Evidence for enemy release in invasive common dace Leuciscus leuciscus in Ireland: a helminth community survey and systematic review.

Invasive species lose parasites in the process of invasion and tend to be less parasitized than conspecifics in the native range and sympatric native species in the invasive range (enemy release). We evaluated enemy release in an invasive freshwater fish in Ireland, common dace Leuciscus leuciscus, using helminth parasite community surveys at the core and front of the invasive range of common dace. Furthermore, we undertook a systematic literature review of helminth infection in common dace across its native range in Great Britain and Europe and invasive range in Ireland. The helminth parasite community survey revealed that invasive common dace were infected with fewer helminth species at the invasion front than at the core. Four helminth taxa - Acanthocephala, Monogenea, Digenea and Nematoda - were present in dace at the invasion core compared to only a single helminth species (Pomphorhynchus tereticollis) at the front. The systematic review revealed that invasive common dace in Ireland hosted fewer species of helminths than common dace in the native range. We report a total of three helminth species in common dace in Ireland compared to 24 in Great Britain and 84 in Continental Europe. Our results support the hypotheses that invasive populations are less parasitized than native populations and that more recently established populations host fewer parasites. However, we demonstrate that invasive species may continue to experience release from parasites long after initial invasion.

Copper and lead concentrations in salt marsh plants on the Suir Estuary, Ireland.

Concentrations of Cu and Pb were determined in the roots and shoots of six salt marsh plant species, and in sediment taken from between the roots of the plants, sampled from the lower salt marsh zone at four sites along the Suir Estuary in autumn 1997. Cu was mainly accumulated in the roots of monocotyledonous and dicotyledonous species. Pb was mainly accumulated in the roots of monocotyledons, while dicotyledons tended to accumulate Pb in the shoots. In the case of Aster tripolium there was a clear differentiation in the partitioning of Pb within the plant, between low and high salinity sites. At the low salinity sites, Pb accumulated only in the roots while at the high salinity sites there was a marked translocation to the shoots. The increase in Pb concentrations in roots and shoots of A. tripolium was accompanied by a concomitant decrease in sediment concentrations of Pb. This inverse correlation between sediment and plant concentrations of Pb was also recorded for Spartina spp. and Schoenoplectus tabernaemontani but in the case of these species the roots contained higher concentrations of Pb regardless of salinity levels. These differences in accumulation of Cu and Pb in various salt marsh species, and the influence of salinity on the translocation of Pb in A. tripolium in particular, should be taken into account when using these plants for biomonitoring purposes.

Kinetic properties of skeletal-muscle-like high-threshold calcium currents in a non-fusing muscle cell line.

Macroscopic kinetics and single-channel properties of skeletal-muscle-type calcium currents were studied in the non-fusing, clonal muscle cell line, BC3H1. Slowly activating, dihydropyridine(DHP)-sensitive currents, associated with T-tubular DHP receptors and ion channels, could be isolated from rapidly activating, DHP-resistant currents. Description of macroscopic current activation kinetics required only a brief delay term (tau o <1 ms), two ascending exponential terms with voltage-dependent time constants (2 < tau 1 > 20 ms and 10 < tau 2 < 200 ms), and a single exponential decay term (0.5 < tau 3 < or = 5 s). Steady-state activation voltage dependence required description by two Boltzmann distribution terms with V 1/2 and slope factors differing by 20 mV and 3.5- to 4-fold respectively. These two distributions were correlated with the steady-state voltage dependence of the two ascending kinetic terms described by tau 1 and tau 2 respectively. Rundown of the DHP-sensitive slow current was correlated with a negative shift in the voltage dependence of current decay (tau 3). Three conductance levels (4.5 pS, 8 pS and 12 pS) were detected in single-channel records, two of which (the 8-pS and 12-pS events) were prolonged by BayK8644 and thus associated with DHP-sensitive single-channel events. Description of single-channel open time distributions required a minimum of two exponential terms (2.5 +/- 0.9 ms and 10.3 +/- 5.4 ms at -10 mV). Slow transitions among closed states results in biexponential latency-to-first-event distributions (47 +/- 12 ms and 470 +/- 123 ms at -10 mV).

Three types of sodium channels in adult rat dorsal root ganglion neurons.

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.

A tentacle gel simplifies the purification of ceruloplasmin.

The mechanism of how chloroethylamine-treated agarose greatly simplifies the purification of ceruloplasmin (Cp) by preferentially binding this protein from blood plasma has been investigated. Chloroethylamine readily cyclizes to ethylenimine under alkaline conditions which then polymerizes to polyethylenimine (PEI). Ethylenimine and PEI were detected in the reaction mixture used to generate the resin. PEI polymers are grown from the matrix and form "tentacle" ligands, while PEI-silica and PEI-cellulose which are not tentacle gels did not bind Cp as readily. Spermine and spermidine, naturally occurring polyamine compounds, were attached to CNBr activated-agarose and showed weak affinity for Cp. [It should be noted that ethylenimine is a potential human carcinogen and presents an inhalation hazard.]

Ca2+ and filopodial responses to glutamate in cultured astrocytes and neurons.

Neurons and glia exhibit complex homeostatic interactions via shared extracellular space which can involve metabolites, inorganic ions, and neurotransmitters. Focal application of glutamate to both human and rat central nervous system astrocytes in primary culture produced a rapid, transient increase in both cytoplasmic and nuclear Ca2+. These Ca2+ waves can propagate at up to 15-20 micron/s for long distances (millimetres) through the astrocyte syncitium. Oscillatory Ca2+ signals were frequently observed under control conditions and were enhanced by glutamate application. These Ca2+ signals were paralleled by rapid extensions of filopodia from the astrocyte cell margin and apical surface near the point of glutamate application. Focal application of glutamate to rat hippocampal neurons also elicited rapid, transient increases in intracellular Ca2+. Levels of Ca2+ signals were consistently two- to three-fold greater in pyramidal neurons cultured from CA1 than in those from CA3. Filopodial extension was extensive in CA1 neurons, but rare in CA3 neurons, and in either case observable only during the first few days of primary culture. Diversity of glial and neuronal responses to binding the glutamate receptors may reflect their roles in homeostatic interactions.

Hemolysis of rabbit erythrocytes in the presence of copper ions. Inhibition by albumin and ceruloplasmin.

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.

Dihydropyridine receptor gene expression is regulated by inhibitors of myogenesis and is relatively insensitive to denervation.

To evaluate developmental and physiological signals that may influence expression of the dihydropyridine-sensitive "slow" Ca2+ channel, we analyzed dihydropyridine receptor (DHPR) mRNA abundance in mouse skeletal muscle. Using synthetic oligonucleotide probes corresponding to the rabbit skeletal muscle DHPR, a 6.5 kb DHPR transcript was identified in postnatal skeletal muscle and differentiated C2 or BC3H1 myocytes, but not cardiac muscle or brain. DHPR gene expression was reversibly suppressed by 0.4 nM transforming growth factor beta-1 or by transfection with a mutant c-H-ras allele, nominal inhibitors of myogenesis that block the appearance of slow channels and DHPR. In contrast, both BC3H1 and C2 myocytes containing the activated ras vector expressed the gene encoding the nicotinic acetylcholine receptor delta subunit, demonstrating that not all muscle-specific genes are extinguished by ras. Denervation stimulated DHPR gene expression less than 0.6-fold, despite 8-fold upregulation of delta-subunit mRNA and reciprocal effects on the skeletal and cardiac alpha-actin genes. Thus, DHPR gene induction is prevented by inhibitors of other muscle-specific genes, whereas, at most, relatively small changes in DHPR mRNA abundance occur during adaptation to denervation.

Vitamin D3 metabolites modulate dihydropyridine-sensitive calcium currents in clonal rat osteosarcoma cells.

A slowly inactivating inward calcium current was identified in the rat osteosarcoma cell line ROS 17/2.8 using a combination of ion flux and electrophysiological techniques. Voltage dependence, dihydropyridine sensitivity, divalent cation selectivity, and single channel properties identified this current as a high threshold, "L-type" calcium current. Ion flux experiments using 45Ca2+ confirmed that calcium uptake through these channel represents a major pathway for calcium entry into osteosarcoma cells. In resting cells, i.e. at negative membrane potentials, stimulation of both calcium current and rapid 45Ca2+ influx could be elicited by concentrations of 1,25-(OH)2-vitamin D3 between 0.1 and 3 nM. At these concentrations, 1,25-(OH)2-vitamin D3 shifted the threshold for activation of inward calcium current to more negative potentials. At higher concentrations (5-10 nM), inhibitory effects became predominant. These opposing effects are functionally similar to those of the dihydropyridine BAY K 8644. Other vitamin D3 metabolites (25-(OH)-D3 and 24,25-(OH)2-D3) exhibited less potent stimulatory effects and greater inhibition of calcium current than 1,25-(OH)2-D3. These results suggest that (i) vitamin D3 acts as a potent modulator of calcium channel function in osteosarcoma cells, and (ii) intracellular Ca2+-dependent signaling processes may be affected acutely by physiological concentrations of vitamin D3 metabolites.

Ca2+ and Na+ currents in developing skeletal myoblasts are expressed in a sequential program: reversible suppression by transforming growth factor beta-1, an inhibitor of the myogenic pathway.

We have analyzed the biophysical and developmental properties of Ca2+ and Na+ currents in C2 muscle cells, whose morphological and biochemical phenotype closely resembles differentiated skeletal muscle. Both fused and unfused C2 myocytes possessed: (1) membrane capacitance consistent with the presence of complex sarcotubular invaginations, (2) tetrodotoxin-sensitive Na+ channels, and (3) "fast" and "slow" Ca2+ channels that inactivated at holding potentials of -40 and -20 mV, respectively. Thus, the passive electrical properties, Na+ currents, and Ca2+ currents expressed in C2 cells each differed from those found in the nonfusing muscle cell line, BC3H1, and corresponded more precisely to characteristic findings observed in skeletal muscle fibers. In further contrast to BC3H1 cells, C2 muscle also expressed "transient" Ca2+ channels similar to those reported in embryonic or neonatal skeletal muscle, which were detected within 12-24 hr of mitogen withdrawal, up to 60 hr before appearance of "fast" and "slow" currents. Na+ channels also were induced 12-24 hr after mitogen withdrawal. Unlike the "fast" and "slow" Ca2+ currents, which were maximally expressed at 8-14 d of serum withdrawal, "transient" Ca2+ channels became down-regulated upon prolonged differentiation (as found in postnatal skeletal muscle in vivo) and were no longer expressed at 14 d. Despite their divergent kinetic and developmental properties, all components of Ca2+ and Na+ current in C2 myocytes were suppressed reversibly in the presence of transforming growth factor beta-1, a purified growth factor that inhibits the myogenic phenotype. The results indicate that fusion is not essential for skeletal myoblasts to produce developmentally regulated voltage-gated channels that resemble those of intact muscle and demonstrate that the formation of diverse Ca2+ and Na+ channels can be mediated by a single peptide that affects the myogenic pathway.

Electron transfer between heme proteins and ceruloplasmin.

Reduced cytochrome-c, reduced myoglobin and oxyhemoglobin respectively have been oxidized to oxidized cytochrome-c, metmyoglobin and methemoglobin by ceruloplasmin. Metmyoglobin and methemoglobin formation was stoichiometric while oxidized cytochrome-c reacted catalytically. Only 50% methemoglobin was formed which suggested that two hemes out of four could transfer electrons. Hydrogen peroxide was formed in the reaction of reduced cytochrome-c with ceruloplasmin.

A monoclonal antibody specifically modulates dihydropyridine-sensitive calcium current in BC3H1 myocytes.

The nonfusing muscle cell line BC3H1 expresses functional Na and Ca channels similar to those found in skeletal muscle (1). We have utilized a monoclonal antibody that cross-reacts with a 45-50-kDa glycoprotein in differentiated BC3H1 myocytes but not in cardiac or neuronal cells. This antibody, MAb1223, specifically modulates 45Ca influx and slowly activating, dihydropyridine-sensitive Ca currents when added to BC3H1 myocytes. Low-threshold, dihydropyridine-insensitive Ca currents and Na and K currents are unaffected. MAb1223 action is voltage dependent. At very negative holding potentials, similar to those of the resting cell, MAb1223 increases slow Ca current without significant changes in the voltage dependence of activation. At more positive potentials, at which channel opening probability is reduced by inactivation, exposure to MAb1223 reduces current. Agonist and antagonist dihydropyridines modulate the action of MAb1223 on the slow Ca channel in a manner that suggests that their respective binding sites may interact directly with the channel.

Control of expression of the 1,4-dihydropyridine receptor in BC3H1 cells.

To determine whether expression of the 1,4-dihydropyridine receptor of skeletal muscle Ca2+ channels is regulated by signals that impinge on muscle-specific gene expression, BC3H1 muscle cells were analyzed using (+)[3H]PN200-110 as a probe for the receptor. No dihydropyridine binding sites were detected in proliferating cells. Binding site density increased following serum withdrawal, peaking at day six, with little or no change in Kd (approximately equal to 250 pM, similar to that seen in skeletal muscle). No DHP binding sites were detected in BC3H1 cells bearing an activated c-H-ras oncogene. Induction of the dihydropyridine receptor was reversibly blocked by 200 pM transforming growth factor beta. The results indicate that formation of dihydropyridine-sensitive Ca2+ channels may require up-regulation of the dihydropyridine receptor itself, and that transforming growth factor beta is a potent, reversible inhibitor of this receptor in BC3H1 muscle cells.

Monoclonal antibody to the alpha 1-subunit of the dihydropyridine-binding complex inhibits calcium currents in BC3H1 myocytes.

A monoclonal antibody, mAb 1A, previously shown to recognize the dihydropyridine (DHP)-binding complex in rabbit muscle transverse tubules, inhibits voltage-sensitive calcium channel activity in a mouse muscle cell line. mAb 1A applied externally to BC3H1 muscle cells produced a concentration-dependent attenuation of the slowly-activating, DHP-sensitive, high-threshold calcium current. mAb 1A had no direct effect on either the rapidly-activating, DHP-insensitive, low-threshold calcium current or the delayed outward potassium current. However, sodium current kinetics were altered. On Western blot analysis of immunoaffinity-purified protein solubilized from BC3H1 membranes, mAb 1A recognized a Mr 210,000 polypeptide that is an analogous to the alpha 1 subunit of the DHP-binding complex from skeletal muscle. These results suggest that an extracellular segment of the alpha 1 subunit of the DHP-binding complex plays a functionally important role in voltage-gated calcium currents.

A comparative study of copper ion induced lysis of vertebrate red blood cells.

1. Erythrocytes from different vertebrate classes were tested for susceptibility towards copper ion-induced lysis under identical copper ion concentration and per cent cell volumes. 2. The susceptibility towards lysis was found to be correlated with the rate of copper ion entry into the erythrocytes. 3. GSH levels decline in red blood cells at a rate proportional to the rate of copper ion entry. 4. Hemolysis does not seem to be causally related to the level of GSH in the erythrocytes.

Expression of single calcium channels in Xenopus oocytes after injection of mRNA from rat heart.

Oocytes of Xenopus laevis, after microinjection with mRNA from rat heart, display typical high-threshold calcium (Ca) whole cell currents. To prepare to study structure-function relationships of the cardiac Ca channel molecule, we examined the fidelity of expression of biophysical and pharmacological properties at the molecular level. Cell-attached gigaseal recordings in five K-depolarized oocytes injected with adult rat heart mRNA showed single channel Ba currents with mean amplitude 1.3-1.5 pA at 0 mV, slope conductance 18-25 pS, and extrapolated reversal potential 57-68 mV. Openings were predominantly brief (mean 1.2 ms) but longer openings (mean 9 ms) were greatly enhanced in 10(-6) M BAY-K 8644, increasing the ensemble average current at 0 mV by more than fivefold. These features are typical of high-threshold cardiac Ca channels. In two patches from one injected oocyte, we saw multiple Ca channel conductances, as recently observed in other preparations. We conclude that X. laevis oocytes injected with adult rat heart mRNA produce high-threshold cardiac Ca channels with molecular properties identical to native cells.

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels.

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.

The effect of copper ion on glutathione and hemolysis in rabbit erythrocytes.

The rate of hemolysis and the decline in glutathione (GSH) in rabbit erythrocytes caused by copper (Cu) ions were determined. Prior investigations have proposed that the oxidative stress induced by Cu ion depleted the normal cell protective mechanisms. The decline in GSH has been proposed as a necessary prerequisite for hemolysis. We have observed that both GSH decline and hemolysis are Cu dependent, but are two concurrent and independent processes. We have confirmed that oxygen is a necessary reactant for hemolysis and responsible for a major portion of GSH decline. However, in the presence of Cu ion, a slow decline in GSH occurs even in a deaerated system.