Bernard S. Gould

Collagen biosynthesis the formation of hydroxyproline and soluble-ribonucleic acid complexes of proline and hydroxyproline by chick embryo in vivo and by a subcellular chick-embryo system

Abstract Experiments have been carried out aimed at determining whether the source of collagen-bound hydroxyproline is non-protein-bound or protein-bound proline. Free hydroxy[ 14 C]proline formation from injected [ 14 C]proline was found to be essentially normal in chick embryos whose collagen-forming ability had been almost completely suppressed by puromycin. Following the injection of [ 14 C]proline into chick embryos, hydroxy[ 14 C]proline bound to s-RNA could be isolated. Similarly, bound hydroxy[ 14 C]proline could be found when chick-embryo s-RNA was incubated in vitro with amino acid-activating enzymes from chick embryo. Supplementation of such a system with s-RNA and activating enzymes from Escherichia coli stimulated the binding of [ 14 C]proline to s-RNA but was ineffective in the hydroxylation of proline to hydroxyproline. The results have led to the conclusion that proline can be converted to hydroxy-proline without prior incorporation of the proline into protein.

Collagen Formation and Fibrogenesis with Special Reference to the Role of Ascorbic Acid

Publisher Summary This chapter discusses the collagen formation and fibrogenesis with special reference to the role of ascorbic acid. Collagen is present in almost all multicellular animals ranging from the primitive poriferans and coelenterates, through the annelids and echinoderms up to the vertebrates. It is one of the most abundant of the mammalian proteins and constitutes about 25–30% of the total body protein. The fibroblast, like the osteoblast and odontoblast, is profoundly influenced by ascorbic acid deficiency. In the absence of the vitamin, these cells characteristically fail to produce their respective fibrous proteins—collagen, osteoid, and dentin. In most respects, the cytochemical properties of these cells are analogous (except for the extremely high phosphatase production by the osteoblast), and that ascorbic acid deficiency brings about a reversal of the differentiated cells to more immature cell types. The administration of ascorbic acid promptly corrects the defect. The chapter discusses the origin of the collagen-forming cell, morphological studies of collagen formation, influence of ascorbic acid deficiency on collagen-forming cells, collagen formation in tissue culture, and collagen synthesis.

SOME ASPECTS OF COLLAGEN FORMATION

Despite the fact that the deficiency state known as scurvy had been recognized since antiquity, and had been classically described by Walter (1746) as involving the healing of wounds as well as the maintenance of wounds that have already healed, it was not until 1919 that Aschoff and Koch and then Hojer in 1924 carried out detailed histological studies. These were soon followed by those of Wolbach and Howe (1926), which related the action of the accessory factor vitamin C to the formation of proper connective tissue, particularly of collagen. A number of investigations based on histological examination or tensile strength studies have established beyond question the relationship between ascorbic acid and collagen formation. Nevertheless, little is known concerning the exact mechanism by which ascorbic acid acts; both the celluhr physiology and the biosynthetic mechanisms await clarification. Two early theories have been the basis of much of the work attempting to elucidate the mechanism of the interaction between ascorbic acid and collagen fiber formation. Aschoff and Koch (1919) suggested that the defect in scurvy involves an inability to produce extracellular substance. Wolbach and Howe (1926) suggested that the secretion of intercellular ground substance and of collagenous precursor materials proceeds quite normally, but that some factor is lacking that normally causes gelling and fibrillation of a precursor in the extracellular material, and that this factor is not involved in the formation of fibroblasts. They found, from histological examination of scorbutic teeth and bones, an apparent accumulation of a fluid substance presumably secreted by the odontoblasts and osteoblasts that remained fluid due to the absence of a gelling factor. When vitamin C was administered, gelation occurred with such rapidity that they believed there had not been sufficient time for a new formation of collagen and suggested that “the failure of cells to produce intercellular substances in scorbutus is due to the absence of an agent common to all supporting tissues which is responsible for setting or gelling of a liquid product.” Soon after the administration of vitamin C to depleted animals, an apparently homogeneous, amorphous substance that stained blue with Mallory’s connective tissue stain formed around the cells. This was followed rapidly by the formation of reticulin fibers embedded in the amorphous material that Wolbach (1933) believed to be amorphous collagen. Opposed to this concept of normal precursor formation in the absence of some * This investigation was supported in part by Grants A-1270 and D-320 from the National Institute of Arthritis and Metabolic Diseases and the National Institute of Dental Research, Public Health Service, Bethesda, Md., and in part by a generous grant from Eli Lilly and Company, Indianapolis, Ind. to whom we express our thanks.

The polyribosomal synthesis of collagen

Abstract The formation of collagen, on polyribosomes has been studied. Collagen was identified its characteristic content of hydroxyproline, which is derived metabolically by from proline. [ 14 C]Proline was injected into chick embryos and nascent collagen was measured in sucrose density gradients by the isolation of protein-bound [ 14 C]hydroxyproline. The results indicate that collagen is synthesized on very large polysomes. Ribonuclease, puromycin and dodecyl sulfate studies indicate that the nascent peptide chains already contain hydroxyproline suggesting that hydroxylation occurs prior to complete peptide chain assembly. There is suggestive evidence that the large polysomes may result from the aggregation of nascent collagen peptide chains.

γ globulin synthesis on ribosomes and ribosomal aggregates

Abstract 1. 1. The formation of γ-globulin at the ribosomal level in the rat lymph node has been studied by density-gradient techniques. 2. 2. Characteristic relatively small ribosomal aggregates as well as single ribosomes appear to be involved in γ-globulin synthesis. 3. 3. These aggregates are resistant to nucleases but sensitive to proteolytic enzymes suggesting that they are held together by protein or polypeptide. 4. 4. The aggregates appear not to be held together by nascent polypeptide chains. 5. 5. No qualitative or quantitative differences could be discerned between the distribution and characteristics of ribosomal material in the primary and secondary immunological responses. 6. 6. Direct binding of antigen to the protein-synthesizing sites does not appear to occur, suggesting that the antigen does not have a template function.

The action of bisphenolic compounds on succinoxidase, cytochrome C oxidase and lactic dehydrogenase of animal tissue.

Abstract A study has been made of the effect of certain chlorinated bisphenolic compounds, particularly hexachlorophene [2,2′-methylenebis(3,4,6-trichlorophenol)], on the succinoxidase system of animal tissues and on the activities of purified cytochrome oxidase and purified lactic dehydrogenase of animal origin. Probable interactions of the bisphenolic compounds with cytochrome and with diphosphopyridine nucleotides have also been explored. At levels of between 10 −4 and 10 −6 M , hexachlorophene effectively inhibits heart, kidney, and liver succinoxidase systems. At similar levels it inhibits the purified cytochrome oxidase as well as the purified lactic dehydrogenase systems. There appears to be no detectable interaction between the chlorinated bisphenols and either reduced or oxidized diphosphopyridine nucleotide, nor does there appear to be any impairment of the oxidizability or reducibility of cytochrome in spite of the strong binding power of cytochrome for hexachlorophene and certain other bisphenols.

Effect of hexachlorophene and related bisphenolic compounds on the dehydrogenases and cytochrome system of Bacillus subtilis and Escherichia coli

Abstract A study has been made of the effect of hexachlorophene (2, 2′-methylenebis [3,4,6-trichlorophenol]) and certain related compounds on the dehydrogenases and cytochrome oxidase systems of Bacillus subtilis and Escherichia coli . Hexachlorophene (G-11), the dichloro analog (G-4), and the compound G-11S, which contains a sulfur bridge rather than a CH 2 -bridge, all at very low concentrations effectively inhibit the glucose, lactic, and succinic dehydrogenases as well as the cytochrome oxidase systems of both organisms. The absence of chlorine from the molecule results in a profound decrease in inhibitory power against the dehydrogenases with a lesser decrease in activity against the cytochrome oxidase. The inhibitory action of these compounds does not appear to be exerted through their action on the sulfhydryl groups of the enzymes nor could evidence be obtained that they compete with the cytochrome system as hydrogen acceptors. Both organisms bind relatively large amounts of inhibitor.

Products of proline peroxidation.

Proline can be oxidized to hydroxyproline in vitro by a system containing Fe2+, Lascorbate and EDTA, and employing 02 or Hz02 as oxidant [ 1,2] . The system has been suggested as a model for collagen-proline hydroxylation [3-S] . We report here on a study of products from H202 hydroxylation of proline and prolylpeptides. It is shown that to be hydroxylated by H202, proline must have its ring nitrogen unsubstituted. Peroxidation of free proline yields a variety of products, including hydroxyprolines, whereas N-substituted proline yields mainly a non-hydroxylated end product. A pathway of in vivo hydroxylation mechanistically similar to Fe2+/H202 hydroxylation of proline is not consistent with our results.

Pigment Production in Certain of the Aspergillus Glaucus Group

SUMMARY(1) Auroglaucin and flavoglaucin are major end products of carbohydrate metabolism among members of the Aspergillus glaucus group. Striking quantities of these pigments are produced in both ...

Ocular tooth germ implant in the ascorbic acid-deficient guinea pig

Abstract Examination of cross sections of teeth in the jaw and of tooth germs implanted in the eyes of scorbutic guinea pigs revealed typical changes of ascorbic acid deficiency in the former while normal tooth development was observed in the implant. Good collagen formation, as well as bone, was observed in the implants. The results suggest that odontoblasts, dentine production, and bone in developing embryonic tissue may not have the same ascorbic acid requirements for their development as has collagen formation in tissue repair.