A covalently cross-linked gel derived from the epidermis of the pilot whale Globicephala melas.

The rheological properties of the stratum corneum of the pilot whale (Globicephala melas) were investigated with emphasis on their significance to the self-cleaning abilities of the skin surface smoothed by a jelly material enriched with various hydrolytic enzymes. The gel formation of the collected fluid was monitored by applying periodic-harmonic oscillating loads using a stress-controlled rheometer. In the mechanical spectrum of the gel, the plateau region of the storage modulus G' (<1200 Pa) and the loss modulus G" (>120 Pa) were independent of frequency (omega = 43.98 to 0.13 rad x s(-1), tau = 15 Pa, T = 20 degrees C), indicating high elastic performance of a covalently cross-linked viscoelastic solid. In addition, multi-angle laser light scattering experiments (MALLS) were performed to analyse the potential time-dependent changes in the weight-average molar mass of the samples. The observed increase showed that the gel formation is based on the assembly of covalently cross-linked aggregates. The viscoelastic properties and the shear resistance of the gel assure that the enzyme-containing jelly material smoothing the skin surface is not removed from the stratum corneum by shear regimes during dolphin jumping. The even skin surface is considered to be most important for the self-cleaning abilities of the dolphin skin against biofouling.

Transepithelial potential differences and Na(+) flux in isolated perfused gills of the crab Chasmagnathus granulatus (Grapsidae) acclimated to hyper- and hypo-salinity.

We studied the transepithelial potential difference (TEPD) and (22)Na flux across isolated perfused gills (anterior pair 5 and posterior pairs 6-8) of the crab Chasmagnathus granulatus acclimated to either hypo- or hyper-osmotic conditions. The gills of crabs acclimated to low salinity, perfused and bathed with 10 per thousand saline solutions, produced the following TEPDs (hemolymph side with respect to bath side): 0.4+/-0.7, -10.2+/-1.6, -10.8+/-1.3 and -6.7+/-1.3 mV for gills 5, 6, 7 and 8, respectively. Gills 6, 7 and 8 did not differ significantly. Reducing the saline concentration of bath and perfusate from 30 per thousand to 20 per thousand or 10 per thousand increased significantly the TEPDs of these gills. TEPDs of gill 6 (representative of posterior gills) were reduced by 69+/-5 % and 60+/-5 % after perfusion with ouabain or BaCl(2) (5 mmol l(-1) each), respectively. The same gill showed a net ouabain-sensitive Na(+) influx of 1150+/-290 microequiv g(-1) h(-1). Gill 6 of crabs acclimated to high salinity produced TEPDs of -1.5+/-0.1 and -1.3+/-0.09 mV after perfusion with 30 per thousand or 40 per thousand salines, respectively. Perfusion with ouabain or BaCl(2) reduced TEPDs by 76+/-7 % and 86+/-4 %, respectively. A net ouabain-sensitive Na(+) efflux of 2282+/-337 microequiv g(-1) h(-1) was recorded in gill 6 perfused with 38 per thousand saline.

A zymogel enhances the self-cleaning abilities of the skin of the pilot whale (Globicephala melas).

Enzyme activity in the stratum corneum of the pilot whale Globicephala melas was investigated employing colorimetric enzyme screening assays combined with NATIVE PAGE, size exclusion chromatography (SEC) and histochemical staining procedures. Applying different substrates, several enzymes were detected. The histochemical demonstration of some selected hydrolytic enzymes enriched in the stratum corneum showed high extracellular accumulation. As demonstrated by size exclusion chromatography, high molar mass aggregates were built up from a glycoprotein-rich 20-30-kD fraction. Using NATIVE PAGE experiments under non-reducing conditions, a selection of five degrading enzymes was recovered within the above-reported aggregates. Activity of extracellular aggregate-attached enzymes in the superficial layer of the stratum corneum exhibited no remarkable decrease potentially resulting from self-degradation. We thus conclude that due to their enclosure within the microenvironment of aggregates, a zymogel is formed and autolysis of the stratum corneum is reduced. With respect to the skin surface, the zymogel with hydrolytic activities covering major parts of it enhances the self-cleaning abilities of the skin of the pilot whale based on physical pre-requisites by hydrolyzing adhesive glycoconjugates of settling biofouling organisms considered as primary steps in fouling.

Ionic balance in the freshwater-adapted Chinese crab, Eriocheir sinensis.

Ionic regulation by the gills of the freshwater-adapted Chinese crab, Eriocheir sinensis, was examined. The balance of uptake and loss of NaCl in crabs living in freshwater was established. Urine production was measured directly by cannulating the nephropores. Daily urinary loss of Na+ is equivalent to 16% of the haemolymph Na+ content and is substantially higher than that based on data from indirect measurements reported in the literature. Weight and area of anterior and posterior gills are proportional to body weight. The role of the gills in compensating urinary loss by uptake was determined by analysing changes in Na+ and Cl- concentrations in the external medium in which isolated perfused gills were suspended. In posterior gills, salt loss is quantitatively balanced by NaCl net uptake from an external concentration of 1.3 mmol l(-1) NaCl upwards. The transport constant (Kt) for half maximum saturation of net uptake and saturation of NaCl uptake are 1.5 mmol l(-1) and 4 mmol l(-1), respectively. In contrast to previous studies in which tracer fluxes or transepithelial short-circuit currents were determined, our method of direct ion determination shows that no net uptake of Na+ or Cl- occurs in posterior gills in the absence of the respective counter ion, or when uptake of one ion is blocked by a specific inhibitor. Net uptake of Na+ and Cl- was about equal. We conclude that the uptake of the two ions is coupled. The properties of the branchial ion uptake of E. sinensis correlates with the distribution of this crab in river systems.

Molecular characterization of V-type H(+)-ATPase (B-subunit) in gills of euryhaline crabs and its physiological role in osmoregulatory ion uptake.

The vacuolar-type H(+)-ATPase (V-ATPase) has been implicated in osmoregulatory ion uptake across external epithelia of a growing variety of species adapted to life in fresh water. In the present study, we investigated whether the V-ATPase may also function in a euryhaline species that tolerates brackish water (8 salinity) but not fresh water, the shore crab Carcinus maenas. cDNA coding for the regulatory B-subunit of the V-ATPase was amplified and sequenced from C. maenas gills and partially sequenced from four other crab species. Two isoforms differing in the 3'-untranslated region were found in C. maenas. In this species, the abundance of B-subunit mRNA was greater in the respiratory anterior gills than the ion-transporting posterior gills and was not increased by acclimation to dilute salinity. Immunocytochemical analysis showed that the B-subunit protein is not targeted to the apical membrane but is distributed throughout the cytoplasmic compartment. Physiological studies of isolated perfused gills indicated that the V-ATPase inhibitor bafilomycin had no effect on transepithelial potential difference. Thus, in contrast to the freshwater-tolerant Chinese crab Eriocheir sinensis, in which the V-ATPase appears to play an important osmoregulatory role, the V-ATPase in C. maenas probably functions in acidification of intracellular organelles but not in transbranchial NaCl uptake.

Active osmoregulatory ion uptake across the pleopods of the isopod Idotea baltica (Pallas): electrophysiological measurements on isolated split endo- and exopodites mounted in a micro-ussing chamber.

The mechanism of active, osmoregulatory ion uptake was investigated in the pleopods of the marine isopod Idotea baltica (Pallas). Using isolated split half-podites of isopods acclimated to brackish water (20 salinity) mounted in a micro-Ussing chamber and symmetrically superfused with identical haemolymph-like salines, a mean short-circuit current I(sc) of -445 microA cm(-)(2) was measured in endopodites 3-5, corresponding to an inwardly directed transcellular movement of negative charge. Application of ouabain (5 mmol l(-)(1)) to the basolateral superfusate resulted in the almost total abolition of the I(sc) (reduced from -531 to -47 microA cm(-)(2)), suggesting that the Na(+)/K(+)-ATPase is the driving force for active, electrogenic uptake of NaCl. In contrast, mean I(sc) values close to zero were found in preparations of all exopodites and in endopodites 1 and 2. The specific activities of Na(+)/K(+)-ATPase corresponded with these results. Specific activities were highest in posterior endopodites 3-5 and depended on ambient salinity. In all other rami, the activities were much lower and independent of ambient salinity. Activities in posterior endopodites 3-5 were lowest in isopods acclimated to 30 salinity (2-4 micromol P(i )mg(-)(1 )protein h(-)(1)), increased in individuals kept in 20 salinity (8.4 micromol P(i )mg(-)(1 )protein h(-)(1)) and were highest in isopods acclimated to 15 salinity (18.2 micromol P(i )mg(-)(1 )protein h(-)(1)). When specimens were transferred from 30 to 40 salinity, Na(+)/K(+)-ATPase activity increased in the posterior endopodites. The electrophysiological and Na(+)/K(+)-ATPase activity measurements show that active electrogenic ion transport in this species occurs almost exclusively in posterior endopodites 3-5. The endopodite of the fifth pleopod of I. baltica exhibited a microscopic structure remarkably similar to that described for the lamellae of the phyllobranchiae of brachyurans. It is composed of two opposed epithelial monolayers of ionocytes, each covered by cuticle. Bundles of pillar cells are located within the ionocyte layers, which are separated by a fenestrated lamellar septum of connective tissue. The results obtained in this study indicate that endopodites 3-5 play the main role in osmoregulatory ion uptake of the isopod I. baltica. Moreover, the Na(+)/K(+)-ATPase is the only driving force behind active electrogenic ion uptake across the epithelial cells.

Active absorption of Na+ and Cl- across the gill epithelium of the shore crab Carcinus maenas: voltage-clamp and ion-flux studies

Mechanisms of active NaCl uptake across the posterior gills of the shore crab Carcinus maenas were examined using radiochemical and electrophysiological techniques. In order to measure short-circuit current (Isc), transepithelial conductance (Gte) and area-related unidirectional fluxes of Na+ and Cl-, single split gill lamellae (epithelium plus cuticle) of hyperregulating shore crabs were mounted in a modified Ussing chamber. The negative short-circuit current measured with haemolymph-like NaCl saline on both sides of the epithelium could be inhibited by application of basolateral ouabain (ouabain inhibitor constant KOua=56±10 µmol l-1), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; KNPPB=7.5±2.5 mmol l-1) or Cs+ (10 mmol l-1). From the apical side, Isc was nearly completely blocked by Cs+ (10 mmol l-1) or Ba2+ (15 µmol l-1), whereas apical addition of furosemide (1 mmol l-1) resulted in only a small current decrease. Cl- influxes were linearly related to negative Isc. The ratio between net influxes of Cl- and Na+ was found to be approximately 2:1. With a single membrane preparation, achieved by permeabilizing the basolateral membrane with amphotericin B, Cl- influxes which were driven by a concentration gradient were shown to depend on the presence of apical Na+ and K+. On the basis of these observations, we propose that active and electrogenic absorption of NaCl across the gill epithelium of hyperregulating shore crabs proceeds as in the thick ascending limb of Henle's loop in the mammalian nephron. Accordingly, branchial NaCl transport is mediated by apical K+ channels in cooperation with apical Na+/K+/2Cl- cotransporters and by the basolateral Na+/K+-ATPase and basolateral Cl- channels.

Biochemical responses and environmental contaminants in breams (Abramis brama L.) caught in the river Elbe.

Adult breams (Abrama brama L.) were caught in October 1992 at seven stations in the river Elbe and at one nonpolluted reference site, the Belauer See. The locations of the sampling stations extended from the city of Steti (Tschechien Republic) to the city of Hamburg. Indices of biochemical effects in microsomal and cytosolic fractions of livers were studied by measuring cytochrome P450-dependent monooxygenases and glutathione-S-transferase (GST) activities. In addition, levels of mercury and 35 polychlorinated biphenyl (PCB) congeners were analyzed in livers of breams. Fish caught in the River Elbe exhibited a significant increase of cytochrome P450-mediated monooxygenase activities and the detoxifications enzyme GST compared to the reference site. At two stations of the river Elbe (Steti and Dresden) elevated activities of ethoxyresorufin-O-deethylase (EROD) were analyzed. These effects were discussed as effects from the pulp mill industries at station Steti and high concentrations of PCBs in the livers of breams at station Dresden. A significant reduction of GST activities was observed at station Dresden compared to those at Steti. These findings were probably a synergistic effect of high mercury concentrations at Dresden. The results presented in this study suggest that breams can be successfully employed for monitoring biological effects in the river Elbe.

Induction of the hepatic biotransformation system of golden ide [Leuciscus idus (L.)] after exposure in the River Elbe.

The xenobiotic-metabolizing enzyme system of hatchery golden ide [Leuciscus idus (L.)] were tested for applicability in biological monitoring in rivers. The golden ide, cyprinid fish used for toxicity testing, were caged for 1 to 3 weeks in flowthrough systems at various locations of the river Elbe. The activities of 7-ethoxycoumarin-O-deethylase (ECOD) and the cytochrome P450 (P450) concentrations in the livers were analyzed. Exposure in the river Elbe increased the ECOD activity and the P450 concentration. In a flowthrough basin located in the harbor of Hamburg the ECOD activity increased up to 3.5 times, and the P450 concentration increased slightly by a factor of 1.5. During exposure of the golden ide at two Elbe stations upstream and downstream the city of Hamburg, levels of P450 in the livers did not change significantly when compared with laboratory controls. There was, however, a significant increase of ECOD activity between stations Bunthaus (upstream) and Blankenese (downstream). The ECOD activity in the livers of golden ide caged downstream were significantly higher compared to those at the upstream station. The results presented in this study suggest that golden ide can be employed as experimentals for routine measurements in limnic habitats using biochemical assays.

Do amiloride and ouabain affect ammonia fluxes in perfused Carcinus gill epithelia?

The effects of inhibitors on the efflux of ammonia (from the basolateral to the apical side, Jb----a) were studied in preparation of isolated Carcinus gills immersed in dilute seawater (DSW) that was identical to the perfusion solution. Adding 10(-4) M amiloride to the solution bathing the gill preparations reduces the efflux of ammonia by 29% relative to the control value. Under experimental conditions, it appears that only about 1% of the amiloride-sensitive influx of Na+ (Ja----b) can be exchanged with NH4+ on an equimolar basis. The ammonium ion is apparently transported at the basolateral side by a carrier-mediated process. Kinetic analyses of the influx of ammonium ions revealed a Km of 36.99 microM and a maximum velocity (Vmax) equal to 19.6 mumol g-1.h-1. Basolaterally applied ouabain (5 x 10(-3) M) and NaCN (10(-3) M) reduced the efflux of ammonia by 46.7 and 42.2%, respectively, suggesting an interaction of NH4+ with the basolaterally located Na+/K+ exchanger in which NH4+ appears to be able to substitute for K+.