Testing rates improved over the study period from less than one in every 300 patients to, on introduction of POCT, just under half of attendees having an HIV test. The prevalence of hitherto undiagnosed HIV infection in our clinic is almost 1% (with an additional 0.8% of patients declining POCT because of known HIV-positive status). This could be a model for other acute medical settings where HIV prevalence is similar. The high rates of uptake of testing, and the reasons given for declining a test, indicate that offering HIV POCT in such settings is acceptable to patients (and staff). We recognize that our mechanism for measuring acceptability was limited by being contemporaneous, but over this period

we received only one adverse ICG-001 in vivo APO866 comment in our anonymous feedback questionnaire from an already HIV-positive man concerned about counselling for new reactives; he was reassured once our process of referral was explained. In addition, other studies in similar settings show that offers of HIV tests are acceptable in community and hospital clinics [14]. Although the higher uptake with POCT than with laboratory testing did not translate into a statistically greater rate

of new diagnoses, our data support previous evidence that POCT, specifically, overcomes additional barriers to testing, by demonstrating a significant increase in acceptance rate compared with a laboratory-based protocol, presumably as a consequence of the perceived reduction in the delay

in receiving a result [8, 9]. Furthermore, rapid HIV POCTs offer an economical advantage in HIV screening programmes [17]. Targeted testing strategies based on dissemination of guidelines and protocols have limited benefit [3, 18]; universal testing strategies, which can be relatively easily provided by a range of healthcare staff, are more effective [19-22]. Reasons for this include the destigmatization of testing, as well as less reliance on busy clinicians (from a range of specialties) to prioritize HIV testing where clinical diagnosis and management are focussed on alternative, more pressing, matters. Cytidine deaminase This is particularly important if the increased international focus on testing is to identify patients with less advanced (and therefore often asymptomatic) disease. Although the numbers were limited, we have demonstrated that POCT screening may identify patients with higher CD4 cell counts, without clinically significant HIV disease. One would certainly expect more patients diagnosed with preserved immune status using a universal testing strategy than a targeted testing strategy based partly on indicator diseases, which are associated with varying degrees of immunosuppression [9]. A universal offer of an HIV test in this setting gives patients who may not attend conventional settings for HIV testing the opportunity to be tested.

3B and C). These effects were also observed after application of guanfacine. When guanfacine was washed out from the medium, the

speed of interneuron migration significantly increased and gradually reached control values (P < 0.01 at the first time interval after the drug wash when comparing guanfacine vs. no-wash medetomidine, one-way anova, Tukey’s multiple comparison test; Fig. 3C). Interestingly, we observed that although the migratory speed of GAD65-GFP+ cells was gradually restored during the removal of either medetomidine or guanfacine, the directionality of GAD65-GFP+ cells was modified by adra2 stimulation (Fig. 3B). Quantification revealed that during Everolimus order the washout period a significant proportion of GAD65-GFP+ cells modified their directionality following medetomidine or guanfacine application. The percentage of GAD65-GFP+ interneurons that made directionality changes in the range > 120–180° after the medetomidine wash or the guafancine wash was significantly increased compared to control GAD65-GFP+ interneurons (P < 0.01 for guanfacine compared to control check details and P < 0.05 for medetomidine vs. control, one-way anova Tukey’s multiple comparison

test; Fig. 3D), suggesting that adrenergic stimulation of cortical interneurons may alter their responsiveness to guidance cues. To determine whether cortical interneuron migration is altered in adra2a/2c-ko mice, we analysed the cortical distribution of GAD65-GFP+ interneurons Pembrolizumab clinical trial at postnatal day 21 in GAD65-GFP mice and in adra2a/2c-ko GAD65-GFP mice. Quantification revealed that the distribution of GAD65-GFP+ cortical interneurons in the somatosensory cortex was significantly altered in adra2a/2c-ko mice (n = 6) compared to the

control mice (n = 6; P < 0.05, χ2 test; Fig. 4). A significant increase in the percentage of GAD65-GFP+ cells was observed in upper cortical layers II/III in adra2a/2c-ko mice (P < 0.05, unpaired t-test), indicating that adrenergic receptors are necessary for the proper positioning of cortical interneurons in vivo. Quantification of the distribution of GAD65-GFP+ cells at P21 in the somatosensory cortex of adra2a-ko or of adra2c-ko mice was not significantly different from control GAD65-GFP+ mice (data not shown), suggesting that constitutive deletion of adra2a or adra2c during development may be compensated for by the presence of the other subtype. In this study we found that migrating cortical interneuron subtypes preferentially derived from the caudal ganglionic eminences express a specific pattern of adrenergic receptors and that pharmacological activation of these receptors affects the dynamic migration of cortical interneurons as they invade the developing cortical plate. Effects of adrenergic stimulation were most effective after adra2 stimulation, and they were concentration-dependent and reversible. Furthermore, effects of adra2 activation on the migration of cortical interneurons were significantly reduced in adra2a/2c-ko mice.

We recommend therapy-naïve patients start combination buy Venetoclax ART containing TDF and FTC as the NRTI backbone (1A). We suggest ABC and 3TC is an acceptable alternative NRTI backbone in therapy-naïve patients who, before starting ART, have a baseline VL≤100 000 copies/mL

(2A). ABC must not be used in patients who are HLA-B*57:01 positive (1A). Three RCTs have compared TDF-FTC with ABC-3TC as the NRTI backbone in combination with different third agents: ATV/r or EFV [2-6], EFV [7-9] and LPV/r [10]. Assessment of virological efficacy as a critical outcome was complicated by different definitions across the three studies. In our analysis for GRADE (see Appendix 3.1), there was no difference in rates of virological suppression at Selleck Pexidartinib 48 weeks or 96 weeks but the analysis excluded the largest of

the three trials (ACTG 5202) and the quality of evidence for this outcome was assessed as low or very low. Assessment of the risk of protocol-defined virological failure at 48 weeks favoured TDF-FTC (RR 0.76, 95% CI 0.53–1.07); the effect was not statistically significant and heterogeneity in the analysis was relatively high (I2 46%). Assessment of protocol-defined virological failure at 96 weeks showed a significant difference favouring TDF-FTC (RR 0.73, 95% CI 0.59–0.92). Data were only available from one study [4] for this analysis; however, this was by far the largest of the three trials and the quality of evidence

assessment for this outcome was rated as high. The difference in virological failure was assessed by the Writing Group to be large enough to be above the clinical threshold for decision-making. The difference equates to a Resminostat number needed to treat to prevent one case of virological failure of approximately 20 patients treated for 1 year. The results of ACTG 5202 [2-4] are complicated by early termination of those individuals with a baseline VL >100 000 copies/mL at the recommendation of the data and safety monitoring board due to significantly inferior performance in those subjects receiving ABC-3TC. No difference in virological efficacy between the TDF-FTC and ABC-3TC arms was seen in those in the lower VL stratum (baseline VL <100 000 copies/mL). The subsequent 96-week analysis, after discontinuation of those subjects in the higher VL stratum, may therefore underestimate the difference between the two backbones. HLA-B*57:01 screening was not routine in ACTG 5202 and this potentially may have influenced some of the safety endpoints, but appears not to have influenced the primary virological outcome. In the higher VL strata the number of patients with suspected hypersensitivity reactions was equal between both arms and virological failure in these patients was infrequent.

The other phenotypic properties of strain KU41ET are stated in the genus and species descriptions, and those characteristics that differentiate strain KU41ET from phylogenetically related taxa are listed in Table 1. Q-9 (79%), Q-8 (21%) The G + C content of the genomic DNA was 48.6 mol%, an check details intermediate value

among members of the order Alteromonadales (Bowman & McMeekin, 2005). The major lipoquinone was ubiquinone-8, as with the members of the order Alteromonadales (Bowman & McMeekin, 2005). The major cellular fatty acids of strain KU41ET were summed feature 3 (C15:0 ISO 2OH and/or C16:1 ω7c, 28.4%), C18:1 ω7c (19.8%), C16:0 (17.0%), C10:0 3-OH (9.4%), C10:0 (6.4%), and C17:1 ω8c (5.6%) (Table 2). Fatty acid composition could differentiate strain KU41ET from P. isoporae SW-11T, T. turnerae T7902T, E. nigra 17X/A02/237T, and S. degradans 2-4, the phylogenetically related taxa, indicating that strain KU41ET

probably represents an independent genus of the order Alteromonadales within the class Gammaproteobacteria. As shown by the 16S rRNA gene sequence analysis, strain KU41ET belongs to the order Alteromonadales within the class Gammaproteobacteria and forms a distinct lineage from the related genera. Furthermore, strain KU41ET can be differentiated from closely related genera by fatty acid composition and phenotypic characteristics. On the basis of data from the polyphasic study, we suggest that strain KU41ET represents Dasatinib in vitro a novel species of a new genus, for which the name Maricurvus nonylphenolicus gen. nov., sp. nov. is proposed. Maricurvus (Ma.ri.cur’ vus. L. neut. n. mare the sea; L. masc. adj. curvus bent; N.L. masc. n. Maricurvus a bent bacterium from the sea). Cells are Gram-negative, aerobic, motile by a single polar flagellum, and curved rods. Sodium ions are required for their growth. The predominant fatty acids are summed feature 3 (C15:0 Ribose-5-phosphate isomerase iso 2OH and/or C16:1 ω7c), C18:1

ω7c, C16:0, C10:0 3-OH, C10:0, and C17:1 ω8c. The predominant respiratory quinone is Q-8. The type species is M. nonylphenolicus. Maricurvus nonylphenolicus (no.nyl.phe.no’li.cus. N.L. n. nonylphenolis nonylphenol; L. suff. -icus -a -um suffix used with the sense of belonging to; N.L. masc. adj. nonylphenolicus referring to the substrate nonylphenol that can be utilized by the species). The description is identical to that for the genus, with the following additions. Cells are 1.0–2.5 μm in length and 0.3–0.8 μm in width. Colonies are pale yellow, circular, smooth, convex, 1.0 mm in diameter, and with an entire margin after incubation on MA after 7 days. Growth occurs at 20–35 °C (optimally at 25–30 °C), at pH 7.0–8.0, and with 1.0–4.0% NaCl (optimally at 2–3%). Degrade p-n-nonylphenol, p-n-octylphenol, and p-n-heptylphenol.

Differences in biofilm formation and aggregation by X. fastidiosa in xylem fluids from grapevine cultivars of varying susceptibility to PD have been correlated with specific differences in the nutritional components of the xylem fluid (Andersen et al., 2007). We were interested in the underlying genetic basis of the differential responses of X. fastidiosa to differences in xylem chemistry in different hosts. Therefore, we began an analysis of the effects of xylem fluid, from the grapevine host of a PD strain and from nonhost

citrus species, on OSI744 the expression of X. fastidiosa genes. Genes predicted to be involved in virulence regulation, such as the virulence regulator xrvA, transcriptional regulator algU, two-component regulator gacA, and post-transcriptional regulator hsq, check details were expressed at greater levels in grapevine xylem fluid vs. citrus xylem fluid (Table 1, Fig. 5). The regulatory genes algU and gacA were previously shown to play roles in controlling several potential virulence factors in X. fastidiosa. An algU defective mutant (Shi et al., 2007) and a gacA defective mutant (Shi et al., 2009) had decreased cell aggregation, biofilm formation, and pathogenicity

in grapevine compared with the wild type. Hsq, an RNA-binding protein, may indirectly affect biofilm formation in X. fastidiosa through a complex hfq/rsmB/rsmA-mediated system (Shi et al., 2007). Genes predicted to be involved in surface structures and attachment components, such as PD0312, hsf, and xadA, were expressed more vigorously in the xylem fluid of grapevine than that of citrus (Table 1, Fig. 5). hsf of X. fastidiosa is similar to the adhesion gene hsf in Haemophilus influenza, and xadA encodes a putative afimbrial outer membrane protein involved in adhesion. An xadA defective mutant in xadA of X. fastidiosa is surface adhesion-deficient, which reduces X. fastidiosa adhesion in the early stages of attachment to the surface of its host (Feil et al., 2007). The expressions of hsf and xadA were increased in grapevine xylem fluid, likely contributing to an enhanced ability to adhere to xylem vessel walls. In

this study, the lower percent aggregation of X. fastidiosa cells and lower biofilm formation in citrus xylem fluid might be related to decreased expression of adhesion-related genes, mafosfamide such as hsf and xadA. In contrast, increased expression of hsf and xadA in grapevine may be related to the higher biofilm formation and percent aggregation of cells. In addition, we reported previously that xadA and hsf were positively regulated by gacA in X. fastidiosa (Shi et al., 2009), suggesting that these adhesion functions are influenced by the gacA regulatory pathway. Genes involved in the biogenesis and of type I and IV pili in X. fastidiosa, such as fimT, fimA, pilI, pilT, pilU, pilY1, pilE, pilG, pilZ, and pilH, showed a higher expression in the xylem fluid of grapevine than of citrus (Table 1, Fig. 5).

For RNA isolations, NT-26 was grown heterotrophically with and without arsenite until the mid log, late log and stationary phases. Arsenite oxidation was measured as reported previously (Santini et al., 2007). DNA sequence upstream of the arsenite oxidase gene aroB was obtained by a primer walking method using a previously constructed genomic DNA library (Santini & vanden Hoven, 2004). To identify putative genes, the sequence results obtained

were submitted to the database search engines smart (Schultz et al., 1998), pfam (Bateman et al., 2002) and tmhmm (Krogh et al., 2001). Sequence alignments were performed using either blastp (Camacho et al., 2008) or clustalw (Larkin et al., 2007). The aroR and aroS sequences have been deposited in GenBank under the accession number AY345225. AroS and AroR genes were PCR amplified using genomic DNA (Santini & vanden Hoven, 2004) as a template. The digested amplified products were ligated into NcoI- and HindIII-digested Alectinib pEMBL His-GST vector. Site-directed mutagenesis was performed using the QuikChangeTM Site-Directed Torin 1 chemical structure Mutagenesis Kit (Stratagene, La Jolla, CA) protocol. All genes were sequenced (Eurofins MWG Operon) to verify cloning and to ensure that the correct mutations had been introduced. The constructs allowed for the overexpression of genes with an N-terminal

polyhistidine affinity tag and a tobacco etch virus (TEV) protease cleavage site to allow for removal of the affinity tag. Mutagenesis of aroR and aroS was performed by targeted gene

disruption as described previously for aroA (Santini & vanden Hoven, 2004) and cytC (Santini et al., 2007). Portions of the aroR and aroS genes were amplified using the following primers: AroRFor (binds click here to nucleotides 31–50) 5′-GCGGATCCCTCGAAGATGATCCGATCAT-3′ (the recognition sequence for EcoR1 is underlined) and AroRRev (binds to nucleotides 709–728) 5′-GCGAATTCGCTGCATGACGCCAATCTCG-3′ (the recognition sequence for BamH1 is underlined); AroSFor (binds to nucleotides 222–242) 5′-GCGGATCCCTATGATCTGCTCGACCGTAC-3′ (the recognition sequence for EcoR1 is underlined) and AroSRev (binds to nucleotides 1082–1102) 5′-GCGAATTCTGCTCATGCACGTCAATGTCT-3′ (the recognition sequence for BamH1 is underlined). The PCR products were digested with EcoR1 and BamH1 and cloned into the suicide plasmid pJP5603 (KmR) and transferred into NT-26 by conjugation (Santini & vanden Hoven, 2004; Santini et al., 2007). One aroR and one aroS mutant were chosen for further study. Mutants were tested for their abilities to grow chemolithoautotrophically and heterotrophically. As no growth was detected with either mutant when grown chemolithoautotrophically with 5 mM arsenite, growth experiments were only conducted under heterotrophic conditions. Growth experiments were conducted with two replicates on two separate occasions in batch cultures in the MSM with 0.04% yeast extract with and without 5 mM arsenite.

For RNA isolations, NT-26 was grown heterotrophically with and without arsenite until the mid log, late log and stationary phases. Arsenite oxidation was measured as reported previously (Santini et al., 2007). DNA sequence upstream of the arsenite oxidase gene aroB was obtained by a primer walking method using a previously constructed genomic DNA library (Santini & vanden Hoven, 2004). To identify putative genes, the sequence results obtained

were submitted to the database search engines smart (Schultz et al., 1998), pfam (Bateman et al., 2002) and tmhmm (Krogh et al., 2001). Sequence alignments were performed using either blastp (Camacho et al., 2008) or clustalw (Larkin et al., 2007). The aroR and aroS sequences have been deposited in GenBank under the accession number AY345225. AroS and AroR genes were PCR amplified using genomic DNA (Santini & vanden Hoven, 2004) as a template. The digested amplified products were ligated into NcoI- and HindIII-digested Regorafenib chemical structure pEMBL His-GST vector. Site-directed mutagenesis was performed using the QuikChangeTM Site-Directed selleck compound library Mutagenesis Kit (Stratagene, La Jolla, CA) protocol. All genes were sequenced (Eurofins MWG Operon) to verify cloning and to ensure that the correct mutations had been introduced. The constructs allowed for the overexpression of genes with an N-terminal

polyhistidine affinity tag and a tobacco etch virus (TEV) protease cleavage site to allow for removal of the affinity tag. Mutagenesis of aroR and aroS was performed by targeted gene

disruption as described previously for aroA (Santini & vanden Hoven, 2004) and cytC (Santini et al., 2007). Portions of the aroR and aroS genes were amplified using the following primers: AroRFor (binds Dimethyl sulfoxide to nucleotides 31–50) 5′-GCGGATCCCTCGAAGATGATCCGATCAT-3′ (the recognition sequence for EcoR1 is underlined) and AroRRev (binds to nucleotides 709–728) 5′-GCGAATTCGCTGCATGACGCCAATCTCG-3′ (the recognition sequence for BamH1 is underlined); AroSFor (binds to nucleotides 222–242) 5′-GCGGATCCCTATGATCTGCTCGACCGTAC-3′ (the recognition sequence for EcoR1 is underlined) and AroSRev (binds to nucleotides 1082–1102) 5′-GCGAATTCTGCTCATGCACGTCAATGTCT-3′ (the recognition sequence for BamH1 is underlined). The PCR products were digested with EcoR1 and BamH1 and cloned into the suicide plasmid pJP5603 (KmR) and transferred into NT-26 by conjugation (Santini & vanden Hoven, 2004; Santini et al., 2007). One aroR and one aroS mutant were chosen for further study. Mutants were tested for their abilities to grow chemolithoautotrophically and heterotrophically. As no growth was detected with either mutant when grown chemolithoautotrophically with 5 mM arsenite, growth experiments were only conducted under heterotrophic conditions. Growth experiments were conducted with two replicates on two separate occasions in batch cultures in the MSM with 0.04% yeast extract with and without 5 mM arsenite.

Thiopurine catabolism via the XO pathway leads to the production of the inactive metabolite 6-thiouric acid. TPMT methylates 6MP to form 6-methylmercaptopurine (6MMP). 6MMP levels do not correlate with thiopurine efficacy and in high levels are associated with hepatotoxicity. Metabolism via the HPRT pathway leads buy Fulvestrant to the production of 6-thioguanine nucleotides (6TGN), the active metabolites responsible for thiopurine efficacy, but are also potentially myelotoxic

at supra-therapeutic levels.[4] 6TGN, which comprises 6-thioguanine monophosphate (6TGMP), diphosphate (6TGDP) and triphosphate (6TGTP), has several actions.[5] First, 6TGN, a purine analogue, triggers apoptosis and arrests the cell cycle by being incorporated into DNA in place of adenosine and guanine, leading to chromatid damage and arresting DNA replication.[6, 7] Second, 6TGN-incorporated base pairs show reduced stability, causing small changes in local DNA structure, and increased levels of methylation, activating the DNA mismatch repair

system.[8, 9] Third and most importantly, 6TGTP is a direct antagonist of Rac1, which blocks the activation of Vav to dampen the inflammatory cascade involving nuclear factor (NF)-κB and signal transducer and activator transcription 3 (STAT-3).[10, 11] These three mechanisms selleck screening library lead to apoptosis, and prevent activation and proliferation of T-lymphocytes implicated in the pathogenesis of IBD (Fig. 1). For over 30 years, thiopurine therapy has been a mainstay of induction and maintenance of remission

in patients with IBD. Using a conventional weight-based dosing regimen (1.0–1.5 mg/kg/day for 6MP and 2.0–2.5 mg/kg/day for AZA), response rates in original studies vary between 42% and 75%.[12, 13] Thiopurines have also been extensively used in the treatment of SLE and RA. In lupus nephritis, 2.0 mg/kg/day of AZA has been shown to prevent flares in up to 75% of patients.[14] In RA, Carnitine palmitoyltransferase II AZA reduced joint swelling by at least 50% in 33% of patients treated with 2.0–2.5 mg/kg/day.[15] AZA is also efficacious in the treatment of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis and polyarteritis nodosa (PN).[16, 17] As IBD and rheumatic diseases are chronic relapsing conditions where disease activity leads to significant disability and impaired quality of life, the proportion of patients achieving adequate efficacy using weight-based approaches would appear far from ideal. The use of thiopurine metabolites has enabled optimization of thiopurine therapy to achieve maximal outcomes for patients. The thiopurine metabolites, 6TGN and 6MMP, can be quantified in human blood using high performance liquid chromatography. Most laboratories measure the red cell (RBC) concentrations of 6TGN and 6MMP. Values are expressed in pmol/8 × 108 RBCs.[18] While leucocyte concentration is preferable, this is seldom performed as purification is tedious and requires a greater volume of blood.

Thiopurine catabolism via the XO pathway leads to the production of the inactive metabolite 6-thiouric acid. TPMT methylates 6MP to form 6-methylmercaptopurine (6MMP). 6MMP levels do not correlate with thiopurine efficacy and in high levels are associated with hepatotoxicity. Metabolism via the HPRT pathway leads this website to the production of 6-thioguanine nucleotides (6TGN), the active metabolites responsible for thiopurine efficacy, but are also potentially myelotoxic

at supra-therapeutic levels.[4] 6TGN, which comprises 6-thioguanine monophosphate (6TGMP), diphosphate (6TGDP) and triphosphate (6TGTP), has several actions.[5] First, 6TGN, a purine analogue, triggers apoptosis and arrests the cell cycle by being incorporated into DNA in place of adenosine and guanine, leading to chromatid damage and arresting DNA replication.[6, 7] Second, 6TGN-incorporated base pairs show reduced stability, causing small changes in local DNA structure, and increased levels of methylation, activating the DNA mismatch repair

system.[8, 9] Third and most importantly, 6TGTP is a direct antagonist of Rac1, which blocks the activation of Vav to dampen the inflammatory cascade involving nuclear factor (NF)-κB and signal transducer and activator transcription 3 (STAT-3).[10, 11] These three mechanisms Z-VAD-FMK solubility dmso lead to apoptosis, and prevent activation and proliferation of T-lymphocytes implicated in the pathogenesis of IBD (Fig. 1). For over 30 years, thiopurine therapy has been a mainstay of induction and maintenance of remission

in patients with IBD. Using a conventional weight-based dosing regimen (1.0–1.5 mg/kg/day for 6MP and 2.0–2.5 mg/kg/day for AZA), response rates in original studies vary between 42% and 75%.[12, 13] Thiopurines have also been extensively used in the treatment of SLE and RA. In lupus nephritis, 2.0 mg/kg/day of AZA has been shown to prevent flares in up to 75% of patients.[14] In RA, DCLK1 AZA reduced joint swelling by at least 50% in 33% of patients treated with 2.0–2.5 mg/kg/day.[15] AZA is also efficacious in the treatment of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis and polyarteritis nodosa (PN).[16, 17] As IBD and rheumatic diseases are chronic relapsing conditions where disease activity leads to significant disability and impaired quality of life, the proportion of patients achieving adequate efficacy using weight-based approaches would appear far from ideal. The use of thiopurine metabolites has enabled optimization of thiopurine therapy to achieve maximal outcomes for patients. The thiopurine metabolites, 6TGN and 6MMP, can be quantified in human blood using high performance liquid chromatography. Most laboratories measure the red cell (RBC) concentrations of 6TGN and 6MMP. Values are expressed in pmol/8 × 108 RBCs.[18] While leucocyte concentration is preferable, this is seldom performed as purification is tedious and requires a greater volume of blood.

ENA homologues exist in all so-far sequenced yeast genomes. Mainly from studies in the model Omipalisib mw yeast S. cerevisiae, it is commonly accepted that the role of the Ena ATPase is crucial for sodium detoxification at high external pH values, where the antiporter system cannot effectively exchange Na+ for protons. However, ENA ATPases are not specific for

sodium (or lithium) extrusion, but they also transport K+, as it was initially deduced from the characterization of the Ena1 ATPase activity in S. cerevisiae (Benito et al., 1997). Further support for this notion came from the discovery of two ATPases (encoded by ENA1 and ENA2 genes) with different functions in D. occidentalis (Banuelos & Rodriguez-Navarro, 1998). These two genes complement the Na+ sensitivity of an S. cerevisiae ena mutant strain. The expression of DoENA2 was increased by high pH, but both high pH and high sodium were required for the DoENA1 expression. Remarkably, whereas D. occidentalis mutants lacking ENA1 were less sodium tolerant, the mutation of ENA2 did not PD-166866 cell line alter sodium tolerance, but resulted in sensitivity to high pH and decreased potassium efflux. From these results, it was concluded that both genes exhibit different cation specificities and that ENA ATPases can mediate the efflux of potassium (Banuelos

& Rodriguez-Navarro, 1998). Besides D. occidentalis, the ENA ATPases have been characterized in several other 4��8C halotolerant yeast species. Two ENA genes have been identified so far in D. hansenii. DhENA1 was expressed in the presence of high Na+ concentrations, while the expression of DhENA2 also required high pH. Heterologous expression of the DhENA genes in an S. cerevisiae mutant indicated their function in

sodium detoxification and extrusion (Almagro et al., 2001). Similarly, a gene encoding the Ena ATPase from Z. rouxii (ZrENA1) was isolated and characterized (Watanabe et al., 1999, 2002). Remarkably, although the expression of ZrENA1 was observed, it was not upregulated by NaCl stress. However, the protein was efficient at extruding sodium cations, because upon overexpression in a salt-sensitive S. cerevisiae strain, its presence increased NaCl tolerance. Nevertheless, it appears that in Z. rouxii cells, the extrusion of Na+ might be carried out mainly via the Na+/H+ antiporter. The extremely halotolerant black yeast Hortaea werneckii appears to contain two ATPases, HwEna1 and HwEna2, that are important for maintaining low intracellular Na+ and K+ content in this organism (Gorjan & Plemenitas, 2006). Although both genes are responsive to salt, the expression of HwENA1 is higher shortly after salt stress, whereas the expression of HwENA2 appears more prominent in adapted cells. The presence of ENA ATPases has also been investigated in another stress-tolerant fungus, Torulaspora delbrueckii.