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There is evidence that spaceflight poses acute and late risks to the central nervous system. To explore possible mechanisms, the proteomic changes following spaceflight in mouse brain were characterized. Space Shuttle Atlantis (STS-135) was launched from the Kennedy Space Center (KSC) on a 13-day mission. Within 3⁻5 h after landing, brain tissue was collected to evaluate protein expression profiles using quantitative proteomic analysis. Our results showed that there were 26 proteins that were significantly altered after spaceflight in the gray and/or white matter. While there was no overlap between the white and gray matter in terms of individual proteins, there was overlap in terms of function, synaptic plasticity, vesical activity, protein/organelle transport, and metabolism. Our data demonstrate that exposure to the spaceflight environment induces significant changes in protein expression related to neuronal structure and metabolic function. This might lead to a significant impact on brain structural and functional integrity that could affect the outcome of space missions.

Little data exist on the structure and function of compressible elastomeric proteins such as abductin. An understanding of the underlying structural features of these proteins may lead to the development of a new class of highly tailored "compressible" hydrogels. To that effect, in this work, the structure of abductin was investigated by means of studies on several synthetic peptides corresponding to the most frequent sequences of abductin. In particular, the 10 amino acid abductin peptide sequence FGGMGGGNAG, tandem repeated in the protein, and two related 25 and 40 amino acid polypeptides were synthesized. These peptides were studied with regard to secondary structure, self-assembly, and polymer morphology. The results obtained with these peptides allow us to propose a preliminary structure-elasticity relationship for abductin not dissimilar from that currently accepted for elastin.A possible mechanism of elasticity relating abductin to elastin.

Background: Antibodies (Abs) to alpha-elastin (elastin breakdown product) and tropoelastin (elastin precursor) are found in the serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs, peptides, and elastin metabolism were altered in autoimmune diseases by detecting a changing ratio of serum anti-alpha:tropoelastin Ab levels., Methods: Serum from patients with a variety of connective tissue diseases, including 28 with systemic lupus erythematosus (SLE), 24 with scleroderma, 18 with rheumatoid arthritis (RA), 10 with polymyositis, and 39 with vasculitis, was compared with serum from 19 age-matched healthy subjects for levels of antitropoelastin and anti-alpha-elastin Abs., Results: We found an increase in IgG anti-alpha-elastin and a decrease in antitropoelastin Abs in the sera of patients with scleroderma (p < .02 and .00005) and SLE (p < .006 and .011). There was also a marked increase in anti-alpha-elastin Abs in patients with polyarteritis nodosa (p < .0005) and decreases in antitropoelastin Abs in patients with RA (p < .05), polymyositis (p < .01), and a variety of other vasculidities (p < .0003)., Conclusions: Abnormal variations in elastin metabolism may be detected in several connective tissue diseases by measuring ratios of alpha- and tropoelastin IgG Abs as markers of elastin degradation and synthesis.

High-resolution solid-state (13)C NMR spectra are presented for samples of alpha-elastin prepared from the aorta of normal and copper-deficient pigs. Chemical shifts of the various peaks indicate that both the normal and undercross-linked peptides have similar overall structures. However, (13)C T(1), (13)C T(1 rho), and (1)H T(1 rho) measurements indicate that the alpha-elastin peptides obtained from the abnormal elastic fibers samples exhibit altered mobilities, particularly in their side chains. Results from spectra taken with a range of contact times and from dipolar dephasing experiments are consistent with conclusions reached with the relaxation measurements. Namely, the loss of function associated with the undercross-linked sample is correlated to a small but measurable difference in relative mobility., (Copyright 2001 John Wiley & Sons, Inc. Biopolymers 59: 266-275, 2001)

Solid-state spectral editing techniques have been used by others to simplify 13C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance 13C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with alpha-elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical 13C CPMAS spectra. Spectral editing is particularly useful for simplifying he aliphatic region of the NMR spectrum of this elastin preparation.

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.

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.

Together with the 31-kDa microfibril-associated glycoprotein (MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa), MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in tissue extracts designed specifically to solubilize the microfibrillar component of elastic fibers. In the present study, both MP78 and MP70 were shown to be forms of a protein which is closely related to the human protein beta ig-h3, and MP340 was confirmed to be the bovine form of fibrillin-1. Peptide sequences from MP25 proved to be unique, and affinity-purified anti-MP25 antibodies were shown, by immunofluorescence and immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. This confirmed that MP25 was a distinct component of these structures. Expression screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770 base pairs) which encodes amino acid sequences matching those of the MP25 peptides. Further library screening with cM10A identified cDNAs which encode the complete primary structures of bovine and human MP25. Bovine and human MP25 were found to be around 80% homologous and contain 170 and 173 amino acids, respectively. Data base searches revealed that MP25 had significant similarity of structure only with MAGP, indicating that the two proteins form a new family of microfibrillar proteins. In acknowledgment, MP25 has been formally renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity between the two proteins (57%) is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. Elsewhere the MAGP-2 molecule is rich in serine and threonine residues and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and tyrosine-rich sequences and a hydrophobic carboxyl terminus, characteristic of MAGP-1. These structural differences suggest that MAGP-2 has some functions which are distinct from those of MAGP-1. The locus of the human MAGP-2 gene was identified on chromosome 12 in the region of 12p12.3-12p13.1.

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.

We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay. The major nonvolatile components effluxed were methionine, proline, histidine, and lysine. In studies of the release of volatile radioactivity in chemotaxis by B. subtilis cells that had been labeled with [3H]methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioactivity in fractions following addition of 0.1 M aspartate. However, methanol was confirmed to be released after aspartate addition and, in lesser quantities, after aspartate withdrawal. Methanol and methanethiol were positively identified by derivitization with 3,5-dinitro-benzoylchloride. Amino acid efflux but not methanol release was observed in response to 0.1 M aspartate stimulation of a cheR mutant of B. subtilis that lacks the chemotaxis methylesterase. The amino acid efflux could be reproduced by withdrawal of 0.1 M NaCl, 0.2 M sucrose, or 0.2 M xylitol and is probably the result of changes in osmolarity. Chemotaxis to 10 mM alanine or 10 mM proline resulted in methanol release but not efflux of amino acids. In behavioral studies, B. subtilis tumbled for 16 to 18 s in response to a 200 mosM upshift and for 14 s after a 20 mosM downshift in osmolarity when the bacteria were in perfusion buffer (40 mosM). The pattern of methanol release was similar to that observed in chemotaxis. This is consistent with osmotaxis in B. subtilis away from an increase or decrease in the osmolarity of the incubation medium. The release of methanol suggests that osmotaxis is correlated with methylation of a methyl-accepting chemotaxis protein.

We have constructed several rat tropoelastin minigene recombinants encoding the complete sequence of rat tropoelastin, two isoforms of rat tropoelastin and a truncated tropoelastin lacking the domains encoded by exons 19-31 of the rat gene. Coding and non-coding domains in all these recombinants were placed under the transcriptional control of 3 kb of the promoter domain of the rat tropoelastin gene. These minigenes were used to prepare a total of 28 separate founder lines of transgenic mice. A species-specific reverse-transcriptase polymerase chain reaction (RT-PCR) assay was established to demonstrate the synthesis of rat and mouse tropoelastin mRNA in several tissues obtained from both neonatal and adult transgenic mice. Thermolytic digestion of insoluble elastin isolated from several neonatal mouse tissues revealed the presence of rat tropoelastin peptides in progeny from all those founder mice in which detectable levels of rat tropoelastin mRNA were noted. Phenotypic and histopathological assessment of transgenic and non-transgenic animals revealed the development of two diverse elastic tissue disorders. The progeny of two separate founder lines overexpressing the rat tropoelastin isoform lacking exon 33, developed an emphysematous phenotype in early adulthood. In contrast, transgenic mice, in which expression of the truncated rat tropoelastin minigene lacking exons 19-31 had been observed, died of a ruptured ascending aortic aneurysm. Tropoelastin gene mutations, therefore, will result in heritable disorders of elastic tissue. Moreover, different mutations in the tropoelastin gene will be responsible for very different abnormalities in elastic tissue function.

Thermolysin digests of human elastins were examined for reliable elastin peptide markers as determined by HPLC followed by amino acid sequencing of promising peaks. The tetrapeptide VAPG was found to occur in the early portion of the chromatogram in a highly reliable fashion. The peptide appears to be significantly amplified, when compared with the other peptides, in that it is derived from the hexapeptide repeat in elastin, VGVAPG, which repeats itself in two three-piece segments in the c-terminal portion of the tropoelastin molecule. VAPG serves as a highly reliable quantitative measure for human elastins, allowing sensitivities to less than a microgram. Thus, it is a significantly more accurate measure than other existing methods. Precision also appears to be enhanced because of the directness of the measurement. The use of VAPG as a quantitative marker for human elastin has clinical application in the study of elastin-based connective tissue diseases.

Thirty-nine 40-day old male Sprague-Dawley rats (average wt. 115g) were divided into 4 groups and fed diets A, control; B, essential fatty acid (EFA) deficient; C, protein deficient; D, combined protein and EFA deficient. At the end of 5 weeks, lungs were removed from the animals for collagen and elastin quantitation and for morphometric measurements. The collagen content of the lungs which ranged from 95-100 micrograms/mg dried fat-free (D.F.F.) tissue, was not altered by protein or EFA deficiencies. The elastin content of lungs was markedly increased in diets C and D while the cross-linking (Isodesmosine-desmosine content) expressed as residues per 1,000 (R/1000) was not different in the four groups. The elastin content of lungs from group D animals was greater than group C suggesting an additive effect from the EFA deficiency in diet D. The morphometric measurements indicated no change in alveolar linear diameter (Lm) while the total alveolar surface area (ISAA1V) was decreased by the deficient diets C and D. The protein deficiency and the combined protein and EFA deficiencies produced an increased elastin content in lung. Elastin cross-linking and collagen quantity was not affected by the dietary treatments. The morphometric measurements indicated that protein deficiency in these animals did not produce structural changes in the lungs as indicated by alveolar dimensions.

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.

Dietary lipid modulation of structural and passive mechanical properties of isolated rat abdominal aortic segments were assessed during the early developmental period. Rats were raised from conception to 90 days of age on semisynthetic diets containing various types and amounts of lipids. Aortic segments from three groups of rats fed high-fat diets (15%, wt/wt) consisting of olive oil, corn oil, or lard as the sole lipid sources were compared with those from rats fed a low-fat control diet containing corn oil (5%, wt/wt). Morphometric analysis of the tunica media demonstrated that rats raised on diets with a relatively low polyunsaturated fatty acid content (olive oil and lard) had greater numbers of elastic lamellae than rats raised on diets with opposite fatty acid indexes (high- and low-fat corn oil). Changes in elastin content of the tunica media, determined biochemically, paralleled those seen by morphometric analysis of the elastic lamellar number. Altered dietary fatty acid ratios were also associated with changes in smooth muscle cell number. In this regard, a decreased cellular density was observed in the olive oil and lard diets compared with the corn oil diet. The olive oil diet was unique amongst the dietary lipid regimens in raising, whereas the lard-containing diet lowered, indexes of aortic tissue elasticity. These results demonstrate an effect of chronic feeding of high dietary fat on the composition and biomechanical properties of the connective tissue matrix of abdominal aortic rings from young Sprague-Dawley rats.

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.

Affinity-purified antibodies to microfibril-associated glycoprotein (MAGP) were used to screen a random-primed, bovine nuchal ligament cDNA library in lambda gt11. A 303-base pair clone, cM5, was isolated which encoded an amino acid sequence homologous with that determined directly from a Lys-C peptide of MAGP. A 936-base pair cDNA clone, cM32, was identified in an oligo(dT)-primed cDNA library using plaque hybridization with clone cM5. Clone cM32 encoded amino acid sequences corresponding to sequences obtained from three Lys-C peptides of MAGP, indicating that the clone was an authentic cDNA for the glycoprotein. The cDNA coded for the entire MAGP polypeptide (21 kDa) of 183 amino acids including a putative signal peptide of 17-19 amino acids. This was confirmed by in vitro translation of synthetic mRNAs transcribed from cM32. The amino acid composition of the encoded protein was virtually identical to that previously published for MAGP. DNA sequence analysis of cM32 indicated that MAGP contains two structurally dissimilar regions, an amino-terminal domain containing high levels of glutamine, proline, and acidic amino acids and a carboxyl-terminal domain containing all 13 of the cysteine residues and most of the basic amino acids. Northern blot hybridization of poly(A+) RNA from fetal nuchal ligament with clone cM32 identified a single mRNA species for MAGP of approximately 1.1 kilobases. The evidence indicates that MAGP is a distinct component of 12-nm microfibrils and that it is not derived from a larger microfibrillar glycopolypeptide.

The rat is notoriously resistant to vascular disease, but administration of haloperidol prenatally to the pregnant dam results in hyperlipidemic changes in F1 and F2 progeny. Total serum cholesterol concentrations in the F1 males reach levels three or more times greater than controls by one year of age. The phenomenon indicates a link between dopaminergic elements of the autonomic nervous system and cholesterol metabolism, a phenomenon heretofore unrecognized. Transfer of the hypercholesterolemia into the second generation further suggests that haloperidol induces a permanent change in the genetic control of lipoprotein metabolism. Although the observations are preliminary, they warrant consideration when administering the drug to pregnant women.

Neonatal rat pups were treated either with ciclosporin at 10 mg/kg/day dissolved in olive oil (experimental) or with pure olive oil (control). Lung protein biosynthesis was evaluated in a protocol which involved the measurement of total accumulated protein, collagen and elastin. Four time points were studied in the first 21 days of life, 12 animals contributing to each point (6 control and 6 ciclosporin). Ciclosporin levels in the treated group ranged widely (2,000-4,000 ng/ml). There were significant differences in total body weight and lung weight in treated vs. controls during and after the first week. DNA contents per unit wet weight varied significantly during the second week of life, indicating increased cellularity of the ciclosporin-treated animals. Associated with this was an increase in the lung protein/DNA ratio as well as the elastin/DNA ratio in the control animals, but not in the treated ones. The lung collagen/DNA ratio was not as dramatically affected by the ciclosporin treatment. However, the collagen content per unit wet weight of lung tissue was increased in the ciclosporin-treated animals at 15 days of life. We conclude that ciclosporin has a marked effect on lung connective tissue metabolism in early life, the long-term effects of which are unappreciated and undocumented but may well be of vital importance in the lungs of long-surviving organ transplant patients.

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.

Both rat and sheep elastin can be quantified by measurement of discrete peptides released from the insoluble protein by thermolysin digestion. These peptides are easily visualized and measured by HPLC. With the sheep the tallest peak on the chromatogram represents the VGVPG pentapeptide derived from a repeating sequence seen in elastin from many species. This repeating sequence allows for amplification of the signal significantly above background so that accurate quantitation can be carried out. The measurement is reproducible over a wide range of protein concentrations. With the rat however the pentapeptide is not present but appears to be replaced by other repeating sequences. We quantitated and determined amino acid sequence on 8 peaks present in the early portion of the chromatogram for purposes of quantifying rat elastin. That signal most reliably present over a range of concentrations was tyrosyl-glycine (YG) which eluted at 8.5 minutes. We have used YG as a basis for quantitation of rat elastin both from tissues and tissue culture. We have also shown that the desmosine crosslinks are not constant in elastin produced in a neonatal rat smooth muscle culture system but vary with the age of the culture. We thus propose that an index of maturation be considered for a given elastin in the form of mumoles of crosslink per gram of elastin so as to better define its quality.

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.

Immunization of rabbits with either porcine tropoelastin, lung alpha-elastin, or aortic alpha-elastin resulted in the production of antibodies against the respective antigens. The assay of antibody activity with a radioimmunoassay indicated a high degree of cross reactivity between these three soluble elastin preparations. Through the use of competitive protein binding experiments it was possible to detect antigen-specific differences between tropoelastin and the two alpha-elastins. No antigenic differences were observed between lung or aortic alpha-elastin by any of the assay procedures uded in this investigation. Absorption of the various antisera with either insolubilized tropoelastin, lung elastin or aortic elastin allowed the preparation of non-crossreactive antibodies and provided further evidence for the antigenic differences between tropoelastin and the soluble alpha-elastins. The implications of the findings, as they relate to our current understanding of the molecular and structural arrangement of elastin and the elastin precursor, are discussed.

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.

An enzyme-linked immunoassay (ELISA) procedure has been developed to quantitate the amount of elastin produced by cultured porcine aortic smooth muscle cells. ELISA was used to determine both titer and specificity of antisera raised in rabbits against porcine aortic alpha-elastin conjugated with key-hole limpet hemocyanin. Under optimum conditions (1: 3000 dilution of antiserum, 20 ng alpha-elastin per assay well), sensitivity averaged 60 ng/ml). Specificity was confirmed by immunoprecipitation of [125I]-tropoelastin, radial immunodiffusion, Western blotting and lack of cross-reactivity with serum proteins or collagen. Extensive cross-reactivity was found with both human alpha-elastin and porcine beta-elastin, while porcine tropoelastin was able to compete with 80% of the alpha-elastin determinants. Affinity of anti-porcine antisera for sheep alpha- elastin was significantly lower. When the ELISA was made specific for tropoelastin by coating wells with 60 ng of this antigen, a time-dependent and serum-dependent rate of production of tropoelastin was observed in the culture medium of primary and secondary cultures of smooth muscle cells. Comparison of elastin production in cultures of porcine smooth muscle cells suggests that porcine aortic elastin production varies as a function of cell density and phase of growth.

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.

Lysyl oxidase, the enzyme responsible for mediating crosslink formation in collagen and elastin, requires copper for its activity. In this study, lysyl oxidase activity and insoluble elastin content were unchanged in lungs from copper-deficient hamsters compared to controls. The lack of dramatic diminution in lysyl oxidase activity in animals who demonstrate significant structural alterations in the lung suggests that other mechanisms in addition to inhibition of crosslink formation are operative in this model.

The amino acid sequences of 16 large tryptic fragments of aortic tropoelastin have been determined establishing the presence of several repeating structures: GVP, GGVP, PGVGV, PGVGVA, and AGVPGFGVG. The methodologies for achieving these results by solid phase sequencing are reviewed and also the possible biologic significance of the unusual primary structures of elastin are discussed.

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.

A mild form of emphysema was produced in pigs raised on a copper-deficient, zinc-supplemented diet. The copper-requiring enzyme, lysyl oxidase, catalyzes the cross-linking of tropoelastin into mature elastin. Zinc further inhibits the activity of lysyl oxidase. Lungs from animals raised on copper-deficient, zinc-supplemented diets of demonstrate perforations in alveolar walls and diminished amounts of elastin bronchi and pulmonary arteries. Mean linear intercepts are greater and alveolar internal surface areas are less than those in control animals, fulfilling the generally accepted definition of emphysema. Physiologic confirmation is provided by a leftward shift of the saline volume-pressure curves when compared with those in control animals. Ultrastructurally, the alveolar walls are effaced and pores of Kohn are enlarged. There are areas in which elastin is absent leaving remnant microfibrils, and there are other changes consistent with active elastin synthesis. Biochemical data demonstrate no difference in elastin content as micrograms/ml of fat-free dry weight but do demonstrate increased collagen content in experimental animal lungs compared with that in control lungs. Ultrastructural similarities to enzyme-induced models of emphysema suggest the presence of elastin degradation in our model. We speculate that although the copper-deficient, zinc-supplemented state may stimulate protein synthesis in general, elastin is being degraded by endogenous means, but collagen is not.

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.

Digestion of insoluble porcine elastin with thermolysin produces a number of discrete small peptides. That present in highest concentration is the pentapeptide valyl-glycyl-valyl-prolyl-glycine (VGVPG) derived from the portion of the polymer containing extensive repeats of this sequence. Among eukaryotes, this sequence appears to be found only in elastin and its precursor tropoelastin. In the pig this is represented by peptide W4 of a tropoelastin tryptic digest (Sandberg, L.B., et al. Path. Biol. 33, 266-274, 1985). Quantitation of this peptide by HPLC separation, monitoring its absorption at 212 nm, offers a simple reliable means of measuring purified insoluble elastin as well as non-purified elastin in fat-free tissue samples. Digestion times and incubation temperatures are discussed. The method is sensitive enough to accurately quantitate elastin at the 2 to 3 microgram level.

An attempt to use the relative concentrations of major plasma proteins for clinical assessment of severe malnutrition is described. Quantitative two-dimensional gel electrophoresis was used to measure the concentrations of 24 major proteins in small aliquots of plasma obtained from children, aged 0 to 3 years, who were patients and outpatients in Liberian hospitals. Fifteen had a clinical diagnosis of kwashiorkor, 36 were diagnosed with marasmus, and 18 were controls. There were also 5 controls from the United States. The individuals were placed in six groups; kwashiorkor, kwashiorkor who died during treatment, marasmus, marasmus who died, Liberian controls, and U.S. controls. The amount of protein in each spot in the two-dimensional gels was estimated by measuring bound stain using a laser scanner and computerized image analysis. We found very low serum transferrin levels in malnourishment, in agreement with reports from other investigators. All of the data for 24 protein variables were pooled for factor analysis; the mean factor scores for each group differed, with the kwashiorkor groups furthest from the controls. Results of discriminant analysis using the amounts of different numbers of protein variables (3 to 24) were compared for posterior assignment of individuals to groups. The validity of the method was tested by analysis of plasma aliquots obtained from patients following initiation of therapy and which were not a part of the training set. Predictive performance (prognosis of patient survival) depended upon the number of protein variables used. Although artifactual fitting of the data is expected to contribute to performance as the number of variables is increased, use of as many as 7 variables may be justified, even with our small patient groups. Possible use of these results for development of a practical clinical test is discussed.

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.

Neonatal pig and rat aortas were studied for their ability to synthesize elastin in an in vitro situation. Smooth muscle cells from the rat aorta produced excellent multilayered cultures and produced soluble elastin (tropoelastin), insoluble elastin, and small amounts of collagen. BAPN proved to be toxic to these cells, adversely affecting the level of extracellular protein production. Tissue minces from pig aorta continued to synthesize elastin for two hours after removal. However, a 24 hour study indicated that elastin synthesis had almost completely shut down and that collagen synthesis continued in an apparently normal fashion. It is concluded that in vitro elastin synthesis is an extremely sensitive process easily altered by culture conditions and the addition of extraneous substances such as BAPN, and also highly influenced by the past history of the smooth muscle cells involved.