Heavy Metal Resistance Patterns of Frankia Strains

The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag¹⁺, AsO₂¹⁻, Cd²⁺, SbO₂¹⁻, and Ni²⁺, but most of the strains were less sensitive to Pb²⁺ (6 to 8 mM), CrO₄²⁻ (1.0 to 1.75 mM), AsO₄³⁻ (>50 mM), and SeO₂²⁻ (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu²⁺, four strains were resistant to elevated levels of Cu²⁺ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO₂²⁻ resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb²⁺ resistance and Cu²⁺ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.     

Metal Ions May Suppress or Enhance Cellular Differentiation in Candida albicans and Candida tropicalis Biofilms

Candida albicans and Candida tropicalis are polymorphic fungi that develop antimicrobial-resistant biofilm communities that are characterized by multiple cell morphotypes. This study investigated cell type interconversion and drug and metal resistance as well as community organization in biofilms of these microorganisms that were exposed to metal ions. To study this, Candida biofilms were grown either in microtiter plates containing gradient arrays of metal ions or in the Calgary Biofilm Device for high-throughput susceptibility testing. Biofilm formation and antifungal resistance were evaluated by viable cell counts, tetrazolium salt reduction, light microscopy, and confocal laser scanning microscopy in conjunction with three-dimensional visualization. We discovered that subinhibitory concentrations of certain metal ions (CrO₄²⁻, Co²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, AsO₂⁻, and SeO₃²⁻) caused changes in biofilm structure by blocking or eliciting the transition between yeast and hyphal cell types. Four distinct biofilm community structure types were discerned from these data, which were designated “domed,” “layer cake,” “flat,” and “mycelial.” This study suggests that Candida biofilm populations may respond to metal ions to form cell-cell and solid-surface-attached assemblages with distinct patterns of cellular differentiation.     

Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag⁺, HgCl₂, etc.) can induce a marked, transitory stimulation of O₂ uptake (QO₂) in Egeria densa leaves, insensitive to CN⁻ and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O₂ consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H⁺-ATPase. In this investigation we compared the FC-induced increase in O₂ consumption with those induced by NEM and Ag⁺, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO₂. The results show (a) the different nature of the FC- and NEM- or Ag⁺-induced increases of QO₂; (b) that FC counteracts the NEM- (and Ag⁺)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN⁻-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.     

Identification of OmpT as the Protease That Hydrolyzes the Antimicrobial Peptide Protamine before It Enters Growing Cells of Escherichia coli

The influence of extracytoplasmic proteases on the resistance of Escherichia coli to the antimicrobial peptide protamine was investigated by testing strains with deletions in the protease genes degP, ptr, and ompT. Only ΔompT strains were hypersusceptible to protamine. This effect was abolished by plasmids carrying ompT. Both at low and at high Mg²⁺ concentrations, ompT⁺ strains cleared protamine from the medium within a few minutes. By contrast, at high Mg²⁺ concentrations, protamine remained present for at least 1 h in the medium of an ompT strain. These data indicate that OmpT is the protease that degrades protamine and that it exerts this function at the external face of the outer membrane.     

Resistance to Mucosal Lysozyme Compensates for the Fitness Deficit of Peptidoglycan Modifications by Streptococcus pneumoniae

The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM^+/+) and -deficient (LysM^−/−) mice. The wild-type strain out-competed the double mutant in LysM^+/+, but not LysM^−/− mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM^+/+ and LysM^−/− mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM^+/+ but not LysM^−/− mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.     

Escherichia coli Strains Lacking Protein HU Are UV Sensitive due to a Role for HU in Homologous Recombination

hupA and hupB encode the α and β subunits of the Escherichia coli histone-like protein HU. Here we show that E. coli hup mutants are sensitive to UV in the rec⁺ sbc⁺, recBC sbcA, recBC sbcBC, umuDC, recF, and recD backgrounds. However, hupAB mutations do not enhance the UV sensitivity of resolvase-deficient recG ruvA strains. hupAB uvrA and hupAB recG strains are supersensitive to UV. hup mutations enhance the UV sensitivity of ruvA strains to a much lesser extent but enhance that of rus-1 ruvA strains to the same extent as for rus⁺ ruv⁺ strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity of hup mutants cannot be suppressed by overexpression of wild-type or mutant gyrB, which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Low-Concentration Kinetics of Atmospheric CH4 Oxidation in Soil and Mechanism of NH4+ Inhibition

NH₄⁺ inhibition kinetics for CH₄ oxidation were examined at near-atmospheric CH₄ concentrations in three upland forest soils. Whether NH₄⁺-independent salt effects could be neutralized by adding nonammoniacal salts to control samples in lieu of deionized water was also investigated. Because the levels of exchangeable endogenous NH₄⁺ were very low in the three soils, desorption of endogenous NH₄⁺ was not a significant factor in this study. The K_m(app) values for water-treated controls were 9.8, 22, and 57 nM for temperate pine, temperate hardwood, and birch taiga soils, respectively. At CH₄ concentrations of ≤15 μl liter⁻¹, oxidation followed first-order kinetics in the fine-textured taiga soil, whereas the coarse-textured temperate soils exhibited Michaelis-Menten kinetics. Compared to water controls, the K_m(app) values in the temperate soils increased in the presence of NH₄⁺ salts, whereas the V_max(app) values decreased substantially, indicating that there was a mixture of competitive and noncompetitive inhibition mechanisms for whole NH₄⁺ salts. Compared to the corresponding K⁺ salt controls, the K_m(app) values for NH₄⁺ salts increased substantially, whereas the V_max(app) values remained virtually unchanged, indicating that NH₄⁺ acted by competitive inhibition. Nonammoniacal salts caused inhibition to increase with increasing CH₄ concentrations in all three soils. In the birch taiga soil, this trend occurred with both NH₄⁺ and K⁺ salts, and the slope of the increase was not affected by the addition of NH₄⁺. Hence, the increase in inhibition resulted from an NH₄⁺-independent mechanism. These results show that NH₄⁺ inhibition of atmospheric CH₄ oxidation resulted from enzymatic substrate competition and that additional inhibition that was not competitive resulted from a general salt effect that was independent of NH₄⁺.     

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.     

The Respiratory Burst and Electrolyte Leakage Induced by Sulfhydryl Blockers in Egeria densa Leaves Are Associated with H2O2 Production and Are Dependent on Ca2+ Influx

In leaves of Egeria densa Planchon, N-ethylmaleimide (NEM) and other sulfhydryl-binding reagents induce a temporary increase in nonmitochondrial respiration (ΔQO₂) that is inhibited by diphenylene iodonium and quinacrine, two known inhibitors of the plasma membrane NADPH oxidase, and are associated with a relevant increase in electrolyte leakage (M. Bellando, S. Sacco, F. Albergoni, P. Rocco, M.T. Marré [1997] Bot Acta 110: 388–394). In this paper we report data indicating further analogies between the oxidative burst induced by sulfhydryl blockers in E. densa and that induced by pathogen-derived elicitors in animal and plant cells: (a) NEM- and Ag⁺-induced ΔQO₂ was associated with H₂O₂ production and both effects depended on the presence of external Ca²⁺; (b) Ca²⁺ influx was markedly increased by treatment with NEM; (c) the Ca²⁺ channel blocker LaCl₃ inhibited ΔQO₂, electrolyte release, and membrane depolarization induced by the sulfhydryl reagents; and (d) LaCl₃ also inhibited electrolyte leakage induced by the direct infiltration of the leaves with H₂O₂. These results suggest a model in which the interaction of sulfhydryl blockers with sulfhydryl groups of cell components would primarily induce an increase in the Ca²⁺ cytosolic concentration, followed by membrane depolarization and activation of a plasma membrane NADPH oxidase. This latter effect, producing active oxygen species, might further influence plasma membrane permeability, leading to the massive release of electrolytes from the tissue.     

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a +/? Mouse Model

Background

Loss-of-function mutations in SCN5A, the gene encoding Na_v1.5 Na⁺ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a ^+/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A.

Methodology/Principal Findings

Based on ECG, 10-week-old Scn5a ^+/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na⁺ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a ^+/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a ^+/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a ^+/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a ^+/− mice had similar Na_v1.5 mRNA but higher Na_v1.5 protein expression, and moderately larger I_Na current than severely affected Scn5a ^+/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a ^+/− mice than in mildly affected ones.

Conclusions

Scn5a ^+/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a ^+/− mice, phenotype severity correlates with wild-type Na_v1.5 protein expression.     

Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores

An assay to detect UO₂²⁺ complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe³⁺ dye and the other containing a CAS-UO₂²⁺ dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO₂²⁺ agar to the discoloration on the CAS-Fe³⁺ agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO₂²⁺ chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO₂²⁺ chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Choice of Mouse Strain Influences the Outcome in a Mouse Model of Chemical-Induced Asthma

Background

The development of occupational asthma is the result of interactions between environmental factors and individual susceptibility. We assessed how our model of chemical-induced asthma is influenced by using different mouse strains.

Methodology/Principal Findings

On days 1 and 8, male mice of 7 different strains (BALB/c, BP/2, A/J, C57Bl/6, DBA/2, CBA and AKR) were dermally treated with toluene-2,4-diisocyanate (TDI) (0.3%) or vehicle (acetone/olive oil, AOO, 2∶3) on each ear (20 µl). On day 15, they received an oropharyngeal instillation of TDI (0.01%) or AOO (1∶4). Airway reactivity to methacholine, total and differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE and IgG_2a levels were measured. Lymphocyte subpopulations in auricular lymph nodes and in vitro release of cytokines by ConA stimulated lymphocytes were assessed. In TDI-sensitized and challenged mice, airway hyper-reactivity was only observed in BALB/c, BP/2, A/J and AKR mice; airway inflammation was most pronounced in BALB/c mice; numbers of T-helper (CD4⁺), T-activated (CD4⁺CD25⁺), T-cytotoxic (CD8⁺) and B- lymphocytes (CD19⁺) were increased in the auricular lymph nodes of BALB/c, BP/2, A/J and CBA mice; elevated concentrations of IL-4, IL-10, IL-13 and IFN-γ were detected in supernatant of lymphocytes from BALB/c, BP/2, A/J, C57Bl/6 and CBA mice cultured with concanavaline A, along with an increase in total serum IgE.

Conclusion

The used mouse strain has considerable and variable impacts on different aspects of the asthma phenotype. The human phenotypical characteristics of chemically-induced occupational asthma were best reproduced in Th2-biased mice and in particular in BALB/c mice.     

The Strength of Selection Against the Yeast Prion [PSI+]

The [PSI⁺] prion causes widespread readthrough translation and is rare in natural populations of Saccharomyces, despite the fact that sex is expected to cause it to spread. Using the recently estimated rate of Saccharomyces outcrossing, we calculate the strength of selection necessary to maintain [PSI⁺] at levels low enough to be compatible with data. Using the best available parameter estimates, we find selection against [PSI⁺] to be significant. Inference regarding selection on modifiers of [PSI⁺] appearance depends on obtaining more precise and accurate estimates of the product of yeast effective population size N_e and the spontaneous rate of [PSI⁺] appearance m. The ability to form [PSI⁺] has persisted in yeast over a long period of evolutionary time, despite a diversity of modifiers that could abolish it. If mN_e < 1, this may be explained by insufficiently strong selection. If mN_e > 1, then selection should favor the spread of [PSI⁺] resistance modifiers. In this case, rare conditions where [PSI⁺] is adaptive may permit its persistence in the face of negative selection.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca²⁺]_cyt response. Cyclic-nucleotide-mediated elevations in [Ca²⁺]_cyt involved both internal and external Ca²⁺ stores. Both cAMP- and cGMP-mediated [Ca²⁺]_cyt elevations could be inhibited by the Ca²⁺-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca²⁺]_cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca²⁺]_cyt elevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca²⁺ signaling.     

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg²⁺ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li⁺ (50% inhibitory concentration [IC₅₀] = 3.6 mM) but only slightly sensitive to Na⁺ (IC₅₀ = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li⁺ and Na⁺, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li⁺ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.     

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.     

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR⁺) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR⁺ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR⁻) cells. The decreased proton motive force (PMF) of ATR⁺ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR⁺ cells were ~7-fold higher than those in ATR⁻ cells. This suggested a role for the F_oF₁ ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F_oF₁ ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR⁻ but not in ATR⁺ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR⁺ listeriae, the downregulation of the proton-translocating c subunit of the F_oF₁ ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F_oF₁ ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

Background

Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice.

Methodology/Principal Findings

We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1–2 months, 3–8 months and 9–12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5^th and 97.5^th percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05–0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K⁺, Ca⁺⁺, PO₄ ⁻ (p<0.05) and for TAG, Chol, AST, Fe⁺⁺ (p<0.001) in 4–8 month-old animals; (2) for CK, Crea, Mg⁺⁺, Na⁺⁺, K⁺, Cl⁻ (p<0.05) and BUN (p<0.001) in 2- and in 10–12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span.

Conclusion/Significance

Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.