Lung function assessment and its association with blood chromium in a chromate exposed population.

Hexavalent chromium [Cr(VI)] and its compounds have been associated with various respiratory diseases, while few studies have attempted to determine its adverse effect on lung function. To explore the potential early indicators of health surveillance for respiratory diseases induced by chromate exposure, a longitudinal cohort study including 515 workers with 918 measurements across 2010-2017 was conducted to investigate the impact of individual internal exposure on lung function. Inductively coupled plasma mass spectrometry (ICP-MS) and spirometry were used to measure whole blood chromium (blood Cr) and lung function respectively. In the linear mixed-effects analysis, each 1- unit increase in Ln- transformed blood Cr was significantly associated with estimated effect percentage decreases of 1.80 (0.35, 3.15) % in FEV1, 0.77 (0.10, 1.43) % in FEV1/FVC, 2.78 (0.55, 4.98) % in PEF, and 2.73 (0.59, 4.71) % in FEF25-75% after adjusting for related covariates. Exposure- response curve depicted the reduction of lung function with blood Cr increase, and the reference value of blood Cr was proposed as 6 µg/L considering the lung function as health outcome. Based on the repeated-measure analysis, compared with the low frequency group, subjects with high frequency of high exposure across 2010-2017 had an additional reduction of 5.65 (0, 11.3) % in FVC. Subjects with medium frequency showed more obvious declines of 9.48 (4.16, 14.87) % in FVC, 8.63 (3.49, 13.97) % in FEV1, 12.94 (3.34, 22.53) % in PEF and 10.97 (3.63, 18.30) % in MVV. These findings suggested that short- term high exposure to Cr associated with obstructive ventilatory impairment, and long- term exposure further led to restrictive ventilatory impairment.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe3+, H2O2, K2CrO4, and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites.

Effect of Interaction between 17ß-Estradiol, 2-Methoxyestradiol and 16α-Hydroxyestrone with Chromium (VI) on Ovary Cancer Line SKOV-3: Preliminary Study.

Ovarian cancer is the leading cause of death from gynecologic malignancies. Some estrogens, as well as xenoestrogens, such as chromium (VI) (Cr(VI)), are indicated as important pathogenic agents. The objective of this study was to evaluate the role of estradiol and some its metabolites upon exposure to the metalloestrogen Cr(VI) in an in vitro model. The changes in cell viability of malignant ovarian cancer cells (SKOV-3 resistant to cisplatin) exposed to 17ß-estradiol (E2) and its two metabolites, 2-methoxyestradiol (2-MeOE2) and 16α-hydroxyestrone (16α-OHE1), upon exposure to potassium chromate (VI) and its interactions were examined. The single and mixed models of action, during short and long times of incubation with estrogens, were applied. The different effects (synergism and antagonism) of estrogens on cell viability in the presence of Cr(VI) was observed. E2 and 16α-OHE1 caused a synergistic effect after exposure to Cr(VI). 2-MeOE2 showed an antagonistic effect on Cr(VI). The examined estrogens could be ranked according to the most protective effect or least toxicity in the order: 2-MeOE2 > E2 > 16α-OHE1. Early pre-incubation (24 h or 7 days) of cells with estrogens caused mostly an antagonistic effect-protective against the toxic action of Cr(VI). The beneficial action of estrogens on the toxic effect of Cr(VI), in the context of the risk of ovarian cancer, seems to be important and further studies are needed.

Response to vanadate exposure in Ochrobactrum tritici strains.

Vanadium is a transition metal that has been added recently to the EU list of Raw Critical Metals. The growing needs of vanadium primarily in the steel industry justify its increasing economic value. However, because mining of vanadium sources (i. e. ores, concentrates and vanadiferous slags) is expanding, so is vanadium environmental contamination. Bioleaching comes forth as smart strategy to deal with supply demand and environmental contamination. It requires organisms that are able to mobilize the metal and at the same time are resistant to the leachate generated. Here, we investigated the molecular mechanisms underlying vanadium resistance in Ochrobactrum tritici strains. The highly resistant strain 5bvl1 was able to grow at concentrations > 30 mM vanadate, while the O. tritici type strain only tolerated < 3 mM vanadate concentrations. Screening of O. tritici single mutants (chrA, chrC, chrF and recA) growth during vanadate exposure revealed that vanadate resistance was associated with chromate resistance mechanisms (in particular ChrA, an efflux pump and ChrC, a superoxide dismutase). We also showed that sensitivity to vanadate was correlated with increased accumulation of vanadate intracellularly, while in resistant cells this was not found. Other up-regulated proteins found during vanadate exposure were ABC transporters for methionine and iron, suggesting that cellular responses to vanadate toxicity may also induce changes in unspecific transport and chelation of vanadate.

Role of Toll-like receptor 4/oxidant-coupled activation in regulating the biosynthesis of omega-3 polyunsaturated fatty acid derivative resolvin D1 in primary murine peritoneal macrophage.

We have previously shown that reactive oxygen species (ROS) as prooxidants can activate Toll-like receptor 4 (TLR4) with the potential to initiate, propagate and maintain "sterile" inflammation of innate immunity, which plays a mediatory role in a host of human disease states. We now present new evidence that ROS can also activate TLR4 to counter the inflammatory phenotype by increasing the production of resolvin D1 (RvD1), which is a specialized anti-inflammatory and pro-resolving lipid mediator. We used primary murine peritoneal macrophages (pM) derived from both TLR4-WT and TLR4-KO mice as a cellular model. We used potassium peroxychromate (PPC) as a direct in vitro source of exogenous ROS. PPC treatment increased intracellular ROS levels, which decreased intracellular total antioxidant capacity, thus suggesting an enhanced cellular oxidative stress. PPC and LPS-EK (a TLR4-specific agonist) increased pro-inflammatory TNFα production with noeffect on IL-10, an anti-inflammatory cytokine. Treatment with the prooxidant increased the expression of 12 lipoxygenase (12-LOX) and 5-lipoxygenase (5-LOX) only in pM derived from TLR4 WT but not in pM from TLR4-KO mice. 5-LOX and 12-LOX are the key enzymes in the RvD1 biosynthetic pathway. In addition, PPC increased the expression of RvD1 receptor, a member of G-protein-coupled receptor only in pM from TLR4-WT mice. Our data support the involvement of TLR4-mediated oxidant-induced pro-inflammatory phenotypes that are in opposition to the production of anti-inflammatory/pro-resolution phenotypes in macrophages. Now, we show that through TLR4 activation, exogenous oxidants can play a role both in producing proinflammatory phenotypes at the same time that it enhances resolution of inflammation to maintain a state of cellular homeostasis and prevent tissue damage/disease.

ChrASO, the chromate efflux pump of Shewanella oneidensis, improves chromate survival and reduction.

The chromate efflux pump encoding gene chrASO was identified on the chromosome of Shewanella oneidensis MR1. Although chrASO is expressed without chromate, its expression level increases when Cr(VI) is added. When deleted, the resulting mutant ΔchrASO exhibits a chromate sensitive phenotype compared to that of the wild-type strain. Interestingly, heterologous expression of chrASO in E. coli confers resistance to high chromate concentration. Moreover, expression of chrASO in S. oneidensis and E. coli significantly improves Cr(VI) reduction. This effect could result either from extracytoplasmic chromate reduction or from a better cell survival leading to enhanced Cr(VI) reduction.

[Characterization of chromate resistance in genetically engineered Escherichia coli expressing chromate ion transporter ChrA].

OBJECTIVE: To construct a genetically engineered Escherichia coli expressing chromate (Cr) ion transporter ChrA and test its Cr resistance capacity. METHODS: ChrA gene was cloned by PCR from the DNA template of Serratia sp. S2 and linked with the prokaryotic vector pET-28a (+). The recombinant vector was transformed into E.coli BL21 (DE3) cells for expression of ChrA protein. Cr (VI) risistance and Cr (VI) uptake and efflux of the engineered bacteria were tested, and the effects of Cr loading time, oxyanions (ulfate, molybdate, vanadate, tungstate), and respiratory inhibitors (valinomycin, CN-, oligomycin, and NADH) on Cr (VI) efflux were examined to analyze the pathway of Cr (VI) transport by ChrA protein. RESULTS: The engineered E. coil strain was successfully constructed. Experiments using cell suspensions showed a lowered Cr2O72- uptake but an increased efflux capacity of ChrA-engineered bacteria compared with the control strain (P<0.05). The engineered E. coil cells in exponential growth incubated for 30 min in the presence of 50 mg/L Cr2O72- showed a total displacement of Cr (VI) of 20% after resuspension in PBS at 10 min, but chromate efflux decreased subsequently as the incubation time extended. Oxyanions sulfate and molybdate significantly inhibited chromate efflux in the engineered bacteria (P<0.05), whereas tungstate and vanadate did not obviously affect chromate efflux; chromate efflux was significantly inhibited by K+ ionophore valinomycin and CN-, enhanced by NADH (P<0.05), but not affected by oligomycin, suggesting the role of chromate transporter ChrA as a chemiosmotic pump that extrudes chromate using the proton-motive force. CONCLUSION: ChrA can efficiently transport chromate ions from the cytoplasm to enhance chromate resistance of the genetically engineered E. coli.

Effect of chromate ions on marine microalgae Phaeodactylum tricornutum.

Effect of chromate ions on the culture of a marine diatom Phaeodactylum tricornutum was studied using an M-PEA-2 fluorimeter, which carries out simultaneous measurement of fluorescence induction and redox transformations of the P700 pigment within a millisecond range. Chromate ions were shown to inhibit electron transport in PS II and decrease the rate of QА reduction. This results in decreased values of the quantum yield of electron transport in PS II (ϕEo) and performance index (PI ABS), lower rates of P700 reduction, and increased energy (DI0/RC) and ΔpH-dependent nonphotochemical quenching (q E ). Emergence of the slow component of P700 reduction was observed, indicating the activation of cyclic transport in the presence of chromate. Performance index (PI ABS), which was the most sensitive parameter, may be recommended for detection of chromate ions at early stages of their toxic action. The fluorescence parameter F O is promising application in biotesting to assess the algal growth rates.

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress.

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

A Cr(VI)-reducing Microbacterium sp. strain SUCR140 enhances growth and yield of Zea mays in Cr(VI) amended soil through reduced chromium toxicity and improves colonization of arbuscular mycorrhizal fungi.

Pot culture experiments were conducted in a glasshouse to evaluate the effects of four efficient Cr(VI)-reducing bacterial strains (SUCR44, SUCR140, SUCR186, and SUCR188) isolated from rhizospheric soil, and four arbuscular mycorrhizal fungi (AMF-Glomus mosseae, G. aggregatum, G. fasciculatum, and G. intraradices) alone or in combination, on Zea mays in artificially Cr(VI)-amended soil. Presence of a strain of Microbacterium sp. SUCR140 reduced the chromate toxicity resulting in improved growth and yields of plants compared to control. The bioavailability of Cr(VI) in soil and its uptake by the plant reduced significantly in SUCR140-treated plants; the effects of AMF, however, either alone or in presence of SUCR140 were not significant. On the other hand, presence of AMF significantly restricted the transport of chromium from root to the aerial parts of plants. The populations of AMF chlamydospores in soil and its root colonization improved in presence of SUCR140. This study demonstrates the usefulness of an efficient Cr(VI)-reducing bacterial strain SUCR140 in improving yields probably through reducing toxicity to plants by lowering bioavailability and uptake of Cr(VI) and improving nutrient availability through increased mycorrhizal colonization which also restricted the transport of chromium to the aerial parts.

Identification and characterization of the transcriptional regulator ChrB in the chromate resistance determinant of Ochrobactrum tritici 5bvl1.

Ochrobactrum tritici 5bvl1 is able to resist to high concentrations of chromate through the expression of an inducible chromate-resistant determinant, found in a mobile element (TnOtChr), which carries the genes, chrB, chrA, chrC and chrF. The regulation of chr operon present in TnOtChr, which is controlled by a transcriptional regulator, ChrB, was characterized in the current work. Fusions of chr promoter, or chr promoter and chrB gene, upstream of a gfp reporter gene, identified the most probable promoter sequence within the tnpR-chrB intergenic region. This region contains an AT-rich imperfect inverted repeat sequence, which overlaps a part of the -10 sequence. The results of the in vitro DNA-binding assays with purified ChrB (His- or no-tagged) showed that the protein binds directly to the chr promoter region. In order to identify the ChrB functional domain for sensing chromate stress and for DNA-binding, site-directed mutagenesis of ChrB was performed. Among several single amino acid mutants, three mutants (R180; R187 and H229) prevented chromate induction without any modification to the protein's stability. Interestingly, two ChrB mutants (R18 and R23) were constitutively active, regardless of chromate stress conditions, indicating that the residues most probably belong to the protein-DNA binding site. As such, the ChrB was classified as a transcriptional regulator that recognizes a specific DNA sequence, regulating the expression of a chromate resistance determinant.

Toll-like receptor 4-mediated nuclear factor kappa B activation is essential for sensing exogenous oxidants to propagate and maintain oxidative/nitrosative cellular stress.

The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1ß and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments.

Comparative evaluations on bio-treatment of hexavalent chromate by resting cells of Pseudochrobactrum sp. and Proteus sp. in wastewater.

Two marine bacterial strains, B5 and H24, were isolated from long-term Cr(VI) contaminated seawater and identified as Pseudochrobactrum and Proteus, respectively, based on 16S rRNA gene sequence analyses. Both strains were examined for their tolerance to Cr(VI) and other metal salts and their abilities to reduce Cr(VI) to trivalent chromium [Cr(III)]. Growing cells of Pseudochrobactrum sp. B5 and Proteus sp. H24 could tolerate Cr(VI) at a concentration of 2000 and 1500 mg/l and completely reduce 1000 mg/l Cr(VI) in LB medium within 96 and 144 h, respectively. Resting cells of the two strains were able to reduce 200mg/l Cr(VI) in Tris-HCl buffer within 16 and 24h, respectively. Furthermore, resting cells of both strains were able to reduce Cr(VI) in industrial wastewaters three times consecutively. Overall, this study provides evidence of the potential for application of chromate-reducing bacteria to direct Cr(VI) decontamination of industrial effluents.

Synechococcus elongatus PCC 7942 is more tolerant to chromate as compared to Synechocystis sp. PCC 6803.

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC50 of 12 ± 2 and 150 ± 15 µM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/10(8) cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using (35)S labeled sulfate. IC50 values of chromate for (35)sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC50 values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 µM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 µM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.

Prooxidant-induced c-Src/nuclear factor kappa B-coupled signalling in sensory ganglia mediates cutaneous hyperalgesia.

BACKGROUND: Persistent pain resulting from peripheral injury/inflammation is associated with altered sensitivity to cutaneous stimuli, which can manifest as hyperalgesia. The role of oxidant stress in the development, progression and maintenance of hyperalgesia is still not understood. Furthermore, there appears to be a relationship between c-Src kinase in the pain pathway and oxidative stress. METHODS: We have used a novel prooxidant inflammatory pain model that involves potassium peroxychromate (PPC), a unique prooxidant that produces the same reactants as activated phagocytes. This model was used to investigate the role of oxidant-activated c-Src in mediating hyperalgesia. We compared the effects of PP2 (a Src family kinase inhibitor) and c-Src siRNA on behavioural hyperalgesia with sodium stibogluconate (SSG) (a non-receptor tyrosine phosphatase inhibitor) and AG 1478 (a receptor tyrosine kinase inhibitor). RESULTS: PP2 and c-Src siRNA attenuated PPC-induced thermal hyperalgesia, while SSG enhanced it. AG 1478 had no effect. PP2 decreased the levels of IL-1ß, c-Src/inhibitory kappa B kinase complex formed and prostaglandin E2 produced in the dorsal root ganglia (DRG) ipsilateral to the inflamed paw, while SSG increased the levels of these parameters. c-Src siRNA decreased Src expression and activity in the DRG ipsilateral to the inflamed paw. CONCLUSIONS: These results confirm that prooxidant-activated c-Src plays a role in initiating and maintaining hyperalgesia by regulating a stimulus-response coupling between the inflamed tissue and the DRG in the pain pathway. Our data also suggest that oxidant-induced dysregulation of c-Src/nuclear factor kappa B coupling may contribute to our understanding of the transition from acute to chronic dysfunctional pain state seen in many human diseases.

Reactive oxygen species mediate Cr(VI)-induced S phase arrest through p53 in human colon cancer cells.

Compounds that contain chromate (Cr(VI)) are well-known carcinogens that are present in both industrial settings and the environment. The mechanism of carcinogenesis associated with these compounds is not well understood. This study focused on Cr(VI)-induced cell cycle arrest in human colon adenocarcinoma DLD1 cells. Treatment of the cells with Cr(VI) at 2.5 µM caused a growth arrest at the S phase. An increase in Cr(VI) concentration enhanced the growth arrest. Superoxide dismutase did not alter the Cr(VI)-induced S phase arrest. Catalase inhibited S-cell growth, indicating that H(2)O(2) is an important mediator in Cr(VI)-induced S phase arrest. Electron spin resonance spin-trapping measurements showed that incubation of cells with Cr(VI) generated hydroxyl radical (·OH). Catalase inhibited ·OH generation, indicating that H(2)O(2) was generated from cells stimulated by Cr(VI) and that H(2)O(2) functioned as a precursor of ·OH radical generation. p53, an oxidative response transcription factor, was activated upon Cr(VI) stimulation. Inhibition of p53 by introducing small hairpin RNA decreased S phase arrest induced by Cr(VI). These results support the following conclusions: (1) reactive oxygen species (ROS) are generated in Cr(VI)-stimulated DLD1 cells; (2) among the ROS generated, H(2)O(2) played a major role in causing S phase arrest in DLD1 cells; and (3) ROS mediated S phase arrest through a p53-dependent pathway.

The Neurospora crassa chr-1 gene is up-regulated by chromate and its encoded CHR-1 protein causes chromate sensitivity and chromium accumulation.

The ChrA membrane protein belongs to the CHR superfamily of chromate ion transporters, which includes homologues from bacteria, archaea and eukaryotes. Bacterial ChrA homologues confer chromate resistance by exporting chromate ions from the cell's cytoplasm. The Neurospora crassa strain 74-A chr-1 gene encodes a putative CHR-1 protein of 507 amino acid residues, which belongs to the CHR superfamily. RT-PCR assays showed that expression of the chr-1 gene was up-regulated by chromate exposure of N. crassa cultures. Introduction in N. crassa of sense and antisense fragments of the chr-1 gene, as part of a silencing module within the pSilent-1 vector, produced transformants with a phenotype of resistance to chromate and diminished accumulation of chromium, as compared with the control strain containing only the vector. A chromate-resistance phenotype was also observed in N crassa strains deleted in the genomic chr-1 gene, thus confirming that the absence of CHR-1 protein confers chromate resistance to the fungus. The cDNA from N. crassa chr-1 gene (Ncchr-1) was cloned into the pYES2 vector under the control of a GAL promoter and the resulting recombinant plasmid was transferred to the yeast Saccharomyces cerevisiae. Galactose-induced S. cerevisiae transformants expressing Ncchr-1 were more sensitive to chromate and accumulated 2.5 times more chromium than the induced strain containing only the vector. Excess sulfate, a chromate analog, was unable to protect S. cerevisiae chr-1 transformants from chromate toxicity. These data indicate that the N. crassa CHR-1 protein functions as a transporter that takes up chromate; it also appears that this transport occurs in a sulfate-independent fashion. This is the first report assigning a role as a chromate transporter to a nonbacterial CHR protein.

Chromate-resistance genes in plasmids from antibiotic-resistant nosocomial enterobacterial isolates.

The presence of chromate-resistance genes in enterobacteria was evaluated in a collection of 109 antibiotic-resistant nosocomial isolates from nine major cities in México. Results were compared with the presence of mercury-resistance genes. Susceptibility tests showed that 21% of the isolates were resistant to chromate (Cr(R)), whereas 36% were resistant to mercury (Hg(R)). Cr(R) levels were high in Klebsiella pneumoniae (61%), low in Enterobacter cloacae (12%) and Escherichia coli (4%), and null in Salmonella sp. isolates. Colony hybridization demonstrated that the majority of metal-resistant isolates hybridized with chrA gene (87% of Cr(R) isolates), encoding a CHR transporter homologue, and merA gene (74% of Hg(R) isolates), encoding MerA mercuric reductase, suggesting that most isolates expressed these widespread metal-resistance systems. Southern blot hybridization of Cr(R) isolates showed that plasmids of 80, 85, and 95 kb from K. pneumoniae isolates, and of 100 kb from an E. cloacae isolate, contained chrA-related sequences. These plasmids belonged to IncN or IncP incompatibility groups, and conferred Cr(R), as well as multiple antibiotic resistance, when transferred by conjugation to an E. coli standard strain. These data indicated that Cr(R) genes may be distributed among clinical enterobacteria via conjugative plasmids, probably by coselection with antibiotic-resistant genes.

Cations but not anions regulate the responsiveness of kainate receptors.

Kainate-selective ionotropic glutamate receptors are unique among ligand-gated ion channels in their obligate requirement of external anions and cations for activation. Although it is established that the degree of kainate receptor (KAR) activation is shaped by the chemical nature of the agonist molecule, the possible complementary role of external ions has yet to be examined. Here we show that external cations but not anions regulate the responsiveness to a range of full and partial agonists acting on rat GluK2 receptors. This observation is unexpected as previous work has assumed anions and cations affect KARs in an identical manner through functionally coupled binding sites. However, our data demonstrate that anion- and cation-binding pockets behave discretely. We suggest cations uniquely regulate a pregating or flipping step that impacts the closed-cleft stability of the agonist-binding domain (ABD). This model departs from a previous proposal that KAR agonist efficacy is governed by the degree of closure elicited in the ABD by ligand binding. Our findings are, however, in line with recent studies on Cys-loop ligand-gated ion channels suggesting that the "flipping" mechanism has been conserved by structurally diverse ligand-gated ion channel families as a common means of regulating neurotransmitter behavior.

Tn5045, a novel integron-containing antibiotic and chromate resistance transposon isolated from a permafrost bacterium.

A novel antibiotic and chromate resistance transposon, Tn5045, was isolated from a permafrost strain of Pseudomonas sp. Tn5045 is a compound transposon composed of three distinct genetic elements. The backbone element is a Tn1013-like Tn3 family transposon, termed Tn1013∗, that contains the tnpA and the tnpR genes, encoding the transposase and resolvase, respectively, the res-site and four genes (orfA, B, C, D) related to different house-keeping genes. The second element is class 1 integron, termed InC∗, which is inserted into the Tn1013∗ res-region and contains 5'-CS-located integrase, 3'-CS-located qacE∆1 and sulfonamide resistance sulI genes, and a single cassette encoding the streptomycin resistance aadA2-gene. The third element is a TnOtChr-like Tn3 family transposon termed TnOtChr∗, which is inserted into the transposition module of the integron and contains genes of chromate resistance (chrB, A, C, F). Tn5045 is the first example of an ancient integron-containing mobile element and also the first characterized compound transposon coding for both antibiotic and chromate, resistance. Our data demonstrate that antibiotic and chromate resistance genes were distributed in environmental bacteria independently of human activities and provide important insights into the origin and evolution of antibiotic resistance integrons.