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Biomedical subjects

M Costell

Publications and source records attributed to M Costell.

At least 19 recordsLinked to original sources
PubMed2003

Development of the coronary arteries in a murine model of transposition of great arteries.

Transposition of great arteries in humans is associated with a wide spectrum of coronary artery patterns. However, no information is available about how this pattern diversity develops. We have studied the development of the coronary arteries in mouse embryos with a targeted mutation of perlecan, a mutation that leads to ventriculo-arterial discordance and complete transposition in about 70% of the embryos. The perlecan-deficient embryos bearing complete transposition showed a coronary artery pattern consisting of right and left coronary arteries arising from the morphologically dorsal and ventral sinuses of Valsalva, respectively. The left coronary artery gives rise to a large septal artery and runs along the ventral margin of the pulmonary root. In the earliest embryos where transposition could be confirmed (12.5 d post coitum), a dense subepicardial vascular plexus is located in this ventral margin. In wild-type mice, however, capillaries are very scarce on the ventral surface of the pulmonary root and the left coronary artery runs dorsally to this root. We suggest that the establishment of the diverse coronary artery patterns is determined by the anatomical arrangement and the capillary density of the peritruncal vascular plexus, a plexus that spreads from the atrio-ventricular groove and grows around the aortic or pulmonary roots depending on the degree of the short-axis aortopulmonary rotation. This simple model, based on very few assumptions, might explain all the observed variation of the coronary artery patterns in humans with transposition, as well as our observations on the perlecan-deficient and the normal mice.

Animals↗
PubMed2001

Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization.

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.

Animals↗
PubMed1999

Perlecan maintains the integrity of cartilage and some basement membranes.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Animals↗
PubMed1998

Inhibition of glycosaminoglycan modification of perlecan domain I by site-directed mutagenesis changes protease sensitivity and laminin-1 binding activity.

Glycosaminoglycan attachment to perlecan domain I (173 residues) was completely prevented by site-directed mutagenesis of Ser-65, Ser-71 and Ser-76 as shown by recombinant production in mammalian cells. This did not interfere with the proper folding of the domain's SEA module but enhanced its sensitivity to neutral proteases. Lack of substitution also abolished binding to the two major heparin binding sites of laminin-1.

Amino Acid Sequence↗
PubMed1997

Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides.

Recombinant mouse perlecan domain 1(173 residues) was produced in transfected embryonic kidney cells and purified from the culture medium on DEAE-cellulose. It was shown to be modified by glycosaminoglycans and could be partially separated into two protein pools which were either substituted with heparan sulfate (fragment IA) or, to a smaller extent (20%), with chondroitin/dermatan sulfate or a mixture of both glycosaminoglycans (fragment IB). The average molecular mass of the glycosaminoglycans was about 8-10 kDa and, thus, smaller than in tissue-derived perlecans. Sequence and carbohydrate analyses localized the heparan sulfate attachment site to three Ser residues within SGD consensus sequences. Furthermore, the N-terminal part of fragment IA contained six Thr/Ser residues substituted by branched galactosamine-containing oligosaccharides and an N-substituted Asn residue. Fragment I was also shown to contain unique immunological epitopes which are not dependent on glycosaminoglycans and are shared by tissue-derived perlecan. Circular dichroism demonstrated a distinct alpha helix (20%) and beta structure (60%) in fragment IA, consistent with predictions of a novel SEA protein module located in the C-terminal part of domain I.

Amino Acid Sequence↗
PubMed1996

Structural characterization of recombinant domain II of the basement membrane proteoglycan perlecan.

Mouse perlecan domain II (325 residues), consisting of four cysteine-rich LA modules, one IG module and a link region, was obtained in purified form from a stably transfected mammalian cell clone. Rotary shadowing electron microscopy demonstrated a globular domain connected to a short rod-like segment of variable length. This suggested that tandem arrays of LA modules form rod-like elements. Folding into a native structure was indicated by the sharing of immunological epitopes with tissue perlecan, a CD spectrum demonstrating 37% beta structure and a limited susceptibility to proteolysis. The domain also showed N-glycosylation of a single acceptor site and 7-8 O-linked oligosaccharides. The latter were located mainly in the link region within proline-rich sequences.

Amino Acid Sequence↗
PubMed1996

Structural and cell-adhesive properties of three recombinant fragments derived from perlecan domain III.

Domain III of the basement membrane proteoglycan perlecan was produced as three overlapping fragments in stably transfected mammalian cell clones. These recombinant fragments (43-48 kDa) were obtained in purified form and showed complete immunological cross-reactivity with perlecan, indicating their native structure. Rotary shadowing electron microscopy of each fragment demonstrated a small globular structure connected to a short rod. These data were interpreted to indicate that domain III has an elongated shape of 30 nm in length and consists of alternating globular domains (L4 modules) and short connecting segments attributed to tandem arrays of LE (laminin-type of EGF-like) modules which form rod-like segments in laminins. Sequence analyses of pepsin fragments were consistent with the disulfide-bonding patterns known for these modules from studies with laminin fragments, but two additional disulfide loops were also identified. Several cell lines which attached to mouse perlecan and/or human fibronectin failed to adhere to the domain III fragments, despite the fact that one of them contained an RGD (Arg-Gly-Asp) site in the L4 module. Furthermore, no significant binding was observed in solid phase binding assays with alpha 5 beta 1 and alpha v beta 3 integrins underscoring the low activity or accessibility of the RGD site.

Amino Acid Sequence↗
PubMed1993

Effect of carnitine feeding on the levels of heart and skeletal muscle carnitine of elderly mice.

Aging has been associated with an increase in muscle dysfunction and weakness. We found a decrease in muscle carnitine with age [Biochem. Biophys. Res. Commun., 161 (1989) 1135-1143]. Prolonged oral administration to both young (2-month-old) and adult (7-month-old) mice with L-carnitine increased its content in blood by 50%. The levels of carnitine in skeletal and heart muscle of old treated animals became higher than in untreated mice of the same age. However, this extensive restoration did not reach the maximum values present in skeletal muscle of young mice. Our findings indicate that an alteration of the carnitine carrier in the sarcolemma could be responsible for the decrease with age of carnitine in skeletal but not in heart muscle.

Administration, Oral↗
PubMed1990

A smaller initial dose protects mice against several lethal doses of ammonium acetate.

The synthesis of urea in the liver is the main mechanism for the elimination of excess ammonia. Rapid stimulation of the synthesis of urea (e.g. by administration of carbamyl glutamate, the analog of the physiological activator of carbamyl phosphate synthetase I) protects animals given lethal doses of ammonia. Since ammonia enhances the activity of the urea cycle, we tested and show here that administration of small doses of ammonium acetate supresses the mortality induced by a series of repeated LD100 of ammonium acetate separated by one hour, when the first LD100 is injected i.p. starting from 30 min to 5 hours after the initial smaller dose of ammonium acetate. Under these conditions, the levels of ammonia in blood are elevated more than ten times, but in spite of the greater amount of ammonia administered, the ammonemia is much lower than in mice dying after a single LD100. The enhanced synthesis of urea observed is correlated with an increase in the intramitochondrial content of N-acetyl glutamate. These findings are of interest as far as the short-term regulation of urea cycle, the mechanism of ammonia toxicity and have clinical implications.

Acetates↗
PubMed1990

New roles of carnitine metabolism in ammonia cytotoxicity.

High levels of ammonia in blood and brain due to metabolic disorders are associated with neurological abnormalities. Although the mechanism of ammonia toxicity at the CNS level is still unknown, alterations in brain energy metabolism, in neurotransmitter function and direct effects on nervous impulse have been proposed. In most hyperammonemic conditions morphological changes in the liver and brain have been demonstrated, especially in mitochondria, endoplasmic reticulum and lysosomes, together with an accumulation of intracellular lipids. The treatment of hyperammonemias is uncertain and mostly directed to reduce the level of circulating ammonia; there is no current therapy aimed to counteract the molecular effects of ammonia. Administration of carnitine prevents acute ammonia toxicity and enhances the efficacy of ammonia elimination as urea and glutamine. In addition the cytotoxic effects of ammonia, possibly arising from lipid peroxidation, are ameliorated by carnitine. These data indicate the feasibility of utilization of carnitine in the therapy of human hyperammonemic syndromes, both for reducing the levels of ammonia and preventing its toxic effects.

Ammonia↗
PubMed1989

Age-dependent decrease of carnitine content in muscle of mice and humans.

Carnitine deficiency associated with impairment of muscle function has been described in infants and young adults as a result of inherited or acquired disease but there are no reports on carnitine levels in healthy aging people. In this paper we show a marked reduction of carnitine and its derivatives in muscle, and of long-chain acyl carnitine in heart of 6-month-old mice, when compared to animals of 6 weeks of age. Analysis of muscle samples of healthy humans of different ages showed a drastic reduction of carnitine and acetyl carnitine in the older subjects with a strong reverse correlation between age and carnitine levels.

Acetylcarnitine↗
PubMed1989

Carbamyl glutamate prevents the potentiation of ammonia toxicity by sodium benzoate.

Sodium benzoate has been recommended for the treatment of hyperammonaemia in humans. However, benzoate potentiates ammonia toxicity and reduces urea synthesis in vitro and in vivo by decreasing the intramitochondrial levels of N-acetyl glutamate. Pretreatment of mice with carbamyl glutamate, a structural analogue of N-acetyl glutamate, decreases mortality induced by ammonium acetate and sodium benzoate administration. The protective effect of carbamyl glutamate is accompanied by an increase in urea production and of carbamyl phosphate synthetase activity.

Ammonia↗
PubMed1987

Effect of L-carnitine on ketone bodies, redox state and free amino acids in the liver of hyperammonemic mice.

L-Carnitine stimulates urea synthesis in mice given a LD100 of ammonium acetate. Unprotected mice show decreased levels of hepatic ketone bodies and lowered NADH/NAD+ ratio in both cytosol and mitochondria. L-Carnitine enhances markedly the production of beta-hydroxybutyrate and raises the NADH/NAD+ ratio in mitochondria. The alterations induced by ammonium acetate in the free amino acid pool are prevented by L-carnitine. The results shown in this paper indicate that L-carnitine stimulates fatty acid oxidation as well as flux through the Krebs cycle in hyperammonemic mice and that these effects may be responsible for the increase in urea synthesis in these animals.

Acetates↗
PubMed1987

The potentiation of ammonia toxicity by sodium benzoate is prevented by L-carnitine.

Sodium benzoate has been recommended and even been used for the treatment of hyperammonemia in humans. More recently, a note of caution was raised since it has been shown that in experimental animals, sodium benzoate potentiates ammonia toxicity and inhibits urea synthesis in vitro. This has been further confirmed in the work presented here and the mechanism by which benzoate increases mortality and the levels of blood ammonia in mice given ammonium acetate have also been studied. In hyperammonemia, urea production and N-acetylglutamate levels were decreased by sodium benzoate. Pretreatment of mice with L-carnitine suppressed mortality following ammonium acetate plus sodium benzoate administration. Under these conditions L-carnitine lowered blood ammonia and increased urea production and N-acetylglutamate levels.

Ammonia↗
PubMed1987

Effect of hyperammonemia on the levels of carnitine in mice.

Decreased carnitine levels have been noted in conditions of hyperammonemia. We have measured carnitine and its derivatives in acute and sustained hyperammonemia in mice and studied the effect of carnitine administration thereon. Sustained hyperammonemia decreased carnitine in liver and muscle. Acetylcarnitine was decreased in liver and muscle in both acute and sustained hyperammonemia but increased in brain. Long-chain acylcarnitines decreased in brain and muscle in acute hyperammonemia and in liver and muscle is sustained ammonia intoxication. Intraperitoneal administration of carnitine increased the levels of free carnitine and acyl derivatives, especially in liver, but sustained hyperammonemia significantly affected the distribution of exogenous carnitine. The importance of these findings relative to the alterations of lipid metabolism observed in Reye's syndrome and inherited hyperammonemias, as well their implication in the protective effect of carnitine on hyperammonemia, are discussed.

Ammonia↗
PubMed1985

Decarbamoylating activity of ornithine transcarbamoylase.

We have purified from beef liver an enzyme which decarbamoylates carbamoyl-hemoglobin and to a much lesser extent carbamoyl histones. Carbamoyl casein was a poor substrate while carbamoyl trypsin, fibrinogen and ovoalbumin were not affected. The optimal pH is 7.4. Addition of Mg++, Mn++ or Ca++ was without effect. On testing citrulline as a substrate we found high activity leading us to suspect that the activity of the decarbamoylase preparation was due to contaminating ornithine transcarbamoylase activity. Evidence for this is the similar ratio of transcarbamoylase to decarbamoylase activities of both ornithine transcarbamoylase and of the purified preparation of decarbamoylase from beef liver. Also, delta-PALO, the specific inhibitor of ornithine transcarbamoylase inhibited both preparations to the same extent. Interestingly, ornithine transcarbamoylase from bacteria also has decarbamoylase activity while aspartic transcarbamoylase does not.

Amino Acids↗
PubMed1984

Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice.

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.

Ammonia↗