Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.     

Enhanced biosorption of mercury(II) and cadmium(II) by cold-induced hydrophobic exobiopolymer secreted from the psychrotroph <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> BM07

The cells of psychrotrophic Pseudomonas fluorescens BM07 were found to secrete large amounts of exobiopolymer (EBP) composed of mainly hydrophobic (water insoluble) polypeptide(s) (as contain approximately 50 mol% hydrophobic amino acids, lacking cysteine residue) when grown on fructose containing limited M1 medium at the temperatures as low as 0-10 degrees C but trace amount at high (30 degrees C, optimum growth) temperature. Two types of nonliving BM07 cells (i.e., cells grown at 30 degrees C and 10 degrees C) as well as the freeze-dried EBP were compared for biosorption of mercury (Hg(II)) and cadmium (Cd(II)). The optimum adsorption pH was found 7 for Hg(II) but 6 for Cd(II), irrespective of the type of biomass. Equilibrium adsorption data well fitted the Langmuir adsorption model. The maximum adsorption (Q (max)) was 72.3, 97.4, and 286.2 mg Hg(II)/g dry biomass and 18.9, 27.0, and 61.5 mg Cd(II)/g dry biomass for cells grown at 30 degrees C and 10 degrees C and EBP, respectively, indicating major contribution of heavy metal adsorption by cold-induced EBP. Mercury(II) binding induced a significant shift of infrared (IR) amide I and II absorption of EBP whereas cadmium(II) binding showed only a very little shift. These IR shifts demonstrate that mercury(II) and cadmium(II) might have different binding sites in EBP, which was supported by X-ray diffraction and differential scanning calorimetric analysis and sorption results of chemically modified biomasses. This study implies that the psychrotrophs like BM07 strain may play an important role in the bioremediation of heavy metals in the temperate regions especially in the inactive cold season.     

Polymeric C3dg primes human B lymphocytes for proliferation induced by anti-IgM

Polymeric C3dg (pC3dg), having an average m.w. of approximately 400,000 and saturating complement receptor type 2 (CR2) on B lymphoblastoid cells at 1 micrograms/ml, was preincubated with tonsillar B cells for 24 h, after which anti-IgM was added and proliferation assessed by thymidine incorporation. Preculture of B cells with 0.01 to 1.0 micrograms/ml of polymerized C3dg (pC3dg) caused a dose-dependent enhancement of proliferation and accelerated entry into S phase after addition of anti-IgM. The continued presence of pC3dg during stimulation by anti-IgM was not required. pC3dg alone did not induce proliferation and preculture of B cells with C3dg monomer had no effect on the subsequent response to anti-IgM. The priming effect of pC3dg required at least 6 h and was greatest after 24 h of preculture. Preincubation with pC3dg did not lower the concentration of anti-IgM necessary for induction of proliferation, but did enhance proliferation at all concentrations above this threshold. Augmented proliferation occurred only in B cells of higher density in Percoll gradients, and neither T cells nor monocytes were required. Thus, independent interaction of CR2 with its natural ligand primes the B cell for subsequent stimulation through the Ag receptor, an effect that might synergize with the previously described CR2 function of lowering the threshold for B cell activation when crosslinked to membrane IgM.     

Removal of cadmium(II) ion from aqueous system by dry biomass, immobilized live and heat-inactivated Oscillatoria sp. H1 isolated from freshwater (Mogan Lake)

Oscillatoria sp. H1 (Cyanobacteria, microalgae) isolated from Mogan Lake was used for the removal of cadmium ions from aqueous solutions as its dry biomass, alive and heat-inactivated immobilized form on Ca-alginate. Particularly, the effect of physicochemical parameters like pH, initial concentration and contact time were investigated. The sorption of Cd(II) ions on the sorbent used was examined for the cadmium concentrations within the range of 25-250 mg/L. The biosorption of Cd(II) increased as the initial concentration of Cd(II) ions increased in the medium up to 100 mg/L. Maximum biosorption capacities for plain alginate beads, dry biomass, immobilized live Oscillatoria sp. H1 and immobilized heat-inactivated Oscillatoria sp. H1 were 21.2, 30.1, 32.2 and 27.5 mg/g, respectively. Biosorption equilibrium was established in about 1 h for the biosorption processes. The biosorption was well described by Langmuir and Freundlich adsorption isotherms. Maximum adsorption was observed at pH 6.0. The alginate-algae beads could be regenerated using 50 mL of 0.1 mol/L HCl solution with about 85% recovery.     

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4-3.3 μg ml(-1)). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ～90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.     

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.     

Comparative study of Cd(II) and Cr(VI) biosorption on Staphylococcus xylosus and Pseudomonas sp. in single and binary mixtures

Biosorption of Cd(II) and Cr(VI) ions in single solutions using Staphylococcus xylosus and Pseudomonas sp., and their selectivity in binary mixtures was investigated. Langmuir and Freundlich models were applied to describe metal biosorption and the influence of pH, biomass concentration and contact time was determined. Maximum uptake capacity of cadmium was estimated to 250 and 278 mg g(-1), whereas that of chromium to 143 and 95 mg g(-1) for S. xylosus and Pseudomonas sp., respectively. In binary mixtures with Cd(II) ions as the dominant species, there is a profound selectivity for cadmium biosorption, reaching 96% and 89% for Pseudomonas sp. and S. xylosus, respectively, at 10 mg l(-1) Cd(II) and 5 mg l(-1) Cr(VI). Interesting, when chromium (VI) ions are the dominant species, there is selectivity towards chromium around 92% with S. xylosus only.     

Role of the yeast ABC transporter Yor1p in cadmium detoxification

Growth of yeast strains, either deleted for the vacuolar ABC transporter Ycf1 or deleted for the plasma membrane ABC transporter Yor1p or overexpressing Yor1p, were compared for their sensitivity to cadmium. On solid medium cell death (or growth inhibition) was observed at cadmium concentrations higher than 100 microM when yeasts were grown at 30 degrees C for 24 h. However, for all tested strains cell death (or growth inhibition) was already observed at 40 microM cadmium when incubated at 23 degrees C for 60 h. Thus cadmium is more toxic to yeast at 23 degrees C than at 30 degrees C. At 23 degrees C, the Deltayor1 strain grew more slowly than the wild-type strain and the double Deltayor1, Deltaycf1 deleted strain was much more sensitive to cadmium than each single Deltayor1 or Deltaycf1 deletant. Overexpression of Yor1p in a Deltaycf1 strain restores full growth. Cadmium uptake measurements show that Deltaycf1 yeast strains expressing or overexpressing Yor1p store less cadmium than the corresponding Deltaycf1, Deltayor1 strain. The strains expressing Yor1p display an energy-dependent efflux of cadmium estimated for the yeast overexpressing Yor1p to be about 0.02 nmol 109Cd/mg protein/min. Yeast cells loaded with radiolabeled glutathione and then with radioactive cadmium displayed a twice-higher efflux of glutathione than that of cadmium suggesting that Yor1p transports both compounds as a bis-glutathionato-cadmium complex. All together, these results suggest that in addition to being accumulated in the yeast vacuole by Ycf1p, cadmium is also effluxed out of the cell by Yor1p.     

Effect of cadmium on cell cycle progression in Chinese hamster ovary cells

Chinese hamster ovary K1 (CHO K1) cells are very sensitive to cadmium (Cd) toxicity. They were used to investigate the effect of Cd on cell cycle progression. Cells were cultured with 0.1, 0.4, 1 or 4 microM Cd for various time intervals. There was no difference in growth rate when less than 0.4 microM Cd was given within 24 h. A dose-dependent reduction of cell proliferation was observed when more than 0.4 microM of Cd was given. The cells were pulse-labeled with 5-bromodeoxyuridine (BrdU), and the labeled cells were cultured in the presence of increasing concentrations of Cd. Cell cycle progression was retarded as a function of Cd concentration. G2/M arrest was observed when the BrdU-labeled cells were treated with 1 microM Cd for 8h, whereas cells receiving 4 microM Cd stopped at the S phase within 4 h. Cell cycle analysis of cells treated with Cd for 24 h showed that G2/M arrest occurred only when cells received 0.8 to 2 microM Cd. Despite the occurrence of G2/M arrest in the Cd treatment, only a limited proportion of the cells were blocked in the M phase. However, the increase in M phase cells coincided with an elevation in the cyclin-dependent kinase 1 activity. To examine whether Cd acts on cells at a specific cell stage, they were synchronized at the G1 or G2/M phase then treated with 1 microM Cd for 12 h. The cells were blocked at the G2/M and G1/S phase, respectively. This finding indicates that Cd toxicity is global and not cell phase specific. We also investigated the involvement of Cd-induced reactive oxygen species (ROS) with the occurrence of G2/M block and found a lack of correlation between cell cycle arrest and ROS production. We measured the Cd content that caused G2/M arrest from a series of Cd treatments and determined the ranges of cumulative Cd concentrations that could result in cell cycle arrest.     

Overexpression of human Raf-1 enhances radiosensitivity in fission yeast, Schizosaccharomyces pombe

Recently we isolated Rad24, a 14-3-3 homologue, which is essential for DNA damage checkpoint, as a Raf-1 interacting protein by screening a Schizosaccharomyces pombe (S. pombe) cDNA library. Raf-1 was also found to recognize Cdc25 that is sequestered and inactivated by Rad24. In the present study, experiments were performed to determine the effect of overexpression of Raf-1 proteins on asynchronously growing S. pombe cells. The overexpression of Rad24 induced elongated cell morphology and reduction in growth rate, resulting in cell cycle arrest while the overexpression of catalytically active Raf-1 led to a decrease in cell size at division in S. pombe. However, the active Raf-1 failed to rescue the growth arrest induced by Rad24 overexpression. In addition, the cells carrying catalytically active Raf-1 were significantly more radiosensitive than those from a normal control as assessed by ultraviolet sensitivity assay, suggesting that constitutive overproduction of Raf-1 kinase can revert DNA replication checkpoint arrest caused by UV irradiation. Taken together, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 in DNA repair, bypassing the checkpoint pathway through Cdc25 activation.     

Accumulation of cadmium by the fourth instar larva of the fly Chironomus thummi

Accumulation and effects of cadmium were investigated in Chironomus thummi larvae exposed to 10, 100 and 250 mug radiolabeled Cd/1 for up to 4 days. (1) After 4 days, average cadmium accumulation was 6.6 ng Cd/mg dry weight (10 mug Cd/1 exposure) and 177 ng Cd/mg dry weight (250 mug Cd/1 exposure). (2) Dissection studies showed that by 32 hr of exposure to both cadmium concentrations, 63.5-81.4% of accumulated cadmium was confined to the posterior midgut epithelium. Light microscope autoradiography similarly showed accumulations of cadmium in posterior midgut epithelium and smaller amounts in fat body and muscle. Little cadmium was associated with Malphigian tubules, haemocoel, anterior midgut or exoskeleton. (3) After exposure to 10 or 250 mug Cd/1, 60-75% of cadmium in ultracentrifuged homogenates of whole animals or dissected guts was associated with the resulting supernatant. When supernatants were further analyzed by gel chromatography, cadmium eluted with both a high and low molecular weight peak. The relative proportions of cadmium in the two peaks varied with concentration and length of exposure. (4) Transmission electron microscopy of posterior midgut cells from animals exposed to cadmium demonstrated frequent mitochondrial lesions. Exposure to high cadmium concentrations caused some posterior midgut cells to undergo generalized structural degeneration.     

Metal tolerance and biosorption potential of filamentous fungi isolated from metal contaminated agricultural soil

Heavy metal analysis of agricultural field soil receiving long-term (>20 years) application of municipal and industrial wastewater showed two- to five-fold accumulation of certain heavy metals as compared to untreated soil. Metal-resistant fungi isolated from wastewater-treated soil belonged to genera Aspergillus, Penicillium, Alternaria, Geotrichum, Fusarium, Rhizopus, Monilia and Trichoderma. Minimum inhibitory concentrations (MIC) for Cd, Ni, Cr, Cu, and Co were determined. The MIC ranged from 0.2 to 5 mg ml(-1) for Cd, followed by Ni (0.1-4 mg ml(-1)), Cr (0.3-7 mg ml(-1)), Cu (0.6-9 mg ml(-1)) and for Co (0.1-5 mg ml(-1)) depending on the isolate. Aspergillus and Rhizopus isolates were tested for their metal biosorption potential for Cr and Cd in vitro. Biosorption experiments were conducted with initial metal concentrations of 2, 4, 6 and 8 mM with a contact time of 4 h and wet fungal biomass (1-5 g) at 25 degrees C. Maximum biosorption of Cr and Cd ions was found at 6 mM initial metal concentration. Aspergillus sp.1 accumulated 1.20 mg of Cr and 2.72 mg of Cd per gram of biomass. Accumulation of these two metals by very tolerant Aspergillus sp.2 isolate was at par with relatively less tolerant Aspergillus sp.1 isolate. Rhizopus sp. accumulated 4.33 mg of Cr and 2.72 mg of Cd per g of biomass. The findings indicated promising biosorption of cadmium and chromium by the Rhizopus and Aspergillus spp. from aqueous solution. There is little, if any, correlation between metal tolerance and biosorption properties of the test fungi.     

A comparison of 60, 70, and 90 kDa stress protein expression in normal rat NRK-52 and human HK-2 kidney cell lines following in vitro exposure to arsenite and cadmium alone or in combination

Arsenite and cadmium are two potent nephrotoxicants and common Superfund site elements. These elements are included among the stress protein inducers, but information regarding relationships between toxicity produced by combinations of these agents to the stress protein response is lacking. In this study, the immortalized cell lines normal rat kidney NRK-52E and human kidney HK-2 were exposed in vitro to arsenite (As(3+)), cadmium (Cd(2+)), or to equimolar As(3+) plus Cd(2+) mixture combinations for 3 and 5 h over a concentration range of 0.1-100 microM. After a 12-h recovery period, cultured cells were then evaluated for expression of the 60, 70, and 90 kDa major stress protein families. Results indicated that expression of stress proteins varied depending on the species of kidney cells exposed, the exposure concentrations, and the length of exposure to each element on an individual basis and for combined mixtures. For the HK-2 kidney cell line, increased levels of the 70 kDa stress protein was observed for single and combined element exposures whereas there was no change or a decrease of stress proteins 60 and 90 kDa. Increased 70 kDa expression was observed for 10-microM doses of single elements and for a lower dose of 1 microM of the As plus Cd mixture at 3- and 5-h exposures. NRK-52 kidney cells exposed to equivalent doses of As(3+) and Cd(2+) alone or in combination showed increased levels of all stress proteins 60, 70, and 90 kDa. This increase was seen for 10 microM of the As plus Cd mixture at 3 h whereas for single element exposures, increased stress protein levels were generally observed for the 100-microM doses. At 5 h- exposure, 60 and 90 kDa levels increased for 10 microM of Cd(2+) and 60 kDa levels increased for 1 microM of As(3+). However, exposures to 10 microM of the As plus Cd mixture decreased 60 kDa protein expression to control levels at 5 h. For both kidney cell lines, there was a decrease in the stress protein expression levels for all three stress protein families for 100-microM doses of the mixture combination for 3- and 5-h exposures. These data indicate a dose- and combination-related correlation between depression of the stress protein response and the onset of overt cellular toxicity and/or cell death. The threshold for these changes was cell line specific.     

Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937.

Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.     

Effect of cadmium on the growth of Chlorella vulgaris and Stichococcus bacillaris

The growth of Chlorella vulgaris and Stichococcus bacillaris cultures in media containing from 20 to 100 mg Cd/l was studied. The examined strains were found to be highly resistant to the action of cadmium since the highest concentration of the metal used limited the production of dry weight (during 5 days of cultivation) by less than 50%. The lower production of chlorophyll a by S. bacillaris cultures in media containing from 60 to 100 mg Cd/l and 2-fold elongation of the cells point to lower tolerance of the strain to cadmium than that shown by C. vulgaris.     

CeHMT-1, a putative phytochelatin transporter, is required for cadmium tolerance in Caenorhabditis elegans

Phytochelatins (PCs), (gamma-Glu-Cys)n Gly polymers that were formerly considered to be restricted to plants and some fungal systems, are now known to play a critical role in heavy metal (notably Cd2+) detoxification in Caenorhabditis elegans. In view of the functional equivalence of the gene encoding C. elegans PC synthase 1, ce-pcs-1, to its homologs from plant and fungal sources, we have gone on to explore processes downstream of PC fabrication in this organism. Here we describe the identification of a half-molecule ATP-binding cassette transporter, CeHMT-1, from C. elegans with an equivalent topology to that of the putative PC transporter SpHMT-1 from Schizosaccharomyces pombe. At one level, CeHMT-1 satisfies the requirements of a Cd2+ tolerance factor involved in the sequestration and/or elimination of Cd x PC complexes. Heterologous expression of cehmt-1 in S. pombe alleviates the Cd2+-hypersensitivity of hmt- mutants concomitant with the localization of CeHMT-1 to the vacuolar membrane. Suppression of the expression of ce-hmt-1 in intact worms by RNA interference (RNAi) confers a Cd2+-hypersensitive phenotype similar to but more pronounced than that exhibited by ce-pcs-1 RNAi worms. At another level, it is evident from comparisons of the cell morphology of ce-hmt-1 and cepcs-1 single and double RNAi mutants that CeHMT-1 also contributes to Cd2+ tolerance in other ways. Whereas the intestinal epithelial cells of ce-pcs-1 RNAi worms undergo necrosis upon exposure to toxic levels of Cd2+, the corresponding cells of ce-hmt-1 RNAi worms instead elaborate punctate refractive inclusions within the vicinity of the nucleus. Moreover, a deficiency in CeHMT-1 does not interfere with the phenotype associated with CePCS-1 deficiency and vice versa. Double ce-hmt-1; ce-pcs-1 RNAi mutants exhibit both cell morphologies when exposed to Cd2+. These results and those from our previous investigations of the requirement for PC synthase for heavy metal tolerance in C. elegans demonstrate PC-dependent, HMT-1-mediated heavy metal detoxification not only in S. pombe but also in some invertebrates while at the same time indicating that the action of CeHMT-1 does not depend exclusively on PC synthesis.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Proteins induced by cadmium in soybean cells

The cadmium (Cd)-induced changes in protein pattern and identification of metal-stimulated polypeptides were analyzed in soybean cell suspension culture. The cell cultures were treated with various concentrations of Cd(2+) (3-10microM) for 24, 48 and 72h. The synthesis of [(35)S]-labeled proteins and their accumulation were analyzed by SDS-PAGE, whereas the identification of selected protein bands was performed by mass spectrometry. It is shown that Cd induced the appearance of the following proteins in soybean cells: superoxide dismutase, histone H2B, chalcone synthase and glutathione transferase.     

Phytochelatin-cadmium-sulfide high-molecular-mass complexes of Euglena gracilis

High-molecular-mass PC complexes (PC-HMWCs) constituted by phytochelatins (PCs), cadmium and sulfide are synthesized by several organisms after exposure to cadmium. In this study, PC-HMWCs were isolated from photoheterotrophic Euglena gracilis and purified to homogeneity, resulting in compounds of molecular mass 50-380 kDa depending on the CdCl2 and sulfate concentrations in the culture medium. In contrast with plants and some yeasts, PC-HMWCs from E. gracilis mainly comprise (57-75%) monothiol molecules (Cys, gamma-glutamylcysteine, GSH) and, to a lesser extent (25-43%), PCs. A similar acid-soluble thiol compound composition was found in whole cell extracts. The -SH/Cd2+ and S2-/Cd2+ ratios found in purified PC-HMWCs were 1.5 and 1.8, respectively; the (-SH + S2-)/Cd2+ ratio was 3.2. PC-HMWCs of molecular mass 60 and 100 kDa were also localized inside Percoll-purified chloroplasts, in which cadmium and PCs were mainly compartmentalized. Cadmium and sulfur-rich clusters with similar sulfur/cadmium stoichiometries to those of the purified PC-HMWCs were detected in the chloroplast and throughout the cell by energy dispersive microanalysis and atomic resolution electron microscopy. The presence of PC-HMWCs in primitive photosynthetic eukaryotes such as the protist, E. gracilis, suggests that their function as the final cadmium-storage-inactivation process is widespread. Their particular intracellular localization suggests that chloroplasts may play a major role in the cadmium-resistance mechanism in organisms lacking a plant-like vacuole.