The purpose of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in isolates and simultaneously differentiate between and gene targets from wild-type and wild-type strains (13 were PZA-resistant strains) and 21 strains (8 were BCG strains). 28). Although cases of PZA-resistant isolates with no mutations have been reported, mutations of and its putative promoter remain the major mechanism of PZA resistance (15, 20). Over 40 different mutations in either the structural gene or its putative promoter associated with PZA resistance in have been described. The changes are either mutations that involve substitution of nucleotides or mutations in the form of nucleotide insertions or deletions (15, 20, 27). In contrast, the natural resistance to PZA demonstrated by strains is uniformly due to a unique single-point mutation (C169G) in (9), the increasing frequency of tuberculosis infections following intravesical instillation of the naturally PZA-resistant BCG strain for the treatment of superficial bladder cancer (1, 17, 19), and the increasing incidence of zoonotic tuberculosis in developing countries due to naturally PZA-resistant (6, 16, 24). Conventional mycobacterial susceptibility testing for PZA is 51-21-8 manufacture dependent on growth of the organism in the presence of the drug. This technique is both time-consuming and potentially unreliable due to the poor growth of in the highly acidic medium required for PZA activity (7, 12). Automated testing systems, such as the BACTEC 460TB and BACTEC MGIT 960 systems, are more sensitive than conventional testing but require from 8 to 12 days to determine antibacterial susceptibility and have the potential for cross-contamination (12, 14, 31). Genotypic assays for the detection of drug resistance have been applied to both cultured isolates and direct patient specimens. Included in these are amplification methods, DNA sequence evaluation, PCR-single-strand conformation polymorphism electrophoresis, and structure-specific cleavage and DNA probe recognition assays, which can handle detecting mutations connected with medication level of resistance (8, 22, 30). Temperature-mediated heteroduplex evaluation (TMHA) using denaturing high-performance liquid chromatography (DHPLC) was originally put on the recognition of particular gene polymorphisms (21). The technology was lately put on the recognition of mutations connected with antituberculosis medication level of resistance (5). The technique used differential retention of homoduplex and heteroduplex DNAs under incomplete denaturing conditions for the identification of mutations in that are responsible for rifampin, isoniazid, streptomycin, ethambutol, and PZA resistance, respectively. Additionally, a separate genetic element (and was found to be problematic. The difficulty of detecting mutations was attributed to the diverse natures of the mutations and their distribution throughout the gene and its putative promoter. It was proposed that the potential for highly stable DNA helices due to increased GC content within specific regions of the gene represented a major technical challenge for TMHA methodology (5). To overcome these difficulties, the analysis conditions of the TMHA assay were reengineered, and a second probe was added. In 51-21-8 manufacture combination, ZNF914 these changes allowed the rapid identification of mutations associated with PZA resistance and the ability to distinguish between the two closely related species of the complex, and complex were studied: 48 strains, 13 of which were PZA resistant, and 21 strains, 8 of which were BCG strains. The PZA-resistant isolates were obtained from either the Tuberculosis Diagnostic Laboratory of the Centers for Disease Control and Prevention (CDC) (20) or the Tuberculosis Diagnostic Section of the Michigan Public Health Laboratory. The genes from 51-21-8 manufacture the 13 PZA-resistant strains had been sequenced and found to contain different mutations distributed throughout the open reading frame, as well as the promoter region (Fig. ?(Fig.1).1). The study isolates included six reference BCG strains (ATCC 35743, ATCC 35744, ATCC 35739, ATCC 35731, ATCC 35738, and ATCC 35748) from the CDC collection. Fifty clinical isolates were obtained from either Creighton University Medical Center (5 and 5 isolates), CDC (4 isolates), or the University of Nebraska Medical Center (UNMC) (4 BCG, and 30 isolates). Clinical isolates were identified as either or as previously described (6, 32) using the standard biochemical reactions, including nitrate reduction, niacin accumulation, and Pzase activity. PZA susceptibility was previously determined for all isolates, with resistance defined by an MIC of >25 g/ml using the proportion method with Middlebrook 7H10 medium (4). Two reference strains were used as probes in the TMHA study, H37Rv,.
P-glycoprotein (P-gp) is usually a membrane-bound efflux pump that actively exports a wide range of compounds from your cell and is associated with the phenomenon of multidrug resistance. fibroblasts. Collectively our SU6668 findings reveal a key and previously undocumented role of P-gp in host-parasite conversation and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells. Introduction P-glycoprotein (P-gp ABCB1 MDR1)2 is one of the most intensively analyzed users of the ABC transporter superfamily. With remarkably broad substrate acknowledgement P-gp drives the ATP-dependent efflux of SU6668 harmful metabolites and xenobiotics from your cell (1) and is thus a central mediator of drug bioavailability. Importantly P-gp overexpression following drug treatment is responsible for the multidrug resistance (MDR) phenotype a major reason for chemotherapy failure not only in malignancy cells (2) but also in pathogenic microorganisms (3 4 Aside from its well known role in drug efflux P-gp is also expressed at basal levels in many different tissues yet the normal physiological functions of the protein remain poorly comprehended. The possibility that physiological levels of host P-gp play a role in host-pathogen conversation other than mediating drug resistance has not been investigated so far. We resolved this question using as a model pathogenic parasite. is the causative agent of toxoplasmosis a potentially fatal disease not only for immunocompromised patients and fetuses but according to SU6668 recent insights also emerging as a life threatening contamination in immunocompetent individuals (5). infects virtually all nucleated host cells and resides in a highly specialized vacuole called the parasitophorous vacuole (PV) which is usually created by invaginating the host cell membrane at the time of invasion. The PV is not qualified for lysosome fusion thus avoiding acidification (6) but it is usually closely associated with host organelles including lysosomes mitochondria and endoplasmic reticulum (examined in Ref. 7). Even though the PV does not intersect directly with host vesicular traffic remains dependent on host SU6668 cells for a number of critical nutrients. Significant progress has been made in our understanding of the mechanisms uses to scavenge nutrients from its host especially in the case of lipid molecules. An important recent example was the identification of H.O.S.T. (host organelle-sequestering tubulo-structures) a unique system of tubular structures formed by the parasite to sequester cholesterol-containing endo-lysosomes from your host cytoplasm into the PV (8). However the molecular mechanisms of the traffic from your host cell to the PV are not completely elucidated and the presence of transporters has been proposed frequently. To analyze whether the P-gp transporter plays a role in biology ZNF914 we compared parasite replication in wild type (WT) mouse embryonic fibroblasts with double knock-out (DKO) fibroblasts in which neither of the two murine P-gp isoforms are expressed (9). In parallel we also analyzed DKO cells complemented with the human P-gp homologue (DKO/P-gp) (10) which restored P-gp functionality to DKO cells and allowed P-gp expression levels higher than those found in WT cells (supplemental Fig. S1). In this way our model did not depend on either drug-selected P-gp-overexpressing cells which may acquire adaptation mechanisms different from P-gp overexpression during the development of the MDR phenotype or P-gp inhibitors several of which are known to have side effects on host metabolism. EXPERIMENTAL PROCEDURES Biochemical Reagents Unless normally stated all chemicals were purchased from Sigma cell culture reagents were from Invitrogen and radiolabeled lipids were from Amersham Biosciences. Anti-P-gp monoclonal antibody C219 was purchased from Alexis Biochemicals; anti-Lamp1 1D4B antibody was obtained through the Developmental Studies Hybridoma Lender (University or college of Iowa Iowa City IA); anti-giantin and anti-tubulin were a kind gift from J. Rohrer and M. A. Hakimi respectively. Conjugated secondary antibodies were from Invitrogen. Reconstituted high density lipoproteins and apolipoprotein A-I (apoA-I) were a kind gift from P. Lerch (CSL Behring Bern Switzerland). NDB-cholesterol was from Avanti Polar Lipids. Mammalian Cell and Parasite Culture Mouse embryonic fibroblasts double knocked out for P-gp (77.1 Mdr1a?/?/Mdr1b?/?) (9) triple knocked out for P-gp and MRP1 (3.8 Mdr1a?/?/Mdr1b?/?/Mrp1?/?) (11) and parental.