Electrophoretic analysis of type I collagen from bovine Achilles tendon: comparison between the extracted raw material and the freeze-dried product.

The present study was aimed at verifying if the freeze-drying process has any effect on the polypeptide composition of the collagen extracted from bovine Achilles tendon in an acetic acid gelified form. Data from our laboratories showed that the freeze-drying process renders the collagen gel essentially insoluble; under these conditions the collagen sample can no longer be analyzed by gel electrphoresis. We found that treatment of the sample with pepsin in acid environment, followed by precipitation with ammonium sulphate yields an insoluble fraction that is susceptible of being analyzed by polyacrylamide gel electrophoresis. The The electrophoresis, run under standard conditions, shows that six major subunits, corresponding to alpha, beta and gamma polypeptides, can be revealed in the sample treated in this way. So the freeze-dried collagen exhibits a polypeptidic composition that is basically identical to that shown by the collagen gel, with regard to the fraction precipitated with ammonium sulphate. Otherwise the pattern of the enzymatic hydrolysis was investigated by measuring the hydroxyproline content, and so the collagen content using the 7.46 conversion factor from hydroxyproline to the scleroprotein collagen, in the various steps of the hydrolysis itself: the analytical results showed no differences between the freeze-dried collagen and the gelified form; this confirms that the lyophilization process does not alter the polypeptidic composition of the collagen in any way.

Fibronectin and laminin in Achilles tendon.

Ten intact and 22 ruptured Achilles tendons were studied by immunohistochemical techniques. In intact tendons, both fibronectin and laminin were found in vascular walls, myotendineal junction, and endomysium, but not in the connective tissue of the tendons. In ruptured tendons the distribution of laminin was not changed. Fibronectin deposits, on the contrary, could be detected on the tear surface and in the collagen fibers of the ruptured tendons.

[Effect of ionizing radiation on the structure of the collagen fibers of human tendons]. / Vliianie ioniziruiushchei radiatsii na strukturu kollagenovykh volokon sukhozhilii cheloveka.

In studying the effect of ionizing radiation on the properties of human Achilles tendon collagen fibres, the following parameters were analyzed: hydrothermal contraction temperature, module of elasticity, the number of cross-links, free and bound water levels, acids-soluble fraction content, and ultrastructure. With radiation doses of 2-10 Gy no changes in the collagen status were noted. An increased (from 5 to 25 Gy) radiation dose caused changes in physicochemical properties which was indicative of the formation, in the connective tissue collagen, of radiation-induced intermolecular cross-links stabilizing the biopolymer structure.

Polyneuropathy with lipid deposits in Schwann cells and axonal degeneration in cerebrotendinous xanthomatosis.

The present paper is a histological, histochemical and electron microscopic study of biopsied specimens from both right and left Achilles tendon, sural nerve and gastrocnemius muscle in a case of peripheral neuropathy with decreased sensory conduction velocity within a cerebrotendinous xanthomatosis confirmed biochemically in a 29-year-old woman. The tendon specimens contained large deposits of complex, non-homogeneous lipids, distributed intra- and extracellularly. The right sural nerve specimen showed a very severe neuropathy with massive diffuse myelinated fiber loss, presence of foamy macrophages and lipid droplets in Schwann cells. Segmental de- and remyelination was noted in 17% of the teased myelinated fibers. No onion bulbs were observed. Two years later, the left sural nerve specimen revealed a mild diffuse myelinated fiber loss, a more active segmental de- and remyelination (23%) without onion bulbs, and an active regeneration. Lipid storage aspects were absent. The gastrocnemius muscle specimens exhibited slight alterations of neurogenic origin. The pathogenesis of this neuropathy is discussed.

Molecular dynamics of collagen side chains in hard and soft tissues. A multinuclear magnetic resonance study.

We have prepared samples of (a) intact calvaria collagen (cross-linked and mineralized), (b) intact tendon collagen (cross-linked but not mineralized), and (c) reconstituted chick calvaria collagen (not cross-linked and not mineralized) containing [methyl-2H3]methionyl, [4,4-2H2]pyrrolidinyl, (4-fluorophenyl)alanyl, and [6-15N]lysyl residues. Using multinuclear magnetic resonance spectroscopy, we have investigated the molecular dynamics of the labeled amino acids. Guided by model compound studies, we reached the following conclusions regarding collagen side chain dynamics from our analysis of line shapes and relaxation rates. At 22 degrees C, imino residues in all samples have flexible rings with root mean square angular fluctuations in the 11-30 degree range. Nearly all labeled amino acid side chains reorient about at least two side chain single bonds. At temperatures below -30 degrees C, most of these side chain motions are absent in all the samples. Surprisingly, in contrast with results obtained for backbone motions, side chain motions are only marginally more hindered in mineralized samples as compared with nonmineralized samples, a result we discuss with reference to collagen-mineral interactions. We also discuss the possible relationship between collagen dynamics and function.

Experimental treatment of tendon injury with heparin.

The effect of heparin on the progress of healing in the injured tendon was examined after a standard injury had been created in the Achilles tendons of New Zealand White rabbits. Half of the rabbits were then treated by the intratendinous injection of heparin. The diameters of the injured right hind and the uninjured left hind Achilles tendon and overlying skin were measured using vernier calipers before injury and at one month after injury. At this time histological analysis was carried out on all Achilles tendons and tendon dry weight, collagen concentration and DNA concentration were measured. Orientation of collagen fibres was more pronounced in treated than untreated tendons while cellularity and blood vessel density were less pronounced. Analysis by Students t-test demonstrated a statistically significant difference between treated and untreated tendons in terms of leg diameter (p less than 0.005), dry weight (p less than 0.01) and DNA concentration (p less than 0.01).

The influence of exercise in the healing of the rabbit Achilles tendon.

Effect of exercise on tendon healing was studied in partially tenotomized rabbit achilles tendons by post-operatively applying a plaster splint to the right leg from the knee to the end of the toe, but no external fixation to the left leg. The animals were serially sacrificed, and new tendon tissues generated at the tenotomy site were examined. Collagen type analysis of the new tendon tissue showed a nearly constant ratio of type III to type I collagen of about 10% in the fixed side, but in the non-fixed side it showed an increase to 19% 2 weeks after the operation followed thereafter by a gradual decrease. Ultrastructural examination revealed an earlier increase in the diameter of collagen fibers suggesting their faster maturation in the non-fixed side.

Isolation and characterization of a proteolytic factor from the venom of Vipera palaestinae.

A proteolytic enzyme which is active on collagen and gelatin was isolated from the venom of Vipera palaestinae. The enzyme showed an optimal temperature of 45 degrees C and an optimal pH of 8.0. It was inhibited by snake blood serum, but not by EDTA or trypsin inhibitors. The enzyme was completely separated from one of the venom hemorrhagins, which accompanied it through the purification procedure. The possible evolution of hemorrhagins from proteolytic enzymes is discussed.

Neutron diffraction studies of collagen in fully mineralized bone.

Neutron diffraction measurements have been made of the equatorial and meridional spacings of collagen in fully mineralized mature bovine bone and demineralized bone collagen, in both wet and dry conditions. The collagen equatorial spacing in wet mineralized bovine bone is 1.24 nm, substantially lower than the 1.53 nm value observed in wet demineralized bovine bone collagen. Corresponding spacings for dry bone and demineralized bone collagen are 1.16 nm and 1.12 nm, respectively. The collagen meridional long spacing in mineralized bovine bone is 63.6 nm wet and 63.4 nm dry. These data indicate that collagen in fully mineralized bovine bone is considerably more closely packed than had been assumed previously, with a packing density similar to that of the relatively crystalline collagens such as wet rat tail tendon. The data also suggest that less space is available for mineral within the collagen fibrils in bovine bone than had previously been assumed, and that the major portion of the mineral in this bone must be located outside the fibrils.

Collagen hydrolysis by a new Streptomyces species.

A soil streptomycete hydrolysed collagen extracted from bovine Achilles tendon, calf skin, carp swim bladder and rat tail tendon. Enzyme activity was highest with rat tail tendon collagen as substrate. The taxonomy of the streptomycete was studied according to internationally approved methods. Comparative studies revealed that it resembled Streptomyces humidus , S. rochei and S. distatochromogenes in some respects, but differed in many cultural and physiological characteristics. Based on these differences the organism was given a new identity as Streptomyces species A.

[Calorimetric study of collagen hydration]. / Kalorimetricheskoe issledovanie gidratsii kollagena.

Water in human Achilles tendon was studied by differential scanning calorimetry. It was shown that in the postmortem period there takes place a redistribution of free and bound water. The quantity of free freezing water in the tissue increases, whereas that of bound unfreezing water diminishes. The data obtained can be used for organ and tissue conservation and in forensic studies.

Collagen cross-linking compounds in human urine.

We report the isolation and chemical characterization of collagen cross-linking compounds, 3-hydroxypyridinium and dihydroxylysinonorleucine, from human urine.

[Structure and properties of the collagen complex of tendons following disorganization of the connective tissue ground substance]. / Struktura i svoistva kollagenovogo kompleksa sukhozhiliia pri dezorganizatsii osnovnogo veshchestva soedinitel'noi tkani.

Scanning and transmission electron microscopy, as well as physicochemical research methods were used to study the structure and characteristics of the tendon collagen complex during enzymatic distintegration of basal substance of connective tissue. Disintegration of basal substance was shown to be accompanied by substantial derangement of the microstructure of collagen fibers and to entail changes in the modulus of elasticity and residual deformation of the collagen complex with its ability to hydration in a aqueous medium to be increased. The physiochemical and rheological characteristics of the collagen complex were found to be affected to a considerable degree by structural changes occurring in collagen fibers. These changes consist in the formation of a retiform structure containing numerous macrospores.

Spontaneous rupture of the Achilles tendon - a sign of hyperlipoproteinaemia (HLP) type II.

In biopsies from spontaneously ruptured Achilles tendons a serum lipid and lipoprotein analysis was performed in fifty nine patients. In nine males considerable histological signs of lipid deposition were detectable. The tendon fibers were found to be partly desintegrated. Only in these cases LDL-cholesterol values were found to exceed the normal range indicative of type II hyperlipoproteinaemia (HLP). None of the fifty remaining patients presented biochemical or clinical signs of disorders of lipid metabolism. The observations demonstrate that a spontaneous rupture of Achilles tendom may indicate a primary type II HLP.

[Polyploidisation - a phenomenon of aging? (author's transl)]. / Polyploidisierung-ein Alternsphänomen?

By means of cytophotometric determination of DNA in different cellular systems an increase in polyploidisation correlated to age could be demonstrated. The latter is well demonstrable if compensatory hyperplasia or hypertrophy occurs. It is discussed that cellular systems of older individuals can increase their DNA - content, but they are not able to carry out a caryo-and cytocinesis.

The effects of zinc on ectopic bone formation.

The effect of dietary zinc deficiency was studies in ectopic bone formation subsequent to Achilles tenotomy and also following the implantation of demineralized bone matrix in the muscle of rats. Three experiments were performed. The first was designed to investigate the relationship between zinc and calcium concentration during the formation of ectopic bone in rats fed a commercial laboratory ration, the second concerned the effects of dietary zinc deficiency on ectopic bone formation, and the third studies the subsequent effects of dietary zinc repletion on ectopic bone formation. The results indicated that, with the commercial ration, zinc increased concomitantly with calcium during ectopic bone formation in rats. Dietary zinc deficiency caused a retardation of ectopic bone formation and a significant reduction of in situ zinc and calcium concentration. Dietary zinc repletion to zinc-deficient animals restored the zinc concentration in ectopic bone to a level comparable to that of zinc-sufficient animals. Thus, these experiments present strong evidence that zinc plays an active role in bone metabolism.