Increased type-III/type-I collagen ratios in diabetic human conjunctival biopsies

The ratio of type-III to type-I collagen is measured in human conjunctival biopsies from control and diabetic subjects. The tissue is digested by CNBr and the resulting peptides are quantified by SDS polyacrylamide gel electrophoresis. The peptides used are alpha 1-(I)CB7 and alpha 1-(III)CB8. In control population, of type-III collagen slightly increases with age. In two diabetic populations, (juvenile onset diabetes and maturity onset diabetes), the percentage of type-III collagen is significantly higher than in age-matched control groups. These data plus those previously obtained on genetically diabetic mice indicate that diabetes mellitus affects the expression of interstitial collagen phenotype. Preliminary results on prediabetic subjects suggest the role of genetic factors in such alterations.     

Thermal stability of human-fibroblast-collagenase-cleavage products of type-I and type-III collagens.

In neutral solutions, desferrioxamine (Desferal) can react with the superoxide free radical, O2.- (possibly through its protonated form HO2.), to form a relatively stable nitroxide free radical, which can have a half-life of approx. 10 min at room temperature. The formation of the radical can be largely prevented by the presence of superoxide dismutase. The radical reacts rapidly with cysteine, methionine, glutathione, vitamin C and a water-soluble derivative of vitamin E. It also reacts rapidly with alcohol dehydrogenase, causing a loss of enzyme activity. The implications of these findings for mechanistic free-radical biochemistry and iron-chelation therapy could be considerable.     

Comparative electron-microscope studies on type-III and type-I collagens.

Long-spacing-segment crystallites prepared from type III collagen with the chain composition [alpha1(III)]3 and type I collagen with the chain composition [alpha(I)]i12alpha2 have been compared in the electron microscope after positive. staining with phosphotungstic acid and uranyl acetate. The comparison revealed several differences in intensities of the cross-striation bands as well as significant differences in band positions. The latter occur most prominently in three distinct regions of the crystallites. Further, crystallites prepared from type III collagen contain an additional intensely staining band in an area corresponding to the carboxy-terminal end of the molecule. The latter band is still observed following limited pepsin digestion and presumably represents a slight elongation of the helical portion of the type III molecule when compared to the type I molecule. In spite of the somewhat altered distribution of charged groups as indicated in studies on the long-spacing-segment crystallites, type III molecules are capable of forming fibrils of the native type with a cross-striation pattern and periodicity virtually identical to that observed when type I molecules are precipitated as native fibers.     

Age-related thermal stability and susceptibility to proteolysis of rat bone collagen.

The shrinkage temperature (Ts) and the pepsin-solubilizability of collagen fibrils in bone matrix obtained from decalcified femur diaphysis from 2-, 5-, 15- and 25-month-old rats were found to decrease with age. Digestion with human fibroblast collagenase dissolved less than half of the collagen, whereas sequential treatment by pepsin followed by collagenase resulted in its complete dissolution. This result shows that collagenase and a telopeptide-cleaving enzyme, when acting in an appropriate sequence, have a great potential for the degradation of bone collagen. The 'melting' profile of the pepsin-solubilized collagen showed a biphasic transition with transition peak at 35.9 degrees C and 40.8 degrees C. With increasing age an increasing proportion of the collagen 'melted' in the transition peak at 35.9 degrees C (pre-transition), and the 'melting' temperature (Tm) of the collagen decreased in parallel with Ts in relation to age. Both Ts and Tm decreased by 3 degrees C in the age span investigated. The age-related change in Ts could therefore be accounted for by the decrease in molecular stability. The collagenase-cleavage products of the bone collagen obtained by the sequential treatment with pepsin and collagenase showed only one peak transition (at 35.1 degrees C), and the Tm for the products was independent of age. The results indicate that the pre-transition for the pepsin-solubilized collagen is due to an age-related decrease in thermal stability may have implications for the mechanical strength and turnover of the bone collagen. In contrast with bone collagen, soft-tissue collagen showed neither the age-dependency of thermal stability nor the characteristic biphasic 'melting' profile.     

Integrated predictive modeling of JET H-mode plasma with type-I and type-III ELMs

Edge plasma parameters influence plasma performance in many different ways (profile stiffness is probably one of the best known examples). In the ELMy H-mode plasma, a thin region with improved transport characteristics (the edge transport barrier) links the core and the scrape-off layer. There is a strong coupling between these three areas, so that even a modest variation of plasma parameters in one region can lead to a dramatic change in the overall plasma performance. A systematic MHD stability analysis and self-consistent integrated predictive modeling of a series of JET ELMy H-mode plasmas, including scans in gas fueling and triangularity, are presented. The main conclusion is that plasma performance indeed sensitively depends on the edge plasma parameters, which should be modeled in a self-consistent way.     

Stability of rat skin collagen during recovery from under-nutrition.

The growth of young rats was arrested for 6 weeks from 48 h after receiving an injection of L-[5-3H]proline. The 3H in the hydroxyproline of the newly synthesized skin collagen remained steady during under-nutrition and did not decrease during the subsequent recovery period. It was concluded that in this animal model the renewed growth did not induce degradation of the pre-existing collagen fibres.     

Size-dependent rheology of type-I collagen networks

We investigate the system size-dependent rheological response of branched type I collagen gels. When subjected to a shear strain, the highly interconnected mesh dynamically reorients, resulting in overall stiffening of the network. When a continuous shear strain is applied to a collagen network, we observe that the local apparent modulus, in the strain-stiffening regime, is strongly dependent on the gel thickness. In addition, we demonstrate that the overall network failure is determined by the ratio of the gel thickness to the mesh size. These findings have broad implications for cell-matrix interactions, the interpretation of rheological tissue data, and the engineering of biomimetic scaffolds.     

Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.     

Characterization of collagen precursors found in rat skin and rat bone.

Two genetic types of collagenous proteins, type I and type III, were isolated by extraction and differential salt precipitation from rat skin. The yield of collagen precursors was increased by injecting animals with colchicine 30 min before sacrifice to inhibit secretion of collagen. DEAE-cellulose chromatography was used to separate collagen from collagen precursors. Although these preparations contained more type I collagen than type III collagen, there were always more type III than type I precursors. The precursor chains of type I fractions were separated on CM-cellulose chromatography after denaturation. Three precursor forms were found for each collagen alpha chain, a complete chain (proalpha chain), and a precursor chain with only an amino-terminal (pNalpha chain) and carboxy-terminal extension (pCalpha chain). Species differences were demonstrated between rat collagen precursors and other species using rat calvaria (frontal and parietal) bones extracted with either 0.5 N acetic acid or neutral salt buffers containing protease inhibitors. Native rat procollagen elutes earlier than chicken or human procollagen on DEAE-cellulose chromatography and does not separate significantly from the pC collagen form. The collagenase resistant amino terminal peptides of rat pNalpha1 and pNalpha2 were the same size (16 000) but could be separated by DEAE-cellulose chromatography.     

Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen

We have purified collagen from two distinct sources; the vertebrate, rat tail tendon and an invertebrate, sea urchin adult tissue, the peristome. The collagenous nature of the purification products was confirmed by amino acid compositional analysis. Both preparations had high contents of glycine and proline residues and hydroxyproline was also present. The total pyrrolidine (proline+hydroxyproline) content decreased from 17.9 mole% in rat tail collagen to 12.9 mole% in peristome collagen. Distinctly different circular dichroic spectra were measured for these collagens. Analyses of spectra, measured as a function of temperature, revealed distinct thermal denaturation profiles. The melting temperature for rat tail collagen was 38.5 degrees C, while the corresponding value for peristome collagen was significantly lower at 27 degrees C. A similar thermal denaturation profile was obtained for rat tail collagen in digestion experiments using a 41-kDa gelatinase activity, isolated from sea urchin eggs. These results identify structural differences between a typical, vertebrate type I fibrillar collagen and an echinoderm collagen which serves as a constituent of a mutable connective tissue. These differences may relate to the functional roles played by collagen in these distinctly different tissues.     

Pre-adsorbed type-I collagen structure-dependent changes in osteoblastic phenotype

Type-I collagen is the most abundant extracellular matrix in bones and modulates various functions of osteoblasts. We prepared two different structures of type-I collagen on tissue culture grade polystylene (TCPS) surfaces, one is feltwork structure of filamentous molecules from acid solutions (ACs) and the other is network structure of fibrils from neutral solutions (NCs), to examine effects of the structures on the maturation process of osteoblast-like cells. No significant differences of cell proliferation were observed between TCPS and ACs, but NCs delayed the proliferation. In initial cell attachment, the cells on ACs had tense lamellipodia with sharp tips, while those on NCs had loose lamellipodia. No detectable differences in levels of expressed integrin alpha2- and alpha5-subunits were observed between the structures. Although the matrix mineralization in NCs was also delayed in comparison with TCPS and ACs, fully mineralized levels in NCs were the same as those of TCPS and ACs. In addition, although we examined the effects of densities of pre-adsorbed collagen molecules on osteoblast maturation, the effects were less serious than those of the structures. This study suggests that the structures of collagen affect proliferation and mineralization of osteoblast-like cells.     

Hydroxyproline content and location in relation to collagen thermal stability.

A new analysis has been made on studies of the influence of imino acid content on the changes of collagen thermal stability (t_m). It is shown that, for the interstitial vertebrate collagens, there is a strict regularity in the changes of t_m depending on hydroxyproline content. No correlation is observed between t_m and proline content. Also, no correlation between t_m and hydroxyproline content is observed for invertebrate and basement membrane collagens. On the basis of the reported data, the dependence of t_m on hydroxyproline content is considered to be not a correlation between t_m and the total content of hydroxyproline, but only as the correlation between t_m and the content of hydroxyproline occurring at the third position in the sequence (Gly-R₂-R₃)_n. The results agree with the idea that the influence exerted by proline and hydroxyproline on the stabilization of the triple helix of collagen is different.     

Sequence specific thermal stability of the collagen triple helix

Theoretical calculations of the thermal stability of collagen triple helices using empirical values for the contribution of individual tripeptide units are presented and compared with direct measurements of the thermal stability of various types of collagens. Relative stabilities are assigned to the positions of the tripeptide units in the amino acid sequence along the length of the collagen molecule. The sequence specific relative stabilities of type I and type XI collagens are compared. These offer insight into the reasons for the existence of unfolding intermediates in type XI collagen that are absent in type I collagen. The pattern of relative stabilities calculated for mouse type IV collagen is consistent with experimental results which indicate that the amino terminal region is very stable and that the interruptions cause increased flexibility and independently unfolding domains. Mutations in the triple helical domain of human type I procollagen occurring in brittle bone disease (osteogenesis imperfecta) show varying effects on the thermal stability of the molecule. The sequence specific thermal stability calculations shed some light on why some mutations of cysteine for glycine have greater effects on the thermal stability than others.     

Endonuclease (R) subunits of type-I and type-III restriction-modification enzymes contain a helicase-like domain.

A statistically significant amino acid sequence similarity is demonstrated between the endonuclease (R) subunit of EcoK restriction-modification (R-M) enzyme, and RNA and DNA helicases of the so-called 'DEAD' family. It is further shown that all three known sequences of R subunits of type-I and type-III R-M enzymes contain the conserved amino acid sequence motifs typical of the previously described helicase superfamily II [(1989) Nucleic Acids Res. 17, 4713-4730]. A hypothesis is proposed that these enzymes may exert helicase activity possibly required for local unwinding of DNA in the cleavage sites.