potential. Recombination of separated epithelium and granulation tissue from wounds restored collagenase activity to high levels approximating that of unseparated tissues. Exposure of nonlytic granulation tissue to epithelium appeared to induce collagenolytic activity which continued in the mesenchymal tissue following removal of the epithelium. The activity of isolated epithelium decayed more rapidly than did that of the “activated” mesenchyme. Viable cells were required for collagenase production; freeze-thawing inactivated the explants. Enzyme activity could not be detected in tissue homogenates or extracts and whole serum applied daily to cultures blocked collagenolysis. Collagenase activity was not altered by vitamin C deficiency.

Human skin collagenase, isolation and mechanism of attack on the collagen molecule☆

Abstract 1. 1.A collagenolytic enzyme has been isolated from the culture medium of tissue cultures of normal human skin but not from tissue extracts. The enzyme is capable of reducing the specific viscosity of native collagen at 28° and neutral pH in addition to lowering the denaturation temperature by approximately 5° without altering its helical content. 2. 2.Enzymatic attack on the collagen molecule results in the appearance of new, faster moving bands on disc electrophoresis. Electron micrographs of segment long spacings prepared from the enzyme-collagen reaction mixtures reveal the presence of an “A” end fragment (TC A ) 75% the length of the collagen molecule and a “B” fragment (TC B ) representing the remaining one-quarter. 3. 3.Human skin collagenase also acts on collagen in the native fibrillar form at 37° with a pH optimum of 7 to 8. No enzyme activity is present below pH 5.0, above pH 8.0 activity falls off markedly. The enzyme is inhibited by low concentrations of EDTA, cysteine and human serum but not by soybean trypsin inhibitor.

The amino acid composition and morphology of some invertebrate and vertebrate collagens.

Abstract 1. 1. The complete amino acid analysis of collagens from a mammal (steer hide), an elasmobranch (shark clastoidin), an echinoderm ( Thyone body wall), two coelenterates ( Metridium body wall and Physalia float), and two structurally distinct collagens from the sponge are presented along with a description of some morphological characteristics of the tissues from which they were derived. 2. 2. Structural observations include a typical collagen axial repeating period in fibrils of the body wall of Thyone , thin (200–300 A) unbranched fibrils revealing periodic axial structure in the connective tissue of Metridium and the float of Physalia , and a reasonably well oriented collagen wide angle X-ray diffraction pattern obtained from water-washed Physalia float. 3. 3. The most striking characteristic of these collagens is the uniformly high glycine content which in each case approaches one-third of the total amino acid residues. This is reflected in the corrected nitrogen values which are all close to 18.6%. The data are consistent with the requirements of the most likely structure of the collagen molecule. 4. 4. A high degree of variability of the other amino acids, including proline, hydroxyproline and hydroxylysine, occurs among the invertebrate phyla. In general the invertebrate collagens when compared to vertebrate collagens have a smaller proportion of imino acids, more total dicarboxylic and hydroxy amino acids, and fewer nonpolar amino acids. 5. 5. Thyone collagen and spongin A have the lowest lysine values yet recorded for collagen, 7.5 and 9.0 residues per 1,000 residues as compared with 25 for steer hide. Metridium, Physalia collagen and spongin B have the highest recorded hydroxylysine content, 25, 30 and 24 residues per 1,000 residues as compared with 6.8 for mammalian collagen. 6. 6. No relationship between amino acid composition and evolutionary development is obvious.

The role of epithelium and mesenchyme in the production of a collagenolytic enzyme and a hyaluronidase in the anuran tadpole

Abstract Remodeling of connective tissue, occurring during amphibian metamorphosis involves the degradation of both collagen fibers and interfibrillar ground substance. The formation of a collagenolytic enzyme by the epithelial cells and the release of a hyaluronidase by the mesenchyme have been demonstrated in cultures of the separated tissue components. Puromycin (which has no direct effect on collagenase) as well as freeze-thawing block the appearance of collagenolytic activity, suggesting either de novo synthesis of collagenase or, alternatively, its activation by some mechanism requiring protein synthesis. Hyaluronidase activity, however, is not inhibited by puromycin and is enhanced by freeze-thawing, indicating that this enzyme is stored in the mesenchymal cells and probably released when needed. A mechanism for connective tissue resorption is proposed in which two different cell types cooperate, each providing a different enzyme capable of attacking extracellular structures in a specific manner.

Animal collagenases: Specificity of action, and structures of the substrate cleavage site☆

Abstract Two highly purified animal collagenases, one derived from homogenates of the rabbit V 2 ascites cell carcinoma growing in muscle and the second isolated from the media of tadpole tissue cultures cleaved isolated non helical α chains from chick and rat skin collagen, and the CNBr peptide CB7 from chick skin α1 chains at one, and the same peptide bond. Although two other Gly-Ile bonds exist elsewhere in the α1 chain they were not cleaved.

Morphologic studies of connective tissue resorption in the tail fin of metamorphosing bullfrog tadpole

Abstract The sequential morphologic changes in bullfrog tadpole tail fin during thyroxine-induced metamorphosis were observed by phase contrast and electron microscopy. The earliest change was fraying and separation of the collagenous layers and fibrils of the basement lamella, soon followed by invasion of the lamella from below by mesenchymal cells. The cytoplasm of these cells filled with bundles of collagen fibrils suggesting phagocytosis of the fibrils. The lamella swelled tenfold from a compact 6–7 μ layer to a diffuse fragmented region filled with large mesenchymal cells. The extracellular collagen fibrils showed great changes in diameter and density, the amount of filamentous material increasing as the fibrils diminished in number. Changes in epithelial cells and adepidermal membrane are also described. The morphologic sequences are correlated with knowledge concerning degradative enzyme systems found in tail fin, resulting in a speculation on the mechanism of connective tissue resorption during metamorphosis.

The collagen of chick embryonic notochord

Abstract Notochords, isolated from 2 1 2 day chick embryos, were cultured in the presence of 3H proline and the labeled proteins co-purified with chick skin carrier collagen. The purified material, most of which eluted from CM-cellulose as a single peak in the region of the carrier collagen α1 chain, contained 41% of the incorporated proline as hydroxyproline and from gel filtration measurements had a molecular weight of approximately 100,000 daltons. When the material was chromatographed on DEAE-cellulose with carrier α1 chains from both skin [α1 (I)] and cartilage [α1 (II)], it eluted predominantly with the cartilage chains.

Collagenolytic activity in regenerating forelimbs of the adult newt (Triturus viridescens)

Summary A collagenolytic enzyme capable of degrading reconstituted collagen fibrils at neutral pH and physiological temperature, not demonstrable in the intact forelimb of the adult newt ( Triturus viridescens ) appears in cultures of healing and regenerating tissues of the amputation stump. Tissues proximal to the regenerating region are inactive. The amount of enzyme produced, measured by visible breakdown of thermally reconstituted mammalian collagen gels, and confirmed by release of radioactivty from 14 C-glycine-labeled collagen gels, rises to a peak at 15 days and remains at a high level during the phases of dedifferentiation of the stump and blastema formation, but gradually falls after 20 days as early digital differentiation occurs. Living cells are required for collagenase production since freezing and thawing inactivates the explants.

Comparative biochemistry of collagen some amino acids and carbohydrates

Abstract Hydroxyproline, proline, glycine, tyrosine and the carbohydrate composition of eleven native and limid collagens and derived gelatins from a mammal, fish, echinoderm, two coelenterates and a poriferan plus the composition of a fish elastin are reported.