Lathyritic Chick Research Articles

Skeletal muscle activity affects the deformity of long bone morphology in lathyritic chick embryo.

Embryonic muscle activity is involved in various aspects of bone morphogenesis and growth. Normal mechanical stimuli of muscle contraction are important in most cases, and when the muscles are immobilized, the developing bones are abnormally shaped. In chick embryos, a characteristic curved deformity is reproducibly induced in the developing tibiotarsus using the bone-weakening agent, beta-aminopropionitrile (bAPN). In this study, we applied decamethonium bromide (DMB), a well-established neuromuscular blocking agent, to embryos treated with bAPN, to test the hypothesis that the deformity is triggered and formed depending on the balance between the decrease in stiffness of the bAPN-affected tibiotarsus and the normal physiological increase in embryonic skeletal muscle activity. The occurrence of curved morphology induced by bAPN administered at 4 or 8 days of incubation (embryonic day [ED]) was temporally consistent with the posterior displacement of the leg muscles, which occurred just before ED8. The displaced muscles were assumed to exert a contraction force comparable to that of untreated normal muscles. When treated with DMB at ED8, the muscles atrophied and exhibited degenerative changes, and the degree of curved morphology was alleviated and reduced to 50% or more in the morphometric evaluation at ED10. These findings indicated that the coordinated development of skeletal element stiffness and muscle activity must be temporally regulated, particularly during the early stages of skeletogenesis.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60-70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.

Nanosecond fluctuations of the molecular backbone of collagen in hard and soft tissues: a carbon-13 nuclear magnetic resonance relaxation study.

We have determined the amplitude of nanosecond fluctuations of the collagen azimuthal orientation in intact tissues and reconstituted fibers from an analysis of 13C NMR relaxation data. We have labeled intact rat calvaria and tibia collagen (mineralized and cross-linked), intact rat tail tendon and demineralized bone collagen (cross-linked), and reconstituted lathyritic (non-cross-linked) chick calvaria collagen with [2-13C]glycine. This label was chosen because one-third of the amino acid residues in collagen are glycine and because the 1H-13C dipolar coupling is the dominant relaxation mechanism. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancements were measured at 15.09 and 62.98 MHz at 22 and -35 degrees C. The measured NMR parameters have been analyzed by using a dynamic model in which the azimuthal orientation of the molecule fluctuates as a consequence of reorientation about the axis of the triple helix. We have shown that if root mean square fluctuations in the azimuthal orientations are small, gamma rms much less than 1 rad, the correlation function decays with a single correlation time tau and T1 depends only upon tau and gamma rms and not the detailed model of motion. Our analysis shows that, at 22 degrees C, tau is in the 1-5-ns range for all samples and gamma rms is 10 degrees, 9 degrees, and 5.5 degrees for the non-cross-linked, cross-linked, and mineralized samples, respectively. At -35 degrees C, gamma rms is less than 3 degrees for all samples. These results show that mineral and low temperature significantly restrict the amplitude of nanosecond motions of the collagen backbone.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine and collagen levels in the lathyritic chick embryos after administration of 48/80.

The studies were undertaken to investigate the influence of compound 48/80 on histamine (Hi) and collagen levels of chick embryos after administration of lathyrogen factor, beta-aminopropionitrile (BAPN). Hi and soluble collagen levels in bones (tibia and femur) and skin were significantly higher in the embryos treated with BAPN. Compound 48/80 administered together with BAPN lowered Hi and soluble collagen in the bones and skin as compared with the embryos treated with BAPN alone. Both, in the skin and bones, 48/80 was found to be a protective factor against the changes in biosynthesis of collagen.

Elastin-proteoglycans association revealed by cytochemical methods.

By using various cytochemical stains, proteoglycans are shown to be present inside elastic fibers in aortas of beta-aminopropionitrile-induced lathyritic chicks. Depending on the characteristics of the dyes, the shape, size and distribution of the proteoglycan-revealing precipitates are described. The monocationic dye toluidine blue O and the tetracationic dye Alcian blue in the presence of 0.3 M MgCl2 give the most detailed results. With these stains the proteoglycans inside lathyritic elastin appear to be lateral branches of matrix proteoglycans, lying on the external surface of the elastic fibers. A possible general biological significance of elastin-proteoglycan association is briefly discussed.

Characterization of the tissue form of type V collagen from chick bone.

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.

Monoclonal Antibodies as Probes for Differentiating Tropoelastin a and b

BALB/C mice were immunized with a mixture of aortic tropoelastin a and b. To ensure the presence of tropoelastin a, the lathyritic chick aortic tissue was first extracted with denaturing and reducing agents. After dialysis, the standard procedure for tropoelastin purification was followed. Following immunization, the lymphocytes were fused with a myeloma cell line. Resulting hybridomas were cloned and tested for reactivity towards tropoelastin a and b. The majority of monoclonal antibodies exhibited crossreactivity between the two tropoelastins. Two of the clones were further characterized because of their ability to differentiate both tropoelastin a and b. As demonstrated by immunoprecipitation and Western blot analyses, one clone reacted preferentially with tropoelastin b and the other with tropoelastin a and b. Significantly, these results demonstrate that tropoelastin a and b can be differentiated by immunological techniques.

Identification of elastin in developing aortic tissue by immunological methods.

Antibodies raised in rabbits to tropoelastin isolated from lathyritic chick aorta were conjugated with ferritin and used to identify and locate elastin-containing elements in sections of aortic tissue from developing chicks. The ferritin-antibody conjugate was associated with a network of fine filaments with a diameter of 3-5 nm which, in the 8-10 day old chick embryo aorta, was distributed between and around small but recognizable elastic fibers. In older aortic tissue this ferritin-labelled material was seen principally in loose association with the periphery of developing elastic fibers. The microfibrils did not interact with the ferritin-antibody conjugate. Treatment of chicks with beta aminopropionitrile did not interfere with the accumulation of ferritin-labelled filaments on these small elastic fibers, suggesting that the mechanism of addition of new material onto growing fibers may be independent of cross-linking.

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.

Improved methodologies for the isolation and purification of tropoelastin

Increased yields of purified tropoelastin were obtained from lathyritic chicks which had been raised on normal commercial starting feed for 1 week followed by 8 days on commercial starting feed supplemented with 0.1% aminoacetonitrile·HCl and 0.05% ϵ-aminocaproic acid. The increased age of chicks prior to lathyrogen administration plus the addition of the trypsin-like enzyme inhibitor, ϵ-aminocaproic acid, to the diet resulted in a 110% increase in the yield of tropoelastin. In addition, a cation-exchange system was developed for the fractionation of tropoelastin and removal of minor acidic protein contaminants.

Type I collagen in solution. Structure and properties of fibril fragments.

We have measured the diffusion coefficient and weight average molecular weight of type I collagen fibril fragments obtained by acid extraction of rat tail tendons and neutral extraction of lathyritic chick skin, using laser light scatttering techniques. The molecular weight and translational diffusion coefficient were found to be 8.05 +/- 0.40 x 10(5) and 0.450 +/- 0.04 x 10 (-7) cm2/s, respectively, for preparations which contain aggregates in 0.01 M HCl, and 2.82 +/- 0.20 x 10 (5) and 0.780 +/- 0.04 x 10 (-7) cm(2)/s for collagen single molecules. Using these data, as well as the theoretical diffusion coefficient for a single collagen molecule, models for various staggering modes and aggregate mixtures were developed in an attempt to understand the structure of the fibril components present in solution. It was found that several models with 1 to 4 D (D = 67 nm) staggers fit the experimental data for the aggregated preparations. Analysis of the end-to-end distance of negatively stained segment long spacing crystallites prepared from solutions containing fibril fragments and the banding pattern of positively stained segment long spacing crystallites suggest that collagen solutions contain linear aggregates with 4 D and possibly 4.4 D staggers in agreement with light scattering data. Thermal denaturation studies demonstrate that the apparent melting point of cross-linked linear aggregates, 33.5 +/- 0.5 degrees C, is identical with that of single molecules at pH 2.0. We conclude that a linear filament with predominant 4 D stagger is a basic unit of type I collagen fibrillar structure.

Native cross-links in collagen fibrils induce resistance to human synovial collagenase.

A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2--3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.

Molecular weights and stokes radii of soluble elastins

Soluble elastin was isolated from lathyritic chick aorta using neutral salt solutions in the presence of β-amino propionitrile. The effect of a carboxymethylation step in conjunction with proteolytic inhibitors was investigated. Hydrodynamic (Stokes) radii of soluble elastins were measured by gel filtration and the molecular size and weight distribution in purified fractions are reported.

Procollagen segment-long-spacing crystallites: their role in collagen fibrillogenesis.

Naturally occurring segment-long-spacing crystallites of procollagen collagen have been found in the culture medium of fibroblasts from chick embryo tendon, human skin, and dermatosparaxic calf skin; in whole-mount preparations of cultured human skin fibroblasts; in homogenates of lathyritic chick embryo tendon, cartilage, and cornea; and in a partially purified preparation of procollagen. Bundles of similar aggregates occurred within secretory vacuoles of collagen-synthesizing fibroblasts and chondrocytes. These observations suggest that fibroblasts and chondrocytes secrete procollagen assemblies that are stable in the extracellular environment. We propose that subsequent enzymatic processing is accompanied by direct incorporation of such structures into the assembling fibril, which then may be considered as an n X 67 nm staggered array of segment-long-spacing crystallites.

Identification of two new collagen α-chains in extracts of lathyritic chick embryo tendons

Two new collagen polypeptide chains have been identified in extracts of lathyritic embryonic chick tendons. The electrophoretic migration of these polypeptides in sodium dodecyl sulfate-polyacrylamide gels indicates that they have about 20% greater apparent molecular weights than α 1 and α 2 chains of Type I collagen. These chains are not held by disulfide bonds since reduction does not affect their electrophoretic behavior. Further, they do not represent incompletely cleaved procollagen since their apparent molecular size remains greater than that of Type I collagen polypeptides after limited proteolytic digestion. Because the ratio of these polypeptides in the purified extracts is not 2:1 it appears that they are components of two separate tropocollagen molecules.

Identification of collagen α1(I) trimer in embryonic chick tendons and calvaria

Collagen with a molecular composition [ α 1(I)] 3 has been identified in acetic acid extracts from lathyritic chick embryo tendons and calvaria. These molecules characteristically have greater solubility than Type I collagen at neutral pH and contain increased amounts of hydroxylysine residues. It is suggested that these molecules represent a separate gene product of embryonic cells which may be important in the process of maturation and development.

A high molecular weight species of soluble elastin-proelastin.

The isolation of a high molecular weight species (130–140,000 daltons) of soluble elastin from the aortae of lathyritic chicks is described. In comparison to chick tropoelastin, the higher molecular weight material possesses an increased amount of acidic and hydroxyl amino acids and in contrast to tropoelastin, contains histidine, methionine and cystine residues. This high molecular weight species of soluble elastin is susceptible to proteolytic degradation and can be shown to readily break down to lower molecular weight components including tropoelastin. Preliminary data suggest that collagen may have a protective effect on the cleavage of the high molecular weight protein pointing to a potentially significant interaction between the two main structural proteins.

A high molecular weight species of soluble elastin

Summary The isolation of a high molecular weight species (130–140,000 daltons) of soluble elastin from the aortas of lathyritic chicks is described. In comparison to chick tropoelastin, the higher molecular weight material possesses an increased amount of acidic and hydroxyl amino acids and in contrast to tropoelastin, contains histidine, methionine and cystine residues. This molecular weight species of soluble elastin is susceptible to proteolytic degradation and can be shown to readily breakdown to lower molecular weight components including tropoelastin.

Inhibition of proline hydroxylation does not inhibit secretion of tropoelastin by chick aorta cells

In vertebrates, the protein elastin is a vital component of major blood vessels, imparting to them their elastic, rubber-like quality. Purified elastin is very inert and insoluble due to extensive crosslinking of the polypeptide chains by desmosines and other cross-linkages [ 1,2] derived from the oxidation of lysine residues by a copper-requiring enzyme [3-51. Sandberg, et al. [6] prepared a soluble protein called tropoelastin, from the aortae of copper-deficient pigs and this protein consisted of a single polypeptide chain with an estimated mol. wt. of about 70 000 and an amino acid composition which closely resembled that of insoluble elastin except for the absence of the desmosines. A significant portion of the tropoelastin molecule has been sequenced [7] and a model for cross-linked elastin has been proposed based on this data [8]. A similar protein has been isolated from the aortae of copper-deficient or lathyritic chicks [9,101. We have previously reported that isolated embryonic chick aortae synthesize a soluble protein whose labeling pattern with radioactive amino acids was suggestive of that of tropoelastin and which migrated close to serum albumin (68 000 daltons) during gel electrophoresis in sodium dodecyl sulfate [ 1 I]. During pulse-chase experiments this soluble protein was incorporated into an insoluble residue. Smith and Carnes [ 121 have demonstrated that when aortae from normal newborn pigs were incubated in vitro, they synthesize a labeled protein which manifests many of the properties of added carrier tropoelastin. Cells isolated from the media of pig aortae can synthesize elastin after they have been in long term culture [ 131. Recently we have shown that cells isolated from embryonic chick aortae can synthesize and secrete procollagen and tropoelastin into the surrounding medium when incubated in suspension culture under the appropriate conditions [ 141. In the present communication we report that incubation of these cells with concentrations of a,cy’-dipyridyl which totally inhibit proline hydroxylation markedly decreases the rate of procollagen secretion but does not affect the rate of tropoelastin secretion.