Modulation of Ca2+ dependent protease activity in fish and invertebrates by weak low-frequency magnetic fields

Results of experiments addressing the effects of weak low-frequency magnetic fields on intracellular Ca²⁺ dependent proteinases (calpains) from invertebrates and fish are discussed. Exposure of live animals to weak low-frequency magnetic fields with parameters chosen to induce the resonance of Ca²⁺ ions led to a significant decrease of calpain activity in the animals investigated. The physical factor studied also caused partial loss of activity in preparations of Ca²⁺ dependent proteinases obtained from invertebrates and fish. The phenomenon discovered is in accordance with the interference model of the effect of weak low-frequency magnetic fields on biological objects.     

Differential diagnosis of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> and <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>: nymphs and larvae

We developed a method for differential diagnosis of nymphs and larvae of sheep (Ixodes ricinus (L.)) and taiga (I. persulcatus Sch.) ticks (Parasitiformes: Ixodidae) which allows to identify live material in the field.     

Calpains expression during Xenopus laevis development

Calpains are cytoplasmic proteases activated by calcium, implicated in cell differentiation and apoptosis. The best characterized enzymes are calpains 1-3. The aim of this work was to localize calpains 1-3 during the development of Xenopus laevis in order to clarify the function of these three proteases. For the first time, we detected the localization of the three proteases at the protein level between one-cell stage and adult age. Their expression was weak at early stages, then increased at tadpole stage and decreased through metamorphosis and adult life. The calpain's expression was maximal during the period characterized by the appearance of organs and modelling process. These observations suggest that calpains play a crucial role during development.     

Characterization of the definitive classical calpain family of vertebrates using phylogenetic,evolutionary and expression analyses

The calpains are a superfamily of proteases with extensive relevance to human health and welfare. Vast research attention is given to the vertebrate ‘classical’ subfamily, making it surprising that the evolutionary origins, distribution and relationships of these genes is poorly characterized. Consequently, there exists uncertainty about the conservation of gene family structure, function and expression that has been principally defined from work with mammals. Here, more than 200 vertebrate classical calpains were incorporated in phylogenetic analyses spanning an unprecedented range of taxa, including jawless and cartilaginous fish. We demonstrate that the common vertebrate ancestor had at least six classical calpains, including a single gene that gave rise to CAPN11, 1, 2 and 8 in the early jawed fish lineage, plus CAPN3, 9, 12, 13 and a novel calpain gene, hereafter named CAPN17. We reveal that while all vertebrate classical calpains have been subject to persistent purifying selection during evolution, the degree and nature of selective pressure has often been lineage-dependent. The tissue expression of the complete classic calpain family was assessed in representative teleost fish, amphibians, reptiles and mammals. This highlighted systematic divergence in expression across vertebrate taxa, with most classic calpain genes from fish and amphibians having more extensive tissue distribution than in amniotes. Our data suggest that classical calpain functions have frequently diverged during vertebrate evolution and challenge the ongoing value of the established system of classifying calpains by expression.     

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.     

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.     

Calpain-dependent Cleavage of N-cadherin Is Involved in the Progression of Post-myocardial Infarction Remodeling

Enzymatic proteolysis by calpains, Ca²⁺-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast^−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast^+/+ mice. The activity of cardiac calpains in Cast^+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast^−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast^−/− and Cast^+/+ mice, Cast^−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast^+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast^−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast^+/+ mice. In the border zone of Cast^−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction.     

Size and settling behaviour in male and female cypris larvae of the parasitic barnacle Sacculina carcini Thompson (Crustacea: Cirripedia: Rhizocephala)

Twenty-one broods from different externae of Sacculina carcini Thompson were cultured to the cypris stage. The size of the cyprids was measured and the larvae subjected to settling upon unparasitized crabs (Garcinus maenas (L.)) and small juvenile externae. The cyprids occur in two sizes that may appear singly or together in the same brood. Small cyprids are of the female sex that settle upon crabs and are infective, while large cyprids are of the male sex and only settle upon juvenile externae. These results are in agreement with other well-studied rhizocephalans.     

The distribution of Zostera and its exploitation by wildfowl in the solent,Southern England

In the Solent estuarine system on the central south coast of England, Zostera marina L. occurs both in mainly sublittoral beds on firm substrate around the low-water mark of spring tides, and in large eulittoral beds on mudflats between the low-water mark of neap tides and the uppermost level of the shore. Zostera noltii Hornem. occurs with eulittoral Z. marina on uppershore mudflats and also on exposed sandy shores near the low-water mark of spring tides. Since 1960 the eulittoral Zostera beds have spread widely on mudflats in Portsmouth, Langstone and Chichester Harbours, where they now occupy 430–450 ha. The Zostera beds at the bottom of the shore remain winter-green, but the mudflat Zostera beds shed most of their leaves between November and January. Comparisons are made with localities further east, which show earlier and greater leaf loss.The uppershore Zostera beds in the three harbours comprise an important and favoured autumn and early winter food for Dark-bellied Brent Geese, (Branta bernicla bernicla (L.)) and Wigeon (Anas penelope L.), which consume the leaves and rhizomes, and Teal (Anas crecca L.) which consume the seed. The pattern of occurrence of these three species of birds is, to some extent, related to the availability of Zostera.     

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov,Nassonova et Cavalier-Smith, 2008

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.     

In situ gross nitrogen transformations differ between temperate deciduous and coniferous forest soils

Despite long-term enhanced nitrogen (N) inputs, forests can retain considerable amounts of N. While rates of N inputs via throughfall and N leaching are increased in coniferous stands relative to deciduous stands at comparable sites, N leaching below coniferous stands is disproportionally enhanced relative to the N input. A better understanding of factors affecting N retention is needed to assess the impact of changing N deposition on N cycling and N loss of forests. Therefore, gross N transformation pathways were quantified in undisturbed well-drained sandy soils of adjacent equal-aged deciduous (pedunculate oak (Quercus robur L.)) and coniferous (Scots pine (Pinus sylvestris L.)) planted forest stands located in a region with high N deposition (north Belgium). In situ inorganic ¹⁵N labelling of the mineral topsoil (0–10?cm) combined with numerical data analysis demonstrated that (i) all gross N transformations differed significantly (p?<?0.05) between the two forest soils, (ii) gross N mineralization in the pine soil was less than half the rate in the oak soil, (iii) meaningful N immobilization was only observed for ammonium, (iv) nitrate production via oxidation of organic N occurred three times faster in the pine soil while ammonium oxidation was similar in both soils, and (v) dissimilatory nitrate reduction to ammonium was detected in both soils but was higher in the oak soil. We conclude that the higher gross nitrification (including oxidation of organic N) in the pine soil compared to the oak soil, combined with negligible nitrate immobilization, is in line with the observed higher nitrate leaching under the pine forest.     

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML–CysPc–C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc–C2L domains of land plant calpains form a separate sub‐clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1‐like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1‐3 mutant using CysPc–C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc–C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1‐3 mutant phenotype. In contrast, neither the CysPc–C2L domains from M. viride nor chimeric animal–plant calpains complement this mutant. Co‐evolution analysis identified differences in the interactions between the CysPc–C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1‐3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺‐binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.     

Impact of weak permanent magnetic field on antioxidant enzyme activities in radish seedlings

The impact of weak permanent magnetic field (PMF) with magnetic flux density of 185–650 μT on activities of antioxidant enzymes in 5-day-old radish (Raphanus sativus L. var. radicula D.C.) seedlings, cv, Rosovo-krasnyi s belym konchikom was demonstrated. In the range of 185–325 μT PMF suppressed superoxide dismutase (SOD) activity in seedling grown in darkness and catalase (CAT) grown in both darkness and light. At the same values of magnetic flux density, all fractions of guaiacol peroxidase were activated and MDA accumulation was enhanced. At the higher values of magnetic flux density, SOD was activated in seedlings grown in darkness and CAT was activated in seedlings grown in both darkness and light. Other indices decreased or remained unchanged. It was concluded that PMF action depends on its intensity: at its low values, antioxidant enzymes are inhibited and at high values — activated.     

Characterization of Ca2+-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.     

Environmental effects of introducing whitefish,Coregonus lavaretus (L.), in Lake Ring

The impact of whitefish (Coregonus lavaretus (L.)) on the trophic structure of eutrophic lakes was studied in Lake Ring, a small eutrophic Danish lake (22.5 ha, mean depth 2.9 m) in which the natural fish fauna is dominated by pike (Esox lucius L.), perch (Perca fluviatilis L.), and eel (Anguilla anguilla (L.)), roach (Rutilus rutilus (L.)) and burbot (Lota lota (L.)) being the only other fish species present. A total of 10993 0+ whitefish were stocked in the lake from October 1989 to July 1990 and the structure of the fish, zooplankton and benthic invertebrate communities studied during the period 1989–1991. Stomach contents analysis revealed that the whitefish mainly ate Daphnia and copepods in 1990–1991, the proportion of copepods decreasing with increasing size of the fish and Daphnia being the overall most important food source. The density of Daphnia in the lake decreased from 72 ind. 1^-1 in 1989 to 9 ind. 1^-1 in 1991; concomitantly the large species Daphnia magna and D. pulex almost disappeared and the density of cyclopoid copepods increased from 72 to 101 ind. 1^-1, presumably because of improved food conditions, while that of calanoid copepods remained virtually unchanged. As a result chl-a increased from 19 to 47 µg 1^-1 and Secchi depth decreased from 2.4 m to 1.7 m, despite there being no change in total P and total N (0.6 mg P 1^-1 and 1.3 mg N 1^-1, respectively). Changes were also observed in the benthic invertebrates; Chaoborus, oligochaetes, and chironomids all decreased, whereas Pisidium increased. It is concluded that the stocking of whitefish in eutrophic lakes for commercial purposes may delay their recovery, or even lead to enhanced eutrophication.     

Fearful symmetry? Intra-individual comparisons of asymmetry in cryptic vs. signalling colour patterns in butterflies

The signalling role of asymmetry has attracted considerable recent interest among evolutionary biologists. Although it has been studied primarily within the context of sexual selection, symmetry of signals may play a role also in inter-specific communication, such as predator–prey interactions. Both theory and experimental evidence suggest that asymmetry may impair the efficacy of visual warning signals used to deter potential predators, but increase the protective value of non-signalling, cryptic colour patterns used to decrease the risk of detection. Here we tested the prediction from this hypothesis by means of intra-individual comparisons of asymmetry in colour pattern elements in three species of moths (Arctia caja (L.), Noctua orbona (L.), Smerinthus ocellata (L.)) that possess cryptic fore wing patterns and signalling hind wing patterns. Mean asymmetries constituted 4.3% (range 2.1–7.0%) of trait size for colour pattern elements, whereas individual asymmetry levels reached as high as 26%. Asymmetry tended to be somewhat larger in cryptic patterns on fore wings than in signalling patterns on hind wings in five of six comparisons, but in only one case was the difference statistically significant. In addition, pattern elements were somewhat more asymmetric on fore wings also in Saturnia pavonia (L.), which possesses identical signalling eyespots on both fore and hind wings. The relatively low levels of asymmetry also in cryptic patterns imply either that selection does not favour increased asymmetry in cryptic patterns, or that the evolution of pronounced asymmetry is developmentally or genetically constrained.     

Attempt at demonstrating the effect of a weak magnetic field onTaraxacum officinale

The exposure ofTaraxacum officinale L. at flowering time to a weak magnetic field resulted in (1) retarded opening and the closing of the inflorescence, (2) wilting and death after prolonged exposure. Controls were unchanged. Plants exposed to the effect of magnetic fields were degenerated in subsequent year.     

Annual variability in the life-history characteristics of brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus) in a subalpine Norwegian lake

The annual variability in growth and life history traits of brown trout (Salmo trutta L.) and Arctic charr (Salvelinus alpinus (L.)) in Lake Atnsjøen, a Norwegian subalpine lake, was studied over a period of 13 years (1985–1997). The extent to which life-history characteristics recorded on one occasion can be regarded as representative for the population was explored. We found inter-cohort variation in growth for both species; estimates of asymptotic length (L _∞) in ten cohorts ranged between 225–305 mm (CV = 10.5%) for brown trout and 273–301 mm (CV = 4.1%) for Arctic charr. However, this variation was much lower than inter-population variation for brown trout based on single samples from 169 populations (CV = 24.6%). In Lake Atnsjøen, annual growth increment correlated highly with the number of days warmer than 7?°C (R ²=0.60–0.89) for brown trout, and days warmer than 10?°C (R ²=0.40–0.58) for Arctic charr. Females of Arctic charr were younger at sexual maturity than males, while no such difference was found in brown trout. Generally speaking, early maturing individuals of both species grew faster, particularly from age-2 and onwards, than immature individuals. Early maturing individuals, however, were smaller at maturity than those maturing one year older. Age and size at maturity were significantly correlated with asymptotic lengths only in Arctic charr females.     

Magnetic and luminescence characteristics of dinuclear complexes of lanthanides and a phenolic schiff base macrocyclic ligand

The magnetic and luminescence characteristics of trimorphic homodinuclear macrocyclic complexes of lanthanides and a 2:2 phenolate Schiff's base L, derived from 2,6-diformyl-p-cresol and triethylenetetramine were determined. The complexes of Pr³⁺ exhibit non-Curie-Weiss temperature dependent magnetic susceptibilities for which satisfactory fits to an axial relationship depends on crystal field splitting and a weak binuclear Pr³⁺Pr³⁺ antiferromagnetic interaction. The exchange interaction parameters are zJ′ = −2.2, −4.4 and −7.0 cm ⁻¹ for ‘off-white’ Pr₂L(NO₃)₄·2H₂O, ‘yellow’ Pr₂L(NO₃)₄, and ‘orange’ Pr₂L(NO₃)₂(OH)₂, respectively. In contrast, magnetic susceptibilities of the Ln₂L(NO₃)₃(OH) complexes (Ln = Dy, Ho) follow Curie-Weiss behavior over the entire temperature range (6 K to 300 K). The complexes of closed shell ions La³⁺, Lu³⁺, Y³⁺ and those of the half filled shell ion Gd³⁺ exhibit a strong ligand fluorescence in the 450 nm to 650 nm range with decay times at 77 K of 5–8 ns for Ln≠Gd or 2–4 ns for Ln = Gd. The complexes of Gd³⁺ also exhibit a phosphorescence at 600 nm (decay time ∼ 200 μs). The complexes containing Ce³⁺, Eu³⁺, Tb³⁺ and Er³⁺ show very weak ligand luminescence indicative of effective quenching processes. Sensitized emission from the lanthanide ion is observed only with the Eu³⁺ complexes (⁵D_o → ⁷F_j transitions). The emission lifetimes are on the order of 250 μs in the pure Eu³⁺ complexes. The emission decay curves from dilute samples of Eu³⁺ in ‘off-white’ La₂L(NO₃)_4nH₂O show a noticeable rise time as well as a biphasic decay (fast component ∼ 400 μs; slow component ∼ 2500 μs). The luminescing states of L and Eu³⁺ have a common excitation spectrum which is similar to the electronic absorption spectrum of L indicating that ligand-to-metal ion energy transfer processes are dominant. Overall the result if this study suggest that the spectral properties of the complexes are determined by the coordination mode of the lanthanide ions to the Schiff base portion of macrocyclic ligand.