Polarized distribution of Na+, K(+)-ATPase alpha-subunit isoforms in electrocyte membranes.

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.

Inhibition of acetylcholinesterase from Electrophorus electricus (L.) by tricyclic antidepressants.

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.

Electrocyte (Na(+),K(+))ATPase inhibition induced by zinc is reverted by dithiothreitol.

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.

A controlled "before-after" study: impact of a clinical guidelines programme and regional cancer network organization on medical practice.

A regional cancer network has been set up in the Rhône-Alpes region in France. The aim of the project is to improve the quality of care and to rationalize prescriptions in the network. In this network, we assessed the impact of the implementation of a clinical practice guidelines project by assessing the conformity of practice with the guidelines and comparing this with the conformity in an external matched control group from another French region without a regional cancer network. Four hospitals (private and public) accepted to assess the impact of the clinical practice guidelines on the management of breast and colon cancer in the experimental group and three hospitals (private and public) in the control group. In 1994 and 1996, women with non-metastatic breast cancer (282 and 346 patients in the experimental group, 194 and 172 patients in the control group, respectively) and all new patients with colon cancer (95 and 94 patients in the experimental group, and 89 and 118 patients in the control group, respectively) were selected. A controlled "before-after" study, using institutional medical records of patients with breast and colon cancer. The medical decisions concerning the patients were analyzed to assess their compliance with the clinical practice guidelines. When medical decisions were judged to be non-compliant, we verified if they were based on scientific evidence in a published article, if they were not, the medical decision was classified as having "no convincing supporting scientific evidence". The compliance rates were significantly higher in 1996 than in 1994 in the experimental group; 36% (126 out of 346) vs 12% (34 out of 282) and 46% (56 out of 123) vs 14% (14 out of 103) (P<0.001) for breast and colon cancer, respectively. Whereas, in the control group the compliance rates were the same for the two periods; 7% (12 out of 173) vs 6% (12 out of 194) (P=0.46) and 39% (49 out of 126) vs 32% (31 out of 96), P=0.19. In the experimental group, in 1994, 101 of the 282 medical decisions (36%) and 27 of the 103 (26%) for breast and colon cancer, respectively, were classified as having "no convincing supporting scientific evidence" compare with 72 out of 346 in 1996 (21%) for breast cancer, and 21 of the 123 (17%) for colon cancer P<0.05. Whereas in the control group these results were 106 out of 194 in 1994 (55%) and 90 out of 172 in 1996 (52%), P=0.65 for breast cancer and 28 out of 96 in 1994 (29%) and 30 out of 126 in 1996 (24%), P=0.36 for colon cancer. The development and implementation strategy of the clinical practice guidelines programme for cancer management results in significant changes in medical practice in our cancer network. These results would suggest that introducing guidelines with specific implementation strategy might also increase the compliance rate with the guideline and "evidence-based medicine".

Denervation alters protein-lipid interactions in membrane fractions from electrocytes of Electrophorus electricus (L.).

Protein-lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.). Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na(+),K(+)-ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na(+),K(+)-ATPase activity, bands a and b. Band b is almost absent in microsomes from the denervated organ, and band a', with the same density as band a has lower Na(+),K(+)-ATPase activity. Band a' presents a larger ratio of protein-interacting lipids than band a. Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein-lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis.

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.

Purification and partial characterization of creatine kinase from electric organ of Electrophorus electricus (L.).

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the electrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish (Torpedo) and human muscle type CK.

Choline Acetyltransferase from the electric organ of Electrophorus electricus (L.)--physicochemical characterization and immunochemical identification.

It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyltransferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 M, pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80%. The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05 M, pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange- Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun's method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 offkDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

[Endovascular closure of a foramen ovale after a right pneumonectomy]. / Fermeture par voie endovasculaire d'un foramen ovale révélé après pneumonectomie droite.

We report the case of a patient, 62-year-old, with a non small cell lung cancer treated by right pneumonectomy followed by chemo and radiotherapy. After surgery appeared a refractory hypoxemia increasing with supine position. Cardiac catheterism showed a right left shunt by reopening of the "foramen ovale". We have performed foramen's occlusion by endovascular method with prothetic material with good result until the death, 14 months later, by neoplasic evolution.

Lipids associated with the (Na+ - K+)ATPase of normal and denervated electric organs of Electrophorus electricus (L.).

The effect of denervation on the lipid metabolism and on the activity of (Na+ - K+)ATPase isoforms from the membrane fraction P3, which corresponds to the innervated electrocyte membrane, was evaluated. On a discontinuous sucrose gradient, normal P3 membranes exhibit a bimodal ("a" and "b bands) distribution of the (Na+ - K+)ATPase activity, which upon denervation changes to an unimodal ("c" band) distribution. Using these fractions, which have a higher (Na+ - K+)ATPase activity, we characterized the lipids at the hydrophobic protein surface boundary, (i.e., the bulk lipids that surround the protein). The results confirm that these lipids consist of phospholipids and cholesterol. The quantitative composition of the phospholipids is similar for both isoform fractions obtained from the discontinuous gradient of normal membranes, with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine representing about 90% of the total phospholipids. Sphingomyelin, phosphatidylinositol, diphosphatidylglycerol and phosphatidic acid were in the minority. However, in the single band obtained after denervation, the three major phospholipid components decreased to 70% of the total, and a significant increase in the other phospholipids and in cholesterol was observed. The high cholesterol content of the denervated fraction may confer membrane stabilization, as it is likely to cause a decrease in the membrane fluidity and consequently in the enzyme activity.

Comparison of the inhibitory effects of mercury and cadmium on the creatine kinase from Electrophorus electricus (L).

We have determined the effects of mercury and cadmium on the creatine kinase activity of the electric organ of Electrophorus electricus (L.) which catalyses the transphosphorylation reaction between phosphocreatine and magnesium adenosine-5'-di-phosphate and has essential sulfhydryl groups. The kinetic effects of these heavy metals, which have high affinity for sulfhydryl groups, on the creatine kinase activity were analysed with the three reaction components: phosphocreatine, adenosine-5'-di-phosphate and magnesium. The kinetic data were analysed with a non-linear regression program (Sigmaplot for Windows). Both metals inhibit creatine kinase activity in the micromolar range, mercury being a more potent inhibitor than cadmium. With phosphocreatine as substrate, mercury behaved as a mixed partial hyperbolic inhibitor, non-competitive inhibitor with adenosine-5'-di-phosphate, and with magnesium mercury behaved as a competitive inhibitor. Cadmium inhibition was shown to be of a classical competitive nature with respect to both substrates, phosphocreatine or adenosine-5'-di-phosphate, and non-competitive when magnesium was the variable in the reaction mixture. The results suggest that the binding site of mercury is at or near the phosphocreatine site, but it is not the same as adenosine-5'-di-phosphate, whereas cadmium competes with these substrates to bind at the same sulphydryl site.

Effect of Mg(2+)-ATP on acetylcholinesterase of Electrophorus electricus (L.).

The effect of Mg(2+)-ATP on purified acetylcholinesterase (AChE) from electric tissue of Electrophorus electricus (L.) was studied. The enzymatic activities were measured with acetylcholine and acetylthiocholine as substrates. The kinetic parameters Vmax, Km and Hill coefficient (nH), for acetylcholine and acetylthiocholine were modified with Mg(2+)-ATP. It was shown that acetylcholinesterase presents an apparent activation at high concentration of substrates and an inhibition in the presence of Mg(2+)-ATP at low concentration of acetylcholine and acetylthiocholine. In addition, the data suggest that Mg(2+)-ATP induced an allosteric modulation of the acetylcholinesterase obtained from Electrophorous electricus (L.), and indicate an active adenosine triphosphate participation during cholinergic activity.

Limited disease small cell lung cancer: alternating combination of doxorubicin, etoposide, ifosfamide and hyperfractionated radiotherapy. Final results of a multicentric pilot study for the Groupe Lyonnias d'Oncologie Thoracique (GLOT).

In order to evaluate the effect on prolonging survival of alternating chemotherapy and radiotherapy schedules in patients with limited disease small cell lung cancer, 89 patients were included in a multi-institutional pilot study between January 1986 and May 1989. Treatment consisted of induction chemotherapy using the combination of doxorubicin, etoposide and ifosfamide (AVI) for four consecutive courses, followed by two cycles of the VI chemotherapy alternating with three hyperfractionated radiotherapy courses and then followed by two additional courses of AVI. Objective response to the four cycles of AVI combination was observed in 65 patients (75%). Thirteen out of 30 patients (44%) who were in partial response (PR) after induction chemotherapy were converted into complete response (CR) after the three alternating courses of chemotherapy and radiotherapy. The principal side effect related to combined modality treatment was acute radiation pneumonitis (21.5% cases) reversible except one which resulted in toxic death, and a second with chronic lung fibrosis with permanent WHO Grade 2 dyspnea (14%). Local relapse was observed in 47% of the patients who were considered in CR at the end of the treatment program and cerebral metastases were the first site of detectable relapse in 25% cases. The 3-year actuarial disease-free survival of the 89 patients is 5%, and the median actuarial survival is 14 months. This study shows that the promising survival rates seen in our previously published interim analysis were not maintained. Reasons for this might include the choice of a non cisplatinum containing induction chemotherapy, the late introduction of thoracic irradiation and/or to the use of non-restrictive criteria for selecting patients.

Conditions for labeling of Schistosoma mansoni cercaria with technetium-99m.

Study of the labeling of Schistosoma mansoni cercaria with technetium-99m (99mTc) at room temperature (25 degrees C) and 37 degrees C shows that the incorporation of radioactivity in this cercaria increases with the increase in stannous chloride concentration, reaching a constant value threshold at 130.00 microM. Strong binding of the 99mTc was obtained since the radioactivity was not washed out. The characteristic motion of the cercaria, labeled at 25 degrees C and 37 degrees C, was only modified at the concentration of 1300 microM for both temperatures, showing that the methodology here described can be applied to living structures. With this technique it is possible to obtain a cercariae suspension labeled with a radionuclide that is very inexpensive and readily available. Furthermore, it is a gamma emitter with a photon energy of 140 keV that would permit one to make scannings of the infected animals and causes less of an environmental impact and present fewer radioactive disposal problems than the long lived radionuclides.

Technetium-99m distribution into Klebsiella pneumoniae.

Bacteria labelled with radionuclide has been the subject of much investigation and has been applied in microbiological research. Technetium-99m (99mTc) may be an alternative radionuclide for the labelling of bacteria employed in various microbiological procedures. This radionuclide is easily available, is not expensive and presents important physical and biological characteristics. 99mTc-labelled bacteria are stable and their cell viability and biological properties are not modified. Study of the distribution of radioactivity in 99mTc-labelled Klebsiella pneumoniae cultures, after homogenization and differential centrifugation of the cells fractions, showed that this radionuclide was present inside the cell, mainly in a ribosomal fraction. Treatment of these fractions with enzymes and detergent revealed a high sensitivity to pronase and Triton X-100. After phenol extraction, a large percentage of radioactivity was detected in the phenol phase. Treatment of the soluble fraction with trichloroacetic acid at different temperatures showed that the concentration of 99mTc in the precipitate was lower at 100 degrees than at 4 degrees C. These results suggest that 99mTc binds mainly to the proteins in Klebsiella pneumoniae.

[Pulmonary histoplasmosis due to Histoplasma capsulatum. A case]. / Histoplasmose pulmonaire à Histoplasma capsulatum. Un cas.

The authors report a case of benign multinodular pulmonary histoplasmosis, occurring in a 65 year old woman coming back from Guatemala. The disease presented with both fever and cough. The diagnosis was made on a lung biopsy (by thoracotomy) that showed granulomas with giant cells, lymphocytes and central necrosis, and histoplasma capsulatum yeasts on Gomori Grocott coloration. The authors recall the main radiological forms of the disease, and the difficulties of the diagnosis. When not disseminated, histoplasmosis usually has a good prognosis and does not require any treatment.

[Late recurrence of a small cell cancer of the lung or a 2nd small cell cancer? Apropos of a case]. / Réévolution tardive d'un cancer du poumon à petites cellules ou deuxième cancer à petites cellules? A propos d'une observation.

We report a case of a patient presenting with a small cell cancer of the lung which was limited to the thorax and treated by radiotherapy and chemotherapy in March 1983. In december 1986 a contralateral small cell cancer still limited to the thorax was discovered and treated with success using chemotherapy. Nevertheless the complete remission obtained did not last beyond 16 months and the patient died in september 1988. We discuss the possibility of late metastasis but we think the occurrence of a second small cell cancer is the more plausible hypothesis.

Quimio-hipertermia intraperitoneal con mitimicina C en cáncer avanzado con carcinomatosis peritoneal: primera experiencia / Intraperitoneal chemo-hypertermia with mytomicina C

La quimiohipertermia intraperitoneal (ITCH) con Mitomicina C (MMC) fue usada en el tratamiento de 7 pacientes con cancer gastrointestinal avanzado y carcinomatosis peritoneal. La reseccion primaria del tumor fue posible en 2 casos. La IPCH se realizo despues del cierre de la pared abdominal durante 90 y 120 minutos bajo anestesia general con previa hipotermia a 32º Celsius, a traves de un circuito cerrado formado con 3 drenajes de silicona intraperitoneal; se uso concentracion de 10 mg/L de MMC en 6000 mL de liquido precalentado a 46-49º Celsius. No observamos ni mortalidad ni morbilidad. Otros autores han informado efectos biologicos secundarios ligeros y temporales; no hubo celulas malignas en el liquido ascitico despues de la IPCH. Igualmente observamos en 6 pacientes un incremento en el indice de Karnofsky, 3 y 7 meses despues de la IPCH. Estos resultados nos permiten confirmar que la IPCH con MMC es un metodo sin riesgo, que puede ser usado en el tratamiento del cancer gastrointestinal avanzado con carcinomatosis peritoneal.