Influence of hypomagnetic conditions on the activities of glycosidases and kinetic characteristics of carbohydrate hydrolysis in juvenile roach, <Emphasis Type="Italic">Rutilus rutilus</Emphasis>

The study on remote consequences of hypomagnetic conditions during roach Rutilus rutilus embryogenesis (at the stages of embryo and prelarva) revealed multidirectional changes in body length/weight, activities of glycosidases (maltase, sucrase, and amylolytic activity), and kinetic characteristics of carbohydrates hydrolysis in the intestine of the yearlings. The exposure of embryos to hypomagnetic conditions leads to the increase in body length and weight in the yearlings; the exposure of prelarvae leads to the decrease in these parameters. The impact of hypomagnetic conditions at the stage of prelarva results in most pronounced changes in the physiological and biochemical parameters in the yearlings. Decline in the Michaelis constant values, reflecting affinity of an enzyme to substrate, may be considered as an adaptive response of the fish digestive system to the lack of magnetic field during embryogenesis.     

Effect of rate of increase of water temperature on sensitivity of fish digestive glycosidases to action of copper and zinc

There was established an increase of sensitivity of glycosidases of intestinal mucosa of juvenile goldfish Carassius auratus, carp Cyprinus carpio, roach Rutilus rutilus, and common perch Perca fluviatilis to action in vitro of heavy metal ions (copper and zinc at concentrations of 0.1–25 mg/l) at heating of water at the summer period. The inhibitory effect of copper and zinc ions as a rule was higher at the heating rate of 50°C/h than at the rate of 4°C/h. The perch glycosidases were less sensitive to the elevation of temperature and to effect of the copper ions as compared with the enzymes of the carp species.     

The effect of weak low-frequency magnetic fields on the intracellular calcium-dependent proteinases of fish

The in vivo and in vitro effects of weak, low-frequency magnetic fields with resonance parameters for calcium ions upon intracellular calcium-dependent proteinases (calpains) in the crucian carp (Carassius carassius (L.)) and roach (Rutilus rutilus (L.)) were studied. It has been revealed that the impact of a weak lowfrequency magnetic field leads to considerable decrease in the activity of calpains in the fish skeletal muscles and brain.     

The effect of organic toxicants on sensitivity of intestinal glycosidases to Cu and Zn in juvenile roach

The chronic effect (duration of exposure 218 days) of polychlorinated biphenyls (PCBs) and the prolonged effect of the short-term action of chlorophos or of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) during embryogenesis upon the sensitivity of intestinal glycosidases to Cu and Zn was studied in roach (Rutilus rutilus (L.) underyearlings. The Cu⁺² and Zn⁺² ions at concentrations ranging from 0.1 to 25 mg/L in vitro cause a 10–77% decrease in amylolytic activity in the intestinal mucosa of control roach. An elevated level of PCBs (50.8 ng/g wet weight of food and 426 ng/g dry weight of ground) increased the sensitivity of glycosidases to Cu and Zn. The embryotoxic action of chlorophos at concentrations of 1 × 10⁻⁶−1 × 10⁻² mg/L in most cases increased the inhibitory effect of Cu but decreased that of Zn. As a rule, MNNG (3 × 10⁻⁷−3 × 10⁻¹ mg/L) reduced the glycosidase sensitivity to the effect of metal ions. The magnitude and direction of the effect depend on the nature and concentration of toxicants.     

On the impact of low concentrations of chlorophos in the period of early ontogenesis on digestive carbohydrases of underyearlings of roach Rutilus rutilus

The remote effects of brief exposure to low doses of chlorophos (1 × 10^?6 ?1 × 10^?2 mg/l) in the period of early ontogenesis of roach Rutilus rutilus have been studied. Diverse changes of the activity of carbohydrases and of the kinetic characteristics of the hydrolysis of di-and polysaccharides in experimental under-yearlings are revealed. Especially interesting are the data on the greater effect of superlow concentrations on changes of the kinetic characteristics; in some cases adaptive changes in the affinity of enzymes to the substrate are recorded.     

Effect of ultralow concentrations of N-methyl-N′-nitro-N-nitrosoguanidine upon early development in roach (<Emphasis Type="Italic">Rutilis rutilus</Emphasis>): Intestine carbohydrase activities and kinetic characteristics of carbohydrate hydrolysis in the intestine of underyearlings

The prolonged effects of short-term action of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in low concentrations 3 × 10^?7 to 3 × 10^?1 mg/l upon roach (Rutilus rutilus) early development have been studied. It was revealed that the treatment resulted in an increase in fish length and weight, in a decrease in total amylolytic and saccharase activities, and in different changes in parameters of di-and polysaccharide hydrolyses. The concentration-effect dependence is nonlinear. Both ultralow and the highest tested MNNG concentrations cause similar changes in carbohydrase activities and kinetic parameters of carbohydrate hydrolysis. The decrease in K _m values of sucrose hydrolysis evidencing the increase in the enzyme to substrate affinity may be attributed to adaptive reactions of roach at early developmental stages in response to damaging embryotoxic action of MNNG in ultralow concentrations.     

Expression of gonadotropin subunits in roach (Rutilus rutilus, Cyprinidae) infected with plerocercoids of the tapeworm Ligula intestinalis (Cestoda)

Plerocercoids of the tapeworm Ligula intestinalis (Cestoda: Bothriocephalidea) have been reported to inhibit gametogenesis of their intermediate fish hosts. However, mechanistic studies are rare and the proximate cues leading to impaired reproduction still remain unknown. In the present study we investigated the effects of infection by L. intestinalis on reproductive parameters of roach (Rutilus rutilus, Cyprinidae), a common fish host of this parasite. Field studies on roach demonstrated that in both genders infection prevented gonad development. As revealed by quantitative PCR, infection was accompanied by essentially lower pituitary expression of follicle-stimulating hormone β-subunit (FSHβ) and luteinizing hormone β-subunit (LHβ) mRNA compared with uninfected roach, providing clear evidence for gonadotropin-insufficiency as the cause of arrested gametogenesis. Under controlled laboratory conditions infected roach showed lower mRNA levels of FSHβ but not of LHβ, despite histology revealing similar gonad stages as in uninfected conspecifics. These findings indicate the involvement of FSH rather than LH in mediating effects of infection early during gonad development in roach. Moreover, the impact of L. intestinalis on reproductive parameters of roach appeared to be independent of the parasite burden. Together, these data provide valuable information on the role of FSH and LH as mediators of parasite-induced sterilization in a vertebrate and implicate the selective inhibition of host reproduction by L. intestinalis as a natural source of endocrine disruption in fish.     

Activity of Peptidases and Glycosidases: Effect of Light Deprivation and Heavy Metals on the Effects of Serotonin and Cholecystokinin in the Carp <Emphasis Type="Italic">Cyprinus carpio</Emphasis>

The dependence of the effects of serotonin and cholecystokinin on the activity of peptidases and glycosidases in the intestine of the carp Cyprinus carpio from the content of zinc and copper ions in different photoperiods (6: 18 and 0: 24 h) was studied. It was shown that the activity of enzymes in the intestine of fish under the influence of both hormones in conditions of light deprivation significantly increases, especially under the action of copper. The mechanisms of the influence of these factors on the activity of peptidases and glycosidases of fish intestines are discussed.     

Activation of Avena coleoptile cell wall glycosidases by hydrogen ions and auxin

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of β-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo β-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.     

Enhanced acylation activity of esterase BioH from Escherichia coli by directed evolution towards improved hydrolysis activity

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.     

Effect of triiodothyronine on the activity and sensitivity of glycosidases to heavy metals (Cu,Zn, and Pb) in juvenile blue bream <Emphasis Type="Italic">Ballerus ballerus</Emphasis> (L.)

The influence of exogenous triiodothyronine (0.25 ng/mL) on the activity glycosidases (maltase, amylolytic activity) and their sensitivity to Cu, Zn and Pb ions (25 mg/L) has been studied in the whole organism of juvenile blue bream Ballerus ballerus (L.). We have found that the treatment of fish with triiodothyronine resulted in an increase in amylolytic activity. Maltase activity is not affected. In addition, the exposure to exogenous triiodothyronine results in a decrease in the sensitivity of glycosidase to the in vitro action of the ions of biogenic metals Cu and Zn and in an increase in the sensitivity of the maltase to the action of Cu, Zn, and Pb ions.     

In Vivo and Ex Vivo Evaluation of L-Type Calcium Channel Blockers on Acid β-Glucosidase in Gaucher Disease Mouse Models

Gaucher disease is a lysosomal storage disease caused by mutations in acid β-glucosidase (GCase) leading to defective hydrolysis and accumulation of its substrates. Two L-type calcium channel (LTCC) blockers—verapamil and diltiazem—have been reported to modulate endoplasmic reticulum (ER) folding, trafficking, and activity of GCase in human Gaucher disease fibroblasts. Similarly, these LTCC blockers were tested with cultured skin fibroblasts from homozygous point-mutated GCase mice (V394L, D409H, D409V, and N370S) with the effect of enhancing of GCase activity. Correspondingly, diltiazem increased GCase protein and facilitated GCase trafficking to the lysosomes of these cells. The in vivo effects of diltiazem on GCase were evaluated in mice homozygous wild-type (WT), V394L and D409H. In D409H homozygotes diltiazem (10 mg/kg/d via drinking water or 50–200 mg/kg/d intraperitoneally) had minor effects on increasing GCase activity in brain and liver (1.2-fold). Diltiazem treatment (10 mg/kg/d) had essentially no effect on WT and V394L GCase protein or activity levels (<1.2-fold) in liver. These results show that LTCC blockers had the ex vivo effects of increasing GCase activity and protein in the mouse fibroblasts, but these effects did not translate into similar changes in vivo even at very high drug doses.     

The effect of temperature,pH, and heavy metals (copper,zinc) upon proteinase activities in the potential preys for typical and facultative piscivores

Separate and combined action of temperature, pH, and heavy metals (copper and zinc) upon whole body activities of casein-and hemoglobinlytic proteinases were studied in perch (Perca fluviatilis), ruff (Gymnocephalus cernuus), roach (Rutilus rutilus), kilka (Clupeonella cultriventris), and common carp (Cyprinus carpio). Also, these activities were determined in cyprinids’ underyearlings (in sum) dominating the food of typical and facultative piscivores. It was revealed that, following a decrease in temperature to 0°C and in the presence of heavy metals (copper and zinc) at concentrations of 10 mg/l, casein-and hemoglobinlytic proteinase activities decreased statistically significantly in all studied fish. Both decrease and increase in pH values lead to less pronounced changes in the enzymatic activities varied in different species. As a rule, decrease in pH values resulted in considerable increase of the hemoglobinlytic activity, while pH increase caused inconsiderable enzyme activation. Combined action of natural factors and, in some cases, combined action of low temperature and metals, changed the caseinlytic activity to a greater extent than at their separate actions. Magnitude of multifactor effects in most cases is lower than of bifactor effects of temperature and metals.     

Effect of thyroid hormones on activity dynamics of enzymes of intestinal mucosa of juvenile roach <Emphasis Type="Italic">Rutilus rutilus</Emphasis>

The effect of thyroid hormones on activity dynamics of enzymes (proteinases and glycosidases) of intestinal mucosa of juvenile roach Rutilius rutilus was investigated. Application of substances increasing and decreasing the level of thyroid hormones in blood plasma significantly influences the growth rate and the activity of proteinases and glycosidases functioning in the intestinal mucosa. In most cases, the activity level of trypsin-like proteinases and the activity of glycosidases in the fish exposed to triiodothyronine were significantly higher than in the control. The activity level of chymotrypsin-like proteinases in fish form the group with exposure of exogenous triiodothyronine only in the end of the experiment surpassed the values of this parameter in the control fish. In the fish developing at deficiency of thyroid hormones, the growth rate and proteinases activity were significantly lower in comparison with the control.     

Effects of copper and cadmium on cholinesterase and glutathione S-transferase activities of two marine gastropods (Monodonta lineata and Nucella lapillus)

With the view of using Nucella lapillus and Monodonta lineata as bioindicators in biomonitoring programs in the NW coast of Portugal, the sensitivity to copper and cadmium of these two common species in Atlantic coasts of Europe was investigated. Assays based on mortality and on the activity of the enzymes cholinesterases (ChE) and glutathione S-transferases (GST) were used, as these biomarkers have been used in biomonitoring studies in the area. ChEs present in foot muscle of both species were characterised and found to show properties of both typical acetylcholinesterase and pseudocholinesterase. Cadmium LC50s for N. lapillus and M. lineata were 2.64 and 2.44 mg/L, respectively, while the lowest observed effect concentration (LOEC) was 1.53 mg/L for both species. LOEC value for cooper was 0.075 mg/L for both. Cadmium in vivo exposure increased ChE activity of N. lapillus, but had no effects on M. lineata ChE. No in vitro effects of cadmium on ChE activity of any of the tested species was observed. Copper had no significant in vivo effects on ChE activity, although it inhibited the ChE of both species in vitro, the IC50s being 5.87 and 12.17 mg/L for N. lapillus and M. lineata, respectively. Cadmium had no significant effects on GST activity of either species, while copper caused a significant reduction of N. lapillus GST (LOEC=0.044 mg/L) but had no effect on M. lineata GST. Results indicate that (i) N. lapillus and M. lineata have a similar acute sensitivity to cadmium and copper; (ii) ChE and GST of these species are sensitive to cadmium (iii) ChEs of both species are inhibited by copper at concentrations in the mg/L range and therefore, its use is limited to heavily polluted sites; (iv) N. lapillus GST does not seem to be a suitable biomarker for copper, at least in the range of concentrations tested, since it was inhibited by copper, but no clear concentration-response relationship was observed.     

Copper accumulation and oxidative stress in the sea anemone, Aiptasia pallida, after waterborne copper exposure

Copper is a common marine pollutant yet its effects on symbiotic cnidarians are largely understudied. To further understand the impact of elevated copper concentrations on marine symbiotic organisms, toxicity tests were conducted using the model sea anemone, Aiptasia pallida, with and without its zooxanthellae symbiont. Symbiotic and aposymbiotic A. pallida were exposed to sublethal copper concentrations (0, 5, 15, and 50 µg/L) for 7 d and copper accumulation, behavior, and the activity of the oxidative stress enzymes, superoxide dismutase (SOD), and catalase (CAT) were measured. Additionally, acute 96-h toxicity tests were conducted to determine LC₅₀ values of the organisms after copper exposure. Both symbiotic and aposymbiotic A. pallida rapidly accumulated copper in a time and dose dependent manner. However, higher copper concentrations accumulated in the aposymbiotic as compared to the symbiotic A. pallida. In response to the highest two copper exposures (15 and 50 µg/L) symbiotic A. pallida upregulated CAT activity to combat the damaging effects of hydrogen peroxide. Contrary to these results, SOD activity significantly decreased during the highest copper exposure, when compared to controls. CAT activity was not detected and SOD was substantially (> 10 fold) reduced in aposymbiotic A. pallida, suggesting that the zooxanthellae are associated with the oxidative stress response. Copper exposure as low as 5 µg/L caused tentacle retraction and increased mucus production in both symbiotic and aposymbiotic anemones. The LC₅₀ values for symbiotic and aposymbiotic A. pallida exposed to copper for 96 h were 148 µg/L (95% confidence interval = 126.4, 173.8) and 206 µg/L (95% confidence interval = 175.2, 242.2), respectively. Understanding the varying responses of symbiotic and aposymbiotic A. pallida to copper stress may advance our comprehension of the functional roles of zooxanthellae and host. Although the mechanism of copper toxicity has not been fully elucidated, it is clear that A. pallida accumulate copper and are sensitive, as effects were detected at environmentally relevant copper concentrations. Likewise, A. pallida may be useful in biomonitoring copper polluted environments.     

Consumption, growth and respiration of bleak, Alburnus alburnus (L.), and roach, Rutilus rutilus (L.), during early ontogeny

Three components of the energy budget, consumption ( C ), production ( P ) and respiration ( R ) in juvenile roach and bleak kept under controlled food ( Anemia salina , 2400 ind. l⁻¹) and temperature (20° C) conditions were measured in a study aimed at defining differences between the two species and elucidating the patterns of energy partitioning during ontogeny.
Daily food consumption rates (J day⁻¹ fish⁻¹) increased allometrically ( C =a W ^b) with body size ( W , mg dry weight) in both species. Covariance analysis indicated no differences in slope or intercept for the two regression lines ( P ≤ 0.05, n = 82). However, the two species grew at significantly different rates, roach faster than bleak.
The dependence of the respiration rate (μmol h⁻¹ fish ⁻¹) on body weight ( W ) can be described by an allometric function: R = a W ^b, where a ± 95% C.L. = 0.17 ± 0.15 for roach and 0.18 ± 0.20 for bleak. The slope for roach (b ± 95% C.L. = 0.78 ± 0.01) is slightly higher than that for bleak (0.69 ± 0.03).
Assimilation efficiency [AE = ( P + R ) C ⁻¹] was significantly higher in roach than in bleak. Different levels of AE correlated with differences in relative gut length (gut length as percentage of body length). Due to the shorter relative gut length above a weight of 5 mg, bleak has lower powers of digestion, which may explain lower production rates. These differences in energetic performance between the two species indicate mechanisms leading to niche differentiation in the early life history of the fishes.     

Cloning,overexpression and characterization of a thermostable β-xylosidase from Thermotoga petrophila and cooperated transformation of ginsenoside extract to ginsenoside 20(S)-Rg3 with a β-glucosidase

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases.     

Quantifying Fish Swimming Behavior in Response to Acute Exposure of Aqueous Copper Using Computer Assisted Video and Digital Image Analysis

Behavioral responses of aquatic organisms to environmental contaminants can be precursors of other effects such as survival, growth, or reproduction. However, these responses may be subtle, and measurement can be challenging. Using juvenile white sturgeon (Acipenser transmontanus) with copper exposures, this paper illustrates techniques used for quantifying behavioral responses using computer assisted video and digital image analysis. In previous studies severe impairments in swimming behavior were observed among early life stage white sturgeon during acute and chronic exposures to copper. Sturgeon behavior was rapidly impaired and to the extent that survival in the field would be jeopardized, as fish would be swept downstream, or readily captured by predators. The objectives of this investigation were to illustrate protocols to quantify swimming activity during a series of acute copper exposures to determine time to effect during early lifestage development, and to understand the significance of these responses relative to survival of these vulnerable early lifestage fish. With mortality being on a time continuum, determining when copper first affects swimming ability helps us to understand the implications for population level effects. The techniques used are readily adaptable to experimental designs with other organisms and stressors.     

Sublethal Toxicity Endpoints of Heavy Metals to the Nematode Caenorhabditis elegans

Caenorhabditis elegans, a free-living nematode, is commonly used as a model organism in ecotoxicological studies. The current literatures have provided useful insight into the relative sensitivity of several endpoints, but few direct comparisons of multiple endpoints under a common set of experimental conditions. The objective of this study was to determine appropriate sublethal endpoints to develop an ecotoxicity screening and monitoring system. C. elegans was applied to explore the sublethal toxicity of four heavy metals (copper, zinc, cadmium and chromium). Two physiological endpoints (growth and reproduction), three behavioral endpoints (head thrash frequency, body bend frequency and feeding) and two enzymatic endpoints (acetylcholine esterase [AChE] and superoxide dismutase [SOD]) were selected for the assessment of heavy metal toxicity. The squared correlation coefficients (R²) between the responses observed and fitted by Logit function were higher than 0.90 and the RMSE were lower than 0.10, indicating a good significance statistically. There was no significant difference among the half effect concentration (EC50) endpoints in physiological and behavioral effects of the four heavy metals, indicating similar sensitivity of physiological and behavioral effects. AChE enzyme was more sensitive to copper, zinc, and cadmium than to other physiological and behavioral effects, and SOD enzyme was most sensitive to chromium. The EC50 of copper, zinc, and cadmium, to the AChE enzyme in the nematodes were 0.68 mg/L, 2.76 mg/L, and 0.92 mg/L respectively and the EC50 of chromium to the SOD enzyme in the nematode was 1.58 mg/L. The results of this study showed that there was a good concentration-response relationship between all four heavy metals and the sublethal toxicity effects to C. elegans. Considering these sublethal endpoints in terms of simplicity, accuracy, repeatability and costs of the experiments, feeding is the relatively ideal sublethal toxicity endpoint of heavy metals to C. elegans.