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With respect to the management of dredged sediments, a crucial issue is whether the removed materials (watered and/or processed) are disposed of or reused in an environmentally sound manner. In this context, the number of studies dealing with hazard or risk assessment has exponentially increased. This emphasis has resulted in the promotion and application of a very large variety of ecotoxicological tests. As a consequence, there is a clear need to highlight the scope and limitations of these tests for their appropriate selection and interpretation. In this paper we discuss the choice, implementation and interpretation of laboratory tests carried out on aquatic organisms at various levels of biological organization. We examine some experimental tools and methods in order to determine how suitable they are in regard to the objectives for which they are employed. To make this discussion more consistent our paper is based on results from research programmes conducted for governmental organizations and industrial partners. The Laboratoire des Sciences de l'Environnement (LSE) was involved with Cemagref de Lyon in a first methodological programme on risk assessment of scenarios of dredged sediment deposition. Another programme for a chemical company was implemented to assess the benefits of a physico-chemical treatment applied to contaminated dredged sediments in a scenario of reuse or deposit in gravel pits. Currently, the LSE is working on a programme of risk assessment for road sediments in valorization scenarios. From these programmes, we expose how single-species tests, as well as more complex bioassays and microcosm tests, can be used in an iterative step of risk assessment. Concerning microcosm tests, we also introduce a more realistic system that has been designed to simulate natural hydraulic conditions of gravel pits to assess the effects of toxicants on gravel pit aquatic biota during the sediment immersion phase and the sediment post-deposition phase (paper in preparation). The benefits of these ecotoxicological approaches are underlined, but limits are discussed with regard to several criteria: ecological relevance, realism, use for decision-making, cost and complexity of methods involved.

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The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special((R)) enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N((R)) enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.

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We have applied flow cytometry to the detection of activated platelets in patients with coronary heart disease. Paraformaldehyde-fixed platelets were incubated with one of the following monoclonal antibodies (MAbs): Bx-1 (anti-GP Ib), AP-2 (anti-GP IIb-IIIa complex), VH10 (anti-GMP-140, a glycoprotein of the alpha-granule membrane), or PAC-1 (directed against an activation-dependent determinant on GP IIb-IIIa complexes). Bound antibody was quantitated after the addition of FITC-conjugated anti-immunoglobulin. This report highlights studies on 16 unstable angina patients undergoing transluminal angioplasty. Blood samples were taken at different periods before and after the angioplasty. Levels of activated platelets were variable, remaining in the 2-4% range of control donors for some, but increasing to 10-30% post-angioplasty for others (despite all patients receiving heparin and aspirin). Maximum numbers of activated platelets were detected at 24 or 48 h. Nonetheless, the amount of antibody bound to individual platelets rarely reached the levels seen when control platelets were stimulated with thrombin in vitro. Results with VH10 and PAC-1 often, but not always, correlated suggesting different pathways of platelet activation.

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Recent advances have resulted in the elucidation of the principal molecular pathways of platelet function. Parallel studies have led to the identification of glycoprotein antigens whose presence at the platelet surface indicates an activated state. Such markers include GMP-140 and other glycoproteins of intracellular membranes whose translocation requires secretion and fusion of granule membranes with those joined to the surface. Other markers include activation-dependent epitopes on the GP IIb-IIIa complex and adhesive proteins bound to the activated GP IIb-IIIa receptor. Such epitopes can be detected by specific monoclonal antibodies. Quantification of their binding by flow cytometry allows an estimation of epitope expression within the whole platelet population. Our studies are designed to answer the question of whether measuring these epitopes is useful for predicting thrombosis in patients with acute cardiovascular disease. For this, we have examined three states where platelet function may be modified and where the risk of thrombosis and/or bleeding is increased. These include (i) patients with severe burns, (ii) patients who have undergone coronary angioplasty, and (iii) patients receiving fibrinolytic therapy following myocardial infarction. Our results show that activated platelets can be detected in the circulation and that their level reflects the degree of the lesion. Nonetheless, we have as yet failed to show a direct correlation between their presence and a future pathological event.

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Experimental studies have demonstrated regression of atheromatous lesions with diet and lipid lowering drugs. In order to confirm these results clinically, reliable angiographic methods of analysis must be developed along two lines: quantitative by consensus between independent "blinded" experts, qualitative by digitalizing radiological images. Given the reproducibility of these methods, a variation of 17 to 20% in the size of the atheromatous plaques should be required to affirm a change. Five studies have been performed in patients with atherosclerosis associated with variable degrees of hyperlipidaemia and compared with a control group. NHLBI type II: 59 out of 146 patients with type II hyperlipoproteinaemia were treated with cholestyramine for 5 years with reduction of the progression of > 50% stenosis but no evidence of regression (6%). CLAS: 80 out of 160 coronary patients were treated with cholestipol and nicotinic acid for 2 years and a reduction of progression and a regression of lesions were observed in 16% of cases. Nikkila: 28 coronary patients with hyperlipidaemia were given clofibrate or nicotinic acid for a 7 year period, stabilising the evolution but with no signs of regression. FATS: 74 of 120 coronary patients with apolipoprotein B concentrations of over 1.25 g/l were given lovastatine-cholestipol or nicotinic acid-cholestipol for 2.5 years: regression of coronary lesions was observed in 32 to 39% of cases depending on the treatment administered. Olsson: reported the same results for femoral atheroma with treatments associating fenofibrate and nicotinic acid: 20% regression and reduction of progression.(ABSTRACT TRUNCATED AT 250 WORDS)

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Hypertrophy is a myocardial adaptation to pathological conditions of overload and leads to a reduction of cardiac afterload. Angiocardiography (the so-called reference method) allows calculation of mass and volume after opacification of the left ventricle in two orthogonal planes, based on different mathematical models: the area-length method: the left ventricle is assumed to be ellipsoid. The short axis diameter and the volume are calculated from measurements of the area of the left ventricle in the long axis; Simpson's method: the ventricle is divided into sections along its long axis. The diameters of the sections are measured in each incidence. Left ventricular volume is calculated from the area of each section and the distance between two sections; myocardial mass: calculation of the myocardial mass requires measurement of the volumes of the left ventricle with the diameters increased by twice the wall thickness. The accuracy of these measurements depends on several factors: geometrical: true parallelism between the long axis of the ventricle and the planes of projection; influence of the contrast medium in the volume and contraction; this is negligible in the first three systoles; detection of the ventricular contours: this may be manual or automatic and requiring techniques of image digitalization. Therefore, the calculation of ventricular mass is directly related to volume determination, and hypertrophy can only really be quantified by mass/volume or wall thickness/diameter ratios.

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The aim of this study was to document the effects of enoximone in congestive cardiac failure. The haemodynamic data (aortic pressure, pulmonary pressures, left ventricular pressure, cardiac output, isovolumic contractility index: Vmax) and left ventricular kinetics of 20 patients with dilated cardiomyopathy (11 ischemic and 9 idiopathic in Stages III or IV of the NYHA Classification before recompensation) were recorded under basal conditions, after 30 minutes infusion of dobutamine (10 micrograms/kg/mn) and after 3 hours infusion of enoximone (total dose: 3.6 mg/kg). The two drugs had an equivalent inotropic effect: ejection fraction + 4 +/- 22% with dobutamine and + 16 +/- 39% with enoximone; Vmax increased from 1.53 +/- 0.5 c/sec to 2.49 +/- 0.8 c/sec with dobutamine and to 1.82 +/- 0.5 c/sec with enoximone. Enoximone induced a greater degree of vasodilation (systemic resistances - 14 +/- 21% with dobutamine and - 21 +/- 27% with enoximone) and a more pronounced fall in ventricular filling pressures (- 35 +/- 42% with dobutamine and - 58 +/- 24% with enoximone). Enoximone was less effective than dobutamine in increasing cardiac output (+ 46 +/- 42% with dobutamine and 16 +/- 33% with enoximone) and stroke volume (+ 23 +/- 47% with dobutamine and + 2 +/- 41% with enoximone). This difference in efficacy may be explained by the major reduction in ventricular preload which enoximone induced after that observed with dobutamine. "Responders" (12 patients) had basal cardiac outputs of less than 2.3 l/mn/m2; the peripheral vasodilatation caused by enoximone was greater. Finally, the reduction in left ventricular end diastolic pressure and the increase in Vmax were significantly less in the 11 patients with ischemic cardiomyopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

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In the absence of thromboembolic risk factors, signs of thrombosis in situ, or disorders of coagulation, two possible diagnoses must be envisaged when acute recurring ischemia is observed in a peripheral area of tissue: extrinsic compression or rupture of an arterial coat, especially when the subject is young and in good general health. The case of a 35 year old patient presenting with recurring ischemia in the popliteal artery of the right lower limb due to sub-adventitial rupture of the popliteal artery suggests three comments: the period of latency (3 months) between initial trauma to the popliteal artery and the operation of reverse saphenous vein bypass, the necessity for dynamic manipulations together with angiographic and ultrasound examination, the probable underestimation of the traumatic or functional causes of this disorder (popliteal artery), and the surgical prognosis remains excellent.

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The discovery of normal coronaries on angiography in patients known and treated for many years for true angina, if reassuring for the physician, raises other problems about the evolution of the patient. Among the 235 patients seen with this diagnosis between january 1980 and may 1986, 68 had healthy coronaries without any associated disease (mean age = 57.4 years). The objective is to determine the evolution of this group with a mean follow-up of 42 months and the advantages of coronary angiography. No infarction or sudden death occurred. 91 p. cent have an unlimited housekeeping activity, 10 p. cent have stopped working for a cardiac reason, 55 p. cent continue to work, 13 p. cent were rehospitalized for the same reason and 25 p. cent developed extra cardiac symptoms (2 neoplasms). But only 35 p. cent do not experience chest pain. On the contrary, failure is obvious concerning the anti-angina treatment since only 10 p. cent are not any longer on medication and the total consumption did not vary. The fact that 84 p. cent of the patients have little or no pain under calcium-blockers, taken alone or in association with other drugs, raises the question of their true efficacy, without any placebo effect, since the beta-blockers consumption decreases slightly.

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The interaction of glyceraldehyde 3-phosphate dehydrogenase with microtubules has been studied by measurement of the amount of enzyme which co-assembles with in vitro reconstituted microtubules. The binding of glyceraldehyde 3-phosphate dehydrogenase to microtubules is a saturable process; the maximum binding capacity is about 0.1 mole of enzyme bound per mole of assembled tubulin. Half saturation of microtubule binding sites is obtained at a concentration of glyceraldehyde 3-phosphate dehydrogenase of about 0.5 microM. Glyceraldehyde 3-phosphate dehydrogenase (between 0.1 and 2 microM) induces a concentration-dependent increase a) in the turbidity of the microtubule suspension without alteration of the net amount of polymer formed and b) in the amount of microtubule protein polymers after cold microtubule disassembly. There is a linear relationship between the intensity of the glyceraldehyde 3-phosphate dehydrogenase-induced effects and the amount of microtubule-bound enzyme. The specificity of the association of glyceraldehyde 3-phosphate dehydrogenase to microtubules has been documented by copolymerization experiments. Assembly-disassembly cycles of purified microtubules in the presence of a crude liver soluble fraction results in the selective extraction of a protein with an apparent molecular weight of 35,000 identified as the monomer of glyceraldehyde 3-phosphate dehydrogenase by peptide mapping and immunoblotting. In conclusion, microtubules possess a limited number of binding sites for glyceraldehyde 3-phosphate dehydrogenase. The binding of the glycolytic enzyme to microtubules shows a considerable specificity and is associated with alterations of assembly and disassembly characteristics of microtubules.

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Glyceraldehyde 3-phosphate dehydrogenase, a tetramer of 140,000 Da, interacts with in vitro reconstituted microtubules. It results in a partial inhibition of the activity of the microtubule-bound enzyme. After cold depolymerization of the microtubule-glyceraldehyde 3-phosphate dehydrogenase complexes, a fraction of the enzyme is recovered in an active form in the disassembly supernatant; the other fraction devoid of activity, identified by polyacrylamide gel electrophoresis, remains associated with the undepolymerizable microtubule protein pellet. The inactivation of the microtubule-bound enzyme is related to the concentration of microtubule protein. Higher the concentration of microtubule protein, lower the fraction of inactivated enzyme; consequently, glyceraldehyde 3-phosphate dehydrogenase is able to copolymerize quantitatively with microtubule protein through one assembly-disassembly cycle, provided that the concentration of microtubule protein is high. Monomeric glyceraldehyde 3-phosphate dehydrogenase (molecular weight: 35,000) devoid of enzyme activity, prepared by reversible dissociation of the tetrameric enzyme, also binds to microtubules and is quantitatively recovered in the undepolymerizable microtubule protein fraction after cold treatment. These results indicate that interacting with microtubules, glyceraldehyde 3-phosphate dehydrogenase partly dissociates into inactive monomers, this process is regulated by the concentration of assembled microtubule protein, and active and inactive glyceraldehyde 3-phosphate dehydrogenase bound to microtubules have different fate at the step of microtubule disassembly. These data suggest that an association of glyceraldehyde 3-phosphate dehydrogenase to microtubules could play a role in modulating the activity of the glycolytic enzyme in intact cells.

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