Evidence of a discrete axial structure in unimodal collagen fibrils

The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.     

Types of collagen fibres in the calcareous sponges Sycon and Leucandra

Collagen from the calcareous sponges Sycon and Leucandra has been studied in thin sections and in sheaths from decalcified spicules. Sections show abundant thin fibrils 8–15 mμ in diameter and, in Sycon juveniles, 20 mμ fibrils with a clear 50 mμ period. The sheaths are also mainly composed of the thin fibrils but giant fibres up to 130 mμ in diameter and many μ in length are also found.     

Histological staining as a measure of stress in collagen fibers

It has been reported previously that collagen fibers will stain either red or green by Masson's and other trichrome methods depending on whether they have been respectively stressed or relaxed prior to fixation. This was shown in skin [1, 2, 3] tendon [4, 5] bone [6] and films of collagen [7]. If this stain-stress dependence is of a unique quantitative nature, then staining could be used as a tension probe for collagen fibers. Relaxed and stressed collagen bundles of rat tail tendon and rat Achilles tendon have been stained using various staining periods, and results indicate that the change in staining may be associated with denser packing of the fibers in the bundle under stress rather than directly due to the stress itself. Denser packing may reduce the rate of penetration of the counterstain thus causing the staining differences. Since this rate of penetration is dependent on a number of other variables (unrelated to stress), it is concluded that collagen staining is not a reliable tension probe.     

Contraction-relaxation of collagen fibers in LiBr-water-acetone solutions

Studies have been made of the contraction of collagen fibers in acetone due to the action of LiBr. No contraction occurs unless a small amount of water is added; on further increase of the water concentration, the fibers relax. The first contraction is irreversible. The effect of water is discussed in terms of hydrate formation.     

Respiratory responses of three Antarctic ascidians and a sea pen to increased sediment concentrations

Glacial retreat and subglacial bedrock erosion are consequences of rapid regional warming on the West Antarctic Peninsula. Sedimentation of fine-grained eroded particles can impact the physiology of filter-feeding benthic organisms. We investigated the effect of increasing concentrations of sediment on the oxygen consumption of suspension feeding species, the ascidians Molgula pedunculata, Cnemidocarpa verrucosa, Ascidia challengeri, and the pennatulid Malacobelemnon daytoni in Potter Cove (South Shetland Islands, Antarctica). In A. challengeri and C. verrucosa, oxygen consumption increased gradually up to a critical sediment concentration (C _crit) where species oxygen consumption was maximal (O _max in mg O₂?g^?1dm?day^?1) and further addition of sediments decreased respiration. C _crit was 200?mg?L^?1 for A. challengeri (O _max of 0.651?±?0.238) and between 100 and 200?mg?L^?1 for C. verrucosa (O _max of 0.898?±?0.582). Oxygen consumption of M. pedunculata increased significantly even at low sediment concentrations (15–50?mg sediment?L^?1). Contrary to the ascidians, sediment exposure did not affect oxygen consumption of the sea pen. The tiered response to sedimentation in the four species corroborates recent field observations that detected a reduction in the abundance of the sensitive ascidian M. pedunculata from areas strongly affected by glacial sediment discharge, whereas sea pens are increasing in abundance. Our investigation relates consequences (population shifts in filter-feeder communities) to causes (glacial retreat) and is of importance for modelling of climate change effects in Antarctic shallow coastal areas.     

Structural and dynamic characterization of a vinculin binding site in the talin rod

Talin is a key protein involved in linking integrins to the actin cytoskeleton. The long flexible talin rod domain contains a number of binding sites for vinculin, a cytoskeletal protein important in stabilizing integrin-mediated cell-matrix junctions. Here we report the solution structure of a talin rod polypeptide (residues 1843-1973) which contains a single vinculin binding site (VBS; residues 1944-1969). Like other talin rod polypeptides, it consists of a helical bundle, in this case a four-helix bundle with a right-handed topology. The residues in the VBS important for vinculin binding were identified by studying the binding of a series of VBS-related peptides to the vinculin Vd1 domain. The key binding determinants are buried in the interior of the helical bundle, suggesting that a substantial structural change in the talin polypeptide is required for vinculin binding. Direct evidence for this was obtained by NMR and EPR spectroscopy. [1H,15N]-HSQC spectra of the talin fragment indicate that vinculin binding caused approximately two-thirds of the protein to adopt a flexible random coil. For EPR spectroscopy, nitroxide spin labels were attached to the talin polypeptide via appropriately located cysteine residues. Measurements of inter-nitroxide distances in doubly spin-labeled protein showed clearly that the helical bundle is disrupted and the mobility of the helices, except for the VBS helix, is markedly increased. Binding of vinculin to talin is thus a clear example of the unusual phenomenon of protein unfolding being required for protein/protein interaction.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.     

Identification of a macrophage-like mSubset in the axial organ sea star Asterias rubens.

The axial organ of the sea star Asterias rubens is a primitive immune organ. In addition to T and B-like cells phagocytic cells were also identified. In this report we demonstrate that these cells can be identified using immunocytochemistry together with the mouse to human monoclonal antibody CD68 KP1, normally used as a macrophage marker.     

X-ray diffraction of reconstituted collagen fibers

The X-ray diffraction of fibers reconstituted from purified rat tail tendon collagen has been compared with that of native rat tail tendon. The axial structure is very similar in the two specimens, while the ordered lateral array found in the native state is only poorly reproduced in the reconstituted fiber. Thus, the axial order is determined by the collagen molecules alone, while the native lateral packing may depend, in part at least, on other tissue components.     

Polytonic studies of reconstituted collagen fibers

The tensile force-contraction characteristics of cross-linked reconstituted collagen fibers have been studied for 30 different water-soluble reagents. The data were obtained via a “polytonic” loading technique, which was neither isometric nor isotonic. Certain data were found to fit a master curve, which did not depend strongly on the denaturant. For many denaturants the contractile force appeared to be a maximum at a denaturant concentration corresponding to all the solvent being consumed in denaturant hydration. Also, the maximum contractile force appeared to vary in the same manner as does the hydration number of the individual cations and anions in the solution (i.e., H > Li > Na > K, and I > Br > Cl > F). Several denaturants were found to be as effective as the well-known LiBr and KSCN in contracting the collagen.     

New perspectives on collagen fibers in the squid mantle

The squid mantle is a complex structure which, in conjunction with a highly sensitive sensory system, provides squid with a wide variety of highly controlled movements. This article presents a model describing systems of collagen fibers that give the mantle its shape and mechanical properties. The validity of the model is verified by comparing predicted optimal fiber angles to actual fiber angles seen in squid mantle. The model predicts optimal configurations for multiple fiber systems. It is found that the tunic fibers (outer collagen layers) provide optimal jetting characteristics when oriented at 31°, which matches empirical data from previous studies. The model also predicted that a set of intramuscular fibers (IM‐1) are oriented relative to the longitudinal axis to provide optimal energy storage capacity within the limiting physical bounds of the collagen fibers themselves. In addition, reasons for deviations from the predicted values are analyzed. This study illustrates how the squid's reinforcing collagen fibers are aligned to provide several locomotory advantages and demonstrates how this complex biological process can be accurately modeled with several simplifying assumptions. J. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

Spatial distribution of collagen and elastin fibers in the lungs

Surface tension forces acting on the thin-wall alveolar septa and the collagen-elastin fiber network are major factors in lung parenchymal micromechanics. Quantitative serial section analysis and morphometric evaluations of planar sections were used to determine the spatial location of collagen and elastin fibers in Sprague-Dawley rat and normal human lung samples. A large concentration of connective tissue fibers was located in the alveolar duct wall in both species. For rats, the tissue densities of collagen and elastin fibers located within 10 microns of an alveolar duct were 13 and 9%, respectively. In human lung samples, the tissue densities of collagen and elastin fibers within 20 microns of an alveolar duct were 18 and 16%, respectively. In both species, bands of elastin fibers formed a continuous ring around each alveolar mouth. In human lungs, elastin fibers were found to penetrate significantly deeper into alveolar septal walls than they did in rat lungs. The concentration of connective tissue elements in the alveolar duct walls of both species is consistent with their proposed roles as the principal load-bearing elements of the lung parenchyma.     

Thermal stability of collagen fibers in ethylene glycol

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.     

Dynamic-elastic investigation of the chemical denaturation of collagen fibers

The dynamic elastic behavior of collagen fibers treated by LiBr solutions was studied by the method of free longitudinal vibrations. The frequency response functions and the stress–strain relationship were evaluated for fibers denatured to different extents by various concentrations of the salt solution. The James and Guth model for rubber elasticity was applied to the experimental data. The elastometric parameter β, which is a measure of the degree of folding of the macromolecular chains, was found to decrease on increasing the salt concentration. It might thus serve as a characteristic of the degree of denaturation of fibrillar proteins.