The induction of cross-links in soluble rat skin collagen in vitro.
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ABSTRACT:
SUBMITTER: Bannister DW
PROVIDER: S-EPMC1184922 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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Project description:This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N(in)-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor alpha-aminoadipic delta-semialdehyde was isolated in its reduced form, in-hydroxynorleucine, together with its acid degradation product in-chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the delta-semialdehyde derived from lysine and hydroxylysine.
| S-EPMC1179041 | biostudies-other
Project description:1. Soluble calf-skin collagen has been denatured thermally between 37 degrees and 60 degrees and the component proteins have been separated on carboxymethylcellulose. 2. Four main fractions have been separated; alpha and beta (in the nomenclature in common usage) and two other fractions. (The alpha and beta components are complex owing to the presence of alpha(1), alpha(2), beta(1) and beta(2) parts). 3. Fractions 3 and 4 undergo rapid denaturation between 39 degrees and 40 degrees whereafter fraction 4 remains virtually unchanged even at 60 degrees . 4. That portion of fraction 4 which remains at 60 degrees is thought to be identical with the fraction designated gamma by other workers, this fraction being composed of three alpha-chains in covalent linkage (such bonds are alkali-labile). 5. The equilibrium between alpha, beta and fractions 3 and 4 is apparently reversible since acid-soluble collagen after denaturation at 45 degrees or 60 degrees followed by cooling to 0 degrees for 30min. was found to contain only fraction 4 when chromatographed at 37 degrees .
| S-EPMC1214329 | biostudies-other
Project description:The purpose of the present study is to determine the effect of Percutaneous Collagen Induction (PCI) on the epidermis and dermis, including the systemic inflammatory response on gene expression level using microarray analysis. PCI Therapy is an alternative for safely treating wrinkles and scars and smoothening the skin. Therefore animal experiments were performed using 31 male Sprague-Dawley rats (350–375 g), age 4 month, randomly assigned into three groups: group (A) (n=24: needling plus skin care), group (B) (n=6: skincare only, controls after 24 h) and group (C) (n=1: negative control). Rats were anesthetized, shaved, and received a 30% total body surface area (TBSA) scald needling (10min) to induce percutaneous collagen, using a medical needling instrument (Environ® Medical Roll-CITTM, Vivida SA cc, Cape Town, South Africa). After needling surgery, the rats were immediately prepared with high levels of vitamin A cream and vitamin C cream, applied topically after cleaning once per day. The control group (C) rats received no injury, no skin care, no treatment, no anesthesia, and no analgesia. Gene expression analyses were performed 1 h, 24 h, 2, 4, and 8 weeks after PCI surgery. To confirm RNA expression in rat skin, self developed microarrays including genes like cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF 2), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and transforming growth factors (TGF ß1, ß2, ß3) were used.
2008-05-31 | GSE11617 | GEO
Project description:A well-characterized three-chain peptide [(Col1)2 X T9] from human type III collagen was a rich source of Ehrlich chromogen. The corresponding two-chain peptide [(Col1)2] was not, implying that the Ehrlich chromogen is a trifunctional cross-link. (Col1)2 X T9 also contained pyridinoline, which is not an Ehrlich chromogen. The 7S domain of type IV collagen also contained an Ehrlich chromogen.
| S-EPMC1154082 | biostudies-other
Project description:Both the type I and type III collagens present in embryonic dermis are stabilized by the intermolecular cross-link, hydroxylysino-5-oxonorleucine, derived from hydroxylysine-aldehyde, although the type I collagen possesses a significant proportion of dehydrohydroxylysinonorleucine. However, concurrent with the change in the proportion of the two types of collagen during postnatal development there is a change-over with both type I and III collagens to the labile cross-link, dehydrohydroxylysinonorleucine, derived from lysine aldehyde. The results indicate that the change in the nature of the cross-link with development is determined primarily by the change in the extent of hydroxylation of the lysine residues in the terminal non-helical regions rather than being due to the change in the type of collagen.
| S-EPMC1172564 | biostudies-other
Project description:New synthetic routes to the reduction products of several collagen cross-links and cross-link precursors are described. By the use of these routes hydroxynorleucine, 5,6-dihydroxynorleucine, hydroxylysinonorleucine and hydroxylysinohydroxynorleucine can be synthesized via one common synthetic intermediate. The synthetic routes provide a convenient source of these unusual amino acids, as well as confirming the structure of hydroxylysinohydroxynorleucine and the other lysine-derived residues found in borohydride-reduced collagens.
| S-EPMC1174044 | biostudies-other
Project description:The periodate-degradation technique was used to demonstrate the mechanism by which the reducible cross-links of collagen are stabilized. In all the tissues examined, Smith degradations of the 3H-labelled cross-links indicated that dihydroxylysinonorleucine is derived solely from hydroxylysino-5-oxonorleucine, the Amadori-rearranged product of the original condensation reaction. Monohydroxylysinonorleucine exists in both keto and aldimine forms, the former being derived from hydroxyallysine and the latter from allysine. Their relative proportions are tissue-dependent and are related to the degree of hydroxylation of the specific lysine residues in both the telopeptides and the triple helix.
| S-EPMC1165631 | biostudies-other
Project description:(1) Proteolytic digests of tissue elastin contain material which reacts with dimethylaminobenzaldehyde in acid solution (Ehrlich's reagent) to give a cherry-pink colour. This Ehrlich chromogen(s) [EC(s)] is similar to but not identical with EC(s) previously demonstrated in tissue collagens [Scott, Hughes & Shuttleworth (1979) Biosci. Rep. 1, 611-618]. Both ECs react with diazonium salts in acid to give coloured products. (2) Diazobenzene linked via a phenolic ester to polyacrylamide beads (Biogel P10) has been used to absorb ECs specifically and almost quantitatively from proteolytic digests. The coupled deeply coloured azo-EC-peptides were then recovered after mild alkaline cleavage from the support and purified by gel chromatography. (3) Using 15N-labelled NaNO2, the collagen azo-EC-peptides were prepared, and 15N abundance measured therein. The molar absorption coefficient of the azo-EC group was calculated (18,700) based on the assumption that each azo-EC group contained one 15N atom. (4) Collagen azo-EC-peptides contained glucose and galactose, whereas elastin azo-EC peptides did not. The amino acid patterns of the two peptides were quite different, the former being rich in polar amino acids, the latter containing much alanine. The patterns were compatible with an origin from the cross-linking regions of collagen and elastin respectively. (5) Quantitative (molar) comparisons of the azo-EC group content with amino acid, amino end-group and sugar contents, and azo-EC peptide molecular mass, suggest that a structure is present in the collagen azo-EC-peptides containing two EC groups shared between four peptide chains. Three peptide chains probably meet at each (cross-linking) EC group. (6) Based on this structure, about 15% of adult bovine skin collagen contains EC groups.
| S-EPMC1149157 | biostudies-other
Project description:The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or 'strength') was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.
| S-EPMC1137721 | biostudies-other
Project description:The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen.
| S-EPMC1147991 | biostudies-other