Henri Bricaud

Phosphorolysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and other 5-substituted-2'-deoxyuridines by purified human thymidine phosphorylase and intact blood platelets.

Various 5-substituted-2-deoxyuridines (dUrd), including 5-ethyl,5-propyl-, 5-trifluoromethyl-, 5-hydroxymethyl-, 5-formyl-, 5-vinyl-, (E)-5-(2-chlorovinyl)-, (E)-5-(2-bromovinyl)-, 5-fluoro-, 5-chloro-, 5-bromo-, 5-iodo-, 5-cyano-, 5-thiocyano-, 5-nitro- and 5-amino-dUrd, were shown to be effective substrates for the thymidine (dThd) phosphorylase isolated from human blood platelets. Some of dUrd analogs, i.e. the highly potent and selective antiherpes agent (E)-5-(2-bromovinyl)-dUrd, were degraded more rapidly than the natural substrates, dUrd and dThd. All dUrd analogs were also readily catabolised by intact human blood platelets. The potent inhibitors of thymidine phosphorylase, 6-amino-thymine and 6-amino-5-bromo-uracil, strongly inhibited the phosphorolysis of (E)-5-(2-bromovinyl)-dUrd by both purified enzyme and intact platelets.

Catabolism of thymidine in human blood platelets purification and properties of thymidine phosphorylase

A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110,000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.

Formation of monohydroxyeicosatetraenoic acids from arachidonic acid by cultured rabbit aortic smooth muscle cells

In addition to the well established cyclooxygenase pathway, cultured aortic smooth muscle cells convert arachidonic acid to several polar metabolites identified by high performance liquid chromatography and gaz chromatography-mass spectrometry. 15-Hydroxyeicosatetraenoic acid, 12-Hydroxyeicosatetraenoic acid and 5-Hydroxyeicosatetraenoic acid are the major products formed. These observations indicate that the rabbit aortic smooth muscle cells are a potential source of lipoxygenase products and raise the possibility that this pathway of arachidonic acid metabolism can influence the biological functions of arterial myocytes under normal and pathological conditions.

Decreased prostaglandin production in cultured smooth muscle cells from atherosclerotic rabbit aorta

Prostaglandin synthesis in aortic smooth muscle cells originating from healthy an atherosclerotic rabbits was studied by incubating [14C]arachidonic acid with intact confluent cells and cell homogenates. In spite of a reduced 6-keto prostaglandin F1 alpha formation, no potentiating effect on the prostaglandin E2 generation occurred. Indeed, both cyclooxygenase and prostaglandin I2 synthetase activities appear to be reduced. These results suggest that an impaired arachidonic acid utilisation in aortic smooth muscle cells may be involved in the course of the atherosclerotic process.

Biochemical and immunological studies of fibroblasts derived from a patient with Ehlers-Danlos Syndrome Type IV

SummaryFibroblasts derived from a skin biopsy of a patient with the Ehlers-Danlos syndrome (EDS) type IV were cultured in monolayer. The amount of collagen synthesized during a 24-h pulse was not different from that found with normal fibroblasts. Chromatographic procedures and immunofluorescence staining showed a normal synthesis of type I procollagen and collagen but a deficiency in synthesis of type III procollagen and collagen. This could be corroborated by radioimmuno assays showing a reduction in type III procollagen by about 90%. The secretion and degradation of collagens was not altered. The results demonstrate that the molecular defect in this particular patient is due to an impairment of the mechanism controlling the gene expression for type III procollagen.ZusammenfassungVon einem Patienten mit den klinischen Symptomen eines Ehlers-Danlos-Syndroms Typ IV wurden Fibroblastenkulturen gezüchtet. Diese synthetisierten dieselbe Menge an Kollagen wie Kontroll-Fibroblasten und zeigten auch ein identisches Sekretions-und Degradations-Verhalten. Biochemische und immunochemische Methoden wiesen jedoch eine signifikante Reduktion des Anteils von Typ III-Kollagen auf etwa 10% des Normalwertes auf.

The human blood platelet: A cellular model to study the degradation of thymidine and its inhibition

Intact platelets catabolize extracellular thymidine into thymine. Studies of the concentration dependent degradation of thymidine by intact platelets indicate a Michaelis mechanism with an apparent Km of about 0.12 mM and a Vmax of 2.5 nmoles/min for 3 X 10(8) platelets. This degradation process is inhibited by various nucleosides, pyrimidine bases and C-5 or C-6 substituted uracils. Cytidine, deoxycytidine, adenosine and deoxyadenosine seem to inhibit thymidine degradation by reducing the intracellular transport of thymidine. Uridine inhibits both the thymidine transport and the activity of the phosphorolytic enzyme, thymidine phosphorylase (EC 2.4.2.4). Some substituted uracils are specific inhibitors of thymidine phosphorylase activity. 6-Amino-5-bromouracil, the most active of them, either with acellular extracts or purified thymidine phosphorylase, is also the best inhibitor of thymidine degradation in intact human platelets. Platelets constitute a new model to study the efficiency of specific inhibitors on thymidine catabolism in an human intact cell which contains only one pyrimidine nucleoside phosphorylase, the thymidine phosphorylase.

Prostacyclin synthesis by proliferative aortic smooth muscle cells A kinetic in vivo and in vitro study

The capacity of arterial SMCs to produce PGI2 when stimulated by exogenous AA was studied in proliferative and confluent cultured cells and at different periods following endothelial denudation in vivo. PGI2 production per cell was doubled during the exponential growth-phase in culture. By contrast, increased PGI2 formation did not correlate with mitotic activity in intimal regeneration tissue but with the presence of SMCs in a synthetic phenotype. The present results suggest a potential role for PGI2 in SMC differentiation and proliferation.

Collagen synthesis in organ culture of normal and atherosclerotic aortas

Using pulse-label experiments in organ culture, collagen synthesis was studied in aortas from healthy and atherosclerotic specimens. Investigations were carried out on human atherosclerotic plaques as well as in the mini-pig in which atherosclerosis occurred spontaneously, and in the rabbit where atherosclerosis was experimentally induced by cholesterol-enriched feeding. When compared to total protein synthesis, the percentage of newly synthesized collagens measured as radioactive pepsin-resistant material, decreased with age in healthy specimens, whereas it remained at a higher level when the aortas were atherosclerotic. Subsequent molecular sieve chromatography of the radioactivity pepsin-resistant material allowed the separation type I collagen from type III collagen and their relative quantification. The results showed that the newly synthesized type III collagen accounted for 16-31% in aortic explants from young animals, for 30-36% when the explants were derived from older specimens and for 35-48% when the tissues were atherosclerotic.

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells.

Abstract Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.

Fibrous long spacing collagen in aortic explants of normal rabbit cultured in hypercholesterolemic serum

Aortic tissues consisting of all three tunics were removed from normal adult rabbits and cultured in a semisynthetic gelosed medium supplemented by 10% serum obtained either from normal or hypercholesterolemic rabbits. Fibrillar cross-striated aggregates appeared with a high frequency (50%) in the extracellular space of explants cultured from four to eight days in medium supplemented by serum from hypercholesterolemic rabbits, but did not appear in explants cultured in serum from control animals (3%). The electron-dense segment was ruthenium red positive and digested by testicular hyaluronidase. The electron-lucent segment, composed of ruthenium red negative thin filaments, was not modified after hyaluronidase treatment but was strongly digested after collagenase treatment. It is believed that this material was fibrous long spacing collagen synthetized under culture conditions, as shown after tritiated proline incorporation.