Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp, Pteromalus puparum in response to environmental stresses

Six heat shock protein (HSP) genes from five HSP families in the parasitoid, Pteromalus puparum, were evaluated for their response to temperature (-15 ~ 3°C , and 30 ~ 42°C for 1 h), heavy metals (0.5 ~ 5 mM Cd(2+) and Cu(2+) for 24 h and 60 h), and starvation (24 h). Compared with other insect HSPs, all conserved motifs are found in P. puparum HSPs, and they are very similar to those of the recently sequenced ectoparasitoid Nasonia vitripennis. The temporal gene expression patterns indicated that these six HSP genes were all heat-inducible, of which hsp40 was the most inducible. The temperatures for maximal HSP induction at high and low temperature zone were 36 or 39°C and -3°C, respectively. In the hot zone, all HSP genes have the same initial temperature (33°C) for up-regulation. Low concentrations of Cd(2+) for a short-term promoted the expression of all HSP genes, but not high concentrations or long-term treatments. Cu(2+) stress for 24 h increased expression of nearly all HSP. Four HSP genes changed after starvation. We infer that all six HSP genes are sensitive to heat. This may help understand the absence of P. puparum during the summer and winter. The expression profiles of six HSP genes in P. puparum under heavy metal stress indicates that HSP is a short-term response to cellular distress or injury induced by Cd(2+) and Cu(2+).     

Transcriptional regulation of selenium-dependent glutathione peroxidase from Venerupis philippinarum in response to pathogen and contaminants challenge

Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post-Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 μg L(-1) Cd and 10, 20 μg L(-1) Cu but depressed by 10 μg L(-1) Cd and 40 μg L(-1) Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.     

Effects of heavy metals on nitrogen uptake by Paxillus involutus and mycorrhizal birch seedlings

The effects of the heavy metals Cu, Cd, Ni, Pb and Zn on [(14)C]methylamine and [(14)C]aminoisobutyric acid uptake were studied in the free-living fungus Paxillus involutus and in mycorrhizal and non-mycorrhizal birch roots. The uptake of both N sources by P. involutus was inhibited by the five metals tested. However, Cu(2+) and Pb(2+) had a greater inhibitory effect. Non-competitive inhibitions were determined between heavy metals and [(14)C]methylamine uptake. [(14)C]Methylamine uptake was reduced by one third by 2 μM Cd(2+) and Cu(2+) in non-mycorrhizal roots, whereas that of mycorrhizal roots was not affected. However, it was reduced by 30 to 80% by 200 μM Cd(2+) and Cu(2+) irrespective of the mycorrhizal status. [(14)C]Aminoisobutyric acid uptake in mycorrhizal roots was not significantly affected by Cd(2+) and Cu(2+), whereas that of non-mycorrhizal roots was decreased by 77% at 200 μM Cu(2+). [(14)C]Aminoisobutyric acid uptake was 4.5 to 6 fold higher in mycorrhizal roots, compared with non-mycorrhizal roots, even under metal exposure. The high efficiency of N acquisition by mycorrhizal birch seedlings under metal exposure might be regarded as a mechanism of stress avoidance.     

Exposure of developing oligodendrocytes to cadmium causes HSP72 induction, free radical generation, reduction in glutathione levels, and cell death

Primary cultures of oligodendrocytes were used to study the toxic effects of cadmium chloride. Cell viability was evaluated by the mitochondrial dehydrogenase activity and confirmed by propidium iodide (PI) fluorescence staining. The expression of the 72 kDa stress protein, HSP72, was assayed by Western blot analysis. The results showed that Cd(2+)-induced toxicity was dependent on the time and dose of exposure, as well as on the developmental stage of the cultures. Oligodendrocyte progenitors were more vulnerable to Cd(2+) toxicity than were mature oligodendrocytes. Mature oligodendrocytes accumulated relatively higher levels of Cd(2+) than did progenitors, as determined by (109)CdCl(2) uptake; treatment with the metal ion caused a more pronounced reduction in intracellular glutathione levels and significantly higher free radical accumulation in progenitors. The latter could explain the observed differences in Cd(2+) susceptibility. HSP72 protein expression was increased both in progenitors and in mature cells exposed to Cd(2+). Pretreatment with N-acetylcysteine, a thiocompound with antioxidant activity and a precursor of glutathione, prevented Cd(2+)-induced (i) reduction in glutathione levels and (ii) induction of HSP72 and diminished (i) Cd(2+) uptake and (ii) Cd(2+)-evoked cell death. In contrast, buthionine sulfoximine, an inhibitor of gamma-glutamyl-cysteine synthetase, depleted glutathione, and potentiated the toxic effect of Cd(2+). These results strongly suggest that Cd(2+)-induced cytotoxicity in oligodendrocytes is mediated by reactive oxygen species and is modulated by glutathione levels.     

Alteration of Phascolosoma esculenta heat shock protein 90 expression under heavy metal exposure and thermal stress

The full-length complementary DNA (cDNA) of heat shock protein 90 was cloned from Phascolosoma esculenta (PeHSP90) using expressed sequence tag and rapid amplification of cDNA end approaches. The cDNA of PeHSP90 was 2521 bp including a 5'-untranslated region of 110 bp, a 3'-untranslated region of 230 bp, and an open reading frame of 2181 bp. All of the characteristic motifs of the HSP90 family were completely conserved in the deduced amino acid of PeHSP90. The expression of PeHSP90 was induced by 3 heavy metals or elevated temperature, under which Zn(2+) displayed effects were more toxic than those of Cd(2+) and Cu(2+). The polyclonal antibodies generated from the recombinant product of PeHSP90 were specifically identified not only in the recombinant product but also in the native protein from hemocytes. These results strongly suggested that PeHSP90 was involved in heavy metal challenge and thermal stress regulation in P. esculenta.     

重金属诱导拟南芥原生质体DNA 损伤的单细胞凝胶电泳检测

以拟南芥原生质体为实验体系, 研究不同浓度的3种重金属离子对拟南芥原生质体的毒性和DNA损伤的差异。结果表明, 用1-5 mmol.L-1的Zn2+、Cd2+ 和Cu2+分别处理的拟南芥原生质体, 2 小时内活力逐渐下降, 并表现出明显的浓度依赖性;与相同浓度的Cd2+ 和Cu2+ 相比, Zn2+对拟南芥原生质体活力的影响程度较小, 表现出较低的毒性。单细胞凝胶电泳检测发现,用0.1-0.8 mmol .L-1的Zn2+、Cd2+ 和Cu2+ 分别处理拟南芥原生质体30 分钟, 以OTM值表示的原生质体DNA损伤量随重金属离子浓度的增加而递增; 相同浓度(0.5 mmol.L-1)的3种重金属离子相比, Zn2+对原生质体的遗传毒性明显低于Cu2+ 和Cd2+。综合原生质体活力和DNA损伤的单细胞凝胶电泳检测结果, 发现Zn2+对拟南芥原生质体的遗传毒性较低, 而Cd2+ 和Cu2+的遗传毒性较高。本研究建立的拟南芥原生质体实验体系, 结合运用单细胞凝胶电泳技术, 能够快速、灵敏地检测重金属对植物细胞的遗传毒性。     

Anti Zn antibodies: cross reactivity and competitive binding with heavy metals

Monoclonal antibodies of IgM class, specific to IDA-Zn were used for evaluating their Zn(2+) binding efficiency in the presence of trace metal ions such as Cr(3+) Cr(6+), Cu(2+) and Cd(2+). In the present work, antibody raised against the hapten IDA-Zn(II) was pre-incubated with different metal ions and the binding capacity to the specific hapten was tested using ELISA and immobilized metal ion affinity chromatography (IMAC) techniques. IMAC was carried out with the free antibody and antibody pre-incubated with selected heavy metal ions using Sepharose IDA-Zn(2+) column and the same samples were tested using a hapten specific ELISA with non-protein hapten carrier. Different effects were observed after pre-incubation with metal ions. Cr(3+) exhibited synergistic binding where as antagonism was detected with Cd(2+). The synergistic effect observed with Cr(3+) suggests involvement of binding sites other than that of zinc and conformational changes that result from Cr(3+) binding. It is probable that, this binding event also increases the accessibility of the zinc binding sites on IgM. On the same lines, the antagonism observed with Cd(2+) could be attributed to structural changes resulting in reduced accessibility to zinc binding sites. In case of Cr(6+), no appreciable change in binding to IDA-Zn was observed while Cu(2+) showed competitive binding.     

Effect of different concentrations of heavy metal ions on the normal physical and chemical characteristics of the hemolymph of Palanorbarius purpura (Mollusca: Bulinidae) in the norm and during a trematode infection

Short-term simultaneous effect of high concentrations (LC25, LC50, LC75) of heavy metal ions. (Cu2+, Zn2+, Cd2+ and Pb2+) and infection with trematode partenites Echinoparyphium aconiatum onto haemolymph of mollusk has been investigated. It was noted that low doses of toxicant (2.5 and 10 maximum admitted concentrations) have variable effect. In infected molluscs the concentration of haemoglobin decreases, while in intact ones it increases. In relation to this index, it was found, that the ion Cu2+ is highly toxic, Zn2+ and Cd(2+)--toxic, Pb(2+)--moderately toxic.     

Nitrate-dependent salicylate degradation by Pseudomonas butanovora under anaerobic conditions

Nitrate-dependent salicylate degradation by the denitrifying Pseudomonas butanovora was investigated and the molar ratio of the cometabolism under anaerobic circumstances was determined. The bacterium was able to utilize salicylate as an electron donor for the reduction of nitrate. Salicylate was eliminated via catechol, which is degraded by means of catechol 2,3-oxygenases (meta-cleavage), forming 2-hydroxymuconic semialdehyde. The molar ratios of NO(3)(-)-N:salicylate existing during the experiment accorded well with the assumed 1:1 molar ratio. The tolerances of the growth, the salicylate degradation and the denitrification of P. butanovora to various heavy metal ions were also studied. Although the strain was tolerant to Pb(2+) and Cu(2+) up to 1 mM in complete medium, salicylate utilization took place only up to a concentration of 0.1 mM for both heavy metal ions. Of the heavy metal ions investigated, Cd(2+) (at a concentration of 0.05 mM) displayed the highest inhibitory effect on salicylate degradation by P. butanovora.     

Equilibrium and kinetics of biosorption of cadmium(II) and copper(II) ions by wheat straw

Biosorption equilibrium and kinetics of Cd(2+) and Cu(2+) ions on wheat straw, Triticum aestivum, in an aqueous system were investigated. Among the models tested, namely the Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich isotherms, the biosorption equilibrium for both Cd(2+) and Cu(2+) was best described by the Langmuir model. The Langmuir biosorption capacity for Cd(2+) was about 27% higher than that for Cu(2+). It was also found that biosorption of Cd(2+) and Cu(2+) by wheat straw followed second-order kinetics. The equilibrium amount of metal ions adsorbed onto the wheat straw increased with increasing of pH from 4.0 to 7.0, and the effect was more pronounced for Cd(2+) than for Cu(2+). The equilibrium adsorbed amount also increased with the initial concentration of the metal ions, as expected. On the other hand, an increase of temperature from 25 to 30 degrees C only enhanced the biosorption of Cd(2+) and Cu(2+) slightly. The apparent temperature independence and the strong pH dependence of the amount of metal ions adsorbed along with moderate mean free energies of biosorption (between 8.0 and 12.9 kJ mol(-1)) altogether indicate that biosorption of Cd(2+) and Cu(2+) by wheat straw might follow a chemisorption mechanism.     

Ruditapes decussatus and Ruditapes philippinarum exposed to cadmium: toxicological effects and bioaccumulation patterns

Since differences in metal accumulation may exist between bivalve species, the aim of this study was to assess the impact of cadmium (Cd) on Ruditapes decussatus and Ruditapes philippinarum. For this, the Cd accumulation, mortality rates and biochemical responses were analysed in the two species after 5days of exposure, under laboratory-controlled conditions. The concentration of Cd that caused 50% of mortality on clams was two-times higher in R. decussatus than in R. philippinarum. For both species, higher percentage (84.5-98.2%) of the Cd was in the insoluble fraction, but the Cd concentration in solution was 3 to 8 times higher in R. decussatus. Nevertheless, R. philippinarum presented higher oxidative stress and higher CAT activity. The paradox observed between the two clams can be explained by the higher capacity of R. decussatus to increase the expression of MTs when exposed to Cd.     

The removal by crab shell of mixed heavy metal ions in aqueous solution

In order to examine the inhibition effect of other heavy metal ions on the removal by crab shell of heavy metal ions in aqueous solutions, three ions (Pb(2+), Cd(2+), Cr(3+)) were used in single, binary and ternary systems. In single heavy metal ion systems, the removals of Cr(3+) and Pb(2+) were much higher than that of Cd(2+). In binary heavy metal ions systems, Cd(2+) did not affect Pb(2+) removal while Cr(3+) had a severe inhibition effect on the removal of Pb(2+). Cd(2+) removal was slightly affected by the presence of Pb(2+); however, it was severely affected by the presence of Cr(3+). The inhibitory effect of Cd(2+) on Cr(3+) was relatively lower than that of Pb(2+).     

Metal toxicity in Chlamydomonas reinhardtii. Effect on sulfate and nitrate assimilation

Cadmium (Cd(2+)) or copper (Cu(2+)) ions are toxic for Chlamydomonas reinhardtii growth, at 300 microM, and the alga may accumulate about 0.90+/-0.02 and 0.64+/-0.02% of its dry weight, respectively. Metal contamination changes the elemental composition of dried alga biomass, which indicates the possibility to use C. reinhardtii as biosensor and bioremediator of the aquatic contamination by heavy metals. Either, Cd(2+) or Cu(2+), inhibits about 20% of the nitrate consumption rate by the cells, while only Cd(2+) increases about 40% the sulfate consumption rate. The presence of 1 mM calcium (Ca(2+)) in the culture medium increases the C. reinhardtii productivity (about 50%), the nitrate uptake rate (about 20%) and the sulfate uptake rate (about 30%). In addition, Ca(2+) overcomes the Cd(2+) (300 microM) toxicity by decreasing (about 35%) the intracellular accumulation of metal. Sulfur-starvation induces in C. reinhardtii the expression of serine acetyltransferase and O-acetylserine(thiol)lyase activities, but decreases 50% the consumption rate of nitrate by the cells. Sulfate is also required for the full expression of the nitrate reductase (NR), nitrite reductase (NiR) and glutamate synthase activities.     

Identification and characterization of heavy metal-resistant unicellular alga isolated from soil and its potential for phytoremediation

A unicellular alga displaying a high growth rate under heterotrophic growth conditions was isolated from soil and identified as Chlorella sorokiniana. The optimal temperature for growth was 35 degrees C and the optimal pH was 6.0-7.0. Glucose, sucrose, galactose, maltose, and soluble starch served as carbon sources supporting growth under dark conditions. The cell yield was 50 g/l (wet weight) in a heterotrophic medium containing 3% glucose. Isolated unicellular algae were highly resistant to heavy metals such as Cd(2+), of which the minimal inhibitory concentration was 4 mM. Algae were capable of taking up the heavy metal ions Cd(2+), Zn(2+) and Cu(2+) at 43.0, 42.0 and 46.4 microg/mg dry weight, respectively. Growth inhibition of Oryza sative shoots by 5 ppm Cd(2+) in hydroponic medium was completely prevented by the addition of 0.25 mg of wet Chlorella cells. These results indicated that this isolate was potentially useful for phytoremediation by preventing environmental dispersion of heavy metals.     

A heavy metal biotrap for wastewater remediation using poly-gamma-glutamic acid

Poly-gamma-glutamic acid (gamma-PGA) obtained from Bacillus licheniformis ATCC 9945 was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Copper (Cu(2+)) was chosen as the model heavy metal used in these studies since it is extensively used by electroplating and other industries, has been the model for many other similar studies, and can be easily assayed through a number of convenient methods. Cu(2+)-gamma-PGA binding parameters under varying conditions of pH, temperature, ionic strength, and in the presence of other heavy metal ions were determined for the purified biopolymer using a specially designed dialysis apparatus. Applying the Langmuir adsorption isotherm model showed that gamma-PGA had a copper capacity approaching 77.9 mg/g and a binding constant of 32 mg/L (0.5 mM) at pH 4.0 and 25 degrees C. Cu(2+)-gamma-PGA adsorption was relatively temperature independent between 7 and 40 degrees C, while an increase in ionic strength led to a decrease in metal ion binding. Cd(2+) and Zn(2+) ions compete with Cu(2+) for binding sites on the gamma-PGA biopolymer. Metal uptake by gamma-PGA was further tested using a tangential flow filtration apparatus in a diafiltration mode in which metal was continually processed through a dilute solution of gamma-PGA without allowing for equilibrium to be established. The circulating polymer solution was able to complex metal as well as successfully prevent passage of unbound copper ions present in solution through the membrane. Using 500 mL of a 0.2% gamma-PGA solution, up to 97% of a 50 mg/L copper sulfate solution processed at a flow rate of 115 mL/min was retained by the polymer. For a 10 mg/L solution of Cu(2+) as copper sulfate, filtrate concentrations of Cu(2+) never rose above 0.6 mg/L while processing 2.5 L of dilute copper sulfate.     

Effects of heavy metals on insect immunocompetent cells

The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals.