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Les canaux potassiques voltage-dépendants forment des tétramères dont chaque sous-unité comporte six segments transmembranaires (S1 à S6). Le pore, formé des segments S5-S6 de chaque sous-unité, est entouré de quatre domaines responsables de la sensibilité au potentiel membranaire, les senseurs de voltage (VS; S1-S4). Lors d’une dépolarisation membranaire, le mouvement des résidus chargés situés dans le VS entraine un mouvement de charges détectable en électrophysiologie, le courant de « gating ». L’activation du VS conduit à l'ouverture du pore, qui se traduit par un changement de conformation en C-terminal du segment S6. Pour élucider les principes qui sous-tendent le couplage électromécanique entre ces deux domaines, nous avons étudié deux régions présumées responsables du couplage chez les canaux de type Shaker K+, soit la région carboxy-terminale du segment S6 et le lien peptidique reliant les segments transmembranaire S4-S5 (S4-5L). Avec la technique du « cut-open voltage clamp fluorometry » (COVCF), nous avons pu déterminer que l’interaction inter-sous-unitaire RELY, formée par des acides aminés situés sur le lien S4-5L et S6 de deux sous-unités voisines, est impliquée dans le développement de la composante lente observée lors du retour des charges de « gating » vers leur état de repos, le « OFF-gating ». Nous avons observé que l’introduction de mutations dans la région RELY module la force de ces interactions moléculaires et élimine l’asymétrie observée dans les courants de « gating » de type sauvage. D’ailleurs, nous démontrons que ce couplage inter-sous-unitaire est responsable de la stabilisation du pore dans l’état ouvert. Nous avons également identifié une interaction intra-sous-unitaire entre les résidus I384 situé sur le lien S4-5L et F484 sur le segment S6 d’une même sous-unité. La déstabilisation de cette interaction hydrophobique découple complètement le mouvement des senseurs de voltage et l'ouverture du pore. Sans cette interaction, l’énergie nécessaire pour activer les VS est moindre en raison de l’absence du poids mécanique appliqué par le pore. De plus, l’abolition du couplage électromécanique élimine également le « mode shift », soit le déplacement de la dépendance au voltage des charges de transfert (QV) vers des potentiels hyperpolarisants. Ceci indique que le poids mécanique du pore imposé au VS entraine le « mode shift », en modulant la conformation intrinsèque du VS par un processus allostérique., Voltage-gated potassium channels are tetramers and each subunit is formed of six transmembrane segments (S1 to S6). The pore, formed by the S5-S6 segments of each subunit, is surrounded by four modules responsible for sensitivity to the membrane potential, the voltage sensors (VS, S1-S4). During membrane depolarization, the movement of charged residues located in the VS causes a detectable charge movement called the gating current. The activation of the VS led to the opening of the pore, resulting in a conformational change in the C-terminal segment of S6. To elucidate the principles underlying the electromechanical coupling between these two domains, we examined two regions presumed responsible for the coupling among channels of the Shaker K + family: the carboxy-terminal region of S6 and the peptide bond linking the transmembrane segments S4-S5 (S4-5L). Using the cut-open voltage clamp fluorometry (COVCF), we have determined that the RELY inter-subunit interaction, formed by amino acids located on the S4-5L linker and S6 of two neighboring subunits, is involved in the development of the slow component observed during the return of the gating charges (OFF-gating) to their resting state. The introduction of mutations in the RELY region modulates the strength of these molecular interactions and eliminates the asymmetry observed in the wild type gating currents. Moreover, we demonstrate that this inter-subunit coupling is responsible for stabilizing the pore in the open state. We have also identified an intra-subunit interaction between residues I384 located on the S4-5L linker and F484 on the S6 segment of the same subunit. The destabilization of this hydrophobic interaction uncouples completely the movement of voltage sensors from pore opening. Without this interaction, the energy required to activate the VS is diminished due to the absence of mechanical weight applied by the pore. Furthermore, this uncoupling also eliminates the "mode shift", defined as an amplified shift of the voltage dependence of gating charge (QV) to hyperpolarizing potentials during prolonged depolarization, thus indicating that the mechanical load of the pore influences the entry of the VS into this shifted mode by modulating the conformation of the VS threw an intrinsic allosteric process.

La confirmation du diagnostic de glycogenose de type III est basee sur des examens specialises appliques sur des biopsies hepatiques et/ou musculaires generalement tres douloureuses pour l’enfant. Ces explorations histologiques necessitent un transfert a l’etranger des fragments biopsiques frais ou congeles qui posent toujours des problemes de delai et de sensibilite. L’interet de ce travail est d’evaluer la technique fluorimetrique pour la determination de l’activite enzymatique de l’amylo-1,6-glucosidase sur des preparations leucocytaires afin de confirmer le diagnostic de glycogenose de type III. Notre methode consiste a mesurer le glucose libere par hydrolyse d’une phosphorylase dextrine limite en presence d’extraits cellulaires des tissus concernes, pour 50 sujets temoins et 18 patients suspects cliniquement d’une atteinte de type glycogenose. L’avantage et l’originalite de cette technique reposent sur sa linearite, sa precision (CV = 1,68 %), son exactitude (CI = 0,17 %), sa haute sensibilite (Sn = 100 %) et sa specificite (Sp = 96,1 %). Elle represente un precieux auxiliaire permettant la mesure des deux activites transferasique (α-1,4) et hydrolytique (α-1,6) de l’enzyme debranchante. La technique testee est pratique, peu coUteuse, fiable, efficace et elle peut etre appliquee dans la majorite des laboratoires de biologie clinique. La comparaison avec la radiometrie et l’immunoblot montre une importante capacite discriminante entre sains et malades, entre sains et heterozygotes et entre glycogenose de type III et les autres types de cette pathologie (I et VI).

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene, which leads to a deficient activity of α-galactosidase A. α-galactosidase A activity can be assayed on dried blood spots on filter paper but the original method has been associated with a number of false positive due in great part to quenching of fluorescence. Here, we describe an adaptation of the original fluorimetric method reducing quenching of the fluorescence.A simple and sensitive fluorimetric method has been described for the determination of the α-galactosidase A activity in dried blood spots on filter paper, convenient for screening of FD in at-risk populations. The procedure uses 4-methylumbelliferyl-α-D-galactose, as a synthetic substrate for the enzyme. In this study, protein precipitation was added after incubation both to stop the enzymatic reaction and eliminate interfering proteins. With the novel method the risk of false-positive due to fluorescence quenching was minimized. A cut-off level of 2.1 μmol.h(-1).L(-1) (control values: 5.6 ± 2.0 μmol.h(-1).L(-1), mean ± SD) was chosen corresponding to 40 % of median control value. In all 60 hemizygotes males, α-gal A activities were below 1.1 μmol.h(-1).L(-1) (0.11 ± 0.2 μmol.h(-1).L(-1)). In the 68 heterozygous females, α-gal A activity ranged from 0 to 7.8 μmol.h(-1).L(-1) (2.2 ± 1.7 μmol.h(-1).L(-1)). Using the improved methodology, one third of the females were not identified using the enzymatic assay, due to significant residual enzyme activity.This improved method for the assay of α-gal A was robust and reduced the number of false-positive results. It can be applied for the screening of at-risk populations. α-galactosidase A enzymatic assay should not be used for screening for FD in women or, if used, should be interpreted cautiously together with the results of genotyping.

Allergic diseases are ranked fourth considering the world health organization classification of diseases. The consequences linked to these ailments are huge for public health economics and the diagnosis is awkward due to clinical polymorphism and multifactorial aetiologies. The allergologic diagnosis is the result of weighing in clinical and biological findings. The biological assessment is made of qualitative specific and multiple-allergen serum IgE test, which once positive drives to skin test and each allergen-specific IgE level determination to conclude. Our study aims at displaying biological analysis results of incoming patients with clinical allergy conditions. We carried out a nine months retrospective study, from June 2007 to March 2008, with 200 outwards patients involved. Blood samples were collected using dry tubes and as recommended we first did screening tests for respiratory (Pharmacia Phadiatop) and food (fx 5) allergens, then for positive samples we proceeded to serum specific IgE assay (UniCap, Phadia). We also realized the total IgE assay on 46 patients using Roche Elecsys 2010 technology. 49% of patients enrolled in the study were positive to aerollergens, d1 Dermatophagoides pteronyssinus being the most incriminated (96.4%), and 2.5% to food allergens. On 13% of patients, we noticed a double sensitization to d1 and g6 (pollen of grasses). Concerning the total IgE dosage, we found 50% of patients tested with normal values, 28% of whom having a positive allergologic screening test. Further studies matching clinical data, skin tests to serum IgE assay are necessary to draw the profile of respiratory and dermatological allergies for our patients.

Gaucher disease is a disease of overload lyosomale which we often met since a score of years. Since 1980 we had to answer several requests for diagnosis of this metabolic disease. Requests emanating primarily from paediatric services. Twelve cases were confirmed within sight of measurement of the intra-leucocytic activity of the beta-glucocerebrosidase, enzyme intervening in the catabolism of the sphingolipides. We report here our experiment in the biochemical diagnosis of Gaucher disease by showing mainly the variability and the extreme heterogeneity of the activity of the beta-glucosidasic during practised measurements. In addition, we expose the problems of diagnosis etiologic which certain patients raise in front of the discordances between the measured enzymatic activity and clinical signs of the disease of left-handed person. In addition, we develop the biological parameters useful to proportion for the monitoring of the treatment which is finally available in our country.

During more than a half of century, numerous compounds have been tested in different models against radiation-induced cataract. In this report, we will review the radioprotectors that have been already tested for non-human crystalline lens protection. We will focus on the most important published studies in this topic and the mechanisms of cytoprotection reported in vitro and in vivo from animals. The most frequent mechanisms incriminated in the cytoprotective effect are: free radical scavenging, limitation of lipid peroxidation, modulation of cycle progression increase of intracellular reduced glutathion pool, reduction of DNA strand breaks and limitation of apoptotic cell death. Amifostine (or Ethyol) and anethole dithiolethione (or Sulfarlem), already used clinically as chemo- and radioprotectants, could be further tested for ocular radioprotection particularly for radiation-induced cataract.

The results of assay of IgE can only be expressed by a very imprecise system of classes. Quantification of the measurement of IgE by the method of the CAP-system of prognosis of allergic patients. Expression of IgE by a measurement of intensity of fluorescence allows the establishment of a decent curve for each allergen. It makes a method of predictive evaluation of risk of the allergy pathology for each allergen. It significantly improves the sensitivity, since it allows detection of IgE at levels less than 0.35 KUA/l. Thus it gives a considerable improvement for the clinician at the level of diagnosis avoiding the "gold standard" provocation test in allergy diagnosis as well at the level of prognosis.

Angiogenesis requires endothelial cell proliferation and their vascular rearrangement. A report of inhibiting effect of spironolactone on smooth muscle cell proliferation led us to study in vitro the effects of this drug on the endothelial cell proliferation and migration. Spironolactone (10 to 100 microM) and one of its active metabolite, canrenone (10 to 100 microM), are added to human umbilical vein endothelial cells (HUVEC). Their effect on cellular proliferation is evaluated by measuring the amount of the cellular nucleic acids using a fluorometric assay (CyQuant). Cell migration is measured using a multiwell chamber assay (Transwell). In further experiments, we investigated their effect on the capillary-like tube formation in vitro generated by HUVEC seeded in a three-dimensional biological gel (Matrigel). The VEGF (10 ng/mL) and the bFGF (10 ng/mL) were used as mitotic and cell differentiation factors. Effect on cell cycle distribution is investigated by flow cytometry analysis. Spironolactone inhibits HUVEC proliferation but canrenone does not have any significant effect. The growth promoters VEGF or bFGF do not modify inhibiting effect of spironolactone. Spironolactone (50 microM) and canrenone (50 microM) are without effect on cell migration. Capillary-like networks on Matrigel is not modified by spironolactone or canrenone. Spironolactone inhibits progression through S phase of the cell cycle. Spironolactone inhibits the proliferation of the endothelial cells in vitro but shows no effect on their migration and their rearrangement in capillary-like structures. These data should be confirmed in models of angiogenesis in vivo.

We investigated nitric oxide (NO) production and inducible NO synthase (iNOS) expression by cultured peripheral blood mononuclear cells (PBMC) in systemic sclerosis (SSc). Eighteen patients with SSc were compared to two control groups: 16 rheumatoid arthritis patients (RA) and 23 mechanical sciatica patients. The sum of nitrites and nitrates was determined by fluorimetry in sera and spectrophotometry in supernatants. Inducible iNOS was detected in cultured PBMC by immunofluorescence, immunoblot and flow cytometry with or without IL-1 beta + TNF alpha, IL-4 or IFN gamma from day 1 to day 5. NO metabolite concentrations in the plasma were lower in SSc (34.3 mumol/l +/- 2.63 SEM) than in RA (48.3 mumol/l +/- 2.2; p0.02) and sciatica (43.3 mumol/l +/- 5.24; p0.03) patients. iNOS was detected in cultured monocytes in the 3 groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus. The concentrations of NO metabolites are decreased in SSc patients and the induction of iNOS in PBMC is delayed. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may suggest therapeutic implications.

In France as well as in most of the other European countries, the prevalence of coeliac disease is underestimated. In order to point out a good screening test, we have determined the most sensitive combination (technique-marker) for the diagnosis of the disease among 81 individuals (50 with coeliac disease and 31 controls). Serum anti-gliadin antibodies were measured using three methods: the qualitative dot-blot (Gliastick-Eurospital) and two quantitative methods Elisa (homemade-Saint-Antoine Hospital and alpha-Gliatest-Eurospital); serum anti-endomysium antibodies (EmA) and anti-reticulin antibodies (ARA) were detected using an indirect immunofluorescence assay. We have shown that the simple and fast Gliastick test can fulfil the selected criterion with a sensitivity of 0.90. Nevertheless, uncertain and positives results have to be confirmed by one of the two more specific quantitative tests. The two other markers (ARA and EmA) have shown a better specificity (1) but they were less sensitive (0.54 and 0.56 for ARA and EmA respectively). Thus, they have both to be used as confirmation tests and for follow-up with supervision of the compliance to recommended diet. In conclusion, the Gliastick can be considered as a good screening test for the detection of anti-gliadin antibodies and it would represent the expected help to determine the prevalence of coeliac disease on a large-scale map.

In vitro lymphocyte mitogenic stimulation by phytohemagglutinin A was determined in 20 subjects, comparing tritiated thymidine incorporation and nuclear propidium iodide fluorescence. Unexpectedly, no correlation could be found between the two measures. The pitfalls of both methods are reviewed and discussed. The inability to validate one test by the other restricts the interpretation of the results obtained with one method and prevents their generalization. Without another gold-standard at the present time, specific and limited method-dependent norms should be defined.

The author has selected and updated four subjects in Plastic Surgery research. All flap monitoring procedures are detailed. They are still insufficient, as none of them can be used in all clinical situations. Free radicals have been implicated in the extension of necrosis after reperfusion of an ischaemic flap. Many substances have been tested for their scavenger action, particularly superoxide dismutase. Continuous intraarterial infusion of fibrinolytics is the treatment of the "no reflow phenomenon". A protocol with urokinasis, lidocaine and enoxaparine for ten days is used in our department with a 75% success rate. Finally, many interesting advances have been made to improve the biotolerance of allogeneic transfers. Cryopreservation of transplanted tissues at -196 degrees C for three weeks seems to be an interesting solution to avoid the rejection phenomenon.

The cornea is frequently associated to hypoxia, whether during residence in the heights or more often when wearing contact lenses. To evaluate the corneal modifications induced by hypoxia at an infraclinical stage, we have used redox fluorometry that enables to study in vivo the metabolic response of the cells while measuring the fluorescence of the flavoproteins (FAD) of the corneal cells.The variations of the corneal fluorescence were studied in 12 healthy subjects, before and after a topically-induced 5-minute corneal hypoxia, submitting 2 eyes to a prehumidified flow of nitrogen 100%. The results were compared to those found in the same subjects after exposure under the same conditions to an ambiant air flow (N2 = 69%; O2 = 21%). The measurements of the corneal fluorescence were carried out with the fluorophotometre Flurotron Master FM2.The authors did not find any statistically significant difference in the corneal fluorescence between the right and the left eyes of these 12 subjects, whether under normal conditions, under hypoxia, or under air flow (wilcoxon T-test, Friedman test).As there are no significant results, these authors suggest that the chosen exposure time, although sufficient in vitro to induce a modification of the fluorecence of the cellular flavoproteins, may be too "short" in vivo. The use of complementary filters with the FM2 system would yield more information. The study of these results led the authors to broaden their search whether by looking for conditions for general hypoxia (hypobarric box) or by increasing local hypoxia (contact lenses).

Time-resolved fluorometric assay is based on lanthanide fluorescence. This time-resolved fluorescence has a narrow-band emission line whose wavelength differs from that of emission-pulsed light and has a long decay-time. These characteristics make it possible to avoid background interference from sample constituents (protein, light-scattering particles, etc). Europium and its chelates are the most commonly used lanthanides. The europium-labelling of antigens or antibodies is followed by immunoassay. In the final step, fluorescence is measured, after enhancement, as counts per second. This assay has several advantages, including a wide working range, high sensitivity and good practicability. The method has widespread applications in the field of immunoassays in both clinical and research laboratories. The use of non-radioactive europium-labelled probes and the development of simultaneous multiple tests are possible future orientations.

We developed a method for the enzymatic assay of glutathione which is easy to practice, rapid, specific, based on the reaction of the thiol group of glutathione with dithiobis-nitrobenzoic acid after the action of glutathione reductase in the presence of NADPH. This spectrophotometric technique allowed, on the one hand, the determination of total glutathione and on the other hand, that of oxidized glutathione (disulfide), after the blockage of reduced glutathione by 2-vinyl-pyridine. The improvements of the assay of blood glutathione concerned the sample preparation, the reaction sensitivity, thanks to a better definition of the optimal pH and a reduction ot the blockage time by 2-vinyl-pyridine in well defined operating conditions. We compared the performances of our technique with a fluorimetric method. We used our method for the determination of total and oxidized blood glutathione in a control population.

In the kidney, angiotensin I-converting enzyme (ACE) is present in the vascular endothelial cells and in the brush border of epithelial cells of the proximal tubule. In spite of this well-known distribution of ACE, little is known of its regulation. In order to elucidate the possible mechanisms of control for brush border ACE, the effects of dexamethasone (DM), (40 micrograms s.c. per day, for 7 days) and triiodothyronine (T3) 0.5 mg/kg s.c. per day, for 10 days) were investigated in male Wistar rats. Plasma and brush border ACE activities were measured by fluorimetry in the presence of an artificial substrate Cbz-Phe-His-Leu and brush border ACE was characterized with a binding assay using 3H-ramiprilat, a specific radiolabelled ACE inhibitor. DM elicited a significant decrease in plasma ACE activity (from 0.46 +/- 0.03 to 0.28 +/- 0.02 nmol His-Leu/min/mg protein) but did not alter enzyme activity in the brush border: 47.12 +/- 5.12 nmol His-Leu/min/mg protein (control, n = 6) and 47.78 +/- 5.63 (DM, n = 6). Administering T3 produced a marked increase in the brush border ACE activity (from 42.87 +/- 4.9 to 81.41 +/- 11.7 nmol His-Leu/min/mg protein). Similarly, the maximum number of 3H-ramiprilat-binding sites increased in the brush border, indicating a good correlation between ACE activity and the quantity of 3H-ramiprilat bound. Thus, the variation in tissue ACE activity corresponded to a change in the enzyme concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Cortinarius poisoning is generally caused by orellanine, a hydroxy bipyridine N, N-dioxide. This intoxication is characterized by acute nephritis which can lead to death without treatment. We reported a highly sensitive and simple fluorimetric technique to analyse orellanine by thin-layer chromatography on the basis of its characteristic photodecomposition into orelline. Using this procedure, we detected and assayed orellanine for the first time in plasma and renal biopsies of a woman who had deliberately ingested two fruit-bodies of Cortinarius orellanus. An early original treatment was carried out based on hemodialysis, combination plasmapheresis-hemoperfusion, and amino acids and diltiazem administration. These results indicate that it is now possible to make a precise diagnosis of orellanine poisoning.

The fluorometric analysis of 5-hydroxy-indoleacetic acid by condensation with o-phthaldialdehyde has been modified by substituting a HCl-H2SO4 mixture at room temperature for hot concentrated HCl. The sensitivity of the method was increased by a factor of 2.7.

The author describes a simple and entirely automatic technique for the estimation of triglycerides in human serum which, in acetic medium, are extracted by a mixture of nonane-isopropanol and saponified. The glycerol is oxidised into formaldehyde which is estimated by fluorimetry after Hantzsch' reaction. Successive stages are discussed together with the precision of the method. The results are in agreement with those obtained by Kessler and Lederer's method.

Laser fluorimetry is a new technique which provides continuous information on tissue metabolism in situ and without destruction. For the moment, it is mainly applied to the study of changes in redox gradients in various organs, including the heart, brain, liver, kidney and skeletal muscle, in cases with imbalance between oxygen supply and oxygen consumption. Other metabolisms, such as that of the crystalline lens with incipient cataract, can also be investigated by this technique.

The authors described a fluorimetric method of estimation of colchicine applicable to biological fluids, during treatment and, especially, during acute poisoning. Blood and urinary concentrations confirm the data in the literature.

Oral administration of large doses of vitamin A permits one to study pos-prandial lipemia. Thanks to a new fluorescimetric and automatic method, estimation of vitamin A is now much easier than formerly. The normal results of the oral vitamin A tolerance test are evaluated with this new method and compared with those given by the previous method.

The authors describe an automatic fluorimetric micromethod based on the reaction of condensation of serotonin with orthophthaldialdehyde forming a fluorescent compound. This precise and sensitive method permits estimation of serotonin on microsamples. The results obtained on capillary blood are identical to those obtained on venous blood.