Gerhard Goffinet

Characterization and supramolecular architecture of the cellulose‐protein fibrils in the tunic of the sea peach (Halocynthia papillosa, Ascidiacea, Urochordata)

Summary— The cellulose‐protein fibrils, which constitute by far the bulk of the fibrous fraction of the sea peach tunic (Halocynthia papillosa), were structurally and chemically characterized, either in situ or after extraction procedures, with the use of classical electron microscoy combined with diffraction contrast imaging and electron diffraction, histochemistry, affinity cytochemistry and chemical analysis. These fibrils exhibit a cross‐sectional shape close to a parallelogram. The cyrstallites forming their core, with lateral dimensions ranging from roughly 5 to 20 nm, are composed of native cellulose of higher crystallinity than that of plant cellulose. They are associated with acid mucopolysaccharidés (amps) and proteins which form a coating material appearing as a continuous sheath enveloping the axial crystallite in the cuticular layer or as patches more‐or‐less periodically distributed around and along the fibre axis in the fundamental layer. Tunicin, the alkali‐insoluble fibrous fraction, is not pure cellulose, yielding only 22–60% of its dry weight as glucose equivalents, depending on the tunical layer. It is suggested that in addition to the high degree of crystallinity of the tunical cellulose, the presence of a significant amount of coating material composed of amino acids and proteoglycans firmly linked to cellulose molecules contributes to tunicins high resistance to hydrolysis.

Stable isotopic composition of chitin from arthropods recovered in archaeological contexts as palaeoenvironmental indicators

Abstract The effects of biodegradation and heating on the stable carbon, nitrogen, hydrogen and oxygen isotope ratios of chitin in arthropods were studied. Chitinous exoskeletons from seven aquatic arthropod species were subjected to anaerobic marine biodegradation in mud, to terrestrial biodegradation in soils, and to thermal degradation under anaerobic and aerobic conditions. The isotope ratios of chromatographically separated D-glucosamine hydrochloride and derivatives from treated and untreated specimens were then compared. Carbon, nitrogen, oxygen and hydrogen stable isotope ratios were all found to be conserved during partial degradation of chitin. Micromorphological comparative studies using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that no fungal chitin or other contaminants were present in the chitins that were chemically isolated from biodegraded substrates. Our results indicate that it will be possible to use stable isotope ratios of archaeological chitin samples for environmental and climatic reconstructions. An illustration of the utility of this approach comes from the observation that the stable isotope ratios of chitin from crustacean exoskeletons recovered from archaeological sites with ages up to 1400 years bp are in good agreement with measurements on modern crustaceans from similar environments.

Elaboration and ultrastructural changes in the pore canal system of the mineralized cuticle of Carcinus maenas during the moulting cycle

Two basic structural components are concerned in the elaboration of the pore canal system in the mineralized cuticle of the decapod crab Carcinus maenas: tubular cytoplasmic extensions originating from epidermis and vertical fibres. These components are present from the moment the first procuticular materials of the new cuticle are laid down but their organization varies according to a precise schedule during the further moult cycle stages. Cytoplasmic extensions form a complicated branching system connecting the epidermal layer with all regions of the cullcular compartment, at least transitorily. During the moult cycle the prolongation of this cellular system appears to result from two concomitant but opposite phenomena. Before ecdysis the growth of cell extensions in the proximal cuticular layers prevails over their regression at the distal level. During the post-moult period these phenomena are reversed in importance so that the pore canal system is without cytoplasmic material as soon as intermoult starts. The depositing of vertical fibres takes place in close contact with the proximal cell extension plasma membrane, which never bears dense plaques. As moult stages progress, they are gradually organized into twisted sheaths that persist throughout the intermoult. Incidentally, some fibres invade the pore canal lumen freed from cell extensions. Some aspects regarding the fine organization, the chemical composition and the functional significance of both epidermal tubular extensions or vertical fibres are also discussed in the light of previous investigations carried out on crustaceans and in other arthropods.

Ultrastructural shape and three-dimensional organization of the intracuticular canal systems in the mineralized cuticle of the green crab Carcinus maenas

Two main self-contained canal systems are present in the crab mineralized cuticle. The first, or fibre canal system, is constituted by simple, unbranched vertical canals containing axially running fibres in close association with myoepidermal junctions. The second, or pore canal system, is composed of procuticular pore canals and epicuticular channels that prolong the procuticular canals. In opposition to widespread opinion, pore canals make up a three-dimensional branched system extending from the apical plasma membrane of the epidermis up to the epicuticle. Branching occurs by projections of lateral horizontal from the vertical canals at the lower level of the pigmented layer and by innumerable ramifications of epicuticular canals. In agreement with Nevilles model for insects, vertical procuticular pore canals of crustacean mineralized cuticle, and also fibre canals, exhibit a twisted ribbon structure reflecting the helicoidal arrangement of the horizontal chitin-protein microfibrils.

Microencapsulation Of Erythromycin And Clarithromycin Using A Spray-Drying Technique

Previous methods of microencapsulation are unable to process particles smaller than 100 microm without organic solvents or the use of multistep processes. The present study investigates the feasiblity of a one-step spray-drying process to microencapsulate erythromycin and clarithromycin, antibiotics known to have an unpleasant, bitter taste. Mixtures of clarithromycin (5% by weight) or erythromycin (30% by weight) with a biodegradable polymer were prepared and spray-dried under specific conditions of temperature and turbine speed. This process resulted in the microencapsulation of 80% of each drug as determined by high pressure liquid chromatography. Particle size ranged from 1 to 80 microm as determined by electron microscopy. These data show that microencapsulation of macrolides using a spray-drying technique is feasible. Spray-drying microencapsulation might be useful in the formulation of palatable oral suspensions of bitter tasting drugs.

Characterization of the primary sonic muscles in Carapus acus (Carapidae): a multidisciplinary approach

Sound production in carapid fishes results from the action of extrinsic muscles that insert into the swim bladder. Biochemical, histochemical and morphological techniques were used to examine the sonic muscles and compare them with epaxial muscles in Carapus acus. Sonic fibres are thicker than red and thinner than white epaxial fibres, and sonic fibres and myofibrils exhibit an unusual helicoidal organization: the myofibrils of the centre are in a straight line whereas they are more and more twisted towards the periphery. Sonic muscles have both features of red (numerous mitochondria, high glycogen content) and white (alkali–stable ATPase) fibres. They differ also in the isoforms of the light chain (LC3) and heavy chain (HC), in having T tubules at both the Z–line and the A–I junction and in a unique parvalbumin isoform (PAI) that may aid relaxation. All these features lead to the expression of two assumptions about sound generation: the sonic muscle should be able to perform fast and powerful contractions that provoke the forward movement of the forepart of the swim bladder and the stretching and ‘flapping’ of the swim bladder fenestra; the helicoidal organization allows progressive drawing of the swim bladder fenestra which emits a sound when rapidly released in a spring–like manner.

PCB CONTAMINATION OF THE COMMON BARBEL, BARBUS BARBUS (PISCES, CYPRINIDAE), IN THE RIVER MEUSE IN RELATION TO HEPATIC MONOOXYGENASE ACTIVITY AND ULTRASTRUCTURAL LIVER CHANGE

To evaluate the impact of PCBs on a wild population of a regressing fish species, we have measured the levels of these toxicants in common barbel (Barbus barbus) from the Belgian part of the river Meuse. We have expressed these levels in terms of the most suitable composition of commercial PCB mixture (Aroclor 1254 and 1260 20/80; v/v), and related them to hepatic xenobiotic-metabolising enzyme activities and to hepatocyte ultrastructure.PCB concentrations in barbel organs were extremely high, with no statistical difference between the two sexes at equal weight. In liver, muscle, and gonads, PCB concentrations increased significantly with age, reaching 20 μg g−1 DW in 12- to 15-year-old individuals. The activities of the monooxygenases (ethoxyresorufin o-deethylase, EROD, and ethoxycoumarin o-deethylase, ECOD) and the cytochrome P-450 content correlated closely with the PCB concentration in fish liver. Moreover, wild fish presented a markedly altered liver ultrastructure, as compared to controls; the rough endoplasmic reticulum (RER) was particularly abundant and the mitochondrial membranes were altered. PCBs thus alter essential metabolic functions in wild barbels, which constitute a highly sensitive tool for biomonitoring wild fish populations. While the effects of PCBs on metabolic pathways may combine additively or synergistically with effects of other xenobiotics, it has been demonstrated elsewhere that they decrease successful reproduction. Their chronic negative effects have thus played a role in reducing barbel populations in highly polluted areas.

Glycoproteins from the cuticle of the Atlantic shore crab carcinus maenas: I. Electrophoresis and western-blot analysis by use of lectins

The protein and glycoprotein content of four different neutral or acidic solvent extracts (0.5 M KCl, 10% EDTA, 0.1 N HCl, or 2% acetic acid) from the mineralized exoskeleton of a decapod crustacean, the Atlantic shore crab Carcinus maenas, were characterized by quantitative analysis of proteins, SDS-PAGE analysis, and probing with lectins on blots. The lectins used were Conconavalin A, Jacalin, soybean agglutinin, Maackia amurensis agglutinin II, and Sambucus nigra agglutinin. The results show that many proteins can be obtained from the crab cuticle without strong denaturants in the extraction medium. Many of the extracted cuticle proteins appeared to be glycosylated, bearing O-linked oligosaccharides and N-linked mannose-rich glycans. N-acetyl-galactosamine and N-acetylneuraminic acids were revealed, for the first time, as terminal residues on N-linked mannose-rich structures of crab cuticle glycoproteins. Sialylated glycoproteins might thus be involved in organic-mineral interactions in the calcified crab exoskeleton. The amount and variety of glycoproteins extracted with the acidic solvents are obviously different from those extracted with neutral solvents. HCl proved to be the best of the tested extraction solvents and a valuable alternative to EDTA.

Coagulocyte Alterations in Clotting Hemolymph of Carausius morosus L

1. The structural changes in the coagulocytes of Carausius morosus during hemolymph coagulation in vitro have been studied under the PCM and in the TEM. 2. In agreement with former PCM observations on Carausius morosus, the coagulocytes are the only hemocytes to induce coagulation of the plasma. Immediately or after a few seconds upon withdrawal of the hemolymph, their structural changes consist of a considerable enlargement of the perinuclear cysterna and of direct ejection into the plasma of nuclear and cytoplasmic substances through microruptures of the cytoplasmic membrane. The other categories of hemocytes do not contribute to the plasma coagulation. Their structural alterations take place without breakage of the cytoplasmic membrane when the plasma reactions are already established. 4. These plasma reactions appear in the form of circular islands of granular material around the coagulocytes, of extension of the coagulum in the channels between the islands and of transformation of the clot into a network of threads. 5. As reported in other studies in the TEM, no specific organelle characteristic of the coagulocyte ultrastructure could be found in the coagulocytes of Carausius. 6. Owing to the absence of any specific structural criterion of identification, the results suggest that the functional difference between coagulocytes and the other categories of hemocytes as regards coagulation of the plasma might be caused in part by differences of permeability of the cell membranes.

Immunocytochemistry of lipids : chemical fixatives have dramatic effects on the preservation of tissue lipids

We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidyl-serine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73–91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.