Rooster Comb Research Articles

Hyaluronic acid synthesis in rooster comb. Effect of testosterone on nucleotide sugar metabolism.

Enzymes involved in the synthesis of UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc), the nucleotide sugar precursors of hyaluronic acid, were assayed in extracts of the mucoid layer of combs of normal roosters, castrated roosters (capons) and capons treated with testosterone. UDP-glucose (UDP-G) dehydrogenase and UDP-GlcNAc pyrophosphorylase activities were found to be present in extracts of both normal and capon comb. Although UDP-G pyrophosphorylase activity could be demonstrated in extracts of normal combs, it was impossible, initially, to demonstrate this activity in capon extracts. A deficiency of this enzyme would result in less UDP-GlcUA available for the synthesis of hyaluronic acid. A protein inhibitor of this enzyme was found in capon extracts which inhibited the activity of the enzyme from normal comb. Glucose 1-phosphate (G1P) was found to overcome this inhibition. By increasing the G1P or UTP concentrations in assays of UDP-G pyrophosphorylase in capon comb extracts,...

Studies on Hyaluronic Acid V. Relationship Between the Protein Content and Viscosity of Rooster Comb Dermis Hyaluronic Acid

Protein accounted for an average of 8.7% w/w of the hyaluronic acid obtained from rooster comb dermis extracts and three types of peptide constituents appeared to be present. A few collagen-like fibers were closely associated with the hyaluronic acid when samples were examined in the electron microscope and collagenase treatment decreased the intrinsic viscosity from 7000-5000 ml/g to 3900-2700 ml/g. The quantities of collagen present, however, were too small to detect chemically with the methods employed. The major peptide consituent was readily separated from the hyaluronic acid by fractionation in a cesium chloride gradient or by treatment with pronase. The viscosity was decreased by the density gradient procedure but not by the pronase digestion. Repeated fractionation in a cesium chloride gradient decreased the intrinsic viscosity still further and a small peptide constituent with a high glycine and serine content remained associated with a hyaluronic acid. The data suggest that an interaction or entanglement with collagen fibers is responsible for the high viscosity of hyaluronic acid in this tissue extract and that the viscosity of purified hyaluronic acid preparations is dependent upon interactions between adjacent polysaccharide chains. Interactions between the major peptide constituent and polysaccharide chains or the small residual peptide component remaining with hyaluronic acid after extensive purification procedures, however, appear to be involved in some organized structure because the presence of the major peptide constituent minimized the decrease in viscosity that occurred when hyaluronic acid samples were lyophilized.

Non-Immunogenicity of Purified Hyaluronic Acid Preparations Tested by Passive Cutaneous Anaphylaxis

The immunogenicity of purified hyaluronic acid (HA) from human umbilical cord and rooster comb, and of some modified HA preparations, was studied in rabbits. HA preparations contained 0.28–0.57% of protein or peptides (Folin’s method). Rabbits received four intramuscular injections at 4-weekly intervals of the following potential immunogens, emulsified in complete Freund’s adjuvant: (1) human umbilical cord HA (HUC-HA), 500 <i>μ</i>g/dose; (2) HUC-HA, precipitated with cetylpyridinium chloride (CPC), 500 <i>μ</i>g/dose; (3) HUC-HA-ovalbumin conjugate, precipitated with CPC, 500 <i>μ</i>g/dose, and (4) rooster comb HA at dose levels of 5, 50 and 500 <i>μ</i>g. Rabbit sera were tested before, during, and after immunization for PCA reactive antibodies against HA in guinea pigs. 11 of 25 pre-immunization sera showed low titres of HA-cross-reactive antibodies. No formation of PCA reactive antibodies against any of the four HA preparations was observed during or after immunization.

Testosterone metabolism in rooster comb

After testosterone was incubated with the cell-free homogenates of rooster comb in air in the presence of NADPH, 5β-androstane-3β,17β-diol was identified as the major metabolite and 5α-dihydrotestosterone, 5β-dihydrotestosterone, 5α-an-drostane-3α, 17β-diol and 5β-androstane-3α,17β-diol were also obtained. Among the subcellular fractions, 5α-reductase activity was mostly detected in the microsomal fraction (10000–105000 × g), while 5β-reductase activity was exclusively found in the cytosol fraction (supernatant fluid at 264000 × g). 3α-Hydroxysteroid dehydrogenase (EC 1.1.1.50) which prefered 5α-dihydrotestosterone as the substrate was localized in the cytosol and microsomal fractions, but the dehydrogenase which prefered 5β-dihydrotestosterone was found in the microsomal and mitochondrial (800–6000 × g) fractions. 3β-Hydroxysteroid dehydrogenase (EC 1.1.1.51) which reduced 5β-dihydrotestosterone but not its 5α-epimer was found mainly in the cytosol fraction. The 3α- and 3β-hydroxysteroid dehydrogenases in the cytosol and microsomal fractions of the comb utilized specifically 4-pro-S-H of NADPH for reduction of the oxo group of 5α- and 5β-dihydrotestosterones. With aging, 5α-reductase activity showed a decrease, whereas 5β-reductase remained unchanged. Among the metabolites, the 5α-hydrogenated steroids showed significant androgenic activities, but the 5β-hydrogenated steroids showed no comparable activities upon cockerel comb. Androgenic activity of 5α-dihydrotestosterone upon the comb was significantly reduced by simultaneous administration of 5β-dihydrotestosterone.

The effect of testosterone on ascorbic acid synthesis in rooster comb

The level of “total” ascorbic acid (ascorbate+dehydroascorbate) has been measured in the mucoid layer of combs from normal roosters, capons and capons treated with testosterone. The “total” ascorbate level in capon comb was lower than the value obtained from combs from normal roosters. This value returned towards normal in combs from capons treated with testosterone. The specific activity of L-gulonate-NADP + oxidoreductase, an enzyme in the pathway of ascorbate biosynthesis, also was measured. The specific activity levels followed a pattern similar to the ascorbate levels in the three types of combs utilized. The results are consistent with the possible role of L-ascorbic acid as a cofactor in the synthesis of collagen, a process which also appears to be dependent on the level of testosterone in the comb mucoid layer.

Hyaluronic acid: Structure of the macromolecule in the connective tissue matrix

Hyaluronic acid was extracted from the mucoid layer of rooster combs with limiting viscosity numbers as high as 13,000 ml per g. This material was shown to contain weak bonds, which were broken, irreversibly, by heating at neutral pH's at temperatures above 65°C. The activation energy of this reaction was 18Kcal per mole. Purified hyaluronic acid did not show this heat dependent irreversible degradation and protein did not seem to be a component of the structure formed by these bonds. These data suggest that the polysaccharide chains of the hyaluronic acid in the connective tissue matrix are organized into a macromolecular structure or network.

Effect of testosterone on β-glycosidases in rooster comb and wattles

Effect of testosterone on β-glycosidases in rooster comb and wattles

Studies on hyaluronic acid II. The protein component(s) of rooster comb hyaluronic acid

1. 1. The amino acid content of hyaluronic acid fractions isolated from the rooster comb has been determined. It was found that five amino acids—serine, glycine, alanine, aspartic acid and glutamic acid—accounted for approx. 65% of the total amino acids present. The amounts of aspartic acid in each fraction were very similar. 2. 2. Expressed as a weight percentage, the amino acid content of the hyaluronic acid fractions was 0.35% and despite large variations in molecular sizes the amino acid contents per mole of hexuronic acid were very similar. 3. 3. During the purification of the hyaluronic acid there was a drop in the limiting viscosity number and also in the amino acid content. The protein(s) removed, however, had an amino acid composition that differed significantly from the component(s) that remained and were thought to be chemically distinct from the hyaluronic acid molecule as such. 4. 4. Alkali treatment of the hyaluronic acid fraction that constituted approx. 90% of the total present in the comb, decreased the serine content but did not significantly reduce the molecular size of the material. 5. 5. The significance of these data is discussed.

Studies on hyaluronic acid: I. The preparation and properties of rooster comb hyaluronic acid

1. 1. Hyaluronic acid has been prepared quantitatively from the rooster comb under mild conditions, with a yield of 6% of the acetone-dried combs. 93% of this hyaluronic acid was extracted with distilled water, while stronger treatments, 1 M NaCl extraction and digestion with pronase, were required to remove the remainder of the hyaluronic acid from the comb tissues. 2. 2. The crude extracts were purified by a modified Sevag's procedure, epichlorohydrin triethanolamine- (ECTEOLA-) chromatography and by fractionation with cetylpyridinium chloride. Hyaluronic acid purified by these procedures was recovered with a yield of greater than 90% with respect to hexuronic acid. 3. 3. Analysis of the purified products by physical and chemical methods showed that the hyaluronic acids in each fraction were identical with respect to their hexuronic acid and glucosamine content, their infrared absorption, optical rotation and electrophoretic mobility. Viscosity measurements and molecular weight determinations using a sedimentation equilibrium method showed the purified hyaluronic acids from each fraction to be of different molecular sizes. 4. 4. The significance of these results is discussed in relation to the known structure and chemical composition of hyaluronic acid and the rooster comb.

Chondroitin sulphate and collagen in the connective tissue of the rooster comb

Chondroitin sulphate and collagen in the connective tissue of the rooster comb

C14 assays and autoradiographic studies on the rooster comb.

The distribution of C(14) was studied in various parts of the rooster comb following treatment with testosterone. The value of gas-phase assay of C(14) in tissue has been demonstrated and the results compared with those of autoradiographic studies on the same tissue. The results of these experiments showed that androgen treatment significantly increases the rate of incorporation of C(14) in various parts of the comb. The specific activity of carbon in the comb, cornea, and liver differed, depending on which precursor, viz. glucose-6-C(14), glucose-1-C(14), and glucuronolactone-U-C(14), was administered. The highest values were obtained after the administration of glucose-6-C(14); glucuronolactone-U-C(14) gave the lowest specific activity. The specific activity of carbon in different parts of the comb showed considerable variation. Carbon assay of serial sections of the comb cut at various planes showed that the specific activity of carbon was highest in the mucoid layer. Both C(14) assays and autoradiograms indicate that C(14) is also present in other parts of the comb. As seen in autoradiography, the concentration of C(14) was highest in the epithelium, in the blood vessel walls, and in the avascular collagenous tissue. These results, and indications from previous studies, suggest that the high specific activity of carbon in the mucoid layer is due mainly to the presence of C(14)-labelled hyaluronic acid. Autoradiograms and PAS staining suggest that a significant amount of C(14) is also incorporated into the glycoproteins associated with the collagen fibers.

DYEBINDING AND MUCOPOLYSACCHARIDE CONTENT IN CONNECTIVE TISSUE

The hyaluronic acid content of the mucoid layer of the rooster comb and the chondroitin sulfuric acid content of bovine nasal cartilage were determined in the fresh tissue, in the water extract of the tissue, and in the water-extracted residue. The binding of cationic dye by the tissue, the tissue extract and the residue was compared with the mucopolysaccharide content. The molar ratio of the bound dye to the anionic sites of the mucopolysaccharides varied between 1.1 and 1.8. In the comb extract this ratio was one. This indicates that, with the exception of the comb extract, anionic sites other than those of the mucopolysaccharides are also responsible for the dyebinding.

Quantitative determination of cationic dyebinding in connective tissue.

Mucoid layer of the rooster comb and cartilage of the nasal septum of steers were used to measure the binding of cationic dyes by unfixed frozen sections. The amount of dye bound was calculated from the decrease of the optical density of the dyebath after equilibrium had been reached between the dyebath and the tissue at the saturation level. Various factors influencing this reaction, such as the tissue-dye ratio, the dyebath concentration, the thickness of the tissue sections and mechanical agitation, have been studied. In order to quantitatively determine the dyebinding, certain conditions must be fulfilled. The dyebath concentration must be high enough to be well in excess of all available binding sites of the tissue. The precipitate formed and the tissue fragments must be removed prior to optical density measurement. The electrolyte concentration of the dyebath must be kept below a certain level. The dyebath should not contain colored substances other than the free dye. Under these conditions, the amount of dye bound by azure A and other cationic dyes is constant for a given tissue, depending only on the pH of the dyebath. Electrostatic interaction between the cationic dye and the anionic sites of the tissue is primarily responsible for dyebinding. The quantitative determination of dyebinding is a measure of the free anionic sites of a tissue.

STUDIES ON THE CONNECTIVE TISSUE OF THE COCK COMB. III. MAST CELLS AND THE EFFECT OF TESTOSTERONE

Although the most obvious structural changes of the comb in response to androgen stimulation take place extracellularly (Champy, 1926, Hardesty, 1931, Ludwig & Boas, 1950, Rizzoli & Rondinini, 1950, Szirmai, 1949, 1954, 1956 b), it seems likely that this process, in one way or another, also involves cellular activity. In several »steroid sensitive connective tissues« (Szirmai, 1954) the accumulation of the ground substance has been found to be accompanied by swelling and enlargement of the fibroblasts (Hardesty, 1931, Aykroyd & Zuckerman, 1938). Alkaline phosphatase activity in the connective tissue cells during testosterone treatment of young chickens has been reported by Ludwig & Boas (1950) and Rizzoli & Rondinini (1950). Using histochemical techniques as well as biochemical assay, we found no significant alkaline phosphatase activity in the comb of testosterone-treated capons (Szirmai, 1949). Ludwig & Boas (1950) and Schiller, Benditt & Dorfman (1952) observed cellular proliferation in young chickens receiving androgen treatment. We found significant mitotic activity in the connective tissue of the comb of young cockerels only during the first few weeks of normal development; later, when the accumulation of the metachromatic ground substance starts, the mitotic activity decreases (Szirmai, 1956 b). Similarly, in the case of testosterone-treated capons, no cell divisions could be observed. Since the main phenomenon of the »expansion growth« is the accumulation and subsequent hydration of the ground substance in the connective tissue of the comb, it may well be asked how the cells in this tissue are eventually concerned in the formation of the ground substance. Fibroblasta have been held responsible for this process, but without definite proof (Garrault, 1934, Hardesty, 1931). It has been suggested by Aykroyd & Zuckerman (1938) that mast cells play a role in the formation of the ground substance in the sex skin of the monkey. Recently Klenermun (1955) described cyclical variations in the cell population of the perineal connective tissue in the female baboon, including changes in the morphology and numerical distribution of mast cells. Mast cells have occasionally been observed in the rooster comb (Rommeney, 1934, Szirmai, 1949), although Fernández Morán (1943) states that such an occurrence is exceptional.