Edward Meilman

Urinary hydroxyproline peptides.

Collagens from widely different animal sources all contain the amino acid hydroxyproline (hypro). Except for a small amount found in elastin, hypro has been found in no other animal protein and therefore may be considered an in vivo label of collagen. In the absence of exogenous sources (diet rich in gelatin or collagen), individuals excrete a more or less constant amount of hypro. This occurs predominantly in the peptide form (1-3). Free hypro in the urine, even when large amounts are ingested, never exceeds a few per cent of the total hypro excretion (1). On a diet free of hypro sources, the amounts of urinary hypro are consistent with the estimated small turnover rates of collagen (4). Ziff, Kibrick, Dresner, and Gribetz (1) reported that the total amount of bound hypro in the urine was essentially unchanged in patients with rheumatoid arthritis or other disorders of connective tissue, as compared to normal controls. High values of bound urinary hypro have been reported present in children (1), Marfans syndrome (5), burn patients (6), and hyperparathN roidism (7). Information concerning the nature and number of the hypro peptides in the urine and their variation in disease states remains fragmentary. Westall (8) demonstrated the presence of a urinary peptide containing equal amounts of proline (pro) and hypro. Mechanic. Skupp, Safier, and Kibrick (9) identified two peptides containing (hypro) 4 (pro)4 (glu) and hypro[ (hypro) (pro) 3(glu) ,], isolated from the urine of a patient with rheumatoid arthritis on a hypro-free diet. Recently, Kibrick, Hashiro, and Safier (10) found no evidence of these two peptides among the five peaks of hypro peptides obtained by ion-exchange chromatography from the urine of three patients with arthritis and two normal individuals.

Absorption of bile acids from the large bowel in man

The absorption of bile acids from the human large bowel was studied in eight patients. All patients had cholecystitis and cholelithiasis and had to undergo cholecystectomy. Cholic acid-(14)C was injected during surgery into the lumen of the cecum, hepatic flexure of the colon, or transverse colon in six patients, under the visual control of the surgeon. Common duct bile was collected by T tube daily for 5 days, and bile acids were extracted. Significant amounts of radioactivity appeared in T tube bile in each patient. T tube bile acids contained a total of 43.6-84.6% of the administered radioactivity; the average for the six patients was 58.9%. The majority of the tracer was excreted during the first 24 hr. In an additional patient cholic acid-(14)C was given in the form of an enema 5 days postoperatively. In this subject 30.8% of the retained radioactivity was excreted through the T-tube in 48 hr. The labeled cholic acid was recovered as both cholic and deoxycholic acid from T tube bile. Thin-layer chromatographic analysis of the bile acid samples indicated that the fraction of radioactivity recovered as deoxycholate increased with time during the postoperative period. Gas-liquid chromatographic analysis showed that the daily total quantity of excreted bile acids increased significantly from the 1st-5th days of the experiment. The amount of cholate excreted in T tube bile increased markedly with time, that of chenodeoxycholate increased moderately, and that of deoxycholate decreased sharply during the 5 days of the experiment. In three patients, injection of radiopaque material mixed with the tracer showed no evidence of regurgitation into the small bowel by serial X-rays. In an additional patient, tube aspirate from the terminal ileum contained no radioactivity. The results indicate that cholic acid is converted to deoxycholic acid in the human colon, and both of these bile acids are absorbed from the human large bowel in significant amounts. These data establish the previously unproved concept that significant absorption of bile acids takes place from the large bowel of man.

Cell-free collagen biosynthesis and the hydroxylation of sRNA-proline☆

Abstract Cell-free incubation systems employing supernatant and ribosomal fractions prepared from chick-embryo homogenates were found to be active with respect to their ability to incorporate proline from free proline-C14 or sRNA-proline-C14 into ribosomal protein. Aerobic conditions enhanced the formation of peptide-bound hydroxyproline-C14 particularly when sRNA-proline-C14 was added. In either case, the incorporation of hydroxyproline lagged behind that of proline. In a separate series of experiments it was found that conditions similar to those which enhance the incorporation of hydroxyproline into ribosomal protein also favor the transformation of portions of sRNA-proline complexes to sRNA-hydroxyproline. Evidence was obtained that factors present in the microsomal supernates and which were associated with the microsomes themselves were involved in the hydroxylation of sRNA-proline. The microsomal-associated factor could be removed by treatment with deoxycholate in which case the RNP-particles were inactive.

Studies with a cell-free system of proline incorporation into protein cleaved by collagenase

Abstract A cell-free incubation system containing skin microsomes was found to incorporate proline-C 14 into ribosomal protein. The collagen content of this protein was low, and the conversion of proline to hydroxyproline could not be demonstrated. However, the ribosomal protein was found to act as a substrate for purified bacterial collagenase with the resultant release of dialyzable peptides. Two of these peptides were shown to be Gly-Pro-Hypro and Gly-Pro-Ala which are known to occur in collagen. Both were found to contain C 14 activity, indicating that the proline-C 14 was incorporated into a collagen precursor or peptide sequences characteristic of collagen.

The Need For Modification of the Polyvinyl Sponge Model of Connective Tissue Growth: Histologic and Biochemical Studies in the Rabbit

Quantitative histopathologic and biochemical comparisons were made between polyvinyl sponge capsular and sponge tissue in the rabbit on different days after subcutaneous implantation. Up to 9 days the predominant cell type in the capsular tissue is the fibroblast and in the sponge it is the neutrophil. During this time period the sponge tissue shows lower rates of (14C) proline and (14C) cytidine incorporation and lower rates of total (14C) collagen synthesis than the surrounding capsule. Different gel electrophoretic patterns of isolated radioactive proteins are found in sponge and capsule at 6 days. These biochemical differences appear to be related to the small number of fibroblasts, relative to granulocytes present in sponges during the first 9 days after implantation. It is suggested that future biochemical investigations of the early phase of connective tissue reactions (first 9 days) in this model utilize sponge capsular tissue within 1 cm of the sponge edge instead of the sponge and its contents.

Hydroxyprolyl-soluble ribonucleic acid and the biosynthesis of collagen.

Abstract Prolyl-U-14C-sRNA and iminoacyl-U-14C-sRNA (containing hyprolyl-14C- and prolyl-14C-sRNA), prepared from chick-embryos, were separately incubated with microsomal and ribosomal systems. The incubation reactions were carried out under atmospheres of N2 (anaerobic) only or N2 followed by 95% O2:5% CO2, and the incorporation of pro-14C into hot trichloroacetic acid-extractable protein (collagen) was determined. If hyprolyl-sRNA is the precursor of collagen hydroxyproline residues, there should have been a direct transfer of hypro, even under anaerobic conditions, from its sRNA linkages to the templates. When either prolyl-14C- or iminoacyl-14C-sRNA was employed, significant incorporation of hypro-14C was obtained only when anaerobic incubation was followed by aerobic conditions. The incorporation into hypro-14C increased with aerobic incubation time, but it did not require, nor was it enhanced by, the presence of hyprolyl-sRNA in the reaction mixtures. In a second series of experiments, chick-embryo sRNA was fractionated on DEAE-Sephadex columns. As a result, prolyl-14C-sRNAs were prepared which could not be (hydroxylated) converted to hyprolyl-14C-sRNA. The same preparations, when incubated with microsomal systems, did serve as efficient precursors of pro-14C and hypro-14C residues which were incorporated into hot trichloroacetic acid-extractable protein. The evidence obtained indicates that prolyl-sRNA and not hyprolyl-sRNA is the precursor of collagen hydroxyproline residues. The results support the thesis that the hydroxylation of proline occurs after its incorporation into microsomal peptide linkages.

Biosynthesis of procollagen in rabbit sponge capsular tissue.

Abstract An in vitro incubation system for the biosynthesis of rabbit ivalon sponge capsule collagen is described utilizing [ 14 C]proline as the labeling compound. Incorporation of isotope was obtained into a non-dialyzable proline-rich protein that was extractable with neutral salt and weak acid. This protein was lysed to the extent of 46–48 % by collagenase and behaved like the α 1 collagen peptide on a carboxymethyl-cellulose column.

Fibroblast DNA Synthesis Activation in Sponge Induced Granulation Tissue: The Effect of Antineutrophil Serum and Cyclophosphamide

DNA synthesis in rat and rabbit polyvinyl sponge induced granulation tissue has been studied using thymidine (methyl-3H). Synthesis was determined by measurement of thymidine incorporation into cold trichloroacetic acid insoluble material and by autoradiography. Granulation tissue was removed and immediately incubated in vitro in the presence of thymidine (methyl-3H) for three hours. The label was incorporated into the nuclei of fibroblasts and, to a lesser extent, of endothelial cells. The labeled material was 93% lysable by DNase and its synthesis was inhibited by hydroxyurea and bleomycin. In this system synthesis was linear for two hours and then ceased. A marked increase in DNA synthesis occurred in tissue harvested at 44 hours after sponge implantation. This rise was confirmed by autoradiographic studies which showed an increase in nuclear labeling at two days after implantation. Neutropenia produced by injections of antineutrophil serum or cyclophosphamide failed to inhibit activation of DNA synthesis in fibroblasts or endothelial cells. Amonocytosis also had no effect on this process. Rates of thymidine incorporation into DNA and thymidine phosphates in vivo were similar to those found during in vitro incubations of granulation tissue.

A quantitative system for the study of collagen and hyaluronic acid synthesis in vitro

Abstract The relationship of various parameters to the biosynthesis of connective tissue proteins and polysaccharides in rabbit sponge capsular tissue in vitro is presented. A linear relationship of isotope incorporation to microsomal RNA and DNA content per flask was found. Enzymatic analysis of 1 M NaCl-soluble products indicates that the [14C]proline-rich macromolecule is at least 50% procollagen and d -[I-14C]glucosamine-containing polymer is 60–70% hyaluronate and 28% glycoprotein. [14C]Tyrosine was found to be rapidly incorporated into 1 M NaCl-soluble, collagenase-resistant proteins. Applications of these methods to the study of the connective tissue requirements for glucose and other simple metabolites is presented. The effects of alcohol, steroid hormones and insulin on connective tissue synthesis is also included.

Activation of Prolyl Hydroxylase in Sponge Induced Granulation Tissue: The Effect of Antineutrophil Serum and Inhibitor Drugs

Prolyl hydroxylase activity in rat and rabbit sponge induced granulation tissue was studied as a function of time after implantation. The method of expression of such data was critical in determination of the shape of the curve. The method of data expression used in this paper was, cpm/μg of fibrocyte and fibroblast (F-DNA) or cpm per μg hydroxyproline. Use of other parameters such as extractable protein, wet weight or total DNA changed the shape of the curve and obscured the rapid increases in prolyl hydroxylase activity occurring during the first 48 hours.Giemsa and fast green staining was found to be a useful method for identification of fibroblasts in tissue sections. The use of this stain facilitated the large number of differential counts necessary for the calculation of F-DNA from total DNA values.Rats were treated with cyclophosphamide or antineutrophil serum to deplete them of neutrophils and monocytes. Such treatment failed to inhibit the usual increase in prolyl hydroxylase occurring in granula...