Much research has been done on the formation of fibrils from solutions of soluble collagen in vitro in order to gain some knowledge of the mechanisms which may occur in vivo. The in vitro formation of fibres from solutions of collagen has been shown to be extremely sensitive to the nature of the solution environment and the presence of added chemical compounds, and thus constitutes an interesting system for the study of collagen-small molecule inter actions. The present study is concerned with the effects of various corticosteroid drugs, used medicinally as anti-inflammatory agents, on collagen in solution. As these corticosteroids are administered to reduce inflammation in conditions such as rheumatoid arthritis and a host of other pathological conditions in which collagen is implicated, this work has been undertaken in order to establish and charac teri ze any binding mechanisms which may be involved. Furthermore, the corticosteroid drugs available commercially in pure form as the free base or as the water-soluble ester salts offer an interesting range of structural and stereochemical variants for the study of their reaction with a complex and biologically important protein molecule such as collagen. A great deal of research on drug- protein interactions (Goldstein, 1949; Meyer and Guttman, 1968a) and more specifically, steroid-protein interactions have been reported over the years (Daughaday, 1959; Sandberg et al., 1966; Villee and Engel, 1961; Westphal , 1971). Comprehensive reports, however, on steroid-collagen interactions in vitro are conspicuously absent from modern scientific literature although relatively superficial accounts have been published (Menczel and Maibach, 1972; Eik-Nes et al., 1954). Although work involving the above has appeared relating specifically to the effects of steroids on collagen biosynthesis both in vivo and in vitro there have been minimal accounts of steroid-collagen interactions tailored to characterize the binding at the molecular level. The effect of corticosteroids on the metabolism of connective tissue has also received special attention (Asboe-Hansen, 1959; Kivirikko, 1953; Nakagawa and Tsurufuji, 1972). Recently, Uitto et al. (1972) reported the effects of several anti-inflammatory corticosteroids on collagen biosynthesis in vitro, whilst Aalto and Kulonen (1972) reported the effects of several antirheumatic drugs on the synthesis of collagen and other proteins in vitro. The interactions between collagen and certain drugs has also been briefly reviewed (Chvapil, 1967). Much data also exists on the binding of a wide range of small molecules and ions with serum albumin (Steinhardt and Reynolds, 1969; Scatchard, 1949; Klotz, 1950). Serum albumin, being specialized for a very general transport function and apparently designed for the purpose of combining with a large range of small molecules, has a proportion of possible reactive sites 'buried' within the molecule itself because of its folded conformation. In addition, serum albumin shows a high degree of cooperative binding in contrast to collagen. The latter molecule, with its larger molecular size and weight is specialized for a biologically structural function and has a higher proportion of possible reactive sites which appear relatively more accessible to ligands. A study of the interactions between corticosteroids and collagen thus provides the opportunity to investigate a protein which is very different from the much studied serum albumin. Because of the limited information available regarding the interaction of steroid drugs and collagen at the molecular level, studies of this nature are relevant to the understanding of the mode of action of steroid compounds which are such an important group of therapeutic substances used in modern medicine.

The collagens are a family of structural proteins which function as an extracellular framework in eukaryotic organisms. They are characterised by a unique protein conformation which consists of three polypeptide chains in a triple helix. At least twelve collagen types encoded by at least twenty non-allelic genes have been identified in vertebrates. There is considerable evidence that each member of the family is represented by a single copy gene. These genes constitute a multi-gene family with a common evolutionary origin. Some of the genes are known to be clustered on certain human chromosomes. The collagens have an extremely important role in development. Alterations in collagen genes can result in a heterogenous group of heritable diseases of connective tissue. There is accumulating evidence that similar phenotypes are due to similar mutations or location of mutations. DNA sequencing studies of cDNA clones of the human type III procollagen gene revealed single base polymorphisms, and one amino acid polymorphism, by comparison with published data. These may be used to generate haplotypes at this locus and increase the polymorphic content for genetic analysis. An attempt to isolate the human type III procollagen gene by cosmid cloning failed. Due to the technical difficulties of performing control experiments, the reasons for this failure could not be identified with certainty. It is possible that this gene is not amenable to cloning using the commonly used cloning reagents. A study of the human type I procollagen gene, COL1A1, revealed the presence of a monomorphic repeat sequence in an intron. This repeat sequence was used to identify a multi-allelic locus in the terminal region of the short arm of chromosome 19. The usefulness of this locus in linkage studies to disorders that map close to this region remains to be analysed.

Abstract The purpose of the study was to evaluate model systems of cancer development and compare some of their critical features with cancer development in vivo. Ovarian and endometrial cancers in man were used as correlates. Tumor development in experimental animals, exposed to carcinogens and UV irradiation, showed the entire spectrum of tumor development as compared to spontaneous carcinomas: hyperplasia, dysplasia, benign papillomas and malignant squamous cell carcinomas. For short-term analysis of differentiated homogenous cell populations, the transplant model proved most useful. For long term analysis of effects of extraneous agents, the skin carcinogenesis model is probably the most rewarding. Analysis of proliferation markers in human tumor samples as studied by immunohistochemistry, showed that an increased expression of PCNA and Ki-67 was associated with poor prognosis in ovarian neoplasms. Analysis of cell proliferation in model tumors showed that the transplant model has a better sensitivity when compared to the animal skin model and the subcutaneous injection model, in that effect of changes in cell-host interaction on the location and extent of the proliferating cell population can be studied therein. The expression of some growth factors, their receptors, oncogenes and suppressor genes were studied in ovarian and endometrial carcinomas and in skin cancer model system in mouse exposed to carcinogens and UV irradiation. Variability in expression and methodological problems precluded detailed analysis of these markers in different models. The expression of TGFβ1, TGFβ2 and TGFβ3 was determined in normal human keratinocytes, and in 7 immortalized and ras-transfected benign and malignant keratinocyte cell lines, maintained as transplants and as subcutaneous tumors in nude mice. By differential immunohistochemical localization of TGFβ isoforms, we demonstrated that each isoform may serve specific roles in tumor development and progression. The complex nature of TGFβ expression prevented detailed analysis of isozymes in different models, the results in this study, however, indicated a similar pattern in the models analyzed. Morphological methods were used to determine relationship between epithelial growth and formation and deposition of collagens in the extracellular matrix in experimental models and human tumors. The composition of the mesenchyme differed in tumors originating from different cell lines reflecting functional interaction between epithelial cells and the mesenchyme in neoplastic development. Tumor-stroma interaction was distinct in human, comparable alterations were observed in experimental models, more so in transplants, less in subcutaneous tumors, affecting tumor growth and differentiation in the different models.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine 2-oxoglutarate 5-dioxygenase, PLOD) catalyzes the hydroxylation of lysine residues in collagens and other proteins. It occurs as a post-translational event. The hydroxylysine residues participate in the formation of covalent cross-links to stabilize the collagenous structure in tissues. The hydroxylysine residues can be glycosylated to galactosyl- or glucosylgalactosylhydroxylysine residues. Novel human lysyl hydroxylases, 2a, 2b and 3 isoforms, were characterized in this study. Lysyl hydroxylases 2a and 2b are alternatively spliced forms of lysyl hydroxylase 2. Lysyl hydroxylase 2b contains an additional exon of 63 nucleotides. The polypeptide size of lysyl hydroxylase 2a is 737 amino acids, lysyl hydroxylase 2b is 758 amino acids and lysyl hydroxylase 3 is 738 amino acids. The putative signal peptide is 25 amino acids in lysyl hydroxylases 2a and 2band 24 amino acids in lysyl hydroxylase 3. Lysyl hydroxylases 2aand 2b contain 7 possible N-glycosylation sites and lysyl hydroxylase3 contains 2 sites. Tissue distribution of novel isoforms were studied on Northern blots. The expression of lysyl hydroxylases 2a, 2b and 3 differ from the expression of previously characterized lysyl hydroxylase 1. Lysyl hydroxylase 1 is expressed constitutively in all tissues whereas the expression of novel isoforms is more strictly regulated. Lysyl hydroxylase 2 is highly expressed in heart, placenta, liver and pancreas. Lysyl hydroxylase 2b expression is highest in heart and skeletal muscle and lysyl hydroxylase 3 expression is highest in heart, placenta and pancreas. Brain, lung and kidney contain the lowest amounts of these isoforms. Novel isoforms were expressed as recombinant proteins in baculovirus expression system in vitro. All these novel isoforms were able to hydroxylate lysine residues in short collagenous peptides. A more detailed kinetic analysis was performed on lysyl hydroxylase 2a and 2b in order to find out if they differed from each other. The binding affinity of ascorbate and peptide substrate is different in lysyl hydroxylase 2a from 2b. Chromosomal assignments were carried out on human lysyl hydroxylases 2 and 3. Lysyl hydroxylase 2 was localized to chromosome 3q23–q24 and lysyl hydroxylase 3 to chromosome 7q36.

Tendons are collagen-based fibrous tissues that connect muscles to bones. Tendon injury is common, but currently available treatments for damaged tendons are limited, and often fail to restore pre-injury function. The cellular and molecular mechanisms underlying tendon formation are not completely understood, and a detailed understanding of the processes involved would facilitate new therapeutic strategies. Work presented in this thesis had two aims: to investigate the role and mechanisms of cell-generated force during tendon development, and to examine the effect of external force applied to tendon tissue. The rationale to investigate micro-mechanical aspects of tendon development came from observations that tendons are intrinsically tensioned, presumably to transmit force efficiently. Tension is also critical for the formation and regulation of the primary cell-matrix interaction structures seen in embryonic tendon, known as fibripositors. In initial experiments cell-generated force was disrupted with the non-muscle myosin II (NMII) inhibitor blebbistatin. NMII inhibition prevented formation of tendon tissue in a 3D cell culture system (tendon-construct). Cell-contraction was also shown to shape the ECM and generate a crimped collagen structure characteristic of tendon. These observations highlight the importance of cell-generated force in tendon development. Further investigation using novel microscopy techniques suggested that fibripositors contained newly formed collagen fibrils, and that fibrils in the ECM were internalised and fragmented in a NMII/MT1-MMP dependent pathway. It is proposed that these fragmented fibrils are re-secreted into the matrix, where they seed fibril growth. This pathway is disrupted in MT1-MMP deficient mice, which have half normal size tendons with fewer collagen fibrils. Investigations using a mechanical rig showed that external application of force to tendon-constructs stimulated matrix maturation that more closely mimicked embryonic development. Taken together these results show that both cell-generated force and external force have important roles during tendon development. Experiments in this thesis aimed to elucidate the role of force at the tissue level (generation of mechanical properties, crimp structure), the microscopic level (cell-matrix interactions) and the molecular level (role of NMII and MT1-MMP in ECM collagen fibril handling). Given the complexity of the developmental system being investigated the multi-level experimental approach used here is necessary to generate a holistic understanding of the important developmental processes involved. Therapeutic tissue regeneration may be facilitated by application of external force or modulation of cell-generated force. Furthermore, the identification of a fibril amplification pathway has implications for medical conditions characterised by excessive collagen fibril formation (fibrosis, some cancers) or by slow or inadequate fibril formation (tendon healing).

No description available.

[Summary] Some properties of zirconium compounds in aqueous solution have been determined using physico-chemical techniques. Zirconium oxychloride was used as the source of zirconium in all detailed investigations; zirconium sulphate was used in a few cases for comparative purposes. The Stokes diaphragm cell method has been used to determine the diffusion coefficient of zirconium in hydrochloric acid solution. It was found that the diffusion coefficient fell progressively with time, a limiting value being reached 4 to 5 weeks after dissolving the salt, and it was demonstrated that particles in the aged solutions were more homogeneous than in freshly prepared solutions. The limiting values were concentration dependent; dilute solutions had a lower diffusion coefficient than the more concentrated solutions when measured at the natural pH. In the presence of added acid the rate of diffusion was increased until a limiting value was reached in 0.5 M acid. The addition of alkali or complexing acids reduced the rate of diffusion.

Organic solvents were used in the present study as a means of investigating the non-covalent interactions involved in the maintenance and pertubation of the three-dimensional structure of the collagen macromolecule in solution. The two main types of non-covalent interaction under consideration are hydrogen bond formation and hydrophobic effects. Elucidation of the relative importance of these factors in the maintenance of the solution structure of proteins is an area of intensive investigation and fundamental significance to biochemistry as a whole. During the past decade, considerable progress has been made towards a clearer understanding of the forces involved, and a number of different theoretical and experimental approaches have emerged. Until about 1960, hydrogen bonding was widely believed to be the dominant non-covalent interaction responsible for the maintenance of secondary and tertiary structure in many proteins. Subsequently, an increasingly important role for apolar (hydrophobic) effects was suggested by a number of authors, and at present there is no satisfactorily definitive interpretation of the available experimental evidence. The current work is based on a comparison of the effects of organic solvents on widely different substrates, namely collagen, cellulose, and the chromatographic reference material, catechin. The chromatographic mobility of catechin on cellulose may be regarded as a phenomenon which is mediated entirely by polar interaction mechanisms. The effects of various organic perturbants and of changing solvent/water ratios are readily interpreted on this basis. In the collagenous systems, however, certain results appear to require the introduction of concepts other than those relating exclusively to polar bonding affinities. The experimental evidence shows that there are cases where the enhancement of the polar interaction potential of solvent/water mixtures, in relation to catechin-cellulose systems, is accompanied by an apparent reduction of polar interaction potential of the same solvent/water mixtures with respect to soluble collagen. The anomaly outlined above will be discussed in terms of two fundamentally different theoretical assumptions. In the first of these, the mechanism of perturbant action in collagenous systems is regarded as essentially similar to that governing catechin-cellulose affinity patterns. Thus, interaction processes are all treated as polar phenomena, in which direct hydrophobic destabilization of the collagen triple helix plays no part. In an attempt to explain the effects of perturbants in both collagen and cellulose-containing systems in terms of the above assumption, two hypotheses are examined involving (1) direct polar interaction between perturbant molecules and functional groups of the protein; (2.) the possibility of an enhanced polar interaction potential of water molecules, due to lowering of the environmental dielectric constant when organic solvents are added to the systems. Within the other broad conceptual division, collagen and cellulose substrates are considered to respond in fundamentally different ways to the action of organic perturbants. As before, cellulose-catechin-solvent interactions are treated as entirely polar phenomena, and perturbant effects interpreted in terms of mechanisms such as direct solvation of the substrate, and the enhanced hydrogen bonding activity of water molecules. In contrast, perturbant lyotropic action with respect to soluble collagen i s viewed as the manifestation of a major contribution by hydrophobic interaction processes to macromolecular stability. Thus, solvents that competitively reduce the assumed entropic contribution to the stability of the collagen triple helix, are seen as potential destabilizers of the native state of the protein and inhibitors of the regeneration of co-operative structures during renaturation. Both of the above approaches are critically assessed in the light of the present work and the dominant trends apparent in the recent literature.

Immobility causes joint contractures, loss in range of motion (ROM), notably in elderly and bed-ridden patients. In a rat knee immobilization flexion contracture (FC) model, the posterior capsule contributes to irreversible limitation of ROM. Through microarray, extracellular matrix and collagen pathways were identified as differentially expressed in the posterior capsule of knees with FC. We hypothesized that intra-articular injection of collagenases in rats with knee FC will interfere with collagen in the capsule and allow increased ROM. After four weeks of hind-limb immobilization, rats develop knee FC; two weeks of remobilization with collagenase treatment showed increased ROM compared to buffer injected knees of 8.043° (p-value=0.046). Histological analysis of knee sections revealed changes in collagen content of the extracellular matrix in posterior capsule. In vitro incubation of rat capsules with collagenases confirmed changes in collagen. Along with current rehabilitation methods, treatment with collagenase may augment ROM recovery from knee joint contractures.

archives@tulane.edu / Rupture of atherosclerotic plaque can cause acute life-threatening events such as myocardial infarction and ischemic stroke; therefore, there is much interest in developing therapies aimed at increasing plaque stability. More stable lesions are characterized as having high collagen content and containing a large number of vascular smooth muscle cells (SMCs) of contractile/differentiated phenotype. In our previous studies using an apolipoprotein E-deficient (Apoe -/-) mouse model of atherosclerosis, we found that insulin-like growth factor-1 (IGF-1)-infusion not only reduced total plaque burden, but also increased collagen expression and the number of alpha-smooth muscle actin (αSMA)-positive cells in plaque. In this study, we identify cellular mechanisms responsible for these observations. We found that in human aortic smooth muscle cells (HASMCs) grown in culture, IGF-1 post-transcriptionally upregulated expression of the procollagen type I alpha-1 subunit (pro-α1(I)) as well as contractile proteins, αSMA and smooth muscle 22-alpha (SM22α), via a PI3K-dependent but Erk1/2- and mTOR-independent signaling mechanism. Furthermore, experiments using an inhibitor of collagen synthesis or a blocking antibody against the alpha2beta1-integrin (α2β1) suggested that interaction with collagen type I promotes HASMC contractile phenotype. To elucidate mechanisms underlying IGF-1 upregulation of collagen synthesis we investigated the effect of IGF-1 on the mRNA-binding protein, la ribonucleoprotein domain family member 6 (LARP6), which had been shown to bind a conserved stem-loop secondary motif in the 5'UTR of COL1a1 and COL1a2 mRNA. IGF-1 rapidly increased LARP6 expression in HASMCs leading to increased COL1a1 and COL1a2 mRNA bound LARP6 and increased synthesis of collagen type I. Mutation of the 5'stem-loop of Col1a1 mRNA (that inhibited binding by LARP6) or overexpression of a 5'stem-loop RNA molecular decoy (that sequesters LARP6) both prevented the ability of IGF-1 to increase pro-α1(I) synthesis as well as mature α1(I) expression in cultured medium. Furthermore, IGF-1-infusion in Apoe -/- mice increased LARP6 and pro-α1(I) expression in aortic lysates, and SMC-specific IGF-1-overexpression in transgenic mice robustly increased collagen fibrillogenesis in atherosclerotic plaque. In conclusion, this work identifies LARP6 as a critical mediator by which IGF-1 augments synthesis of collagen type I in vascular smooth muscle, and uncovers key mechanisms whereby IGF-1 promotes atherosclerotic plaque stability. / 1 / Admin