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Biomedical subjects

J A Trotter

Publications and source records attributed to J A Trotter.

At least 19 recordsLinked to original sources
PubMed2001

STEM/TEM studies of collagen fibril assembly.

Quantitative scanning transmission electron microscopy (STEM), implemented on a conventional transmission electron microscope with STEM-attachment, has been a primary tool in our laboratory for the quantitative analysis of collagen fibril assembly in vivo and in vitro. Using this technique, a precise measurement of mass per unit length can be made at regular intervals along a fibril to generate an axial mass distribution (AMD). This in turn allows the number of collagen molecules to be calculated for every transverse section of the fibril along its entire length. All fibrils show a near-linear AMD in their tip regions. Only fibrils formed in tissue environments, however, show a characteristic abrupt change in mass slope along their tips. It appears that this tip growth characteristic is common to fibrils from evolutionarily diverse systems including vertebrate tendon and the mutable tissues of the echinoderms. Computer models of collagen fibril assembly have now been developed based on interpretation of the STEM data. Two alternative models have so far been generated for fibril growth by accretion; one is based on diffusion limited aggregation (DLA) and the other based on an interface-limited growth mechanism. Inter-fibrillar fusion can also contribute to the growth of fibrils in vertebrate tissues and STEM data indicates the presence of a tight regulation in this process. These models are fundamental for the hypotheses regarding how cells synthesise and spatially organise an extracellular matrix (ECM), rich in collagen fibrils.

Animals↗
PubMed2000

Echinoderm collagen fibrils grow by surface-nucleation-and-propagation from both centers and ends.

Collagen fibrils from sea cucumber (class Holothuroidea) dermis were previously found to grow by coordinated monomer addition at both centers and ends. This analysis of sea urchin (class Echinoidea) collagen fibrils was undertaken to compare the growth characteristics of fibrils from two classes of echinoderms, and to determine whether a single growth model could account for the main features of fibrils from these two taxa. Native collagen fibrils (37-431 micrometer long) from the spine ligaments of the sea urchin Eucidaris tribuloides were studied by scanning transmission electron microscopy and image analysis. The analyses revealed the mass per unit length, and hence the number of molecules in cross-section, along the entire length of each fibril. The fibrils were symmetrically spindle shaped. The maximum mass per unit length occurred in the center of each fibril, where the fibril contains anti-parallel molecules in equal numbers. The two pointed tips of each fibril showed similar linear axial mass distributions, indicating that the two tips retain shape and size similarity throughout growth. The linear axial mass distributions showed that the tips were paraboloidal, similar to those of vertebrate and sea cucumber fibrils. The computed maximum diameters of the fibrils increased linearly with fibril length. The overall shapes of the fibrils showed that they retain geometric similarity throughout growth. Computer modeling showed that the simplest self-assembly mechanism that can account for the features of these fibrils, and of the sea cucumber fibrils that have been described, is one in which the fibril tips produce independent axial growth, while lateral growth takes place through a surface nucleation and propagation mechanism. This mechanism produces coordinated growth in length and diameter as well as geometric similarity, characteristic features of echinoderm collagen fibrils.

Animals↗
PubMed2000

Towards a fibrous composite with dynamically controlled stiffness: lessons from echinoderms.

Sea urchins and sea cucumbers, like other echinoderms, control the tensile properties of their connective tissues by regulating stress transfer between collagen fibrils. The collagen fibrils are spindle-shaped and up to 1 mm long with a constant aspect ratio of approx. 2000. They are organized into a tissue by an elastomeric network of fibrillin microfibrils. Interactions between the fibrils are regulated by soluble macromolecules that are secreted by local, neurally controlled, effector cells. We are characterizing the non-linear viscoelastic properties of sea cucumber dermis under different conditions, as well as the structures, molecules and molecular interactions that determine its properties. In addition, we are developing reagents that will bind covalently to fibril surfaces and reversibly form cross-links with other reagents, resulting in a chemically controlled stress-transfer capacity. The information being developed will lead to the design and construction of a synthetic analogue composed of fibres in an elastomeric matrix that contains photo- or electro-sensitive reagents that reversibly form interfibrillar cross-links.

Animals↗
PubMed1999

Collagen fibril aggregation-inhibitor from sea cucumber dermis.

Collagen fibrils from the dermis of the sea cucumber Cucumaria frondosa are aggregated in vitro by the dermal glycoprotein stiparin (Trotter et al., 1996). Under physiological ionic conditions stiparin appears to be both necessary and sufficient to cause fibrils to aggregate (Trotter et al., 1997). We report here the initial biochemical and biophysical characterization of a sulfated glycoprotein from C. frondosa dermis that binds stiparin and inhibits its fibril-aggregating activity. This inhibitory glycoprotein, which has been named 'stiparin-inhibitor,' has the highest negative charge density of all the macromolecules extracted from the dermis. SDS-PAGE reveals three approximately 31-kDa bands that stain with alcian blue but not with Coomassie blue. Analytical ultracentrifugation indicates a native molecular weight of 62 kDa. Transmission electron microscopy of rotary-shadowed molecules shows curved rods about 22 nm long. The glycoprotein does not bind collagen fibrils, but does bind stiparin with a 1:1 stoichiometry. The binding of stiparin-inhibitor to stiparin prevents the binding of stiparin to collagen fibrils. The carbohydrate moiety produced by papain-digestion of the glycoprotein retains all of its inhibitory activity. The carbohydrate moiety of the inhibitor is dominated by galactose and sulfate.

Amino Acids↗
PubMed1998

Growth of sea cucumber collagen fibrils occurs at the tips and centers in a coordinated manner.

Collagen fibrils are the principle source of mechanical strength in the mutable dermis of the sea cucumber Cucumaria frondosa. To obtain information about the mechanism by which collagen molecules self-assemble into fibrils, we have isolated single intact fibrils with lengths in the range 14-444 microm. These fibrils have been studied by scanning transmission electron microscopy, yielding data that show how cross-sectional mass, and hence the number of molecules in the cross-section, depend on axial location. In an individual fibril, the two ends always display similar mass distributions. The two tips of each fibril must therefore maintain identity in shape and size throughout growth. The linear relationship between cross-sectional mass and distance from the adjacent end shows that a growing tip is (like the tip of a vertebrate collagen fibril) paraboloidal in shape. Comparison of data from many different fibrils, over a wide range of lengths, however, revealed that the paraboloidal tip becomes blunter as the fibril grows in length. In contrast to vertebrate fibrils, those from C. frondosa do not have a central shaft region of constant cross-sectional mass. Rather, the cross-sectional mass increases to a maximum in the center of each fibril. The maximum cross-sectional mass of the fibrils increases exponentially with increasing fibril length. The centrosymmetry, the paraboloidal shape of the tips, and the hyperbolic increase in maximum cross-sectional mass with fibril length, is evidence for a co-ordinated regulation of length and diameter, which differs from the kind of regulation that gives rise to collagen fibrils in vertebrates (chickens and mice).

Animals↗
PubMed1997

Isolation of intact collagen fibrils from healing ligament.

The inability to isolate intact collagen fibrils has limited the study of their growth and structure. Although intact fibrils have been isolated from echinoderms and from embryonic chick tissues, no method has previously succeeded in isolating intact collagen fibrils from a postfoetal vertebrate tissue. Having previously observed that gentamicin weakens rat tail tendon, we hypothesized that gentamicin may weaken interfibrillar bonds and that intact collagen fibrils might be isolated from tissue treated with gentamicin. In this study medial collateral knee ligaments of Sprague Dawley rats were transected and then harvested 24, 48 or 96 h postoperatively. These specimens were placed in gentamicin or phosphate-buffered saline for 72 h, vortexed for 1 h, incubated in gentamicin for an additional 24 h, and vortexed again for 1 h. Negatively stained specimens were examined with a transmission electron microscope. The phosphate-buffered saline specimens yielded only broken fibrils. The gentamicin specimens yielded both broken and intact fibrils. The latter and tapering ends and consisted of molecules orientated such that their amino termini pointed toward the tip and their carboxy termini pointed toward a short central region where the molecular polarity reversed.

Animals↗
PubMed1997

Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis.

The dermis of the sea cucumber Cucumaria frondosa is a mutable collagenous tissue composed of collagen fibrils, microfibrils, proteoglycans, and other soluble and insoluble components. A major constituent of the dermis is a network of 10-14 nm microfibrils which surrounds and penetrates bundles of collagen fibrils. These microfibrils, which are morphologically very similar to the fibrillin microfibrils of vertebrates, were found to be insoluble in protein denaturants, including chaotropic agents and ionic and nonionic detergents, regardless of the reduction of disulfide bonds. The microfibrils are covalently crosslinked by epsilon-(gamma-glutamyl)lysine at a concentration of 3.725 nmol/mg dry weight of purified insoluble material. The network is susceptible to proteolysis by trypsin, chymotrypsin, and pancreatic elastase, but not by bacterial collagenase. Amino acid compositional analysis of the network shows it to be composed of 25% ASX and GLX residues. Comparison with the proteins in the SwissProt database gives the network protein a high probability of being related to the mammalian protein fibrillin. The network is glycosylated: approximately 7% of the mass is constituted by neutral and amino sugars. The intact microfibrillar network cross-reacted with a well-characterized antiserum to mammalian fibrillin.

Amino Acids↗
PubMed1996

Collagen fibril formation.

Collagen is most abundant in animal tissues as very long fibrils with a characteristic axial periodic structure. The fibrils provide the major biomechanical scaffold for cell attachment and anchorage of macromolecules, allowing the shape and form of tissues to be defined and maintained. How the fibrils are formed from their monomeric precursors is the primary concern of this review. Collagen fibril formation is basically a self-assembly process (i.e. one which is to a large extent determined by the intrinsic properties of the collagen molecules themselves) but it is also sensitive to cell-mediated regulation, particularly in young or healing tissues. Recent attention has been focused on "early fibrils' or "fibril segments' of approximately 10 microns in length which appear to be intermediates in the formation of mature fibrils that can grow to be hundreds of micrometers in length. Data from several laboratories indicate that these early fibrils can be unipolar (with all molecules pointing in the same direction) or bipolar (in which the orientation of collagen molecules reverses at a single location along the fibril). The occurrence of such early fibrils has major implications for tissue morphogenesis and repair. In this article we review the current understanding of the origin of unipolar and bipolar fibrils, and how mature fibrils are assembled from early fibrils. We include preliminary evidence from invertebrates which suggests that the principles for bipolar fibril assembly were established at least 500 million years ago.

Animals↗
PubMed1996

Stiparin: a glycoprotein from sea cucumber dermis that aggregates collagen fibrils.

The interactions between collagen fibrils in many echinoderm connective tissues are rapidly altered by the secretions of resident neurosecretory cells. Recent evidence has suggested that a secreted protein is responsible for the interactions that lead to an increase in tissue stiffness (Trotter and Koob, 1995). Structurally intact collagen fibrils have been isolated from such a connective tissue- the dermis of the sea cucumber Cucumaria frondosa- and used in an assay in vitro to identify a protein that binds to them and causes them to aggregate. This protein has been purified by anion-exchange and molecular sieve chromatography. It is eluted from a MonoQ column at approximately 0.55 M NaCl. Its isoelectric point is 5.2. It elutes from a Superose-6 column in a position corresponding to a molecule with a Stokes radius of 11.5 nm. Its native molecular weight estimated from sedimentation equilibrium analysis under non-denaturing conditions is 375,000, and its monomer molecular weight, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is approximately 350,000. Sedimentation velocity measurements indicated for the native molecule a sedimentation coefficient of 11 x 10(-13)s, a diffusion coefficient of 3.274 x 10(-7) cm2s-1, and a frictional ratio of 1.95, which corresponds to a prolate ellipsoid of revolution with an axial ratio of 19. The highly asymmetric structure suggested by the above correlated well with the images obtained by transmission electron microscopy following rotary shadowing, which revealed a flexible structure approximately 125 nm long. Based on its ability to aggregate collagen fibrils, this protein has been named "stiparin," from the Latin stipare, "to pack together."

Animals↗
PubMed1994

Native collagen fibrils from echinoderms are molecularly bipolar.

Collagen fibrils are generally assumed to be cylinders with uniform diameters (except possibly at their ends) and to be composed of molecules all of which have the same polarity. These assumptions have been largely untested because of the extreme difficulty associated with isolating entire native fibrils. Intact collagen fibrils are readily extracted from certain echinoderms, however, and we have therefore analyzed the molecular structure of these fibrils. Our electron microscopic analyses show the above assumptions to be false: echinoderm fibrils, which previously have been shown to be symmetrically spindle shaped, are also molecularly bipolar. Their constituent molecules have their N-termini oriented toward the nearest fibril end, and they are antiparallel in the fibril center. The shape and molecular arrangement of these fibrils have implications for fibrillogenesis.

Animals↗
PubMed1994

The morphology and mechanical properties of endomysium in series-fibred muscles: variations with muscle length.

In the series-fibred muscle architecture commonly found in large muscles of mammals and birds, the intrafasciculary-terminating muscle fibres have no direct tendinous attachments. Contractile force produced in these fibres must be transmitted between adjacent muscle fibres via the endomysial connective tissue which separates them. The endomysium is thus an essential mechanical component in such muscles. Studies of motor end-plate banding patterns and the frequent occurrence of tapering ends of fibres within the fascicles of the bovine sternomandibularis muscle show it to be a series-fibred muscle. Sodium hydroxide digestion of fixed samples of this muscle to remove the myofibrillar apparatus revealed the endomysium to be a disordered planar network of mainly curvilinear collagen fibrils. The orientation distribution of the collagen fibrils in the endomysial network was measured by image analysis of scanning electron micrographs. Analysis of endomysial preparations from muscle fixed at sarcomere lengths between 1-4 microns showed that the orientation distribution of collagen fibrils is quantitatively related to muscle length. At rest sarcomere length the collagen fibril network is not completely random, but has a slight circumferential bias. The orientation distribution shows a progressive shift towards the circumferential direction at short sarcomere lengths and towards the longitudinal direction at long sarcomere lengths. The relationship between the number-weighted mean collagen orientation and sarcomere length was compared to two geometric models of network behaviour, the isoareal and constant shape models. Both fitted the data reasonably, although the constant shape model described the rate of change of mean orientation more closely. From fibrous composites theory, the reinforcement efficiency factor, eta, was calculated from the measured collagen fibril orientation distributions. These calculations predict a non-linearly increasing longitudinal tensile modulus for the endomysium with increasing sarcomere length, in agreement with its known non-linear properties, but confirm that the tensile properties of the endomysium are unsuitable for transmission of tensile force from muscle fibres contracting near rest length. This reinforces a previous interpretation that contractile force is transmitted between neighbouring muscle fibres by trans-laminar shear through the endomysium rather than by in-plane tension.

Animals↗
PubMed1994

Molecular structure and functional morphology of echinoderm collagen fibrils.

The collagenous tissues of echinoderms, which have the unique capacity to rapidly and reversibly alter their mechanical properties, resemble the collagenous tissues of other phyla in consisting of collagen fibrils in a nonfibrillar matrix. Knowledge of the composition and structure of their collagen fibrils and interfibrillar matrix is thus important for an understanding of the physiology of these tissues. In this report it is shown that the collagen molecules from the fibrils of the spine ligament of a sea-urchin and the deep dermis of a sea-cucumber are the same length as those from vertebrate fibrils and that they assemble into fibrils with the same repeat period and gap/overlap ratio as do those of vertebrate fibrils. The distributions of charged residues in echinoderm and vertebrate molecules are somewhat different, giving rise to segment-long-spacing crystallites and fibrils with different banding patterns. Compared to the vertebrate pattern, the banding pattern of echinoderm fibrils is characterized by greatly increased stain intensity in the c3 band and greatly reduced stain intensity in the a3 and b2 bands. The fibrils are spindle-shaped, possessing no constant-diameter region throughout their length. The shape of the fibrils is mechanically advantageous for their reinforcing role in a discontinuous fiber-composite material.

Animals↗
PubMed1993

Functional morphology of force transmission in skeletal muscle. A brief review.

The work done by the contractile proteins of muscle in accelerating, decelerating, or maintaining the positions of skeletal elements requires the efficient transmission of tension across the surface membranes of the fibers. The most widely studied sites of tension transmission are the ends of muscle fibers where they contact either connective or epithelial tissues. In most animals, regardless of phylum, muscle fiber ends are characteristically folded, producing a junctional interface that significantly reduces the absolute value of stress applied to the cell membrane, insures that the principle stress vector at the cell membrane is shear rather than tension, and minimizes stress concentrations. The morphological and molecular similarities of muscle-tendon junctions (MTJs) in different animals suggest that the problem of creating a strong adhesive joint between a muscle fiber and a tissue of dissimilar physical properties is essentially the same for all muscles, and that the solution arose early in evolution. In addition to those muscle fiber ends that occur where fibers contact dissimilar tissues, there are intramuscular fiber terminations that consist either of folded cell-cell junctions similar to the fasciae adherentes of cardiac muscle, or of gradually tapering fiber ends. Both sorts of intramuscular ends occur in those vertebrate muscles in which the individual muscle fibers are too short to reach from the tendon of origin to the tendon of insertion. In series-fibered muscles in which the fiber ends are tapered, tension is transmitted from contractile proteins to endomysial collagen fibrils across the fiber membranes. The endomysium of such muscles is an essential series-elastic element. The existing evidence suggests that tension transmission is a general property of muscle cell surfaces, and that specific junctional morphologies are the results of dynamic interactions between muscle cells and the tissues to which they adhere.

Animals↗
PubMed1992

The composite structure of quail pectoralis muscle.

The twitch fibers of the quail pectoralis muscle were found to have one neuromuscular junction each, located in the middle third of the fiber. The length of isolated fibers varied between 8.8 and 33.2 mm, with mean and median values of 16 and 15.6 mm, respectively. The lengths of the fascicles from which the fibers were isolated varied between 30 and 51 mm. The muscle fibers taper at both ends. The neuromuscular junctions, revealed after histochemically reacting the intact muscle for acetyl cholinesterase activity, were arranged in discrete bands, separated by intervals of between 0.94 and 6.70 mm, with a mean value of 3.14 mm. The quail pectoralis muscle is thus composed of discontinuous, tapered muscle fibers, arranged in an overlapping series. It is therefore a muscle in which tension is transmitted laterally between muscle fibers.

Acetylcholinesterase↗
PubMed1992

Functional morphology of the endomysium in series fibered muscles.

Many skeletal muscles, including the feline biceps femoris, are composed of short, tapered myofibers arranged in an overlapping longitudinal series. The endomysium of such muscles transfers tension between overlapping myofibers, and is thus an elastic element in series with them. The endomysium of the cat biceps femoris contains curvilinear collagen fibrils in an approximately isotropic (random) array. The collagen fibrils undergo only a modest reorientation as the myofibers shorten or lengthen within the physiological range. A geometrical model predicts no change in the thickness of the endomysium on changing muscle fiber length and quantifies the expected collagen fibril reorientation in the endomysium as a function of muscle extension. It is also demonstrated that a high proportion of the collagen fibrils will be curvilinear at all sarcomere lengths. The organization of endomysial collagen is appropriate for the transfer of loads between myofibers by means of shear.

Animals↗
PubMed1992

Organization of cells and extracellular matrix in mesenteric arteries of spontaneously hypertensive rats.

Biochemical studies have been used to assess the quantitative changes in elastin and collagen in hypertensive vs. normotensive arteries. However, the relative distribution and organization of these fibrous proteins is likely to be equal in importance to their absolute amounts. In this study we have used scanning electron microscopy in association with selective digestion techniques to assess the organization of cellular and extracellular components of the tunica media of mesenteric arteries of spontaneously hypertensive rats. Superior and small mesenteric arteries were digested with acid, alkali, or bleach to exposure cells, collagen, or collagen and elastin, respectively. We observed that hypertension does not cause a qualitative change in the 3-dimensional arrangement of cells, collagen, or elastin in spontaneously hypertensive arteries when compared to normotensive arteries. However, cells in the superior artery are significantly different in overall shape and surface features when compared to cells of small arteries. These differences in surface morphology of cells are present in hypertensive and normotensive vessels and suggest that superior and small mesenteric artery cells transmit load to the isotropic matrix in different ways. In the elasto-muscular superior artery, force is transmitted across digitations throughout the cell surface. In the muscular small artery, force is transmitted across the tapered, smooth cell surface.

Animals↗
PubMed1991

Dynamic shape of tapered skeletal muscle fibers.

The muscle fibers of the feline biceps femoris have tapered ends, across which tension is transmitted to the endomysium. The angle of taper of 11 ends, measured on scanning electron micrographs, varied between 0.16 degrees and 1.18 degrees. The muscle fibers are highly variable in cross-sectional shape. The shape of the fibers has been quantified as the ratio (form factor [FF]) of the measured perimeter to the calculated circumference of a circle having an area equal to that contained by the fiber perimeter. The FF for 173 terminal portions of fibers varied between 1.06 and 1.85 and was found to have a highly significant negative correlation with sarcomere length. The slope of the regression line suggests that the fibers maintain both volume and surface area as they change length. These studies suggest that isovolumic muscle fibers maintain a constant surface area by changing shape as they change length.

Animals↗