Physiological comparison of copper toxicity in the lichens <Emphasis Type="Italic">Peltigera rufescens</Emphasis> (Weis) Humb. and <Emphasis Type="Italic">Cladina arbuscula</Emphasis> subsp. <Emphasis Type="Italic">mitis</Emphasis> (Sandst.) Ruoss

Peltigera rufescens (Weis) Humb. with a prokaryotic photobiont Nostoc sp. and Cladina arbuscula subsp. mitis (Sandst.) Ruoss with a eukaryotic photobiont Trebouxia sp. were studied to determine the copper sensitivity of lichens with different algal symbionts. Samples growing on historic copper mine-spoil heaps at Ľubietová–Podlipa, Slovakia were assessed for physiological parameters, including total and intracellular uptake of copper, assimilation pigmentation, activity of photosystem II, ergosterol levels, thiobarbituric acid reactive substances and water-soluble protein content. Our results indicate that P. rufescens was more sensitive to copper exposure than C. arbuscula subsp. mitis.     

Glutamate synthase from Chlamydomonas reinhardtii: Interaction studies with its substrate ferredoxin and molecular cloning

Glutamate synthase (GOGAT) from Chlamydomonas reinhardtii is able to form functional covalent complexes with its substrate ferredoxin (Fd), either wild-type (^WTFd) or recombinant form (^rFd). However, when Fd carboxyl groups were chemically modified (^mdFd), no complexes were detected and its ability to serve as electron donor for glutamate synthase activity was also decreased. By site-directed mutagenesis, we have demonstrated that Fd glu91 and a negative core in the helix α1 are critical for Fd interaction with this enzyme and its functionality as electron carrier for glutamate synthase. As a previous step to elucidate the specific positive charged residues involved in glutamate synthase interaction with Fd, we have isolated a cDNA, CrFG-3, encoding Fd-GOGAT from C. reinhardtii. The cDNA comprised about 60% of the protein and sequence comparison showed that CrFG-3 was structurally more similar to higher plant enzymes than to the corresponding prokaryotic GOGAT. Two conserved domains were present in this protein fragment, the FMN-binding domain and the cysteines involved in the iron–sulfur cluster binding. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of DMT1 knockdown on iron,cadmium, and lead uptake in Caco-2 cells

DMT1 (divalent metal transporter 1) is ahydrogen-coupled divalent metal transporter with a substrate preferencefor iron, although the protein when expressed in frog oocytestransports a broad range of metals, including the toxic metals cadmiumand lead. Wild-type Caco-2 cells displayed saturable transport of leadand iron that was stimulated by acid. Cadmium and manganese inhibitedtransport of iron, but zinc and lead did not. The involvement of DMT1in the transport of toxic metals was examined by establishing clonalDMT1 knockdown and control Caco-2 cell lines. Knockdown cell linesdisplayed much lower levels of DMT1 mRNA and a smaller V_max for iron uptake compared with control celllines. One clone was further characterized and found to display an~50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, butuptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters thesecells through an independent hydrogen-driven mechanism.

     

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in <Emphasis Type="Italic">E. coli</Emphasis> spheroplasts

The spider peptide GsMTx4, at saturating concentration of 5 μM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5–64 μM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein–lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 μM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 μM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity.     

Zinc transport by respiratory epithelial cells and interaction with iron homeostasis

Despite recurrent exposure to zinc through inhalation of ambient air pollution particles, relatively little information is known about the homeostasis of this metal in respiratory epithelial cells. We describe zinc uptake and release by respiratory epithelial cells and test the postulate that Zn²⁺ transport interacts with iron homeostasis in these same cells. Zn²⁺ uptake after 4 and 8 h of exposure to zinc sulfate was concentration- and time-dependent. A majority of Zn²⁺ release occurred in the 4 h immediately following cell exposure to ZnSO₄. Regarding metal importers, mRNA for Zip1 and Zip2 showed no change after respiratory epithelial cell exposure to zinc while mRNA for divalent metal transporter (DMT)1 increased. Western blot assay for DMT1 protein supported an elevated expression of this transport protein following zinc exposure. RT-PCR confirmed mRNA for the metal exporters ZnT1 and ZnT4 with the former increasing after ZnSO₄. Cell concentrations of ferritin increased with zinc exposure while oxidative stress, measured as lipid peroxides, was decreased supporting an anti-oxidant function for Zn²⁺. Increased DMT1 expression, following pre-incubations of respiratory epithelial cells with TNF-α, IFN-γ, and endotoxin, was associated with significantly decreased intracellular zinc transport. Finally, incubations of respiratory epithelial cells with both zinc sulfate and ferric ammonium citrate resulted in elevated intracellular concentrations of both metals. We conclude that exposure to zinc increases iron uptake by respiratory epithelial cells. Elevations in cell iron can possibly affect an increased expression of DMT1 and ferritin which function to diminish oxidative stress. Comparable to other metal exposures, changes in iron homeostasis may contribute to the biological effects of zinc in specific cells and tissues.     

<Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis>: evolution of a metal-resistant bacterium

Cupriavidus metallidurans CH34 has gained increasing interest as a model organism for heavy metal detoxification and for biotechnological purposes. Resistance of this bacterium to transition metal cations is predominantly based on metal resistance determinants that contain genes for RND (resistance, nodulation, and cell division protein family) proteins. These are part of transenvelope protein complexes, which seem to detoxify the periplasm by export of toxic metal cations from the periplasm to the outside. Strain CH34 contains 12 predicted RND proteins belonging to a protein family of heavy metal exporters. Together with many efflux systems that detoxify the cytoplasm, regulators and possible metal-binding proteins, RND proteins mediate an efficient defense against transition metal cations. To shed some light into the origin of genes encoding these proteins, the genomes of C. metallidurans CH34 and six related proteobacteria were investigated for occurrence of orthologous and paralogous proteins involved in metal resistance. Strain CH34 was not much different from the other six bacteria when the total content of transport proteins was compared but CH34 had significantly more putative transition metal transport systems than the other bacteria. The genes for these systems are located on its chromosome 2 but especially on plasmids pMOL28 and pMOL30. Cobalt–nickel and chromate resistance determinants located on plasmid pMOL28 evolved by gene duplication and horizontal gene transfer events, leading to a better adaptation of strain CH34 to serpentine-like soils. The czc cobalt–zinc–cadmium resistance determinant, located on plasmid pMOL30 in addition copper, lead and mercury resistance determinants, arose by duplication of a czcICAB core determinant on chromosome 2, plus addition of the czcN gene upstream and the genes czcD, czcRS, czcE downstream of czcICBA. C. metallidurans apparently evolved metal resistance by horizontal acquisition and by duplication of genes for transition metal efflux, mostly on the two plasmids, and decreased the number of uptake systems for those metals. This paper is dedicated to Dr. Max Mergeay for a long time of cooperation, constructive competition and friendship.     

N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela α2,6-sialyltransferase

Photobacterium damsela α2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16–497 aa or 113–497 aa). Expression and activity assays indicated that the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16–497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST(N), the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5–10.0. A divalent metal ion was not required for the sialyltransferase activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly. Mingchi Sun and Yanhong Li contributed equally to this work.     

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

Summary A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.     

Identification of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> Rad51C: Recombinational characteristics

Unicellular green alga Chlamydomonas reinhardtii is a promising model for fundamental and biotechnological research. However, little is known about its system of homologous recombination underlying recombination repair of double-strand breaks. Sequencing of the C. reinhardtii nuclear genome has revealed many repeats, which account for a low level of nuclear homologous recombination compared to that of nonhomologous recombination. Analysis of C. reinhardtii EST and genomic libraries made it possible to reconstruct and clone the RAD51C cDNA. In this work, this cDNA was expressed, the protein product was purified, and its main biochemical activities were studied. It was shown that Rad51C of lower eukaryote C. reinhardtii is a typical member of the subfamily of higher eukaryotic Rad51-like recombination proteins.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 112–119.Original Russian Text Copyright © 2005 by Shalguev, Kaboev, Sizova, Hagemann, Lanzov.     

Lead and Cadmium Synergistically Enhance the Expression of Divalent Metal Transporter 1 Protein in Central Nervous System of Developing Rats

Divalent metal transporter 1 (DMT1) can transport a large range of ions, including toxic lead (Pb) and cadmium (Cd), across membranes. In this study, a total of 24 rats were divided into four groups for intragastrical perfusion treatment: control, Pb alone, Cd alone, and Pb + Cd. Pb and Cd contents in blood were detected, and the mRNA and protein levels of DMT1 were analyzed in the cerebellum, cortex, and hippocampus. Both Pb and Cd levels were elevated in all groups perfused with Pb and/or Cd, except for Pb level in the Cd-alone group (P < 0.05). The mRNA level of DMT1 did not differ among the four groups (P > 0.05). However, the DMT1 protein expression was significantly increased by 0.9-, 1.0-, and 1.1-fold in cerebellum, cortex, and hippocampus of the Pb + Cd group than in controls, respectively. Pb and Cd exposure can synergistically induce DMT1 protein synthesis and has implications for transportation of toxic ions in the developing rat’s brain. Chengwu Gu and Songjian Chen contributed equally to this work, they are joint first authors.     

Molecular analysis of increased iron status in moderately exercised rats

Although iron plays a critical role in exercise, the regulatory mechanism of iron metabolism remains poorly understood. The aims of the present study were to investigate the effects of different intensity exercise on body iron status and the regulatory mechanism of duodenal iron absorption. Thirty female Sprague-Dawley rats (90–100 g) were randomly divided into three groups: a control group (remained sedentary, CG), a moderately exercised group (swam 1.5 h/day, MG) and a strenuously exercised group (swam with different load, SG). Serum iron status, serum ferritin and Hct were examined after 10 weeks of swimming. Western blot was performed to detect the expression of iron transport proteins: divalent metal transporter1 (DMT1) and ferroportin 1 (FPN1) in duodenal epithelium. The expression of hepcidin mRNA in liver was examined by RT-PCR. The results showed: (1) the body iron status in MG was kept at a high level compared to that of CG and SG, (2) Western blot showed DMT1 with iron responsive element (IRE) and FPN1 in duodenal epithelium which were higher in MG than that of CG and (3) the expression of hepatic hepcidin mRNA was down regulated in MG (p < 0.05). The data suggested that moderate exercise improved iron status and that was likely regulated by increased DMT1 with IRE and FPN1 expression. Hepcidin signaling pathway may involve in the regulation of duodenal iron absorption proteins. Xiang Lin Duan and Yan Zhong Chang share Senior Authorship     

Characterization of a new RNase HII and its essential amino acid residues in the archaeon <Emphasis Type="Italic">Sulfolobus tokodaii</Emphasis> reveals a regulatory C-terminus

The archaea possess RNase H proteins that share features of both prokaryotic and eukaryotic forms. Although the Sulfolobus RNase HI has been reported to have unique structural and biochemical properties, its RNase HII has not yet been investigated and its biochemical properties remain unknown. In the present study, we have characterized the ST0519 RNase HII from S. tokodaii as a new form. The enzyme utilized hybrid RNA/DNA as a substrate and had an optimal temperature between 37 and 50°C. The activity of wild-type protein was stimulated by Mn²⁺, whereas this cation significantly inhibited the activity of C-terminal truncated mutant proteins. A series of mutation assays revealed a regulatory C-terminal tail in the S. tokodaii RNase HII. One mutant, ST0519 (residues 1–195), retained only partial activity, while ST0519 (residues 1–196) completely lost its activity. Based on the presumed structure, the C-terminus might form a short α-helix in which two residues, I195 and L196, are essential for the cleavage activity. Our data suggest that the C-terminal α-helix is likely involved in the Mn²⁺-dependent substrate cleavage activity through stabilization of a flexible loop structure. Our findings offer important clues for further understanding the structure and function of both archaeal and eukaryotic RNase HII.     

Deficiency in the divalent metal transporter 1 increases bleomycin-induced lung injury

Exposure to bleomycin can result in an inflammatory lung injury. The biological effect of this anti-neoplastic agent is dependent on its coordination of iron with subsequent oxidant generation. In lung cells, divalent metal transporter 1 (DMT1) can participate in metal transport resulting in control of an oxidative stress and tissue damage. We tested the postulate that metal import by DMT1 would participate in preventing lung injury after exposure to bleomycin. Microcytic anemia (mk/mk) mice defective in DMT1 and wild-type mice were exposed to either bleomycin or saline via intratracheal instillation and the resultant lung injury was compared. Twenty-four h after instillation, the number of neutrophils and protein concentrations after bleomycin exposure were significantly elevated in the mk/mk mice relative to the wild-type mice. Similarly, levels of a pro-inflammatory mediator were significantly increased in the mk/mk mice relative to wild-type mice following bleomycin instillation. Relative to wild-type mice, mk/mk mice demonstrated lower non-heme iron concentrations in the lung, liver, spleen, and splenic, peritoneal, and liver macrophages. In contrast, levels of this metal were elevated in alveolar macrophages from mk/mk mice. We conclude that DMT1 participates in the inflammatory lung injury after bleomycin with mk/mk mice having increased inflammation and damage following exposure. This finding supports the hypothesis that DMT1 takes part in iron detoxification and homeostasis in the lung.     

Modulation of DMT1 Activity by Redox Compounds

Iron(II) exacerbates the effects of oxidative stress via the Fenton reaction. A number of human diseases are associated with iron accumulation including ischemia-reperfusion injury, inflammation and certain neurodegenerative diseases. The functional properties and localization in plasma membrane of cells and endosomes suggest an important role for the divalent metal transporter DMT1 (also known as DCT1 and Nramp2) in iron transport and cellular iron homeostasis. Although iron metabolism is strictly controlled and the activity of DMT1 is central in controlling iron homeostasis, no regulatory mechanisms for DMT1 have been so far identified. Our studies show that the activity of DMT1 is modulated by compounds that affect its redox status. We also show that both iron and zinc are transported by DMT1 when expressed in Xenopus laevis oocytes. Radiotracer uptake and electrophysiological measurements revealed that H₂O₂ and Hg²⁺ treatments result in substantial inhibition of DMT1. These findings may have a profound relevance from a physiological and pathophysiological standpoint. Present address for D.T.: Department of Neurology, Cecil B. Day Laboratory for Neuromuscular Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA     

Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of three Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. Of six ZIP’s studied, MtZIP1, MtZIP5 and MtZIP6 were the only members from this family determined to transport Zn and were further characterized. MtZIP1 has a low affinity for Zn with a K_m of 1 μM as compared to MtZIP5 and MtZIP6 that have a higher affinity for Zn with K_m of 0.4 μM and 0.3 μM, respectively. Zn transport by MtZIP1 was more sensitive to inhibition by copper (Cu) concentrations than MtZIP5 and MtZIP6, because 3 μM Cu inhibited Zn transport by 80% in MtZIP1 while 5 μM Cu was required to achieve the same inhibition of Zn transport in MtZIP5 and MtZIP6. Cadmium (Cd) had a greater effect on the ability of MtZIP1 to transport Zn than MtZIP5 and MtZIP6, because at a concentration of 3 μM Cd, the Zn transport by MtZIP1 was inhibited 55% and the transport of Zn by MtZIP5 and MtZIP6 was inhibited by 20–30%. However, only MtZIP6 transported Cd at higher rates than those observed in the control plasmid pFL61, demonstrating a low affinity for Cd based on a K_m of 57 μM. These results suggest that Medicago truncatula has both high and low affinity Zn transporters to maintain Zn homeostasis and that these transporters may function in different compartments within the plant.     

Genetic analysis of mutant clones of Chlamydomonas reinhardtii defective in potassium transport

Mutant clones of Chlamydomonas reinhardtii defective for potassium transport were isolated and characterized. Of the four genes identified, three –TRK1, TRK2 and TRK3– encode high-affinity transport functions, and one gene, HKR1, encodes a low-affinity transport function. Characterization of the potassium dependence of recombinants possessing two mutant trk alleles suggests that the protein products of TRK2 and TRK3 interact functionally, and that TRK1 may serve a regulatory function. The mutant clone defective for a low-affinity potassium transporter was isolated by mutagenizing trk2-1 cells, which lack a functional high-affinity transporter, and screening surviving cells for dependence on very high potassium concentrations. The hkr1 phenotype is expressed only in the presence of trk2-1. Received: 24 August 1998 / Accepted: 16 November 1998     

Methionine motifs of copper transport proteins provide general and flexible thioether-only binding sites for Cu(I) and Ag(I)

Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins (Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM, where X can be one of several amino acids. Model peptides corresponding to 15 different Mets motifs were synthesized and determined to selectively bind Cu(I) and Ag(I), with no discernible affinity for divalent metal ions. These are rare examples of biological thioether-only metal binding sites. Effective dissociation constant (K _D) values for the model Mets peptides and Cu(I) were determined by an ascorbic acid oxidation assay and validated through electrospray ionization mass spectrometry and range between 2 and 11 μM. Affinity appears to be independent of pH, the arrangement of the motif, and the composition of intervening amino acids, all of which reveal the generality and flexibility of the MX_1–2MX_1–2M domain. Circular dichroism spectroscopy, ¹H-NMR spectroscopy, and X-ray absorption spectroscopy were also used to characterize the binding event. These results are intended to aid the development of the still unknown mechanism of copper transport across the cell membrane.     

Polyclonal antibodies against the TLA1 protein also recognize with high specificity the D2 reaction center protein of PSII in the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The Chlamydomonas reinhardtii DNA-insertional transformant truncated light-harvesting antenna 1 (tla1) mutant, helped identify the novel TLA1 gene (GenBank Accession # AF534570-71) as an important genetic determinant in the chlorophyll antenna size of photosynthesis. Down-regulation in the amount of the TLA1 23 kDa protein in the cell resulted in smaller chlorophyll antenna size for both photosystems (in Tetali et al. Planta 225:813–829, 2007). Specific polyclonal antibodies, raised against the recombinant TLA1 protein, showed a cross-reaction with the predicted 23 kDa TLA1 protein in C. reinhardtii protein extracts, but also showed a strong cross-reaction with a protein band migrating to 28.5 kDa. Questions of polymorphism, or posttranslational modification of the TLA1 protein were raised as a result of the unexpected 28.5 kDa cross-reaction. Work in this paper aimed to elucidate the nature of the unexpected 28.5 kDa cross-reaction, as this was deemed to be important in terms of the functional role of the TLA1 protein in the regulation of the chlorophyll antenna size of photosynthesis. Immuno-precipitation of the 28.5 kDa protein, followed by LC–mass spectrometry, showed amino acid sequences ascribed to the psbD/D2 reaction center protein of PSII. The common antigenic determinant between TLA1 and D2 was shown to be a stretch of nine conserved amino acids V-F—L(V)LP-GNAL in the C-terminus of the two proteins, constituting a high antigenicity “GNAL” domain. Antibodies raised against the TLA1 protein containing this domain recognized both the TLA1 and the D2 protein. Conversely, antibodies raised against the TLA1 protein minus the GNAL domain specifically recognized the 23 kDa TLA1 protein and failed to recognize the 28.5 kDa D2 protein. D2 antibodies raised against an oligopeptide containing this domain also cross-reacted with the TLA1 protein. It is concluded that the 28.5 kDa cross-reaction of C. reinhardtii protein extracts with antiTLA1 antibodies is due to antibody affinity for the GNAL domain of the D2 protein and has no bearing on the identity or function of the TLA1 protein.     

Extracellular phycoerythrin-like protein released by freshwater cyanobacteria <Emphasis Type="Italic">Oscillatoria</Emphasis> and <Emphasis Type="Italic">Scytonema</Emphasis> sp.

During growth of the freshwater cyanobacteria, Oscillatoria sp. BTCC/A0004, and Scytonema sp. TISTR 8208, a pink pigment is released into the growth medium. The pigment from each source had a molecular weight of approximately 250 kDa and had adsorption maxima at 560 and 620 nm. These results suggest that pink pigment is a phycoerythrin-like protein. It inhibited the growth of green algae, Chlorella fusca and Chlamydomonas reinhardtii, but not other cyanobacteria or true bacteria. The concentration at which growth inhibition 50% occurred was 0.5, 6 and more than 10 mg ml⁻¹, respectively.