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PubMed1986

Reactivity of a functional carbonyl moiety in bovine aortic lysyl oxidase. Evidence against pyridoxal 5'-phosphate.

Previous studies have pointed towards a cofactor role for pyridoxal 5'-phosphate (PLP) in lysyl oxidase, the enzyme that generates the peptidyl aldehyde precursor to the lysine-derived cross-linkages in elastin and collagen. The nature of a carbonyl moiety in purified bovine aortic lysyl oxidase was explored in the present study. A PLP dinitrophenylhydrazone could not be isolated from lysyl oxidase, although corresponding preparations of aspartate aminotransferase, a PLP-dependent enzyme, yielded this derivative, as revealed by h.p.l.c. Analysis of lysyl oxidase for PLP after reduction of the enzyme by NaBH4, a procedure that converts PLP-protein aldimines into stable 5'-phosphopyridoxyl functions, also proved negative in tests using monoclonal antibody specific for this epitope. Lysyl oxidase was competitively inhibited by phenylhydrazine, and inhibition became irreversible with time at 37 degrees C, displaying a first-order inactivation rate constant of 0.4 min-1 and KI of 1 microM. [14C]Phenylhydrazine was covalently incorporated into the enzyme in a manner that was prevented by prior modification of the enzyme with beta-aminopropionitrile, a specific active-site inhibitor, and which correlated with functional active-site content. The chemical stability of the enzyme-bound phenylhydrazine exceeded that expected of linkages between PLP and proteins. The absorption spectrum of the phenylhydrazine derivative of lysyl oxidase was clearly distinct from that of the phenylhydrazone of PLP. It is concluded that lysyl oxidase contains a carbonyl cofactor that is not identical with PLP and that is bound to the enzyme by a stable chemical bond.

Amino Acid Oxidoreductases↗

PubMed2002

Integrative articular cartilage repair: dependence on developmental stage and collagen metabolism.

OBJECTIVES: The objectives of this research were to determine whether the integrative repair of bovine cartilage explants was dependent on developmental stage, and whether observed differences in integration with developmental stage were related to deposition of newly synthesized collagen and lysyl oxidase-mediated collagen cross-linking. METHODS: Pairs of fetal, newborn calf, and adult bovine cartilage blocks were cultured in partial apposition for 2 weeks in medium supplemented with serum, ascorbate, and [3H]proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength in a single-lap shear configuration. Formation and stabilization of newly synthesized protein and collagen was investigated by determination of [3H]proline and [3H]hydroxyproline in tissue digests and guanidine extracts. RESULTS: Calf cartilage exhibited a relatively high integrative repair phenotype, achieving an adhesive strength that was three--four-fold that of adult or fetal specimens. The low and high integrative repair phenotypes appeared related in part to different levels of collagen biosynthesis, which was approximately four--five-fold higher in calf cartilage samples than in the adult. However, fetal cartilage also exhibited a high level of biosynthesis. The different integrative repair phenotypes were not associated with marked differences in the kinetics of chemical stabilization of newly synthesized collagen, as the proportion of incorporated [3H]proline and newly-formed [3H]hydroxyproline that was resistant to extraction by 4M guanidine-HCl following culture was similar for cartilage from all developmental stages. Integration of calf cartilage appeared to depend on lysyl oxidase-mediated collagen cross-link formation, since inclusion of beta-aminopropionitrile (BAPN) in the culture medium completely eliminated development of adhesive strength. BAPN treatment also increased the percentage of newly synthesized protein in the guanidine extracts from 10% to 36% of the total, and that of newly synthesized collagen from 2% to 20%, while having only slight inhibitory effects on overall protein and collagen biosynthesis. CONCLUSION: The finding that cartilage exhibits enhanced integrative repair at a certain developmental stage suggests that it may ultimately be possible to enhance repair when needed in clinical situations.

Animals↗

PubMed1997

Irreversible inhibition of lysyl oxidase by homocysteine thiolactone and its selenium and oxygen analogues. Implications for homocystinuria.

Homocysteine thiolactone, selenohomocysteine lactone, and homoserine lactone were found to be competitive, irreversible inhibitors of lysyl oxidase, with KI values of 21 +/- 3 microM, 8.3 +/- 2.2 microM, and 420 +/- 56 microM, respectively. The first order rate constants for inactivation (k2) of the enzyme varied over a much smaller range, ranging from 0.12 to 0.18 to 0.28 min-1 for the Se-, thio-, and O-lactones, respectively. Mutually exclusive labeling of the enzyme by [1-14C]beta-aminopropionitrile, [U-14C]phenylhydrazine, or [35S]homocysteine thiolactone was observed. These labeling results, together with the closely similar perturbations of the near UV-visible spectra of lysyl oxidase and of a model of its lysine tyrosylquinone cofactor by the thiolactone, indicate that the lactones likely derivatize and reduce the active site carbonyl cofactor. Substitution with deuterium at the alpha-carbon of the thiolactone caused a deuterium kinetic isotope effect on k2 of 3.2 +/- 0.2, consistent with the involvement of rate-limiting alpha-proton abstraction during lactone-induced inactivation of the enzyme. The activities of plasma amine oxidase and diamine oxidase were only minimally reduced at concentrations of the sulfur or selenium lactones that fully inhibited lysyl oxidase. Thus, these lactones constitute a new category of mechanism-based inactivators selective for lysyl oxidase. Further, these results may relate to the development of connective tissue defects seen in homocystinuria.

Animals↗

PubMed2001

Molecular cloning and biological activity of a novel lysyl oxidase-related gene expressed in cartilage.

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by beta-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.

Amino Acid Oxidoreductases↗

PubMed2006

A tissue-specific variant of the human lysyl oxidase-like protein 3 (LOXL3) functions as an amine oxidase with substrate specificity.

The human lysyl oxidase-like 3 (LOXL3) encodes a member of the emerging family of lysyl oxidase (LOX) that functions as a copper-dependent amine oxidase. The LOXL3 protein contains four scavenger receptor cysteine-rich domains in the N terminus in addition to the C-terminal characteristic domains of the LOX family, such as a copper binding domain, a cytokine receptor-like domain and residues for the lysyl-tyrosyl quinone cofactor. Using BLASTN searches, we identified a LOXL3 variant LOXL3-sv1 that lacked the sequences corresponding to exons 1, 2, 3, and 5 of LOXL3. LOXL3-sv1 showed an exon-intron structure distinct from LOXL3, additionally containing an 80-bp sequence corresponding to intron 3 of LOXL3 in the 5'-UTR and a 561-bp sequence corresponding to the 3'-flanking genomic region of exon 14 in the 3'-UTR. LOXL3-sv1 was predicted to encode a polypeptide of 392 amino acids that contains the C-terminal domains required for amine oxidase activity but lacks the N-terminal SRCR domains 1, 2, and 3. The recombinant LOXL3-sv1 protein showed a beta-aminopropionitrile-inhibitable amine oxidase activity toward elastin and collagen with substrate specificity. In RT-PCR assays with various human tissues, LOXL3-sv1 and LOXL3 showed distinct expression patterns. Further, luciferase reporter assays revealed a strong promoter element in intron 3 that probably functions as a regulatory region for the expression of LOXL3-sv1. These findings strongly indicate that LOXL3 encodes two variants, LOXL3 and LOXL3-sv1, both of which function as amine oxidases with distinct tissue and substrate specificities from one another.

Amino Acid Oxidoreductases↗

PubMed1999

Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.

Amine Oxidase (Copper-Containing)↗

PubMed1992

Cigarette smoke exposure does not prevent cadmium-induced alterations in rat lungs.

Cigarette smoke has been demonstrated to suppress the biosynthesis of connective tissue in the lung. To further characterize this suppressant effect, we studied the ability of cigarette smoke to prevent or ameliorate cadmium-induced alterations in rat lungs in vivo. The effects of beta-aminopropionitrile (beta APN), an agent that inhibits the cross-linking of elastin, also were studied. Eighty-eight young female Long-Evans rats were randomly divided into seven groups as follows: control, cigarette smoke, sham smoke, beta APN, cadmium, cadmium + cigarette smoke, and cadmium + beta APN. Each animal in the cigarette smoke group was exposed to mainstream smoke generated from University of Kentucky 2R1 reference cigarettes (10 puffs daily for 12 wk). Sham-treated animals received room air in place of cigarette smoke. beta APN (0.5 g/kg) was injected intraperitoneally twice weekly. In cadmium-treated groups, each rat received intermittently three intratracheal instillations of cadmium chloride (0.15 mumol/kg) over a 5-d period. For the cadmium + cigarette smoke group, smoke exposure began 3 d after the first cadmium instillation and was continued for 12 wk. The beta APN administration began 5 d before cadmium instillation and also was continued for 12 wk. After these treatments, pulmonary function and lung morphometry were examined. Neither cigarette smoke, sham smoke, nor beta APN produced significant changes in lung function or morphometry. Cadmium caused significant decreases in total lung capacity, dynamic and static compliance, and carbon monoxide diffusing capacity, as well as significant increases in lung weight and alveolar wall thickness. In addition, the quasistatic deflation pressure-volume curve showed a rightward shift whereas the mean linear intercept of the alveoli did not change significantly. Efforts to prevent or ameliorate the changes through exposure to cigarette smoke or administration of beta APN were unsuccessful. It is concluded that interventions designed to inhibit the biosynthesis of lung connective tissue do not perforce inhibit the development of cadmium-induced pulmonary changes in the rat.

Animals↗

PubMed1987

Lysyl oxidase activity and elastin/glycosaminoglycan interactions in growing chick and rat aortas.

Hydrophobic tropoelastin molecules aggregate in vitro in physiological conditions and form fibers very similar to natural ones (Bressan, G. M., I. Pasquali Ronchetti, C. Fornieri, F. Mattioli, I. Castellani, and D. Volpin, 1986, J. Ultrastruct. Molec. Struct. Res., 94:209-216). Similar hydrophobic interactions might be operative in in vivo fibrogenesis. Data are presented suggesting that matrix glycosaminoglycans (GAGs) prevent spontaneous tropoelastin aggregation in vivo, at least up to the deamination of lysine residues on tropoelastin by matrix lysyl oxidase. Lysyl oxidase inhibitors beta-aminopropionitrile, aminoacetonitrile, semicarbazide, and isonicotinic acid hydrazide were given to newborn chicks, to chick embryos, and to newborn rats, and the ultrastructural alterations of the aortic elastic fibers were analyzed and compared with the extent of the enzyme inhibition. When inhibition was greater than 65% all chemicals induced alterations of elastic fibers in the form of lateral aggregates of elastin, which were always permeated by cytochemically and immunologically recognizable GAGs. The number and size of the abnormal elastin/GAGs aggregates were proportional to the extent of lysyl oxidase inhibition. The phenomenon was independent of the animal species. All data suggest that, upon inhibition of lysyl oxidase, matrix GAGs remain among elastin molecules during fibrogenesis by binding to positively charged amino groups on elastin. Newly synthesized and secreted tropoelastin has the highest number of free epsilon amino groups, and, therefore, the highest capability of binding to GAGs. These polyanions, by virtue of their great hydration and dispersing power, could prevent random spontaneous aggregation of hydrophobic tropoelastin in the extracellular space.

Amino Acid Oxidoreductases↗

PubMed1997

An extracellular matrix response element in the promoter of the LpS1 genes of the sea urchin Lytechinus pictus.

The extracellular matrix (ECM) has been shown to play an important role in development and tissue-specific gene expression, yet the mechanism by which genes receive signals from the ECM is poorly understood. The aboral ectoderm-specific LpS1-alpha and -beta genes of Lytechinus pictus , members of the Spec gene family, provide an excellent model system to study ECM- mediated gene regulation. Disruption of the ECM by preventing collagen deposition using the lathrytic agent beta-aminopropionitrile (BAPN) inhibits LpS1 gene transcription. LpS1 transcription resumes after removal of BAPN and subsequent collagen reformation. Using a chloramphenicol acetyltransferase (CAT) reporter gene assay, we show that a 125 bp region of the LpS1-beta promoter from -108 to +17 contains an ECM response element (ECM RE). Insertion of the 125 bp region into the promoter of the metallothionein gene of L. pictus, a gene unaffected by ECM disruption, caused the fused promoter to become ECM dependent. As with the endogenous LpS1 genes, CAT activity directed by the fused LpS1-beta promoter resumed in embryos recovered from ECM disruption. A mutation in a cis -acting element called the proximal G-string, which lies in the 125 bp region, caused CAT activity levels in ECM-disrupted embryos to equal that of the wild-type LpS1-bet apromoter in ECM-intact embryos. These results suggest that the intact ECM normally transmits signals to inhibit repressor activity at the proximal G-string in aboral ectoderm cells. Consistent with these results were our findings which showed that in addition to expression in the aboral ectoderm, the proximal G-string mutation caused expression of the CAT gene in oral ectoderm cells. These studies suggested that the proximal G-string serves as a binding site for negative regulation of the LpS1 genes in oral ectoderm during development. We also examined trans -acting factors binding the proximal G-string following ECM disruption. Band shift gels revealed a predominant set of slower migrating nuclear proteins from ECM-disrupted embryos which bound the proximal G-string. This work suggested that ECM disruption initiates signaling that induces a repressor to bind the ECM RE and/or modifies ECM RE binding proteins, which in turn represses LpS1 gene activity.

Animals↗

PubMed1991

Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction.

Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data provide evidence that collagen telopeptide sites play a role in collagen gel lattice contraction.

Collagen↗

PubMed1980

Procollagen I synthesis in human skin fibroblasts: effect on culture conditions on biosynthesis.

Human skin fibroblast culture promises to be a useful system for the investigation of the regulation of collagen biosynthesis and the study of abnormalities in collagen biosynthesis in connective tissue disorders. The effect of culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen biosynthesis were: 10% dialyzed heat-inactivated fetal calf serum, 0.15 mM ascorbate, and 0.078 mM beta-aminopropionitrile. Newly synthesized procollagen I accumulated in the medium at a linear rate for 18 hr. Preincubation of cells in labelling media for 4 hr before adding radioactive proline enhanced synthesis. Collagenase digestion was used to study overall collagen biosynthesis. 94% of all collagen synthesized was found in the medium, and 6% in the cell pellet. Under optimal conditions, collagen comprised 24% of all protein in the medium, and 14% of protein produced by the whole culture.

Adolescent↗

PubMed2006

Investigation of keratinocyte regulation of collagen I synthesis by dermal fibroblasts in a simple in vitro model.

BACKGROUND: Hypertrophic scarring and skin graft contracture are major causes of morbidity after burn injuries. A prominent feature is excessive fibroplasia with accumulation of increased fibrillar collagen relative to normal scar tissue. The application of split-thickness skin grafts or cultured epithelial autografts to burn wounds is known to reduce scarring and contraction. OBJECTIVES: To investigate further how the keratinocyte influences underlying fibroblast behaviour by examining the influence of keratinocytes on fibroblast collagen synthesis, using a new assay for collagen synthesis never previously applied to skin cell biology. METHODS: We investigated the influence of the keratinocyte on fibroblast synthesis of type I collagen using an immunoassay for the aminoterminal propeptide of type I collagen (P1NP) in conditioned medium from monocultures and cocultures of keratinocytes and fibroblasts over 14 days. The importance of the physical presence of the keratinocyte was investigated by comparing cocultures of keratinocytes and fibroblasts against fibroblast monocultures with keratinocyte-conditioned medium. Pharmacological agents known to promote fibroblast proliferation [basic fibroblast growth factor (bFGF)], keratinocyte proliferation [insulin-like growth factor (IGF)-1], modify scarring in vivo[tumour necrosis factor (TNF)-alpha] or modify collagen biochemistry [putrescine, estrone, estradiol and beta-aminopropionitrile (beta-APN)] were then investigated for their effect on collagen synthesis in fibroblasts and in keratinocyte/fibroblast cocultures. RESULTS: Keratinocytes in coculture with fibroblasts, and keratinocyte-conditioned medium, both reduced fibroblast P1NP synthesis. Of the pharmacological agents investigated, bFGF, IGF-1, TNF-alpha and beta-APN all increased collagen synthesis both in monocultures of fibroblasts and in cocultures of keratinocytes and fibroblasts. CONCLUSIONS: Fibroblast collagen synthesis appears to be downregulated by keratinocyte-derived cytokines. Fibroblast growth factors and proinflammatory cytokines appear to be able partially to overcome this downregulation and to increase collagen synthesis.

Cell Communication↗

PubMedDate not supplied

An animal model of cerebral aneurysms.

Cerebral aneurysms were induced experimentally in rats by ligating the common carotid artery on one side, making the animals hypertensive, and feeding them the lathyrogen beta-aminopropionitrile. On analysis of this experimental model, haemodynamic stress, hypertension and metabolic disorder of the connective tissue proved to be of primary importance as aetiological factors in the development of cerebral aneurysms. The experimentally induced cerebral aneurysms resemble those in man both in location and in microscopic structure; they can thus be regarded as a good model for the study of the pathogenesis of cerebral aneurysms. The initial changes leading to the formation of aneurysms occurred on the distal side of major artery branches adjacent to the apex, but distal to the intimal pad. It is proposed that degenerative changes in the internal elastic lamina and muscle cells of the media, caused by haemodynamic stress at vessel branches and intimal pads, are the basis for aneurysm formation.

Animals↗

PubMed2005

Members of the lysyl oxidase family are expressed during the development of the frog Xenopus laevis.

Lysyl oxidase (Lox) is a copper-dependent amine oxidase that catalyzes the cross-linking of collagen and elastin fibers in the extracellular matrix (ECM). In mammals, four closely related Lox-like enzymes have been described that share a highly conserved catalytic domain with Lox. We have characterized Xenopus laevis cDNAs for Lox, Loxl-1, and Loxl-3, and show that they are expressed during early embryonic development. Using RT-PCR we detected maternal transcripts for Xloxl-1, but levels remained low until tailbud stages. Transcripts for Xlox and Xloxl-3 were not detected until early neurulae, although transcripts for Xlox remained at low levels until tailbud stages. Whole mount in situ hybridization showed that transcripts for Xloxl-1 and Xloxl-3 are localized in the notochord, while transcripts for Xlox are found in the notochord, somites, and head. X. laevis Lox-like enzymes were inhibited by incubating embryos, from cleavage stages to tadpole stages, in beta-aminopropionitrile, a specific inhibitor of the catalytic domain. The resulting embryos appeared to differentiate normally but suffered from poor collagen fiber formation. Defects included kinks in the notochord, a posterior shift of the somites, abnormal gut coiling, and the formation of edemas. Our data suggest that Lox-related enzymes are required for the proper formation of the ECM during X. laevis development.

Amino Acid Sequence↗

PubMed2006

Mechanism for covalent binding of rofecoxib to elastin of rat aorta.

We have previously reported that oral administration of [(14)C]rofecoxib to rats resulted in the long retention of radioactivity by the aorta as a consequence of covalent binding to elastin. Treatment of rats with alpha-phenyl-alpha-propylbenzeneacetic acid 2-[diethylamino]-ethyl ester hydrochloride (SKF-525A), a cytochrome P450 inhibitor, significantly decreased the systemic exposure of 5-hydroxyrofecoxib, one of the main metabolites of rofecoxib, whereas there was no statistically significant change in the retention of radioactivity from [(14)C]rofecoxib in the aorta. On the other hand, the aortic retention of radioactivity closely correlated to the systemic exposure of unchanged rofecoxib in the dose range between 2 and 10 mg/kg. A covalent binding study of [(14)C]rofecoxib in vitro using rat aorta homogenate in the presence of d-penicillamine, hydralazine, beta-aminopropionitrile, and sodium borohydride suggested that the aldehyde group of allysine in elastin was relevant to the covalent binding. In a model reaction using benzaldehyde, rofecoxib but not 5-hydroxyrofecoxib reacted with the aldehyde group of benzaldehyde in a manner of condensation reaction under a physiological pH condition. A histopathological examination using an electron microscope demonstrated that multiple oral administration of rofecoxib to rats caused marked degradation of the elastic fiber system of the aorta. These results suggested that rofecoxib as such is reactive in vivo, undergoing a condensation reaction with allysine, thereby preventing the formation of cross-linkages in elastin, i.e., desmosine and isodesmosine, and causing the degradation of the elastic fibers.

Animals↗

PubMed1980

Localization of lysyl oxidase in hen oviduct: implications in egg shell membrane formation and composition.

Lysyl oxidase activity was found in the isthmus (the membrane-forming region) of the hen's oviduct in a copper-rich region proximal to the shell gland. Desmosine and isodesmosine, cross-linking compounds associated with mature elastin, were found in hydrolysates of the shell membrane, confirming the necessity for lysyl oxidase in its biosynthesis. Shell membranes from hens fed a copper-deficient diet or a diet supplemented with beta-aminopropionitrile had a reduced content of desmosine and isodesmosine.

Amino Acid Oxidoreductases↗

PubMed1983

Measurement of the absolute synthesis of soluble and insoluble elastin by chick aortic tissue.

The rate of in vitro synthesis of soluble and insoluble elastin by thoracic aorta of 2-day-old chicks has been measured in absolute terms. In the absence of beta-aminopropionitrile (beta APN), the steady state level of soluble elastin was 120 pmol/100 mg of aortic tissue or 3.7 micrograms/whole aorta segment. The rate of synthesis of elastin in vitro was approximately 130 micrograms/day per whole aorta segment. This is three- to four-fold lower than the estimated rate of in vivo synthesis for a comparable segment of aortic tissue at the same stage of development. Pulse-chase experiments suggested that this difference was not due to in vitro proteolysis of a significant proportion of the newly synthesized soluble elastin, but rather that the conversion of soluble to insoluble elastin was both rapid and efficient. These experiments also indicated the presence in aortic tissue of a substantial pool of elastin of intermediate solubility. Although inclusion of beta APN in the incubation medium resulted in an increase in the amount of soluble elastin in aortic tissue, the rate of accumulation of newly synthesized soluble elastin in the presence of this inhibitor of cross-linking was not linear, but decreased with incubation time. Furthermore, although beta APN effectively suppressed the appearance of insoluble elastin for at least 2 h, some escape from the effect of this inhibitor was seen with further incubation. In general, beta APN significantly depressed elastin synthesis.

Amino Acids↗

PubMed1983

Static elastic studies of lathyritic rabbit carotid arteries and thoracic aorta.

Sixteen New Zealand White rabbits with an average initial weight of 1.6 kg were used to determine if lathyrism altered the static elastic properties of the aorta and carotid arteries. Eight were given 1% beta-aminopropionitrile in their drinking water for the same period. All were fed regular rabbit chow. Blood pressures and body weights were taken twice weekly. Blood pressure was not significantly different between the two groups but the lathyritic animals did lose weight while the controls gained. The rabbits were sacrificed at the end of 4 weeks with an overdose of sodium pentobarbital (Nembutal) and the carotid arteries and thoracic aorta were removed for pressure--volume experiments. From these experiments tension--strain curves, elasticities, and slack of the collagen were obtained. The longitudinal distensibility curves for the aortas and carotid arteries for both groups were almost identical. The longitudinal elasticities of elastin and collagen, and the slack were not significantly different between the two groups. The circumferential-distensibility curves for the same arteries showed few differences, however, the elasticities of elastin and collagen, and the slack decreased indicating an alteration in the elastin and a decrease in collagen fibres or a defect in the intramolecular cross-links of collagen.

Animals↗