The changes in chemical composition during development of the bovine nuchal ligament.

Whole bovine nuchal ligaments, or portions thereof (in the case of commercially valuable animals), were obtained from 45 animals (28 fetal and 17 postnatal) ranging in age from 110 days of gestation to 10 yr. Insoluble elastin was quantitatively prepared from the fresh ligaments by extraction with hot alkali and by a combination of multiple extractions with alkaline buffer and then repeated autoclaving. When adult samples were examined, the yields of insoluble residue by these two methods were very similar, but with young fetal samples the second method gave significantly higher values, because of incomplete purification of the elastin residue. The changes in the concentration of collagen, alkali-insoluble elastin, and DNA have been examined. DNA concentration, and, thus, cell population density, fell progressively during the fetal period of development, to reach a steady value soon after birth. Collagen appeared in appreciable quantities before elastin, but its concentration was rapidly halved at about the time of birth. Insoluble elastin concentration was low until the end of the 7th fetal month, at which time it began to rise rapidly. The rate of increase in elastin concentration remained high throughout the next 10-12 wk, by which time the adult value had been reached. Quantitative studies, on the basis of the whole ligament, showed that the total cell content rises to a maximum at birth, but falls soon after to a level about half that at birth. Total collagen production and elastin deposition continue at a steady, maximal rate over the interval from 235 days of gestation to the end of the 1st postnatal month. It is concluded that the immediate postnatal period would be the most favorable phase in which to attempt the isolation of the soluble precursor elastin.

Histological changes during the development of the bovine nuchal ligament.

The histological changes occurring during the development of the bovine nuchal ligament have been observed in sections of formalin-fixed material from 21 animals ranging in age from 110 days of gestation to 10 yr. The elastic fibers which constitute the bulk of the adult ligament were initially few in number. During fetal development, the fibers showed a rapid increase both in number and in their stainability with the usual elastic stains. The average diameter of these elastic fibers increased only slowly until the last uterine month, at which time it began to increase very rapidly. This rapid rate of increase continued through the first 6 postnatal months, after which the rate of increase slowed markedly. However, the fiber diameter continued to rise steadily throughout the period of the study. During the fetal stage of development, the fibroblastic cells of the ligament exhibited unusual nuclear appearances which distinguish them from other fibroblasts. These consisted of marked clumping of the chromatin and an associated nuclear vacuolation or vesiculation. While these changes seem likely to be artefacts of fixation, their temporal correlation with elastin deposition and their demonstration in other tissue cells engaged in elastin production suggest that the factors responsible for these appearances may be related to elastin synthesis.

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture.

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.

The use of omicron-phthaldialdehyde in the detection of proteins and peptides.

A highly sensitive method utilizing omicron-phthaldialdehyde for an automated fluorescent assay of proteins and peptides is described in this paper. As an example of the utility of omicron-phthaldialdehyde, the separation of bovine serum albumin monomer from bovine serum albumin oligomers is discussed.

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study.

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.

Structure of the elastic fiber: an overview.

Intense research efforts over the past 18 yr have probed deeply into the structure of the elastic fiber. This began with the elucidation of the demosine crosslinks in elastin and the description of the elastin precursor, tropoelastin, derived from copper-deficient animals. Characterization of the precursor material indicates that it is a single polypeptide chain of approximately 800 amino acid residues containing lysine residues in clusters destined to form the desmosine crosslinks. The molecule contains large areas of hydrophobic sequence interspersed with shorter stretches of polyalanine and the lysines. The shorter structures may be folded into alpha-helices. The larger hydrophobic areas appear to form a unique structure known as the beta spiral which possesses elastometric properties. Inside the hydrophobic areas repeating sequences such as the pentapeptide pro-gly-val-gly-val have been observed the exact significance of which is not appreciated, but it appears to be well-conserved between species. Recent studies in the molecular biology of this protein have indicated that it is synthesized on the rough ER with a short leader sequence of about 25 residues. This is lost before the tropoelastin is exported. Diversity in sequence studies in these leaders suggest that there may be two elastins, type A and B, which vary with the maturation of the animal.

Developmental expression of dermatan sulfate proteoglycans in the elastic bovine nuchal ligament.

The nuchal ligament of bovines is a useful system in which to study elastic fibre formation since it contains up to 83% elastin and undergoes a period of rapid elastinogenesis during the last trimester of fetal development and in the first four post-natal months. To identify proteoglycans (PGs) which may be involved in this process we initially investigated changes in the glycosaminoglycan (GAG) profiles during nuchal ligament development. In contrast to the collagenous Achilles tendon, nuchal ligament exhibited: (a) elevated hyaluronan (HA) levels in the peak period of elastin-associated microfibril (fibrillin) synthesis (130-200 days) which precedes elastinogenesis; and (b) markedly increased synthesis of a glucuronate-rich copolymeric form of dermatan sulfate (DS) in the period corresponding to elastin formation (200-270 days). Analysis of DSPGs isolated from 230-day nuchal ligament showed that this copolymer was predominantly associated with a glycoform of biglycan which was specifically elevated at this stage in development. This finding was consistent with Northern blot analysis which showed that steady-state biglycan mRNA levels increased significantly during the elastinogenic period. In contrast, the mRNA levels for decorin, the only other DSPG detected in this tissue, declined rapidly after 140 days of fetal development. In conclusion, the results suggest that HA may play a role in microfibril assembly and that a specific glycoform of biglycan may be associated with the elastinogenic phase of elastic fibre formation.

Extensive alternate exon usage at the 5' end of the sheep tropoelastin gene.

Several overlapping cDNA clones were isolated from a lambda gt10 cDNA library constructed using poly A+ RNA from neonatal sheep lung. DNA sequence analysis of these cDNA recombinants revealed the complete derived amino acid sequence of sheep tropoelastin. A comparison of DNA sequences from individual sheep tropoelastin cDNA also confirmed the presence of several tropoelastin mRNA isoforms in neonatal lung tissue. Coding domains corresponding to exons 13, 14 and 33 were present in several of the sheep tropoelastin cDNA fragments but absent in others. The relative amount of alternate usage of these exons was quantitated by polymerase chain amplification. In confirmation of previous studies in other mammalian species, extensive alternate usage of exon 33 was observed in total RNA isolated from aorta, nuchal ligament and pulmonary artery from neonatal sheep. In striking contrast to all previous studies, however, exons 13 and 14 were shown to be subject to almost the same level of alternate usage as exon 33 in all three neonatal sheep tissues examined.

Improvement in plasma protein concentrations with fibronectin treatment in severe malnutrition.

Severely malnourished young children (n = 72) were treated with intravenous fibronectin to assess its efficacy as an adjunct treatment for kwashiorkor and/or marasmus. The protein was given in a double-blind study during the first 4 d of hospitalization together with standard nutrition and supportive therapy. Fibronectin concentrations as well as albumin, transferrin, prealbumin, and alpha-2-macroglobulin were monitored in samples taken before each dose of fibronectin and in samples taken five times thereafter. Sick individuals had significantly lower concentrations of all five proteins than did healthy control individuals of matching ages. Mean fibronectin concentrations were 98 +/- 7 mg/L (mean +/- SEM) for sick vs 303 +/- 21 mg/L for healthy individuals. Concentrations of all five proteins increased at a greater daily rate in patients treated with fibronectin than in patients who received placebos. Eighty-seven percent of the treated children survived to the end of the treatment and observation periods (mean hospitalization 14.7 d) whereas only 56% of the control subjects survived (P = 0.004). These data support the use of intravenous fibronectin as an adjunct in the treatment of severe malnutrition at a dosage of 7.5 mg.kg-1.d-1 over a 4-d period.

Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

The influences of sample distribution and age on reference intervals for adult males.

This investigation was designed to improve reference information and to evaluate the influences of sample distribution and age on the derived reference intervals. Specimens from 127 men were collected after a 12- to 14-hour fast and analyzed by 60 different laboratory procedures. Differences in the reference intervals derived, using three separate statistical methods, appeared to be unimportant clinically, but the percentile method was preferred because it required no assumptions concerning the frequency distribution. Relationships between age and analyte concentrations were examined by linear regression analysis, and the analytes were placed in one of three groups, according to the significance of this relationship: greatest significance (P less than or equal to 0.01), 18 analytes; intermediate significance (0.01 less than or equal to P less than or equal to 0.05), 12 analytes; and least significance (P greater than 0.05), 30 analytes. The age-related changes for each analyte were evaluated according to analytic variation, population dispersion, and clinical relevance. Age-dependent reference intervals for adult males are recommended for albumin, cholesterol, phosphorus, and sedimentation rate.

Uniqueness of dietary olive oil in stimulating aortic prostacyclin production in post-weanling rats.

Groups of weanling Sprague-Dawley rats developed from conception through gestation, and weanling periods on a formulated diet fed to the dams were continued on the same diet until sacrificed at 30 days of age. The diet groups consisted of control (5% corn oil, w/w) and experimental (15%, w/w) olive, safflower (hi-oleic and hi-linoleic), soy oil, and lard. The object of the study was to identify the effect of high and low fat content and differing proportions of polyunsaturated:saturated (P:S) and mono:polyunsaturated (M:P) fatty acids on arachidonate stimulated aortic prostacyclin (PGI2) production (measured as 6-keto-PGF1 alpha). Neither the amounts of dietary fat or wide ranging P:S or M:P fatty acid ratio levels (P:S or M:P) affected PGI2 production. PGI2 production was, however, markedly enhanced (2x) in aortic segments from rats raised on diets containing olive oil. The unique stimulation of aortic PGI2 production by the olive oil diet suggests an effect of the extraordinarily high M:P fatty acid ratio or, alternatively, of a still-to-be identified substance(s) in this ancient food.

Oxysterol incorporation into rat aorta resulting in elastin compositional changes.

The incorporation of dietary cholestan-3 beta,5 alpha,6 beta-triol (triol) into rat thoracic aortic tissue and changes in amino acid composition of the elastin were investigated to identify the cytotoxic properties of the triol. Weanling male Sprague-Dawley rats were fed the following diets for three months: (i) normal chow, (ii) normal chow with 1% (w/w) cholesterol added, or (iii) normal chow with 0.9% (w/w) cholesterol and 0.1% (w/w) triol added. Triol levels in the blood and in the thoracic aortic tissue were measured. Compositional changes of elastin were also determined. After three months on the triol-containing diet, triol was found in the thoracic aorta but was not detected in the blood. Amino acid analyses of the aortic tissue elastin revealed that the proline levels in the triol-fed animals were significantly greater than in the other two diet groups, while the elastin levels of leucine, aspartate, arginine, and phenylalanine decreased significantly. The mechanism for these observed changes induced by triol may reflect alternate splicing of elastin messenger ribonucleic acid (mRNA) resulting in structual changes in the elastin molecule. Dietary triol does contribute to tissue triol content and is associated with aortic elastin compositional changes. How these changes may contribute to the development of cardiovascular disease is not known.

Serum anti-tropo:anti-alpha-elastin antibody ratio assessing elastin turnover in scleroderma.

Serum antibodies to native (tropo) and denatured (alpha) elastins appear to correlate with the production and breakdown respectively of elastic tissue. Elastin may be degraded as a part of autoimmune diseases. This possibility was tested by measuring IgG antibodies to tropo- and alpha-elastins by ELISA in the sera of 111 patients with a variety of connective tissue diseases compared with 18 healthy individuals. Anti-alpha-elastin antibodies were significantly higher in sera from 18 scleroderma patients than from healthy controls (p less than 0.008). Conversely, anti-tropoelastin antibody levels for scleroderma patients (p less than 0.03) and for patients with a variety of other connective tissue diseases (p less than 0.02) were lower than in healthy controls. Low antibody levels to native elastin and high levels of antibodies to denatured elastin suggest a low synthesis: degradation ratio for elastin in scleroderma. Scleroderma may be a unique model for elastin turnover because of its heretofore unrecognized accelerated elastolysis.