Efficient routes towards a series of 5,5'-bithiazolidinylidenes as π-electron acceptors.

Different approaches have been studied in order to prepare efficiently the sulfur rich electron acceptor, DEBTTT. Among the various routes used, the one going through the synthesis of a bicyclic derivative, where the thiazole-2-chalcogenone is fused with a 1,3-dithiole-2-one, leads to the target molecule under milder conditions and better yield. Thus, this approach has been explored for the synthesis of a series of acceptors either by modifying the substituent on the thiazole core or by changing the exocyclic chalcogen atoms. All these sulfur rich electron acceptors exhibit short intra- and intermolecular SS contacts in the solid state. Electrochemical investigations show that the nature of the exocyclic chalcogen atom of the thiazole ring has a significant influence on the accepting ability as a cathodic shift of about 220 mV is observed just by changing sulfur for oxygen. This structural modification enables the tuning of the redox properties.

Inhibition of marine bacteria by extracts of macroalgae: potential use for environmentally friendly antifouling paints.

Although a total ban on the use of TBT coatings is not expected in the short term, there is a growing need for environmentally safe antifouling systems. A search for new non-toxic antifoulants has been carried out among marine macroalgae. Antifouling activity of aqueous, ethanolic and dichloromethane extracts from 30 marine algae from Brittany coast (France) was examined in vitro against 35 isolates of marine bacteria. About 20% of the extracts were found to be active. The high levels of inhibitory activities against bacteria recorded in some extracts and the absence of toxicity on the development of oyster and sea urchin larvae and to mouse fibroblast growth suggests a potential for novel active ingredients in antifouling preparations.

Inhibition of the development of microorganisms (bacteria and fungi) by extracts of marine algae from Brittany, France.

The inhibitory effects of aqueous, ethanolic and dichloromethane fractions from 16 marine algae from the Atlantic shores of North-East Brittany, France, have been investigated against microorganisms frequently associated with immersed surfaces. The extracts were tested in vitro against isolates of marine fungi, bacteria and yeasts potentially involved at different stages in the formation of biofilms in the sea. The high levels of inhibitory activity of nine extracts against marine fungi and Gram-positive bacteria and their apparent absence of toxicity against larvae of oysters and sea urchins suggests a potential for novel active ingredients.

Coordinated changes of adenylate energy charge and ATP/ADP: use in ecotoxicological studies.

The coordinated variations of the adenylate energy charge and ATP/ADP ratio were modeled and a function that depends on the numerical value of the adenylate kinase-catalyzed reaction has been derived. The model allows sensitive detection of the effects of xenobiotics on adenylate kinase and its cellular environment and offers a robust estimation of the direct or indirect effects of pollutants on the adenylate kinase system: data obtained in laboratory studies on shrimp exposed to cadmium and in field studies on oysters either exposed to polychloro-biphenyl compounds or located in a heavily polluted area indicate that xenobiotics affect the adenylate kinase reaction directly or by changing its cellular environment. These results demonstrate that application of the model to the treatment of ecotoxicological data allows detection of energetic changes that would have been missed by simple analysis of the usual energetic parameters, and should overcome problems encountered in using energetic parameters during assessment of pollution monitoring.

Secretagogues and Growth Factors in Fish and Crustacean Protein Hydrolysates.

: The search for new molecules in fish protein hydrolysates is of great interest in animal feeding as it is in aquaculture, fertilizer, cosmetic, and pharmacologic domains. Different sources of hydrolysates such as shrimp waste (Pandalus borealis), cod (Gadus morhua) head, and head and viscera of sardine (Sardina pilchardus), obtained after hydrolysis or autolysis, were tested on fibroblast cell cultures and by gastrin radioimmunoassay. The level of hydrolysis seems to play an important role in the presence of biological peptides. Elution profile on a gel filtration Sephadex G-50 column was used to estimate the degree of hydrolysis of the fractions studied. Growth-factor-like activities were found in less-hydrolyzed fractions. Conversely, the most-hydrolyzed fractions showed gastrin and cholecystokinin immunoreactivity.

Heparin-binding molecules with growth factor activities in regenerating-tissues of the starfish Asterias rubens.

Regenerating-tissues of the starfish Asterias rubens were studied for the presence of growth factors liable to stimulate the proliferation of fibroblast and epithelial cells (3T3, BHK21 and Hela cells). As a first attempt to isolate growth factors, the extracts were fixed on heparin-affinity column and were eluted by 1-1.2 M NaCl. After separation on a Vydac C18 HPLC column. a fraction that stimulates the proliferation of fibroblast cells was isolated. It contained four different peptides, separated by electrophoresis, and for which the amino acid composition and molecular mass were determined. All the peptides were lysine rich and one presented an amino-acid composition comparable to basic-fibroblast growth factor (b-FGF) while its molecular weight was higher.

A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila.

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.

Isolation of an acid fraction from a fish protein hydrolysate with a calcitonin-gene-related-peptide-like biological activity.

The possibility of obtaining calcitonin and/or calcitonin-gene-related peptide (CGRP) immunorelated molecules from partly digested proteins was investigated with fish and shrimp hydrolysates. These two peptides were quantified by both radioimmunoassay and radioreceptor assay; the positive extracts were partly purified. Different hydrolysates were analysed: cod head, stomach and viscera hydrolysates, a shrimp hydrolysate and two sardine hydrolysates. Although each cod extract interacted in the CGRP radioimmunoassay, none of these extracts was able to displace the CT binding to its antibody. In contrast, shrimp and sardine hydrolysates interacted with both radioimmunoassays. Radioreceptor assays performed on the same extracts demonstrated that only three extracts contained the structural determinants that allowed them to interact in the CGRP radioreceptor assay. No interaction with the calcitonin radioreceptor assay could be demonstrated. Molecular sieving of the two sardine extracts showed that the immunoreactivity was resolved into two main fractions. The higher-molecular-mass fraction interacted only in the CGRP radioreceptor assay. The results obtained suggest the presence of a biologically related CGRP molecule in peptone hydrolysates and requires further investigation into the role of these peptide fragments in the regulation of intestinal function by partly digested proteins.

Effect of ammonia on survival and adenylate energy charge in the shrimp Palaemonetes varians.

Shrimps Palaemonetes varians were submitted to lethal and sublethal concentrations of ammonia in order to measure lethality, ATP and adenylate nucleotide levels, and adenylate energy charge (AEC) index. LC50 was of about 3 mg/liter of ammonia. Adenylate measurements were performed over a period of 14 days and for two different concentrations. A population submitted to 0.5 mg/liter of ammonia exhibited high survival and a marked consumption of ATP, whereas high mortality and disordered ATP metabolism are the characteristics of the population submitted to 3 mg/liter of ammonia. An homeostatic model was applied in order to explain the significance of the AEC signal. Here, AEC is proposed as a measure of the limits of active response of an organism to environmental stress.

Purification and characterization of two pancreatic elastase isoforms from dogfish (Scyliorhinus canicula).

Two elastase isoforms were isolated from activated pancreatic extract of dogfish (Scyliorhinus canicula). The purification procedures for both elastases included ammonium sulfate fractionation, ion exchange on DEAE cellulose and Mono-QHR column followed by gel filtration on PL-GFS (HPLC) column. The two isoenzymes, EI and EII, exhibit a high activity toward the specific elastase substrate succinyl-(ala)3-p-nitroanilide (SANA) with different kinetic parameters at 37 degrees C. However, the two different enzymes have similar properties on the basis of pH, temperature, and molecular weight study. The activity of both variants was completely inhibited by elastatinal, phenylmethyl sulfonyl fluoride, (PMSF), diisopropyl fluorophosphate, (iPr2-FP), but less by p-chloromercuribenzoic acid (PCMB) and soybean trypsin inhibitor. EI and EII have similar amino acid composition; their amino-terminal sequences have 85% homology with human and rat elastase 2.

Catalytic site studies on tuna (Thunnus albacares) pyloric caeca aminopeptidase.

Tuna pyloric caeca aminopeptidase (tAP) is a glycosylated zinc-metalloenzyme containing apparently two identical subunits. The enzyme is reversibly inhibited in a time-dependent manner by amastatin. Slow development of tAP inhibition by this inhibitor could be demonstrated. Dissociation of the complex of tAP with amastatin is also slow. Two molar equivalents of the inhibitor are bound by the enzyme suggesting the presence of one catalytic site in each subunit. Chemical modification of tAP with 1-cyclohexyl-3-(2-morpholinoethyl) carbonyl-metho-p-toluene sulfonate and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone revealed the presence of essential acidic amino acid residues probably located at the active site. Compatible with the presence of arginine and tyrosine residues at the catalytic site of most metalloproteinases, tAP is reversibly inhibited by phenylglyoxal and inactivated by tetranitromethane in a time-dependent fashion. The rate of inhibition by these modifiers could be significantly decreased if the enzyme was previously treated with amastatin suggesting that the modified amino acid residues are located at the catalytic site. Diethylpyrocarbonate did not affect the activity of both native and zinc-depleted tAP suggesting that histidine is not involved in the zinc-ligand formation.

Heart-lung protection from ischemic injury during 8 hour hypothermic preservation.

Optimal techniques of heart-lung preservation are yet to be defined. The aim of this study was to develop, in a canine model, a method of heart-lung preservation which would permit distant procurement of the organs. The animals were divided into 2 groups. In the experimental group (N = 6) the method of preservation consisted of cold cardioplegic arrest of the heart with St. Thomas' Hospital solution containing superoxide dismutase, catalase and deferoxamine, followed by cold pulmonary artery flush with modified Euro-Collins solution to which prostaglandin E1, superoxide dismutase, catalase, deferoxamine and Dextran 40 were added. Following harvesting, the heart-lung block was stored for 8 hours in cold (4 degrees C) Euro-Collins solution containing superoxide dismutase, catalase, deferoxamine, lactobionic acid, raffinose, mannitol, Dextran 40, magnesium sulfate, insulin and penicillin. In the control group (n = 6), the heart-lung block underwent the same treatment as the experimental group except that lactobionic acid, raffinose and insulin were omitted from the storage solution, and that oxygen radical scavengers were excluded from the cardioplegic, pneumoplegic and storage solutions. Histologic and electron microscopic examinations of heart-lung specimens taken before and after 8 hours cold storage of the organs suggested that our preservation technique may be effective in preventing ischemia-induced injury.

Evaluation of a cardiopulmonary preservation method for heart-lung transplantation.

This study was designed to examine whether concomitant administration of anti-free radicals with donorcore cooling on cardiopulmonary bypass (CPB) and hypothermic storage of the heart and lung, could provide successful extended cardiopulmonary preservation. Fourteen sheep heart-lung blocks harvested after core-cooling and cardioplegic arrest were preserved at 4 degrees C for 8.5 hr. Before and during CPB, the animals were perfused with Prostaglandin E1 (PGE1), superoxide dismutase (SOD), catalase (CAT) and deferoxamine (DEF). Cardioplegic arrest was induced with St. Thomas' Hospital solution (Plegisol) to which SOD, CAT and DEF had been added. The preservation solution consisted of Plegisol modified by the addition of K-lactobionate, raffinose, mannitol, SOD, CAT, DEF, a phosphate buffer and penicillin. Histological examination performed on 3 donor heart-lung blocks before and after CPB and cardioplegia, then immediately following cold storage, produced no clear evidence of structural damage in cardiac myocytes and lung parenchyma. Eleven donor organs were therefore transplanted in size-matched recipients with a total mean ischemic time of 12 hr. The combined administration of SOD, CAT, DEF, insulin and glucose during the initial period of reperfusion had no beneficial effect on cardiopulmonary performance. A progressive fall in Pa O2 and mean aortic pressure was observed post-operatively in the 7 animals that were weaned from CPB. Five of them died within 3 to 5 hr after CPB weaning, the remaining 2 animals died of cardiac arrest within the fourth hour. The results of this experiment seem to indicate that: 1. better organ function and improved survival could have been obtained if the duration of anti-radical treatment had been prolonged after reperfusion and, 2. physical manipulation of the donor grafts during harvesting and transplantation may have been partly responsible for the poor post-transplantation cardiopulmonary performance.

Purification and characterization of an aminopeptidase from tuna (Thunnus albacares) pyloric caeca.

An aminopeptidase was purified from a water soluble fraction of tuna pyloric caeca by heat treatment, Zn2+ fractionation, ion exchange on a DEAE cellulose column, gel filtration on Fractogel TSK-55, and immobilized metal ion affinity chromatography (IMAC) on IDA(Cu2+/Zn2+)-Sepharose 6B. The molecular mass of the enzyme was estimated to be 150,000 on Sephacryl S-300 HR, and was found to be near 72,000 by SDS-PAGE. The aminopeptidase, which is a glycoprotein rich in acidic amino acids, is optimally active at pH 8.8 and 65 degrees C. The enzyme activity was not affected by Mg2+, Zn2+, Ca2+, Mn2+, Co2+, PMSF, iPr2FP, 4-hydroxymercuribenzoic acid, iodoacetamide, puromycin, and cysteine but it was strongly inhibited by metal chelators (EDTA and o-phenanthroline), amastatin, Hg2+, Cd2+, and Cu2+. The enzyme was also inhibited by some L-amino acids. Kinetic parameters of the enzyme were determined with some aminoacyl-p-nitroanilides and aminoacyl-beta-naphthylamides. L-Alanine-p-nitroanilide and L-alanine-beta-naphthylamide were hydrolysed most rapidly while the highest hydrolytic coefficient (kcat/Km) value was obtained with L-methionine-p-nitroanilide. The apoaminopeptidase was prepared and reconstitution of an active enzyme was carried out using metal chelating interaction chromatography on an IDA-Sepharose 6B column charged with a metal ion. Full activity was restored with Zn2+, Co2+, Cu2+ and Al3+. Zn(2+)-Enzyme was the most thermostable form of the aminopeptidase. Reversal inhibition by Cu2+ and Cd2+ was also examined. When the aminopeptidase was partially deglycosylated by a treatment with N-glycosidase F some of its physical properties differed from that of the native enzyme: its electrophoretic mobility was reduced and its stability to denaturation by SDS and by ionic strength were lower than those of the untreated enzyme. All together, our results indicate that the tuna pyloric caeca aminopeptidase is distinct from the peptide hydrolases characterized in the literature.

Assessment of industrial sewage impacts by adenylate energy charge measurements in the bivalve Cerastoderma edule.

Laboratory toxicity tests performed on the bivalve Cerastoderma edule submitted to sublethal concentrations of paper mill effluent revealed significant decreases of adenylate energy charge (AEC), and changes in the total adenylate pool were observed in a 24-hr period even for the lowest concentration of pollutant tested. Field transfer experiments of C. edule from a safe zone to polluted areas of the Sado estuary were carried out at two different times of the year. Close proximity to sewage outfall was shown to result in significant decreases in AEC values within 24 hr. One week after transfer, either normal AEC values were found or the organisms died, depending on the location of the sampling station.

[Techniques involved in the protection of the lung and heart-lung for the purpose of transplantation]. / Techniques de protection du poumon et du bloc coeur-poumons en vue d'une transplantation.

Impaired healing of the bronchial anastomosis and its subsequent complications has been an important problem following lung transplantation. The decrease in the dosage of steroids, the use of cyclosporine immunosuppressive therapy and the use of an omental flap to revascularize rapidly the bronchial anastomosis, have reduced considerably the incidence of these post-operative bronchial complications. We have recently demonstrated that the application of a pedicled intercostal muscle graft or peritracheal membrane adipose tissue wrap on the bronchial anastomosis improved bronchial healing by restoring a vascular flow across the anastomosis within 7 days after left lung allotransplantation. Since the introduction of heart-lung transplantation at Stanford (USA) in 1981 and Pittsburgh in 1982 for the treatment of Eisenmenger's syndrome and terminal pulmonary vascular diseases, more than 250 combined heart-lung transplantations have been carried out throughout the world. However the present intermediate and long term results obtained with this operation have not yet reached the same level of success as cardiac transplantation alone. While several factors such as selection of candidates and donors have played a role in this difference, a prominent cause has been the lack of a reliable and simple method for pulmonary protection against prolonged ischemia. Most of the techniques proposed against ischemia can be classified as normothermic or static hypothermic cardiopulmonary preservation. The utilization of the normothermic method has not always been successful. For this reason, interest has now been directed towards the potential for hypothermic preservation of the heart-lung bloc and the use of free radical scavenger therapy in the reduction of reperfusion injury.

Electrophoretic study of the caseinolytic activity of a pepsin from the dogfish Scyliorhinus canicula.

Electrophoretic patterns of casein and casein subfractions were studied following proteolysis by dogfish pepsin II or calf chymosin. Both enzymes hydrolyze the kappa casein subfraction with the production of kappa paracasein peptide. alpha S1 and beta subfractions hydrolysis is stronger with dogfish enzyme than with chymosin. It is concluded that, despite a broader specificity, the activity spectrum of dogfish enzyme is, in many respects, similar to that of calf chymosin.

Characterization of a chymosin-like pepsin from the dogfish Scyliorhinus canicula.

1. Pepsin II extracted from the gastric mucosa of Scyliorhinus canicula has been characterized and compared to calf chymosin. 2. The kcat and Km of the dogfish enzyme for the synthetic hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe have been determined. The kcat/Km ratio is close to that of calf chymosin. Its milk-clotting efficiency is however 21-fold lower than that of calf chymosin. 3. The proteolytic activity against haemoglobin is optimal at pH 2.5. It clots the milk up to pH 6.8. 4. The dogfish pepsin II shows relatively better activity at low temperatures than calf chymosin.

[A new technic for examining breast amputation specimens. Results of 11 cases of Paget's disease of breast]. / Une nouvelle technique d'examen des pièces d'amputation mammaire. Résultats concernant onze cas de maladie de Paget mammaire.

Mammectomy specimens fixed to maintain their anatomical shape may be cut in slices parallel to the thoracic wall. The resulting histologic slides (serially cut or single) always show all the ducts. With a comparatively small number of slides, the galactophoric system may be explored as a whole from the nipple to the surgical section. This technic is especially efficient to establish the extent of the intraductal lesions of the Paget's disease, their relation with the nipple and with a possibly associated invasive cancer. It leads to propose three groups of related lesions, the disease of the nipple being constant: 1 - an infiltrating cancer unrelated to the intraductal cancer. 2 - an infiltrating cancer associated to part of the ducts involved by intraductal cancer, several ducts in a distant part of the breast being involved too by intraductal carcinoma. 3 - intraductal cancer without invasive cancer. These facts and the topography of the intraductal carcinoma are inconsistent with the conception of cells from an invasive cancer reaching the epidermis of the nipple by spreading through the ducts.

Exogenous histones and chloroplastic DNA function in Chlamydomonas reinhardii.

The effect of exogenous added histones on chloroplastic DNA and RNA synthesis has been studied in whole Chlamydomonas reinhardii cells and in isolated chloroplasts. The observed inhibition is shown to be mainly due to the H1 histone and to result in drastic modifications of the cell cycle.