A Functional Putative Phytochelatin Synthase from the Primitive Red Alga Cyanidioschyzon merolae

Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins (PCs), which play a detoxification role in higher plants. Heterologous expression of CmPCS, a product of a PCS-like gene from the genomic DNA of the red alga Cyanidioschyzon merolae, rescued Cd²⁺-sensitive yeast from Cd²⁺ toxicity. The fact that these transformed cells synthesized PCs demonstrates that CmPCS is functional.     

Characterization of Cadmium Uptake in Lactobacillus plantarum and Isolation of Cadmium and Manganese Uptake Mutants

Two different Cd²⁺ uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn²⁺ uptake system which also takes up Cd²⁺ and is induced by Mn²⁺ starvation. The calculated K_m and V_max are 0.26 μM and 3.6 μmol g of dry cell⁻¹ min⁻¹, respectively. Unlike Mn²⁺ uptake, which is facilitated by citrate and related tricarboxylic acids, Cd²⁺ uptake is weakly inhibited by citrate. Cd²⁺ and Mn²⁺ are competitive inhibitors of each other, and the affinity of the system for Cd²⁺ is higher than that for Mn²⁺. The other Cd²⁺ uptake system is expressed in Mn²⁺-sufficient cells, and no K_m can be calculated for it because uptake is nonsaturable. Mn²⁺ does not compete for transport through this system, nor does any other tested cation, i.e., Zn²⁺, Cu²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺, or Ni²⁺. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn²⁺-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn²⁺ for growth as the parental strain. Mn²⁺ starvation-induced Cd²⁺ uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn²⁺ or Cd²⁺ accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn²⁺ and Cd²⁺ uptake system.     

Enhanced accumulation of Cd2+ by a Mesorhizobium sp. transformed with a gene from Arabidopsis thaliana coding for phytochelatin synthase

We expressed the Arabidopsis thaliana gene for phytochelatin synthase (PCS(At)) in Mesorhizobium huakuii subsp. rengei B3, a microsymbiont of Astragalus sinicus, a legume used as manure. The PCS(At) gene was expressed under the control of the nifH promoter, which regulates the nodule-specific expression of the nifH gene. The expression of the PCS(At) gene was demonstrated in free-living cells under low-oxygen conditions. Phytochelatin synthase (PCS) was expressed and catalyzed the synthesis of phytochelatins [(gamma-Glu-Cys)(n)-Gly; PCs] in strain B3. A range of PCs, with values of n from 2 to 7, was synthesized by cells that expressed the PCS(At) gene, whereas no PCs were found in control cells that harbored the empty plasmid. The presence of CdCl(2) activated PCS and induced the synthesis of substantial amounts of PCs. Cells that contained PCs accumulated 36 nmol of Cd(2+)/mg (dry weight) of cells. The expression of the PCS(At) gene in M. huakuii subsp. rengei B3 increased the ability of cells to bind Cd(2+) approximately 9- to 19-fold. The PCS protein was detected by immunostaining bacteroids of mature nodules of A. sinicus containing the PCS(At) gene. When recombinant M. huakuii subsp. rengei B3 established the symbiotic relationship with A. sinicus, the symbionts increased Cd(2+) accumulation in nodules 1.5-fold.     

Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif

A gene coding for a de novo peptide sequence containing a metal binding motif was chemically synthesized and expressed in Escherichia coli as a fusion with the maltose binding protein. Bacterial cells expressing the metal binding peptide fusion demonstrated enhanced binding of Cd²⁺ and Hg²⁺ compared to bacterial cells lacking the metal binding peptide. The potential use of genetically engineered bacteria as biosorbents for the removal of heavy metals from wastewaters is discussed.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

Development, Characterization, and Application of a Cadmium-Selective Microelectrode for the Measurement of Cadmium Fluxes in Roots of Thlaspi Species and Wheat

A Cd²⁺-selective vibrating microelectrode was constructed using a neutral carrier-based Cd ionophore to investigate ion-transport processes along the roots of wheat (Triticum aestivum L.) and two species of Thlaspi, one a Zn/Cd hyperaccumulator and the other a related nonaccumulator. In simple Cd(NO₃)₂ solutions, the electrode exhibited a Nernstian response in solutions with Cd²⁺ activities as low as 50 nm. Addition of Ca²⁺ to the calibration solutions did not influence the slope of the calibration curve but reduced the detection limit to a solution activity of 1 μm Cd²⁺. Addition of high concentrations of K⁺ and Mg²⁺ to the calibration solution to mimic the ionic composition of the cytoplasm affected neither the slope nor the sensitivity of the electrode, demonstrating the pH-insensitive electrode's potential for intracellular investigations. The electrode was assayed for selectivity and was shown to be at least 1000 times more selective for Cd²⁺ than for any of those potentially interfering ions tested. Flux measurements along the roots of the two Thlaspi species showed no differences in the pattern or the magnitude of Cd²⁺ uptake within the time frame considered. The Cd²⁺-selective microelectrode will permit detailed investigations of heavy-metal ion transport in plant roots, especially in the area of phytoremediation.     

cadDX Operon of Streptococcus salivarius 57.I

A CadDX system that confers resistance to Cd²⁺ and Zn²⁺ was identified in Streptococcus salivarius 57.I. Unlike with other CadDX systems, the expression of the cad promoter was negatively regulated by CadX, and the repression was inducible by Cd²⁺ and Zn²⁺, similar to what was found for CadCA systems. The lower G+C content of the S. salivarius cadDX genes suggests acquisition by horizontal gene transfer.     

A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme

We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thiosubstituted substrates S11SpS and S11RpS, in which the respective pro-Sp and pro-Rp oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg²⁺ or Ca²⁺ ions the rate of ribozyme-catalyzed cleavage of S11SpS was as high as that of S11O, whereas the corresponding rate for S11RpS was nearly four orders of magnitude lower than that for either S11O or S11SpS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd²⁺ ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the Rp position of the cleavage site with the added Cd²⁺ ion. However, our present analysis demonstrates that (i) the added Cd²⁺ ion binds at the P9 site; (ii) the bound Cd²⁺ ion at the P9 site replaces two Mg²⁺ or two Ca²⁺ ions, an observation that suggests a different mode of interaction with the added Cd²⁺ ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd²⁺ ion does not interact with the sulfur atom at the Rp position of the scissile phosphate either in the ground state or in the transition state.     

atp Mutants of Escherichia coli Fail To Grow on Succinate Due to a Transport Deficiency

Escherichia coli atp mutants, which lack a functional H⁺-ATPase complex, are capable of growth on glucose but not on succinate or other C₄-dicarboxylates (Suc⁻ phenotype). Suc⁺ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H⁺-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc⁺ mutants on succinate, which implicates the yhiF gene product in the regulation of C₄-dicarboxylate metabolism. Indeed, when the E. coli C₄-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc⁻ phenotype of the atp deletion strain reverted to Suc⁺, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C₄-dicarboxylates is insufficient transport capacity for these substrates.     

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd²⁺ uptake as a model system. Radiotracer techniques were used to quantify unidirectional ¹⁰⁹Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for ¹⁰⁹Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd²⁺ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd²⁺ influx across the root-cell plasma membrane. The Cd²⁺ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd²⁺ influx, whereas the maximum initial velocity for Cd²⁺ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H⁺-ATPase did not play a role in the enhanced Cd²⁺ uptake. Expression studies with the Fe²⁺ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd²⁺ and Zn²⁺, in addition to Fe²⁺.     

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons

Low voltage–activated (LVA) T-type Ca²⁺ (I_CaT) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and I_CaT currents. NaN/Nav1.9 was inhibited by inorganic I_Ca blockers as follows (IC₅₀, μM): La³⁺ (46) > Cd²⁺ (233) > Ni²⁺ (892) and by mibefradil, a non-dihydropyridine I_CaT antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni²⁺-resistant I_CaT (AR-I_CaT) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Aδ–like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aα/β cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-I_CaT was restricted to CII-nociceptors, putative D-hair cells, and Aα/β-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aα/β-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.     

Characterization of SKT1, an Inwardly Rectifying Potassium Channel from Potato, by Heterologous Expression in Insect Cells

A cDNA encoding a novel, inwardly rectifying K⁺ (K⁺_in) channel protein, SKT1, was cloned from potato (Solanum tuberosum L.). SKT1 is related to members of the AKT family of K⁺_in channels previously identified in Arabidopsis thaliana and potato. Skt1 mRNA is most strongly expressed in leaf epidermal fragments and in roots. In electrophysiological, whole-cell, patch-clamp measurements performed on baculovirus-infected insect (Spodoptera frugiperda) cells, SKT1 was identified as a K⁺_in channel that activates with slow kinetics by hyperpolarizing voltage pulses to more negative potentials than −60 mV. The pharmacological inhibitor Cs⁺, when applied externally, inhibited SKT1-mediated K⁺_in currents half-maximally with an inhibitor concentration (IC₅₀) of 105 μm. An almost identical high Cs⁺ sensitivity (IC₅₀ = 90 μm) was found for the potato guard-cell K⁺_in channel KST1 after expression in insect cells. SKT1 currents were reversibly activated by a shift in external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺_in channels. Comparative studies revealed generally higher current amplitudes for KST1-expressing cells than for SKT1-expressing insect cells, which correlated with a higher targeting efficiency of the KST1 protein to the insect cell's plasma membrane, as demonstrated by fusions to green fluorescence protein.     

A Saccharomyces cerevisiae Mutant Lacking a K+/H+ Exchanger

The KHA1 gene corresponding to the open reading frame YJL094c (2.62 kb) encoding a putative K⁺/H⁺ antiporter (873 amino acids) in Saccharomyces cerevisiae was disrupted by homologous recombination. The core protein is similar to the putative Na⁺/H⁺ antiporters from Enterococcus hirae (NAPA gene) and Lactococcus lactis (LLUPP gene) and the putative K⁺/H⁺ exchanger from Escherichia coli (KEFC gene). Disruption of the KHA1 gene resulted in an increased K⁺ accumulation and net influx without a significant difference in efflux, as well as an increased growth rate, smaller cells, and twice the cell yield per glucose used. Flow cytometry analysis showed an increase of the DNA duplication rate in the mutant. Kinetic studies of ⁸⁶Rb⁺ uptake showed the same saturable system for wild-type and disruptant strains. Mutant cells also produced a greater acidification of the medium coincident with an internal pH alkalinization and showed a higher oxygen consumption velocity. We speculate that higher K⁺ accumulation and increased osmotic pressure accelerate the cell cycle and metabolic activity.     

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd²⁺) can result in cell death, but the molecular mechanisms of Cd²⁺ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl₂ and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl₂ treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N^G-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd²⁺-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd²⁺ cytotoxicity in plants.Cadmium (Cd²⁺) is a heavy metal with a long biological half-life, and its presence as a pollutant in agricultural soil is due mainly to anthropogenic activities. It is rapidly taken up by roots and enters the food chain, resulting in toxicity for both plants and animals (for review, see Sanità di Toppi and Gabbrielli, 1999). Cd²⁺ inhibits seed germination, decreases plant growth and photosynthesis, and impairs the distribution of nutrients. Overall, the symptoms of chronic exposure to sublethal amounts of Cd²⁺ mimic premature senescence (Rascio et al., 1993; McCarthy et al., 2001; Sandalio et al., 2001; Rodriguez-Serrano et al., 2006). Depending on the concentration, Cd²⁺ treatment of tobacco (Nicotiana tabacum) cell cultures and onion (Allium cepa) roots eventually triggers either necrosis or programmed cell death (PCD; Fojtovà and Kovařik, 2000; Behboodi and Samadi, 2004).Although Cd²⁺ is an environmental threat, the mechanisms by which it exerts its toxic effects in plants are not fully understood. In plant cells, Cd²⁺ is believed to enter through Fe²⁺, Ca²⁺, and Zn²⁺ transporters/channels (Clemens, 2006). Once in the cytosol, Cd²⁺ stimulates the production of phytochelatins (PCs), a glutathione-derived class of peptides containing repeated units of Glu and Cys, which bind the metal ions and transport them into the vacuole (Sanità di Toppi and Gabbrielli, 1999). Strong evidence exists that high (millimolar) concentrations of Cd²⁺ induce reactive oxygen species (ROS) bursts in plants, which might have a role in signaling and/or degenerative steps leading to cell death (Piqueras et al., 1999; Olmos et al., 2003; Cho and Seo, 2005; Garnier et al., 2006). Treatment with a lower, nontoxic Cd²⁺ concentration also caused increase in ROS production in pea (Pisum sativum) leaves and roots (Sandalio et al., 2001; Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006) and Arabidopsis (Arabidopsis thaliana) cell cultures (Horemans et al., 2007).Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signaling role in many developmental and response processes (for review, see Neill et al., 2003; Besson-Bard et al., 2008). In plants, it can be synthesized via several routes, either enzymatically or by chemical reduction of nitrite. Nitrate reductase and a root-specific plasma membrane nitrite-NO reductase also utilize nitrite as substrate. In animals, nitric oxide synthase (NOS) converts l-Arg into NO and l-citrulline. Although no plant NOS has been unambiguously identified yet, activity assays and pharmacological evidence suggests the existence of a NOS-like counterpart in plants. Depending on its concentration and possibly on the timing and localization of its production, NO can either act as an antioxidant or promote PCD, often in concert with ROS (Delledonne et al., 2001; Beligni et al., 2002; de Pinto et al., 2006). Extensive research has shown that NO plays a fundamental role in the hypersensitive response, but its involvement in other types of PCD, such as that resulting from mechanical stress and natural and cytokinin-induced senescence of cell cultures, has also been demonstrated (Garcês et al., 2001; Carimi et al., 2005). Because of its participation in numerous biotic and abiotic responses, NO has been proposed as a general stress molecule (Gould et al., 2003). However, the mechanisms by which NO determines its effects are far from being completely elucidated, and a number of downstream signaling pathways, involving Ca²⁺, cyclic GMP, and cyclic ADP-Rib, are involved (Neill et al., 2003; Besson-Bard et al., 2008). NO can also modulate biological responses by direct modification of proteins, reacting with Cys residues (S-nitrosylation), Tyr residues (nitration), or iron and zinc in metalloproteins (metal nitrosylation; Besson-Bard et al., 2008).The aim of this work is to study the plant responses to various concentrations of Cd²⁺ and, in particular, the role of ROS and NO in the signaling events leading to cell death. Cell cultures of the model plant Arabidopsis were chosen as an experimental system because the homogeneity and undifferentiated state of the cells, combined with the uniform delivery of the treatments, allow a clear and reproducible response. The results point to NO as a master regulator of Cd²⁺-induced cell death. Possible mechanisms that explain this evidence will be discussed.     

Cadmium Ion Biosorption by the Thermophilic Bacteria Geobacillus stearothermophilus and G. thermocatenulatus

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl₂ (as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd²⁺ adsorption experiments to characterize Cd²⁺ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd²⁺ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.     

Mitochondrial Contribution to the Anoxic Ca2+ Signal in Maize Suspension-Cultured Cells

Anoxia induces a rapid elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in maize (Zea mays L.) cells, which is caused by the release of the ion from intracellular stores. This anoxic Ca²⁺ release is important for gene activation and survival in O₂-deprived maize seedlings and cells. In this study we examined the contribution of mitochondrial Ca²⁺ to the anoxic [Ca²⁺]_cyt elevation in maize cells. Imaging of intramitochondrial Ca²⁺ levels showed that a majority of mitochondria released their Ca²⁺ in response to anoxia and took up Ca²⁺ upon reoxygenation. We also investigated whether the mitochondrial Ca²⁺ release contributed to the increase in [Ca²⁺]_cyt under anoxia. Analysis of the spatial association between anoxic [Ca²⁺]_cyt changes and the distribution of mitochondrial and other intracellular Ca²⁺ stores revealed that the largest [Ca²⁺]_cyt increases occurred close to mitochondria and away from the tonoplast. In addition, carbonylcyanide p-trifluoromethoxyphenyl hydrazone treatment depolarized mitochondria and caused a mild elevation of [Ca²⁺]_cyt under aerobic conditions but prevented a [Ca²⁺]_cyt increase in response to a subsequent anoxic pulse. These results suggest that mitochondria play an important role in the anoxic elevation of [Ca²⁺]_cyt and participate in the signaling of O₂ deprivation.     

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca²⁺-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca²⁺]_cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca²⁺]_cyt and inhibition of growth that was similar to the effect of the Ca²⁺ channel blocker La³⁺ and the Ca²⁺ chelator ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid. The Ca²⁺ channel blockers verapamil and nifedipine did not induce a decrease in [Ca²⁺]_cyt in these cells and also failed to inhibit growth. Al and La³⁺, but not verapamil or nifedipine, reduced the rate of Mn²⁺ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca²⁺- and Mn²⁺-permeable channels. These results suggest that Al may act to block Ca²⁺ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.     

Hair loss and defective T- and B-cell function in mice lacking ORAI1

ORAI1 is a pore subunit of the store-operated Ca²⁺ release-activated Ca²⁺ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1^−/− mice. Orai1^−/− mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1^−/− mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1^−/− mice, but B cells showed a substantial decrease in Ca²⁺ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1^−/− T cells showed substantial reductions in store-operated Ca²⁺ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.     

The cdr2+ Gene Encodes a Regulator of G2/M Progression and Cytokinesis in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells respond to nutrient deprivation by altering G₂/M cell size control. The G₂/M transition is controlled by activation of the cyclin-dependent kinase Cdc2p. Cdc2p activation is regulated both positively and negatively. cdr2⁺ was identified in a screen for regulators of mitotic control during nutrient deprivation. We have cloned cdr2⁺ and have found that it encodes a putative serine-threonine protein kinase that is related to Saccharomyces cerevisiae Gin4p and S. pombe Cdr1p/Nim1p. cdr2⁺ is not essential for viability, but cells lacking cdr2⁺ are elongated relative to wild-type cells, spending a longer period of time in G₂. Because of this property, upon nitrogen deprivation cdr2⁺ mutants do not arrest in G₁, but rather undergo another round of S phase and arrest in G₂ from which they are able to enter a state of quiescence. Genetic evidence suggests that cdr2⁺ acts as a mitotic inducer, functioning through wee1⁺, and is also important for the completion of cytokinesis at 36°C. Defects in cytokinesis are also generated by the overproduction of Cdr2p, but these defects are independent of wee1⁺, suggesting that cdr2⁺ encodes a second activity involved in cytokinesis.