, 2006; Nakashima et al, 2011) Consequently, RND-type drug tran

, 2006; Nakashima et al., 2011). Consequently, RND-type drug transporters have been termed ‘cis transporters’. This is in contrast to drug transporters from the major facilitator superfamily and ATP-binding cassette families where drug transport occurs through an alternating access mechanism across the cytoplasmic membrane, the so-called trans-transporters (Eicher et al., 2009; Nikaido & Takatsuka, 2009; Doshi et al., 2011). However, studies on purified and reconstituted AcrD from E. coli revealed that this RND

transporter could efflux the SGI-1776 solubility dmso membrane-impermeable aminoglycoside gentamycin from either side of the liposomes, consistent with the existence of both periplasmic and cytoplasmic drug efflux pathways (Aires & Nikaido, 2005). It is therefore highly likely that, in addition to the periplasmic drug efflux pathway, a pathway exists for removal of drugs directly from the cytosol or inner membrane leaflet. Das and co-workers

(Das et al., 2007) predicted that conserved Phe residues on the small N-terminal helix of AcrB which line a 15 Å opening on the cytoplasmic side of AcrB could play a role in the discrimination, uptake and transport of drug molecules from the cytoplasm through the central cavity formed by the membrane domains of the AcrB trimer. Phenylalanine residues are hugely important in EPZ015666 mouse substrate recognition and binding in the RND-type drug transporters. The binding protomer in the asymmetric AcrB structure forms a hydrophobic pocket lined by phenylalanines 136, 178, 610, 615, 617 and 628 (Murakami et al., 2006; Seeger et al., 2006; Sennhauser et al., 2009) while minocycline, rifampicin and erythromycin are in direct contact with Phe residues (Murakami et al., 2006; Nakashima et al., 2011). Replacement of these phenylalanines with alanines reduced the ability of AcrB to confer resistance to a wide range of compounds (Bohnert et al., 2008; Vargiu et al.,

2011), with the F610A mutation having the greatest effect. Similarly, site-directed mutagenesis of F386, F388, F458 and F459 in AcrB resulted in a decrease in the minimum inhibitory concentration (MIC) for tetracycline, erythromycin, dequalinium and acriflavine (Yu et al., 2005). In this study, we aimed to provide more insight on the role of the conserved phenylalanine residues on the small L-NAME HCl N-terminal helix of MexB in drug efflux. These Phe residues were mutated to Ala residues to generate FAFA MexB. The ability of the mutant protein to confer resistance to toxic compounds and to efflux dyes was compared to that of the wild-type protein. For cytotoxicity and transport assays, plasmids were propagated in E. coli strain BW25113 lacking either AcrB or AcrA and AcrB (a kind gift from Professor Martin Pos, Institute of Biochemistry, Goethe-University, Frankfurt am Main, Germany). The plasmids used were pET41a (+), pUC18 (Novagen) and derivatives expressing MexB with a C-terminal His tag (pMexBH; Barrera et al.

These data are consistent with previous findings on mutL (eg Za

These data are consistent with previous findings on mutL (e.g. Zahrt et al., 1994); here,

we repeated the same kind of experiments to demonstrate the association of greatly elevated mutability with the specific 6-bp lesion of mutL, which will be the basis for the proposal of spontaneous conversion between mutL and 6bpΔmutL as a genetic switch in bacterial evolution. In particular, these experimental results support the presumption that the 6bpΔmutL genotype facilitates homologous recombination and thus provides the bacteria many more chances to incorporate Selleckchem Caspase inhibitor beneficial DNA of foreign sources. To further confirm differences in homologous DNA recombination efficiency between 6bpΔmutL and mutL cells, we carried out crosses between S. typhimurium LT7 (SGSC1417 and 8608F2 series) and E. coli Hfr cells by conjugation, using E. coli Hfr 3000 as the donor (Low, 1973;

Theze et al., 1974). With SGSC1417 or 8608F2mutL as the recipient, the conjugation frequencies were <10−8 per Hfr; with SGSC14176bpΔmutL, 8608F2 or the ΔmutL strain of SGSC1417 as the recipient, the conjugation frequencies were >10−6 per Hfr (Fig. 3). As the 6bpΔmutL genotype significantly facilitated mutability, as shown above, and because the 6-bp tandem repeat structure easily leads to copy number changes through slipped-strand mispairing (Streisinger et al., 1966; Cell Cycle inhibitor Levinson & Gutman, 1987), we hypothesized that bacterial populations dominated with 6bpΔmutL cells under some kind of selective pressure may begin having mutL cells when the pressure is no longer present, and the mutL cells may continuously increase in number or even replace the original 6bpΔmutL population. As the S. typhimurium LT7 mutants had suffered from starvation during stock in sealed agar stab cultures under room temperature

for over 40 years, we cultured the bacteria on LB plates to ‘remove’ such starvation pressures. We started with strain 9052D1, which was the first strain to have the MMR genes sequenced in our laboratory and was found to have the 6bpΔmutL genotype (Gong et al., 2007). During the first plating, a minority of colonies contained both 6bpΔmutL and mutL cells (Fig. 4a, lane 9). When such colonies were restreaked, most of them made only mutL cells (data not shown). The 6bpΔmutL cells continued making both 6bpΔmutL and mutL cells, but very few mutL cells made 6bpΔmutL cells (Fig. 4b), which demonstrates a much crotamiton stronger tendency of the 6bpΔmutL allele to convert to mutL than in the opposite direction. Bacteria use several strategies to increase mutability for acquiring genetic novelty in adaptation to changing environments, involving, in addition to allele conversion of the MMR genes as reported in this paper, the RpoS regulon, SOS responses, DinB error-prone DNA polymerase, RecA, etc., as has been documented widely (Bjedov et al., 2003; Friedman et al., 2005; Ponder et al., 2005; Finkel, 2006; Galhardo et al., 2007, 2009; Sundin & Weigand, 2007; Weigand & Sundin, 2009).

Baseline data collection for this study was made possible by an u

Baseline data collection for this study was made possible by an unrestricted educational grant from GlaxoSmithKline. We acknowledge assistance of all staff, people with HIV infection and assistants. The authors acknowledge G. Arthur, S. Norwood, A. Jayakody, T. Hill and S. Zetler for their contributions. “
“Given the importance of adherence to combination antiretroviral therapy (cART) for the reduced morbidity and improved mortality AZD0530 concentration of people living with HIV infection (PLWH), we set out to determine which of a number of previously investigated personal, socioeconomic, treatment-related and disease-related factors were independently associated with self-reported

Ibrutinib clinical trial difficulty taking antiretroviral therapy (ART) in an Australian sample of PLWH. Using data from a national cross-sectional survey of 1106 PLWH, we conducted bivariate and multivariable analyses to assess the association of over 70 previously investigated factors with self-reported difficulty taking ART. Factors that maintained an association with reported difficulty taking ART at the level of α=0.05 in the multivariable logistic regression analysis were considered to be independently associated with reported difficulty taking ART. A total of 867 (78.4%) survey respondents were taking antiretroviral medication at the time of completing

the HIV Futures 6 survey. Overall, 39.1% of these respondents buy Y-27632 reported difficulty taking ART. Factors found to be independently associated with reported difficulty taking ART included younger age, alcohol and party drug use, poor or fair self-reported health, diagnosis of a mental health condition, living in a regional centre, taking more than one ART dose per day, experiencing physical adverse events or health service discrimination, certain types

of ART regimen and specific attitudes towards ART and HIV. Thirteen previously investigated factors were found to be independently associated with reported difficulty taking ART, reaffirming the dynamic nature of adherence behaviour and the ongoing importance of addressing adherence behaviour in the clinical management of PLWH. Combination antiretroviral therapy (cART) has revolutionized the course of HIV disease, transforming HIV infection from a life-threatening infection to a manageable chronic condition, particularly in developed countries [1–3]. However, a key challenge is the high level of adherence to cART that is required for viral suppression, immunological response and reduced morbidity and mortality in individuals with HIV/AIDS [4–6]. Studies have demonstrated a requirement for adherence levels of at least 95% in order to achieve adequate viral suppression for regimens including unboosted protease inhibitor (PI) therapy [4,7].

, 1999) RecA*, besides assisting in LexA self-cleavage, also fac

, 1999). RecA*, besides assisting in LexA self-cleavage, also facilitates the intermolecular self-cleavage of UmuD2 (Burckhardt et al., 1988; Nohmi et al., 1988; Shinagawa et al., 1988). Cleaved UmuD′2 bound to UmuC (Woodgate et al., 1989) forms DNA polymerase V about 20–40 min after DNA damage (Sommer et al., 1998). Pol V carries out translesion replication of damaged DNA, but lacks 3′-5′ exonuclease activity, and thus is error prone (Tang et al.,

1999), resulting in SOS mutagenesis. Research in non-E. coli species reveals variation in LexA function and number, as well as different SOS genes and SOS boxes bound by LexA. In Acinetobacter baylyi Selleck Dabrafenib strain ADP1, additional differences also exist. In ADP1, recA (Rauch et al., 1996) and ddrR (a gene of unknown function that is unique to the Acinetobacter genus; Hare et al., 2006, 2012) are induced after DNA damage, but only ddrR requires RecA for induction (Whitworth, 2000). The ADP1 recA and ddrR promoters also lack a known or predicted SOS box (Gregg-Jolly & Ornston, 1994; Hare et al., 2006). Additionally, typical DNA damage response genes click here encoding

LexA, SulA, or sigma factor σ38 are not found in A. baylyi or Acinetobacter baumannii (Hare et al., 2006; Robinson et al., 2010), and accordingly, SOS mutagenesis has not been observed in Acinetobacter (Berenstein, 1987) with the notable exception of the emerging pathogens A. baumannii and Acinetobacter ursingii (Hare et al., 2012). Further Mannose-binding protein-associated serine protease differences are centered on the umuDC operon in Acinetobacter. In ADP1, A. baumannii, and seven other Acinetobacter species examined, the umuD homolog (termed umuDAb; Hare

et al., 2012) encodes an extra 59-aa N-terminus region relative to the typical bacterial umuD and is always located adjacent to ddrR. Conversely, umuDC operons similar in size to those found in E. coli are present in only 50% of Acinetobacter species studied, seemingly acquired through horizontal gene transfer (Hare et al., 2012). Also unlike typical UmuD function, this newly described umuDAb allele regulates transcription of the adjacent DNA damage–induced ddrR gene (Hare et al., 2006), as well as other genes (J. M. Hare and J. A. Bradley, unpublished data) in ADP1. This Acinetobacter UmuDAb possesses both the conserved serine–lysine catalytic dyad required by UmuD, LexA, and some bacteriophage repressors for self-cleavage (Paetzel et al., 1997; Walker, 2001) as well as the (Ala/Cys)-Gly cleavage site (Hare et al., 2006, 2012), which suggests that UmuDAb may self-cleave by a similar mechanism. The regulatory activity and possession of an N-terminal domain (Hare et al., 2006) that both UmuDAb and LexA possess further predict that UmuDAb may conduct intramolecular cleavage like LexA, instead of the intermolecular cleavage of UmuD2 (McDonald et al., 1998) that is required for its participation in SOS mutagenesis.

, 2009; Shafiei et al, 2011) In the present study, the β-amylas

, 2009; Shafiei et al., 2011). In the present study, the β-amylase and serine protease from S. halophilum strain GDC-0980 ic50 LY20 showed excellent thermostable, alkalitolerant, halotolerant, and surfactant-stable properties. Also, considering their high activity and stability in the presence of organic solvents, they could be potentially useful for practical applications in biotechnological processes with nonconventional media. This work was financially supported by Shanxi Provincial Science and Technology Foundation (grants no. 20110021) and Natural Science Fund of Shanxi Province (grants no. 2011021031-4). “
“The aim of the studies was to identify immunogenic proteins of Streptococcus

agalactiae (group B streptococcus; GBS) isolates. Investigation of the immunoreactivity with human sera allowed us to determine major immunogenic proteins which might be potential candidates for the development of vaccine. For the study, we have selected 60 genetically different, well-characterized GBS clinical isolates. The proteins immunoreactivity with 24 human sera from patients with GBS infections, carriers, and control group without GBS was detected by SDS-PAGE and Western blotting.

As a result, some major immunogenic proteins were identified, of which four proteins with molecular masses of about 45 Daporinad manufacturer to 50 kDa, which exhibited the highest immunoreactivity features, were analyzed by LC-MS/MS. The proteins were identified by comparative analysis of peptides masses using MASCOT and statistical analysis. The results showed known molecules such as enolase (47.4 kDa),

aldehyde dehydrogenase (50.6 kDa), and ones not previously described such as trigger factor (47 kDa) and elongation factor Tu (44 kDa). The preliminary results indicated that some GBS proteins that elicit protective immunity hold promise not only as components in a vaccine as antigens but also as carriers or adjuvants in polysaccharide conjugate vaccines, but more studies are needed. “
“Endophytic bacterial communities of tomato leaves were analyzed by 16S-rRNA gene pyrosequencing and compared to rhizosphere communities. Leaf endophytes mainly Nintedanib (BIBF 1120) comprised five phyla, among which Proteobacteria was the most represented (90%), followed by Actinobacteria (1,5%), Planctomycetes (1,4%), Verrucomicrobia (1,1%), and Acidobacteria (0,5%). Gammaproteobacteria was the most abundant class of Proteobacteria (84%), while Alphaproteobacteria and Betaproteobacteria represented 12% and 4% of this phylum, respectively. Rarefaction curves for endophytic bacteria saturated at 80 OTUs, indicating a lower diversity as compared to rhizosphere samples (> 1700 OTUs). Hierarchical clustering also revealed that leaf endophytic communities strongly differed from rhizospheric ones. Some OTUs assigned to Bacillus, Stenotrophomonas, and Acinetobacter, as well as some unclassified Enterobacteriaceae were specific for the endophytic community, probably representing bacteria specialized in colonizing this niche.

3) Thus, XrvB may regulate not only hrp gene


3). Thus, XrvB may regulate not only hrp gene

expression but also the expression of genes involved in bacterial growth. H-NS protein binds preferentially to curved DNA, which is commonly associated with promoters, via its conserved C-terminal domain (Tendeng & Bertin, 2003; Dorman 2004). Generally, the binding of H-NS leads to repression of gene expression, while its release leads to gene activation. We found that XrvB has an amino acid sequence similar to the ABT 888 core motif in the C-terminal domain of H-NS (Fig. S1). We, therefore, investigated whether XrvB has DNA-binding activity and whether the protein binds to the promoter region of hrpG. The XrvB protein tagged with six histidine residues at its C-terminus was extracted and purified from E. coli transformed with pETXrvB harboring the coding region of xrvB (Fig. 4a),

and then incubated with SspI-, PvuII- and BamHI-digested pBSHrpG-Pro, in which the putative promoter region of hrpG (−686 to +56) is contained. Electrophoresis, followed by staining with ethidium bromide revealed that, like other H-NS proteins reported previously (Zuber et al., 1994; Tendeng et al., 2003), XrvB bound to a 500-bp SspI–PvuII fragment containing the bla promoter Selleck AZD0530 with a curved structure, along with the 1900-bp fragment derived from the vector sequence (Fig. 4b). The electrophoretic mobility of the fragment was completely retarded at a protein concentration of 1.8 μM. Under the same conditions, the 740-bp BamHI fragment containing the hrpG promoter was not retarded as much as the 500- and 1900-bp fragments. When the predicted promoter region of hrpG was examined using the bend-it computer program (http://www.icgeb.org/dna/bend_it.html), the

possibility that it possesses curved regions Flavopiridol (Alvocidib) was low (data not shown). The results suggest that XrvB possesses DNA-binding activity, but that it does not bind to the hrpG promoter. It is likely that the regulation of hrpG expression by XrvB is indirect and that some unknown gene(s)/protein(s) mediate the regulation. Although many researchers have contributed to identifying various hrp regulatory genes in Xoo and other Xanthomonas spp., the entire scheme of the complicated hrp-regulatory cascade remains unclear (Tsuge et al., 2006; Lee et al., 2008; Zhang et al., 2008; Feng et al., 2009; Huang et al., 2009). Here, our study suggests that the H-NS-like, DNA-binding protein XrvB is involved in the negative regulation of hrp gene expression in Xoo by repressing the expression of a key hrp regulatory gene hrpG. Besides the regulation of hrp gene expression, XrvB is likely to be involved in the regulation of various genes because the growth of the mutant decreased under the culture conditions. Moreover, virulence of the XrvB mutant on rice decreased compared with the wild type (data not shown).

Microscopic smears of body fluids remain an essential part of TB

Microscopic smears of body fluids remain an essential part of TB diagnosis. Results should be available within 1 working day. Identification of mycobacteria is performed at reference centres, and is based on morphology, growth and biochemical characteristics. M. tuberculosis needs to be distinguished from other mycobacteria, for which treatment may be different and there are no infection-control

concerns. Cultures are central to the confirmation and identification of the mycobacterium, and for drug susceptibility testing. More rapid results are obtained from liquid media, which usually grow M. tuberculosis in 7–28 days. Drug susceptibility tests are usually available within 10–21 days of the laboratory receipt of selleck screening library isolates and are performed using standard assays. When

it is important to differentiate rapidly, gene probes are increasingly used in some laboratories, but are less sensitive than culture and are used mainly on respiratory specimens. Most nucleic acid amplification methods to detect M. tuberculosis are complex, labour-intensive, and technically challenging. The sensitivity and specificity estimates of commercial nucleic acid amplification tests (NAATs) are highly variable, compared with culture [12,13]. All specimens, even those negative for M. tuberculosis on polymerase chain reaction Enzalutamide (PCR), still require culture because a negative PCR does not exclude TB and a positive PCR does not indicate the drug susceptibility profile [14,15]. However, recently a fully automated molecular test for TB identification and drug resistance testing has been evaluated on sputum samples from adult patients with TB or MDR-TB [16]. The Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA), an automated molecular test for M. tuberculosis identification and resistance to rifampin, uses a hemi-nested real-time PCR assay. This assay identifies >97% of all patients with culture-confirmed TB, including >90% of patients

with smear-negative disease. The result can be available in hours. The assay has been developed as a laboratory-based and point-of-care test for developing countries, but may be useful in rapid diagnosis of TB in the United Kingdom. Currently there are no data derived from children Carnitine palmitoyltransferase II or using nonrespiratory specimens in HIV-infected persons. Molecular tests for rifampicin resistance are useful especially when MDR-TB is suspected, as about 95% of isolates that are rifampicin resistant will also be isoniazid resistant. As MDR-TB is defined as TB resistant to at least rifampicin and isoniazid, patients with positive molecular-based rifampicin resistance should be treated as having MDR-TB until the full resistance profile from cultures is available. Tuberculin testing can identify patients with latent infection but there are high false-negative rates in HIV-positive patients, especially in those with low CD4 cell counts [17–23].

Hence, it is possible that the ComS peptide may also function int

Hence, it is possible that the ComS peptide may also function intracellularly without its export and subsequent import into the cell. We have also taken into consideration that conditions tested in complex medium may not be optimal for the expression

of the XIP exporter, which can likely result in the accumulation of ComS inside the cell, making it vulnerable to intracellular cleavage. Our expression analysis combined with LC-MS/MS in CDM demonstrates a negative-regulatory role for the ComDE Selleck Oligomycin A system in XIP production. Kreth et al. (2007) reported that ComDE repressed comC expression prior to CSP stimulation. It is possible that ComDE may prevent premature expression of comS, thereby delaying competence induction in CDM to the latter stages of growth. As compound screening assay observed by Desai et al. (2012), competence in CDM is first observed in mid-logarithmic cells of S. mutans and continues well into the stationary phase. We further observe that the amount of XIP was significantly reduced in ∆SMcomX, suggesting a ComX-mediated positive feedback mechanism for XIP synthesis. Putative ComX binding sites were located within the comR gene, upstream of comS, suggesting that ComX may directly

regulate comS expression (Fig. 6a). This positive autoregulation of XIP production may contribute to the persistence of the competent state in CDM. Based on previous works and our findings presented here, we Etofibrate propose a growth condition–dependent model for genetic competence in S. mutans (Fig. 6b). We thank Kirsten Krastel for technical assistance. We are thankful to Dr. Donald Morrison for his review of our manuscript and helpful suggestions provided along with Dr. Lauren Mashburn-Warren and Dr. Mike Federle. D.G.C. is a recipient of the NIH grant R01DE013230-03 and CIHR-MT15431. “
“Bacillus sp. strain CS93, which was previously isolated from Pozol, was previously shown to produce iturin A, bacilysin and chlorotetaine. To investigate the biosynthetic

mechanism of chlorotetaine production, the bac genes were amplified from genomic DNA of Bacillus sp. CS93 by PCR and sequenced. The genes bacABCDE were determined, but no gene that might code for a halogenating enzyme was detected either within the gene cluster or in the flanking sequences. Following further analysis of culture supernatants that were active against bacteria by liquid chromatography-MS, it was not possible to detect bacilysin/chlorotetaine. However, in methanolic fractions containing antibacterial activity, molecular ions characteristic of surfactins and fengycin were detectable by electrospray MS. Using primers complementary for conserved regions of nonribosomal peptide synthase, it was possible to amplify gene fragments that had a high degree of homology with known surfactin and fengycin biosynthetic genes.

Nevertheless, AHS is a potentially fatal condition


Nevertheless, AHS is a potentially fatal condition

which may be preventable. Although the positive predictive value of HLA-B*5801 is low, the test may be useful in patients with Asian ethnic background. Since other hypo-uricemic drugs such as probenecid and febuxostat are available, patients may not wish to take the risk (albeit small) of a serious drug reaction to allopurinol. The option of having this test (on a self-financed basis) should be made available MG-132 clinical trial to patients if routine screening has not been or cannot be implemented. However, it should be stressed that having the HLA-B*5801 test does not result in absolutely no risk of allopurinol-related SJS/TEN. Monitoring for signs and symptoms is still necessary. Other mitigating factors include only prescribing allopurinol for treatment of hyper-uricemia in symptomatic conditions such as gout, urate nephrolithiasis and nephropathy and when cytolytic therapy is considered. Recently, a study by Stamp[21] has shown that the starting dose of allopurinol is an important risk factor for development of AHS. The study suggests a starting dose of 1.5 mg

per unit of estimated glomerular filtration rate, with progressive up-titration of the dose to achieve the target serum uric acid level. Further evaluation of the cost-effectiveness of HLA-B*5801 testing in a population setting should be carried out. This may lead to the development of guidelines which can assist prescribing physicians and ensure that a uniform approach is

adopted when the question about genotype Avasimibe ic50 testing arises in clinical practice. “
“Aim:  Prompted by a clinical question, we critically appraised a meta-analysis of efficacy and safety of mycophenolate mofetil (MMF) versus cyclophosphamide (CYC) in the treatment of proliferative lupus nephritis. Methods:  Systemic reviews and a meta-analysis are introduced to the reader Resveratrol in the perspective of a clinical scenario that raises questions about applicability of certain treatment options in clinical practice. Critical appraisal of meta-analysis addresses three questions. (i) What are the results? (ii) Are the results valid? (iii) How can I apply the results to my patient care? Results:  A meta-analysis paper titled ‘Mycophenolate mofetil is as efficacious as, but safer than, cyclophosphamide in the treatment of proliferative lupus nephritis: a meta-analysis and meta-regression’by Mak et al. (2009) was selected. Our critical appraisal identified several strengths of the paper, such as having a clearly focused clinical question, considering clinically important outcomes, using appropriate inclusion criteria to select primary studies, assessing quality of selected papers, good reproducibility in the assessment of primary studies and performing sensitivity analysis and meta-regression to account for heterogeneity.

Whole-cell patch-clamp

recordings showed that the input r

Whole-cell patch-clamp

recordings showed that the input resistance and membrane capacitance of the EGFP-positive Purkinje cells from mice that underwent IUE at E11.5 www.selleckchem.com/products/dabrafenib-gsk2118436.html were similar to those of wild-type Purkinje cells (Table 1). In addition, there were no significant differences in either the PF– or CF–EPSC kinetics (Table 1). The PF– and CF–EPSCs in the EGFP-positive Purkinje cells showed the typical paired-pulse facilitation and paired-pulse depression, respectively, that were observed in wild-type Purkinje cells (Fig. 2B and Table 1). By the end of the third postnatal week in mice, most wild-type Purkinje cells lose their redundant CFs and become innervated by a single CF. EGFP-positive Purkinje cells electroporated at E11.5 were similarly innervated by a single CF, as shown by their single threshold for excitation (Fig. 2C). Furthermore, the input–output

relationships of the PF–EPSC were not significantly different between the electroporated EGFP-positive and wild-type Selleck ABT-888 Purkinje cells (Fig. 2D), indicating that the PF inputs to Purkinje cells were also intact. Finally, the conjunctive stimulation of PFs and the depolarization of Purkinje cells induced LTD similarly in both wild-type and electroporated Purkinje cells (Fig. 2E; 67 ± 5% at t = 25–30 min, n = 7 from four wild-type mice; 69 ± 6% at t = 25–30 min, n = 7 from four electroporated Purkinje cells; Mann–Whitney U-test, P = 0.947). Together, these results indicate that IUE

did not alter the basic membrane properties, EPSC parameters, or short-term or long-term synaptic plasticity of the transfected Purkinje cells. To examine whether cell-type-specific and inducible promoters were compatible with the IUE method for Purkinje cells, we employed an inducible Cre/loxP system (Matsuda & Cepko, 2007). The Purkinje-specific L7 promoter (Oberdick et al., 1990; Smeyne et al., 1991; Tomomura et al., 2001) was used to express the conditionally active form of Cre recombinase ERT2CreERT2, in which the ligand-binding domain of the estrogen receptor PLEKHB2 was mutated; the Cre recombinase is activated in response to 4OHT (Matsuda & Cepko, 2007). By coexpressing pCALNL-DsRed2, which contains the CAG promoter and a stop signal flanked by loxP sequences, the reporter gene DsRed2 was designed to be expressed in a 4OHT/Cre- and L7-dependent manner (Fig. 3A). To unconditionally label all the electroporated cells, pCAG-EGFP was co-electroporated with the pL7-ERT2CreERT2 and pCALNL-DsRed2. After IUE at E11.5, the mice received an intraperitoneal injection of 4OHT or vehicle at P6 and were fixed at P14 (Fig. 3A). As expected, only mice that received 4OHT displayed DsRed2 signals in the cerebellum (Fig. 3B). Confocal microscopy further confirmed that the DsRed2 signals were observed only in a subset of EGFP-positive Purkinje cells (Fig. 3C).