[ANOCOR registry]. / Le registre ANOCOR.

Anomalous aortic origin of the coronary arteries are congenital anomalies with many anatomical forms. Due to the varying risk of sudden death, these abnormalities must be classified accurately. There are still questions about the mechanism and individual risk of sudden death, the natural history of these abnormalities and the benefits of a surgical correction. Large-scale observational registries may provide more evidence-based data to practitioners caring for the patients concerned. The ANOCOR registry, the largest in size published to date, enrolled 472 patients (mean age 63 years) with 496 coronary abnormalities. The angiographic representation (with invasive coronary angiography or coronary CT angiography) according to the coronary artery and initial ectopic course could be specified with the identification of two main phenotypes: the circumflex artery (n = 235) with a retroaortic course in 97% of cases and the right coronary artery (n = 165) with an interarterial course in 89.7% of cases. Two left coronary anatomical forms have been confused by non-expert cardiologists: those with a retropulmonary or interarterial course. Sudden death related to coronary anomaly was a very rare mode of presentation (3 patients or 0.6% of the cohort) in this population with very few young patients < 35 years (11 cases or 2.3% of the cohort).

[Anomalous origin of coronary arteries in adults]. / Anomalies de connexion proximale des artères coronaires chez l'adulte.

In lack of structural congenital heart disease, anomalous origin of a coronary artery is rare with an angiographic prevalence of approximately 1%. The prognosis depending on anatomical features, initial course of coronary ectopic vessel is important to be considered. Although some anomalies are associated with sudden death, the majority are simply incidental anatomic findings. In patients with high-risk coronary anomalies, the treatment is not clearly codified. Large-scale prospective multicenter studies are needed to define recommendations in the future.

[Gene therapy of restenosis and atherosclerosis: hopes and facts]. / Thérapie génique des resténoses et de l'athérosclérose: espoirs et réalités.

Stents are the main technique of coronary revascularization in France and western countries. However, a better understanding of the pathophysiology of in-stent restenosis and the well-recognized roles played by inflammation and cell proliferation led to the development of drug-eluting stents, which have nearly eliminated the risk of restenosis. In this context, the success of gene therapy will depend on our ability to simplify and optimize current protocols of arterial gene transfer. For the time being, arterial gene therapy remains a powerful tool for deciphering the complex pathophysiology of restenosis and will certainly have far-reaching implications in the fields of vascular biology and therapeutics.

Urokinase and type I plasminogen activator inhibitor production by normal human hepatocytes: modulation by inflammatory agents.

We examined the effects of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha and transforming growth factor-beta) on the plasminogen activator system (urokinase, tissue-type plasminogen activator, type 1 plasminogen activator inhibitor) in primary cultures of human hepatocytes. We show that interleukin-1 beta and tumor necrosis factor-alpha increase urokinase-type plasminogen activator production, reinforcing the concept that increased urokinase production is associated with inflammatory processes. By contrast, the same agents (i.e., interleukin-1 beta and tumor necrosis factor-alpha) do not stimulate plasminogen activator inhibitor type 1 production. This latter observation rules out hepatocytes as a major cellular source of plasmatic plasminogen activator inhibitor type 1 during acute-phase-related responses. Among the inflammatory agents used, transforming growth factor-beta was found to be the most effective modulator of both urokinase-type plasminogen activator and plasminogen activator inhibitor type 1, inducing severalfold increases of activity of urokinase-type plasminogen activator, antigen and the corresponding mRNA and increasing plasminogen activator inhibitor type 1 antigen and mRNA levels. Urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 modulation by transforming growth factor-beta may play a critical role in hepatic pathophysiology.

In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative.

N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR). The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM. The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype. In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of [3H]daunorubicin and blocks the efflux from preloaded cells. It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein. GF120918 effectively competes with [3H]azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action. After i.v. administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10. It is eliminated from organs and blood with a half-time of approximately 2.7 h. It is well absorbed after p.o. administration. In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose (5 mg/kg) of doxorubicin administered 1 h later. A statistically significant effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c. as assessed by tumor size at day 19. GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.

Refractory period phenomenon in the induction of tissue factor expression on endothelial cells.

Tissue factor (TF) is the first factor of the extrinsic pathway of coagulation. Normally, TF is not expressed on the surface of endothelial cells. However, expression of TF can be induced in these cells in response to stimulation by diverse inflammatory mediators such as interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). We have studied the effect of these mediators on the kinetics of the induction of TF-related procoagulant activity (PCA) on human umbilical vein endothelial cells (HUVECs). PCA is transiently induced on HUVECs, attaining a peak some 4 to 8 hours after addition of inflammatory agents, with maximal accumulation of TF messenger RNA (mRNA) occurring 3 to 5 hours earlier. Because the expression of PCA by treated HUVECs returns to basal levels by 20 to 30 hours, we examined the response of these cells to a second inflammatory stimulus. Continuous incubation of cells with a single inflammatory agent for 24 to 48 hours induces a hyporesponsive state with respect to the reinduction of TF expression by the same agent (14% of the initial stimulation for IL-1 beta, 39% for TNF-alpha 30% for LPS, and 7% for PMA). Such a diminution in PCA was also observed in the levels of TF mRNA. By contrast, pretreatment of HUVECs with one agent did not dramatically affect the reinduction of TF by any of the three other factors. We subsequently focused our attention on the induction of the autologous refractory period by IL-1 beta. De novo protein synthesis was not required during the preincubation of ECs for hyporesponsiveness to be observed. The establishment of the refractory state did not depend on the downmodulation of IL-1 beta receptor affinity or expression. Moreover, pretreatment of HUVECs with IL-1 beta increased prostacyclin (PGI2) production in response to a second stimulation by IL-1 beta, although such cells were unable to reexpress TF under the same conditions. This result suggests that distinct secondary messenger pathways are involved in TF induction and PGI2 synthesis by IL-1 beta in HUVECs.

Elimination of serum proteins and potential virus contaminants during hepatitis B vaccine preparation.

As first generation hepatitis B vaccines are derived from human plasma, detailed information is required concerning the elimination of hepatitis B virus and other potential transmissible infectious agents during vaccine preparation. To demonstrate the safety of a hepatitis B vaccine, the efficiency of each of the six main steps used in the preparation process to remove or destroy pathogens was determined for representatives of major groups of animal viruses. Infectivity of all the tested viruses was reduced 10(5)-fold to a factor of 10(9)-fold by the first and last steps, namely PEG fractionation and formalin treatment. The four successive zonal ultracentrifugations decreased virus infectivity by at least 10(7)-fold. Five of these steps tend also to purify the hepatitis B surface antigen (HBsAg) which increases the HBsAg: protein ratio by at least 10(5)-fold. Considering the high degree of purity obtained, checked on each batch, it is concluded that the procedure consistently eliminates any potential virus with a ide safety margin.

Uvomorulin: a nonintegral membrane protein of early mouse embryo.

A monoclonal antibody has allowed the characterization of various forms of uvomorulin, a glycoprotein involved in the process of compaction of mouse morula. In addition to various degradation products, uvomorulin exists as a 120-kilodalton exocellular molecule stable at the cell surface. A short-lived 135-kilodalton precursor of uvomorulin has been detected after 10-min pulse labeling. Uvomorulin-like molecules are found on various tissues at various stages of development of the mouse.

Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium.

Compaction, a process of cell-cell adhesion between mouse blastomeres or between embryonal carcinoma (EC) cells requires calcium ions. A decompaction effect similar to that observed in the absence of Ca2+ is triggered by Fab fragments of rabbit anti-EC IgG. This effect occurs through the recognition of a specific cell-surface glycoprotein named uvomorulin. An 84,000 dalton fragment of uvomorulin (UMt) has been previously extracted by trypsin from EC cell membranes and purified. WE present evidence that effects of Ca2+ on compaction are transmitted through conformational changes in uvomorulin. First, Ca2+ protects UMt from further proteolysis by trypsin. Mn2+ and Sr2+ have similar effects, whereas this protection is reversed by La3+. Second, UMt can bind the monoclonal antibody De1 only in the presence of Ca2+ (half-binding at 10(-5) M Ca2+). This antigenic exposure also takes place in the presence of Mn2+ or Sr2+ and is reversed by La3+. Third, metal ions (Ca2+, Mn2+, Sr2+) that promote trypsin resistance and recognition by DE1 are found to trigger the compaction of morulae and EC cells. Metal ions (La3+) that reduce trypsin resistance and affinity for DE1 result in decompaction.

Interferon enhances the amount of membrane-bound beta2-microglobulin and its release from human Burkitt cells.

Human leukocytes interferon (HuIFN-A) increased the amount of beta2-microglobulin on the surface of human Burkitt lymphoma cells (Ramos) and also increased the amount released into the culture medium. The effect was observed 1 h after addition of IFN. These results suggest that the increase in beta2-microglobulin on the cell surface of IFN-treated cells is not due to a decreased shedding of antigen from the cell surface, nor an "unmasking" of surface antigen, but rather to an increased synthesis of antigen.

A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos.

Fab fragments of rabbit anti-embryonal carcinoma cells IgG dramatically perturb cell-cell interactions between embryonal carcinoma cells and between early mouse embryo blastomeres. These antibodies prevent compaction of preimplantation embryos (or trigger their decompaction) and have similar effects on embryonal carcinoma cells. They probably act through the masking of specific molecules (Fab targets) involved in the mechanisms of recognition between cells during compaction. Fab target molecules have been extracted from embryonal carcinoma cell membranes and purified using their property to inhibit the effects mediated by anti-embryonal carcinoma Fab. The solubilization of the Fab targets could be achieved using both detergent extraction and trypsin treatment of membranes. In the latter case, a glycoprotein of 84,000 daltons could be purified which has all the properties expected from the Fab target and accounts for most of the Fab-inhibiting activity of embryonal carcinoma cell membranes.

Dissociation and exchange of the beta 2-microglobulin subunit of HLA-A and HLA-B antigens.

Human histocompatibility antigens HLA-A, HLA-B, and HLA-C are a complex of two noncovalently associated subunits: a heavy chain glycoprotein (alpha) carrying the genetic polymorphism and an invariant light chain, beta 2-microglobulin (beta 2m). Upon incubation of papain-solubilized HLA with radiolabeled urinary beta 2m, the latter is incorporated into HLA, where it substitutes for the preexisting beta 2m that has dissociated from the complex. The association-dissociation equilibrium that governs this beta 2m exchange reaction was investigated and found to be characterized by a long lifetime of the complex (half-life of 80 min at 37 degrees C) and a relatively low Kd (4 nM). The beta 2m exchange was used as the basis of a radioimmunoassay for HLA antigens with radiolabeled beta 2m as a unique label for all HLA specificities. In a similar fashion, radiolabeled beta 2m can be incorporated into HLA at the cell surface. Although the process is slower and less extensive than in solution, it can be used as a means to tag cells with specific probes for HLA antigens.

Methionyl-tRNA synthetase from Escherichia coli. Absence of interaction between the metal ion and the purine ring of ATP in the L-methionine activation site.

Enhancement of the nuclear relaxation rates by manganese has been used to derive manganese--purine-ring distances in the activation site of methionyl-tRNA synthetase. This is possible with the help of an abortive complex between the enzyme, methionine, adenosine, pyrophosphate and manganese which simulates an intermediate species of the activation reaction. It is found that the distances between the manganese ion and the purine ring are too high (greater than 0.8 nm) to allow interaction between them. Thus, metal-purine interaction is involved neither in the catalytic mechanism nor in the stabilization of abortive synergistic complexes [S. Blanquet, G. Fayat and J. P. Waller (1975) J. Mol. Biol. 94, 1-15].

Methionyl-tRNA synthetase from Escherichia coli: substituting magnesium by manganese in the L-methionine activating reaction.

While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP-PPi isotopic exchange reaction catalysed by methionyl-tRNA synthetase from Escherichia coli, the latter ion was selected for detailed analysis of the L-methionine activation reaction. In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments. The thermodynamic parameters derived from steady-state (ATP-PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped-flow) experiments are compared to those obtained in the presence of Mg2+ [Hyafil et al. (1976) Biochemistry, 15, 3678-3685]. While the standard deltaG for the reaction (E-Met-ATP-Me2+equilibriumE-Met approximately AMP-PPi-Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species. The deltaG for the formation of the E-Met approximately AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex. Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi-Me2+ from the E-Met approximately AMP-PPi-Me2+ species and from its abortive analog E-Met-Ado-PPi-Me2+. Similarly the dissociation constant of ATP-Me2+ from another dead-end analog E-methioninol-ATP-Me2+ is decreased by Mn2+. Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl-tRNA synthetase is ruled out by the use of 7-deaza-adenosine. The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.

Methionyl-tRNA synthetase from Escherichia coli: active stoichiometry and stopped-flow analysis of methionyl adenylate formaiton.

Native dimeric methionyl-tRNA synthetase and its monomeric proteolytic fragment are shown to form and to bind 1 mol of methyionyl adenylate per polypeptide chain. Moreover, at 25 degrees C, each monomer of the dimeric native enzyme behaves independently, exhibiting the same parameters for the methionine activation reaction as does the monomeric modified enzyme. These results were obtained using several independent methods including equilibrium and nonequilibrium dialysis, active site and tryptophan fluorescence titrations, and stopped-flow by fluorescence. Stopped-flow resolution of the reversible methionine activation reaction also demonstrates that methionine and ATP-Mg2+ react without coupling to form a ternary enzyme-methionine-ATP-Mg2+ complex. This complex readily converts to enzyme-methionyl approximately adenylate-PP-Mg2+ with a standard free energy close to zero. It is concluded that the uncoupled enzyme-methionine-ATP-Mg2+ complex may resemble the transition state of the reaction at the expense of the additional state of the reaction at the expense of the additional synergistic binding energy provided by reciprocal coupling, within the site, of the methionine molecule with the adenosine and PP-Mg2+ parts of the ATP-Mg2+ molecule (Blanguet, S., Fayat, G., and Waller, J. P. (1975), J. Mol. Biol. 94, 1.).