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Biomedical subjects

R Timpl

Publications and source records attributed to R Timpl.

At least 433 records · Page 24Linked to original sources
PubMed1976

Simultaneous synthesis of types I and III collagen by fibroblasts in culture.

Specific antibodies against types I and III collagens and procollagens were used to localize these proteins in cultured human cells. These studies indicate that the same cell makes both proteins. No type III procollagen synthesis was observed in cells from two patients with two patients with the Ehlers-Danlos type IV syndrome.

Ascorbic Acid↗
PubMed1976

Isolation and structure of the amino-terminal cross-linking region in insoluble type III collagen.

A collagenous peptide T1X was isolated from a tryptic digest of the insoluble matrix of calf skin. The peptide consists of two identical polypeptide chains each with a length of 72 amino acid residues joined by a cross-link. Absorption spectra obtained from hydrazone and azine derivatives of T1X indicated that the peptide contains an aldol-type of cross-link (X). The sequence of 23 amino acid residues in the amino-terminal region was determined as Glx-Tyr-Glu-Ala-Tyr-Asp-Val-X-Ser-Gly-Val-Ala-Gly-Gly-Gly-Ile-Ala-Gly-Tyr-Hyp-Gly-Pro-Ala. This sequence overlaps the previously described amino-terminal sequence of alpha1 (III) chain obtained from pepsin treated, insoluble type III collagen. Thus, the present data demonstrate a nonhelical segment of 14 amino acid residues in type III collagen important for cross-linking.

Amino Acid Sequence↗
PubMed1976

Requirement for T cells in the antibody response of mice to calf skin collagen.

The antibody response to triple-helical calf skin collagen is apparently regulated by T cells since nude mice (BALB/c strain) responded to this antigen only after transfer of T cells. Syngeneic transfer of B and T cells was required to prepare thymectomized and irradiated normal C57B1/10 recipients for an anti-collagen response. Reconstitution by B cells alone was unsuccessful. The results are compatible with previous evidence on the H-2 linked genetic control of the antibody response to and the non-repetitious nature of helical antigenic determinants in calf collagen.

Animals↗
PubMed1976

Production and specificity of antibodies against the aminoterminal region in type III collagen.

A cross-linked fragment (peptide T1X) with a molecular weight of 13,000 could be isolated from a tryptic digest of insoluble type III collagen of calf skin. Peptide T1X was conjugated on to bovine serum albumin by glutaraldehyde and used for immunization of rabbits. The antisera reacted in passive haemagglutination and radioimmune assay with peptide T1X, type III collagen and its constituent alpha1(III) chain. Little or no reaction was observed with type I collagen and alpha1(I) chain. While rabbit antisera to neutral salt-soluble type III Collagen also showed a strong binding for 125I-labelled peptide T1X much less reaction was observed with antisera to type I collagen. The antigenicity of type III collagen was largely destroyed by pepsin treatment suggesting that it resided in non-helical segments. A fragment of peptide T1X produced by digestion with collagenase retained antigenic activity. The data indicated that the aminoterminal region of type III collagen contains strong antigenic determinants located in a non-helical sequence of about sixteen amino acids. Antibodies to these antigenic determinants were purified and rendered specific for type III collagen by immunoadsorption. The antibodies stained in indirect immunofluorescence tests particularly those regions in various connective tissues which are rich in reticulin fibres. Different staining patterns were observed with antibodies to type I collagen.

Antibodies↗
PubMed1976

Visualization of type I and II collagens in tissue sections by immunohistologic techniques.

Rabbit and rat antibodies were prepared against Type I and II collagens derived from bovine skin and articular cartilage, respectively. As judged by passive hemagglutination and radioimmune assays, these antibodies could be rendered generally specific for the type of collagen used for immunization by immunoadsorption. Thus, antibodies to Type II collagen did not cross-react with Type I and III collagens from skin. However, antibodies to Type I collagen still showed some cross-reaction with Type III collagen. Antibodies to Type I procollagen showed a negligible degree of cross-reaction with Type III procollagen. These purified antibodies reacted strongly with bovine and human tissue collagen as demonstrated by indirect immunoflourescence. Antibodies to Type I collagen stained dermal tissue, perichondral tissue, kidney stroma, aortic tissue, and annulus fibrosus. Antibodies to Type II collagen stained mainly the hyaline matrix of rib cartilage and nucleus pulposus. The staining patterns with anti-Type I procollagen were similar but not identical to that found with antibodies to Type I collagen. Neither of these antibodies reacted with kidney glomerular basement membrane. These antibody reagents are recommended as a sensitive and rapid screening tool for studying tissue distribution of collagen under normal and pathologic conditions.

Animals↗
PubMed1975

Antigenic structure of the cyanogen bromide peptide F-CB3 from fibrinogen alpha-chain.

The antigenic properties of the cyanogen bromide peptide F-CB3 from bovine fibrinogen alpha-chain were studied in radioimmune assays with rabbit antibodies to fibrinogen or to peptide F-CB3. Both fibrinogen and peptide F-CB3 were indistinguishable in inhibition and dissociation tests. Modification of the single disulfide bridge in peptide F-CB3 either by reduction or by cleavage with cyanide was not accompanied by loss of serologic activity. Inhibition studies with three individual fragments obtained after cyanide cleavage (molecular weight range 7000 to 23000) indicated the presence of at least three distinct antigenic determinants in peptide F-CB3. After trypsin digestion of peptide F-CB3 still 75% of its maximal inhibiting capacity was retained. Lack of change in antigenic activity of peptide F-CB3 after release from the fibrinogen molecule by cyanogen bromide and upon further fragmentation is presumably due to the presence of several sequential antigenic determinants but the presence of conformational determinants could not be entirely excluded. Since no cross-reaction was observed between bovine and human peptides F-B3 one may expect considerable variation in their amino acid sequence.

Animals↗
PubMed1975

Antigenic determinants in the disulfide regions of bovine fibrinogen.

Rabbit antibodies to bovine fibrinogen were used to study the antigenic activity of four cyanogen bromide peptides containing the disulfide regions of this molecule. In precipitation tests the highest activity was associated with the peptide F-CB3 which is exclusively derived from the alpha-chain. Reduction of the single disulfide bridge in peptide F-CB3 did not influence its serologic activity. Weaker reactions were observed with the N-terminal multichain peptide F-CB1. The antigenicity of peptide F-CB1 was not affected by removal of fibrinopeptides A and B but it was lost after reduction. The immunological activity of the multichain peptide F-CB2 was even less than that of peptide F-CB1 and the antigenic determinants were destroyed by reduction. A large fragment essentially composed of peptides F-CB1 and F-CB2 could be obtained by limited cyanogen bromide cleavage and showed considerably better immunological activity than peptides F-CB1 and F-CB2 together. Apparently no activity was associated with a mixture of small hydrophobic, disulfide-loop peptides tentatively called peptide F-CB4. The large loss of antigenic activity in cyanogen bromide digests of fibrinogen suggests that the disulfide-stabilized regions do not have an important role in maintaining conformational antigenic determinants of fibroinogen. Changes in noncovalently stabilized conformation requiring uncleaved chains is considered as a possible reason for the findings observed.

Amino Acids↗
PubMed1975

H-2-linked genetic control of antibody response to soluble calf skin collagen in mice.

An autosomal dominant immune response gene could be demonstrated in congenic resistant strains of mice which is linked to the H-2 locus and controls the antibody response to soluble calf collagen. High responsiveness was associated with the H-2 alleles, b and f, low responsiveness with the H-2 alleles, d, k, m and r. Studies with calf procollagen, which contains an additional carrier moiety, indicated that these genetic differences might be expressed at the level of T cells.

Animals↗
PubMed1975

Non-helical sequences of rabbit collagen. Correlation with antigenic determinants detected by rabbit antibodies in homologous regions of rat and calf collagen.

Non-helical peptide fragments were isolated from rabbit skin collagen after cleavage of alpha chains with cyanogen bromide and proteases. Determination of their amino acid sequence indicated a length of 9, 16 and 25 amino acid residues for the non-helical sequences located in the N-terminal region of alpha2 and alpha1 chain and in the C-terminal region of alpha1 chain, respectively. The C-terminal sequence Tyr-Tyr hitherto considered as the genuine end of collagen alpha1 chain is in part of rabbit collagen extended by two residues, alanine and arginine. Rabbit collagen may differ considerably in its non-helical sequences from other vertebrate collagens, particularly in the C-terminal part. Some but not all of these differences are clustered in areas occupied by antigenic determinants which are recognized in the antibody response of rabbits to rat or calf collagen. On the other hand, a high homology to rabbit collagen, e.g. in the N-terminal region of rat collagen alpha1 chain or calf collagen alpha2 chain, probably prevents immunological recognition by the rabbit. The degree of foreignness alone, however, may not necessarily determine whether a particular non-helical area is able to express immunogenic activity.

Amino Acid Sequence↗
PubMed1975

Purified antibodies to collagen: an immunofluorescence study of their reaction with tissue collagen.

Antibodies to soluble calf or rat skin collagen were purified by appropriate immunoadsorption and separated into three subfractions directed either to antigenic sites on unfolded alpha-chains (denatured collagen), to nonhelical sites, or to helical sites exposed on the triple helical molecule. In indirect immunofluorescence tests each of the antibody solutions reacted with collagen of skin and kidney tissue, although the latter two antibody solutions appeared to be more active. Distinct activity was also observed with antibodies to the N-terminal antigenic determinant of rat skin collagen alpha2-chain, a structure usually involved in cross-linking. Indirect immunofluorescence tests with anti-collagen sera on sections of skin resulted in the staining of the whole dermis, while anti-procollagen sera revealed binding only to the uppermost subepithelial layer of the dermis (stratum papillare). On kidney sections only the interstitial connective tissue reacted with purified anti-collagen or anti-procollagen sera. Both skin and glomerular basement membranes remained unstained with either kind of purified antibodies. However, antisera not subjected to immunoadsorption do react with the glomerular basement membrane. Antibodies to noncollagenous contaminants are considered to be responsible for this finding which emphasizes the necessity to use purified antibodies exclusively for this type of immunofluorescence analysis.

Adsorption↗
PubMed1975

Isolation, chemical and electron microscopical characterization of neutral-salt-soluble type III collagen and procollagen from fetal bovine skin.

Extraction of fetal bovine skin at neutral pH and in the presence of protease inhibitors solubilized substantial amounts of type I and type type III collagen and Type III procollagen. Type I and Type III collagen were separated from each other by salt precipitation, and DEAE-cellulose chromatography was used to separate collagen from procollagen. The main chain constituents in type III collagen and procollagen were disulfide-bonded gamma and pgamma components, respectively. Amino acid composition, cross striation banding as observed using electron microscopy, cyanogen bromide peptide patterns in disc electrophoresis gels and resistance of the disulfide regions to pepsin digestion indicated a close similarity to previously described insoluble type III collagen, which was solubilized by limited pepsin digestion. Electron microscopy of long-spacing-segment crystallites and evidence for an extended form of the disulfide-bonded cyanogen bromide peptide suggested that neutral-salt-soluble type III collagon is longer at its C-terminal end by about 10 to 20 amino acid residues than pepsin-treated material. A small elongation was also indicated in the N-terminal portion of the molecule. Procollagen has an additional N-terminal extension with a length of about 160 A, but no difference was observed between collagen and procollagen at their C-terminal ends.

Amino Acids↗
PubMed1975

Demonstration of a unique antigenic specificity for the collagen alpha1 (II) chain from cartilaginous tissue.

The rabbit antibody response to native collagen (chain composition [alpha1(II)3) from cartilaginous tissue, has been examined by agglutination assays, gel diffusion, haemagglutination-inhibition studies, and immunoadsorption. The results show that the rabbit antibody response to the cartilage-type collagen is characterized by considerable reactivity to both helical [alpha1(II)]3 as well as alpha1(II) chains. This is in contrast to the rat antibody response to the same antigens where titres are generated to largely helical antigenic determinants. Similarly to the rat response, rabbit antibodies to [alpha(II)]3 exhibit no strong cross-reaction with the genetically distinct [alpha(I)]2ALPHA2 collagen or its component chains. Strong cross-reactions were, however, observed between bovine and chick alpha1(II) chains. One of the major antigenic sites on [alpha1(II)]3 collagen appears to reside in the sequence represented by CB-11, a peptide derived from the helical portion of the [alpha1(II)]3 molecule after cyanogen bromide cleavage. The data, however, are compatible with the presence of other antigenic determinants which are probably located in the amino- and carbocy-terminal portions of the molecule.

Agglutination Tests↗
PubMed1975

Specificity of the antibody response in inbred mice to bovine type I and type II collagen.

Mouse antibodies to soluble bovine skin (type I) collagen react with determinants which are located in the rigid triple-helical portion of the antigen and become destroyed upon unfolding the molecule. Helical antigenic determinants are dependent on the genuine chain assembly, e.g. alpha[1(I)]2alpha2. Artefactual triplehelical structures of the composition [alpha1(I)]3 or [alpha2]3 or a genetically distinct type II collagen from cartilage showed no or only weak cross-reactivity. Pepsin treatment of type I collagen known to remove short, non-helical sequences at both ends of the molecule had virtually no effect on antigenicity and immunogenic activity. A radioimmunoassay failed to detect antibodies in three congenic resistant mouse strains immunized with denatured type I collagen. These strains had been previously classified as high or low responders to native type I collagen. Agglutination titres vs denatured collagen culd already be demonstrated in nonimmune sera. The agglutinating activity was labile against heating at 56 degrees and could not be increased by immunization. Two out of five inbred strains showed a high response against pepsin-dissolved bovine type II collagen with the chain composition [alpha1(II)]3. Lack of correlation in the responder state to both collagen types indicated control by different immune response genes. Antibodies to type II collagen also reacted against triple-helical antigenic determinants and showed neglible cross-reaction with type I collagen.

Animals↗