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Kinds of Cassette Terms modified by Cassette Selected AbstractsActivity of Azole Fungicides and ABC Transporter Modulators on Mycosphaerella graminicolaJOURNAL OF PHYTOPATHOLOGY, Issue 6 2002I. STERGIOPOULOS The antimicrobial activity of the azole fungicides cyproconazole and propiconazole as single active ingredients and in mixtures with the ATP-Binding Cassette (ABC) transporter modulators rhodamine 6G, quercetin, quinidine, and verapamil and the strobilurin kresoxim-methyl was assessed against the wheat pathogen Mycosphaerella graminicola. Interactions amongst these compounds were evaluated on germination and germ tube growth of pycnidiospores using the Colby and Wadley method. Water agar proved to be the best test medium since all pycnidiospores germinated within 24 h of incubation and apical germ tube growth dominated over bud formation by intermediate cells. Analysis with the Colby method revealed that interactions between the compounds in all mixtures tested on germination of pycnidiospores were additive. With regard to germ tube growth, mixtures of cyproconazole and verapamil or kresoxim-methyl displayed a synergistic interaction. Analysis of mixtures of cyproconazole and kresoxim-methyl with the Wadley method revealed that the interaction between the two compounds was purely additive. These results indicate that the Colby method overestimated the interaction between these two compounds in a mixture. [source] A mass spectrometry based functional assay for the quantitative assessment of ABC transporter activityRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2009Mária Katona ATP-Binding Cassette (ABC) transporters are highly expressed in pharmacological barriers limiting the access of drugs to their targets. Since characterization of a compound as a transporter substrate or inhibitor bears significant consequences in drug development, there is a great need for reliable tools that enable the rapid analysis of the transport susceptibility of drugs. Here we describe a simple but very efficient high-performance liquid chromatography/mass spectrometry (HPLC/MS) assay for measuring the ABC transporter-dependent vesicular transport of compounds. In addition, we provide evidence that the requirement for sample preparation can be minimized using desorption electrospray ionization (DESI)-MS, paving the way for a direct, high-throughput investigation of drug-transporter interactions. Copyright © 2009 John Wiley & Sons, Ltd. [source] Danofloxacin-mesylate is a substrate for ATP-dependent efflux transportersBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2007J A Schrickx Background and purpose: Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin-Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP-Binding Cassette (ABC) superfamily, including P-gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell-membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux-transporters. Experimental approach: Confluent monolayers of Caco-2 cells, grown on microporous membranes in two-chamber devices were used. DM concentrations were measured by fluorimetric assay after HPLC of the culture media. Key results: DM transport across Caco-2 cells was asymmetric, with a rate of secretion exceeding that of absorption. The P-gp inhibitors PSC833 and GF120918 and the MRP-inhibitor MK571 partially decreased the secretion of DM and increased its absorption rate. The BCRP inhibitor, Ko143, decreased secretion only at a concentration of 1 ,M. When DM was applied together with ciprofloxacin, secretion as well as absorption of DM decreased. Conclusions and Implications: DM is a substrate for the efflux transporters P-gp and MRP2, whereas the specific role of BCRP in DM transport needs further evaluation. These findings provide a mechanistic basis for the understanding of the pharmacokinetics of DM in healthy and diseased individuals. British Journal of Pharmacology (2007) 150, 463,469. doi:10.1038/sj.bjp.0706974 [source] Through-Bond Energy Transfer Cassettes with Minimal Spectral Overlap between the Donor Emission and Acceptor Absorption: Coumarin,Rhodamine Dyads with Large Pseudo-Stokes Shifts and Emission Shifts,ANGEWANDTE CHEMIE, Issue 2 2010Weiying Lin Prof. Eine neue Klasse von Cumarin-Rhodamin-Systemen, die auf dem Energietransfer über Bindungen (TBET) beruhen und minimale Überlappung zwischen Donoremission und Akzeptorabsorption aufweisen, zeichnet sich durch große Pseudo-Stokes-Verschiebungen (bis 230,nm) und Emissionsverschiebungen (bis 170,nm) aus. Die Nützlichkeit dieser TBET-Plattform für die Entwicklung von TBET-Sonden wurde mit einer neuen ratiometrischen pH-Fluoreszenzsonde demonstriert. [source] Synthesis of Symmetrical Multichromophoric Bodipy Dyes and Their Facile Transformation into Energy Transfer Cassettes,CHEMISTRY - A EUROPEAN JOURNAL, Issue 21 2010Altan Bozdemir Dr. Abstract Multichromophoric boron-dipyrromethene (Bodipy) dyes synthesized on phenylene-ethynylene platforms have been be converted to energy transfer cassettes in a one-step chemical transformation. Excitation energy transfer processes in these highly symmetrical derivatives were studied in detail, including time-resolved fluorescence spectroscopy techniques. Excitation spectra and the emission lifetimes suggest efficient energy transfer between the donor and acceptor chromophore. These novel energy transfer cassettes, while highlighting a short-cut approach to similar energy transfer systems, could be useful as large pseudo-Stokes shift multichromophoric dyes with potential applications in diverse applications. Fenilenetilen platformu üzerinde sentezlenen multikromoforik Bodipy boyarmaddeleri, tek basamakl, bir kimyasal transformasyonla enerji transferi kasetlerine dönü,türülmü,tür. Zaman ayr,ml, floresans spektroskopisi tekniklerinin de içinde bulundu,u yöntemlerle, yüksek simetri ö,eleri bulunduran bu türevlerdeki eksitasyon enerjisi transferi süreçleri ayr,nt,l, olarak çal,,,lm,,t,r. Eksitasyon spektrumlar, ve emisyon ömürlerindeki de,i,im, donör ve akseptör kromoforlar, aras,nda etkin bir enerji transferi oldu,unu dü,ündürmektedir. Bu yeni enerji transfer kasetleri, benzer enerji transfer sistemlerine kolay bir geçi, yolu göstermekle birlikte, pek çok farkl, alanda potansiyel uygulamalar, olabilecek, büyük pseudo-Stokes kaymas, de,erlerine sahip multikromoforik boyarmaddeler olarak da yararl, olabilirler. [source] Single-cell image analysis to assess ABC-transporter,mediated efflux in highly purified hematopoietic progenitorsCYTOMETRY, Issue 4 2002H.G.P. Raaijmakers Abstract Background Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. Methods We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter,mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). Results The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein,mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 ± 0.35 and 1.14 ± 0.11, respectively; P = 0.01). P-glycoprotein,mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 ± 0.19 and 1.28 ± 0.18, respectively). Conclusion The described method is a valuable tool for assessing ABC-transporter,mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells. Cytometry 49:135,142, 2002. © 2002 Wiley-Liss, Inc. [source] Mutation in the abcb7 gene causes abnormal iron and fatty acid metabolism in developing medaka fishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2008Akimitsu Miyake The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka isolated a unique mutant that develops a fatty liver at larval stages. Positional cloning identified the responsible gene as medaka abcb7. Abcb7, a mitochondrial ABC (ATP binding cassette) half-transporter, has been implicated in iron metabolism. Recently, human Abcb7 was found to be mutated in X-linked sideroblastic anemia with cerebellar ataxia (XLSA/A). The homozygous medaka mutant exhibits abnormal iron metabolism in erythrocytes and accumulation of lipid in the liver. Microarray and in situ hybridization analyses demonstrated that the expression of genes involved in iron and lipid metabolisms are both affected in the mutant liver, suggesting novel roles of Abcb7 in the development of physiologically functional liver. The medaka abcb7 mutant thus could provide insights into the pathogenesis of XLSA/A as well as the normal function of the gene. [source] Increase in multidrug transport activity is associated with oocyte maturation in sea stars,DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2006Troy A. Roepke In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein-am, increased two-fold 3 h post-maturation. Experiments using specific and non-specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P-glycoprotein (P-gp), and an ABCC protein similar to the MDR-associated protein (MRP)-like transporters. Western blots using an antibody directed against mammalian P-gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post-maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP-specific transporter inhibitors. [source] A new evaluation technique for the detection of impurities in purified proteins via CE with native UV-LIFELECTROPHORESIS, Issue 2 2010Audrey Rodat Abstract An analytical methodology for quality control analyses of IgG and their impurities is presented using a new UV-LIF (266,nm) detector inside the cassette of a CE instrument and its performance was evaluated. The observed sensitivity was very close to that obtained by silver staining of slab gels (LOD of 25,ng/mL), while the sensitivity of the analysis is 80 times better than with CE/UV absorption (214,nm). Examples of the analysis of pharmaceutical and other commercial IgGs are provided and the kinetics of the reduction of IgG by ,-mercaptoethanol is reported, demonstrating the ease of performing the analysis. [source] Miniaturized movable contactless conductivity detection cell for capillary electrophoresisELECTROPHORESIS, Issue 12-13 2003Miroslav Macka Abstract A miniaturized capacitively coupled contactless conductivity detector (mini-C4D) cell has been designed which is small enough to allow it to slide along the effective capillary length inside the capillary cassette of an Agilent capiillary electrophoresis system (CE) (or other CE brand of similar construction), including the possibility of positioning it close to the point of optical detection (4 cm), or even putting two such detector cells in one cassette. The cell was tested and the performance characteristics (noise, sensitivity, and peak width) were compared with those obtained with the previously used large C4D cell. No significant differences were observed. The mini-C4D was used in simultaneous separations of common cations and anions where its advantage over a larger C4D cell is the ability to vary the point of detection with the mini-C4D cell continuously at any point along the capillary length, so that the optimum apparent selectivity can be chosen. Other applications include providing a convenient second point of detection in addition to photometric detection, such as to measure accurately the linear velocity of a zone, or to allow placement of two mini-C4D cells in one capillary cassette simultaneously. [source] Improved ,-Glucanase Production by a Recombinant Escherichia coli Strain using Zinc-Ion Supplemented MediumENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2007U. Beshay Abstract In order to investigate the suitability of different metal chelates for affinity chromatography, an expression vector was constructed. It contained a hybrid ,-glucanase as a model protein fused with a His6 -tag and a secretion cassette providing the ability to secrete ,-glucanase into the culture medium. Supplementation of zinc to the medium led to a rapidly increased expression and release of the target protein into the cultivation medium. Results in respect to the supplementation of the commonly used Terrific Broth "TB-medium" with different metal ions are reported with special emphasis on the influence of zinc ions. A concentration of zinc ions in the order of about 0.175 mM led to optimal results. Batch cultivation under well-controlled conditions showed that the growth behavior did not change significantly by adding zinc ions. Growth in a stirred tank bioreactor was much faster in unsupplemented TB-medium compared to shake flask experiments leading to a much higher biomass concentration (15,g/L instead of 3,g/L). The secretion of ,-glucanase under theses conditions started at the transition into the stationary phase and increased to yield an extracellular activity of 1350,U/mL at the end of the fermentation process. An even higher yield of extracellular ,-glucanase (2800,U/mL) was reached when the fermentation was carried out with TB-medium supplemented with 0.175,mM ZnSO4. [source] Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on rootsENVIRONMENTAL MICROBIOLOGY, Issue 4 2003N. Carol Casavant Summary We constructed and characterized a plasmid-based genetic system that reports the expression of a toluene-responsive promoter (PtbuA1) by effecting an irreversible, heritable change in the biosensor cell. Expression of the reporter gene gfp is strongly repressed in the absence of expression from the PtbuA1 promoter, and high level gfp expression in the original cell and its progeny is mediated by the site-specific recombination machinery of bacteriophage P22 to initiate removal of a repressor cassette. The reporter plasmid pTolLHB was functional in two soil saprophytes, Pseudomonas fluorescens A506 and Enterobacter cloacae JL1157, with the efficiency and sensitivity to low toluene concentrations being optimal in P. fluorescens A506. In culture, 80,100% of the A506 (pTolLHB) population expressed gfp following exposure to 0.2 µm toluene for one to three hours. Compared to the response of A506 containing a plasmid-borne PtbuA1 - gfp fusion, the recombination-based biosensor was more sensitive at detecting low toluene and trichloroethylene concentrations. An A506 (pTolLHB) inoculum, which had a background of 2.5% of the cells expressing gfp, was introduced onto barley roots in soil microcosms. If toluene was introduced into the microcosms, after 24 h, 72% of the A506 (pTolLHB) cells recovered from roots expressed gfp, indicating bioavailable toluene to rhizosphere bacteria. When toluene was not introduced, 16.5% of the A506 (pTolLHB) cells recovered from the roots expressed gfp, indicating that natural inducers of the PtbuA1 promoter were present in the barley rhizosphere. When introduced into rhizotrons containing barley plants and toluene vapours, the biosensor allowed localization of the availability of toluene along the seminal roots. In rhizotrons that were not exposed to toluene vapours, the biosensor exhibited high PtbuA1 -promoter activity in distinct regions along the seminal roots, indicating spatial heterogeneity plant- or rhizosphere microbial community-derived inducers of the PtbuA1 promoter. This recombination-based toluene biosensor thus was useful in identifying bacterial exposure to transient or low levels of toluene, or related compounds, directly in the environment. [source] Analysis of a radiographic technique for measurement of equine metacarpal bone shapeEQUINE VETERINARY JOURNAL, Issue S33 2001L. J. WALTER Summary Accuracy and limitations of a radiographic technique were established for measurement of metacarpal bone shape in horses. A radiographic index (RI) has been used to measure changes in third metacarpal (Mc3) bone shape in response to training in young racehorses. The aim of these experiments was to determine the effects of positioning of the radiographic equipment on RI measurements from lateromedial radiographs of a left ex vivo metacarpus. Repeatability of the RI measurement in left lateromedial and right mediolateral view Mc3s were evaluated. The width of the dorsal cortex (DC), palmar cortex (PC) and medulla (M) were measured at a position 25 mm distal to the nutrient foramen and the RI calculated where RI = [(DC+PC)/M] x [DC/PC]. The reference RI values were obtained from 12 standard lateromedial radiographs. Factors analysed included the optimum focus-object distance, the angle at which the x-ray machine was positioned, the distance of the cassette from the limb, and the horizontal angle and vertical tilt of the cassette. The RI values included within the confidence interval (mean ° 1.96 x s.d.) were considered sufficiently accurate. The optimum focus-object distance was 1 m. Accurate measurements were obtained when the cassette was held as close to the limb as possible with the horizontal angle not exceeding 5°. The x-ray machine needed to be orientated within 6° of the lateromedial plane. These findings suggest that the radiographic index can be used to measure Mc3 bone shape, providing there is accurate alignment of the x-ray machine, cassette, and limb, with respect to one another. [source] Functional C3435T polymorphism of MDR1 gene: an impact on genetic susceptibility and clinical outcome of childhood acute lymphoblastic leukemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2004Krzysztof Jamroziak Abstract: The significance of genetic background in childhood acute lymphoblastic leukemia (ALL) is not well understood. Polymorphisms of genes encoding for xenobiotics and drug transporters are potential factors, which can influence the risk of developing ALL and its clinical outcome. P-glycoprotein (P-gp) is an adenosine triphosphate-binding cassette (ABC)-family transporter involved in protection against xenobiotics and multi-drug resistance. Recently, the single-nucleotide polymorphism C3435T of MDR1 gene has been found to be associated with altered tissue expression and function of P-gp. To evaluate whether C3435T MDR1 polymorphism is associated with the occurrence and outcome of ALL, 113 children with ALL (median age 5.1 yr) and 175 healthy individuals of Polish Caucasian origin were studied by polymerase chain reaction-restriction fragment-length polymorhism (PCR-RFLP) assay. The mutant homozygous TT genotype was found to be associated with occurrence of ALL (OR, 95% CI; 1.8, 1.1,3.1; P = 0.037). Besides, the analysis of factors influencing clinical outcome of our ALL patient cohort showed that CC genotype carriers had significantly lower event-free survival probability (pEFS) (0.62 vs. 0.87; P = 0.007) and overall survival probability (pOS) (0.72 vs. 0.91; P = 0.006). The Cox proportional hazards model-based analysis revealed that the hazard ratios for lower pEFS and lower pOS among CC homozygous subjects were 3.9 (P = 0.008) and 3.3 (P = 0.02), respectively. In conclusion, the results of the present study provide evidence that C3435T MDR1 polymorphism may involve both the susceptibility to and the clinical outcome of childhood ALL. Carriers of the TT genotype are more at risk of developing ALL than other individuals, whereas CC genotype carriers are supposed to have worse prognosis. [source] Systemic IFN-, drives kidney nephritis in B6.Sle123 miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2008Anna-Marie Fairhurst Abstract The impact of IFN-, secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental C57BL/6 (B6) congenic partner using an adenovirus (ADV) expression vector containing a recombinant IFN-, gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFN-,. Most IFN-,-induced phenotypes were similar in B6 and B6.Sle123 mice; however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFN-, exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFN-, were identical in both strains, severe glomerulonephritis occurred only in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum anti-nuclear antibody levels. In summary, the predominant impact of systemic IFN-, in this murine model is an exacerbation of mechanisms mediating end organ damage. [source] Deletion of the LIME adaptor protein minimally affects T and B cell development and functionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2007Claude Grégoire Abstract LIME (Lck-interacting membrane protein) is a transmembrane adaptor that associates with the Lck and Fyn protein tyrosine kinases and with the C-terminal Src kinase (Csk). To delineate the role of LIME in vivo, LIME-deficient mice were generated. Although Lime transcripts were expressed in immature and mature B and T cells, the absence of LIME impeded neither the development nor the function of B and T cells. TCR transgenic mice deprived of LIME showed, however, a 1.8-fold enhancement in positive selection. Since B cells and activated T cells express LIME and the related adaptor NTAL, mice lacking both adaptors were generated. Double-deficient mice showed no defect in the development and function of B and T cells, and the lack of LIME had no effect on the autoimmune syndrome that develops in aged NTAL-deficient mice. In contrast to a previous report, we further showed that this autoimmune syndrome develops in the absence of T cells. Therefore, our in vivo results refute all the previous roles postulated for LIME on the basis of studies of transformed B and T cells and demonstrate that LIME has no seminal role in the signaling cassette operated by antigen receptors and coreceptors. [source] Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2004R. J. Lang Abstract Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6,7 days post-plating (D6,7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na+ and tetraethylammonium (TEA)-sensitive K+ channels. Neurons reached their maximal cell capacitance after D6,7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca2+ -free, 2.5 mm Mg2+ saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca2+ levels within ES cell-derived neurons increased in response to glutamate receptor agonists l -glutamate, AMPA, N -methyl- d -aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl, -selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons. [source] Molecular genetics of pseudoxanthoma elasticumEXPERIMENTAL DERMATOLOGY, Issue 4 2001F. Ringpfeil Abstract: Pseudoxanthoma elasticum (PXE), a systemic heritable connective tissue disorder, is characterized by progressive calcification of elastic structures in the skin, the eyes and the cardiovascular system, with considerable intra- and interfamilial phenotypic variability. Recently, underlying genetic defects have been identified in the ABCC6 gene, which resides on the chromosomal locus 16p13.1 and encodes the MRP6 protein, a member of the ATP-binding cassette (ABC) family of proteins. The affected individuals are homozygous or compound heterozygous for a spectrum of genetic lesions, including nonsense and missense mutations, or deletions and splice-site alterations, confirming the autosomal recessive nature of this condition. Analysis of the deduced primary sequence suggests that MRP6 is a transmembrane transporter, but its function has not been delineated yet. Surprisingly, however, MRP6 is expressed primarily, if not exclusively, in the liver and the kidneys, suggesting that PXE may be a primary metabolic disorder with secondary involvement of elastic fibers. Identification of mutations in the ABCC6 gene in PXE provides a means for prenatal and presymptomatic testing in families at risk for recurrence. DNA-based analyses will also identify heterozygous carriers who may be at risk for development of limited manifestations of the disease as a result of compounding genetic factors and/or environmental modifiers. [source] A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situFEBS JOURNAL, Issue 12 2008Fanjie Meng To measure mechanical stress in real time, we designed a fluorescence resonance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts. The probe was composed of a green fluorescence protein pair, Cerulean and Venus, linked with a stable ,-helix. We measured the FRET efficiency of the free cassette protein as a function of the length of the linker, the angles of the fluorophores, temperature and urea denaturation, and protease treatment. The linking helix was stable to 80 °C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected. We modified the ,-helix linker by adding and subtracting residues to vary the angles and distance between the donor and acceptor, and assuming that the cassette was a rigid body, we calculated its geometry. We tested the strain sensitivity of stFRET by linking both ends to a rubber sheet subjected to equibiaxial stretch. FRET decreased proportionally to the substrate strain. The naked cassette expressed well in human embryonic kidney-293 cells and, surprisingly, was concentrated in the nucleus. However, when the cassette was located into host proteins such ,-actinin, nonerythrocyte spectrin and filamin A, the labeled hosts expressed well and distributed normally in cell lines such as 3T3, where they were stressed at the leading edge of migrating cells and relaxed at the trailing edge. When collagen-19 was labeled near its middle with stFRET, it expressed well in Caenorhabditis elegans, distributing similarly to hosts labeled with a terminal green fluorescent protein, and the worms behaved normally. [source] Fast set-up of doxycycline-inducible protein expression in human cell lines with a single plasmid based on Epstein,Barr virus replication and the simple tetracycline repressorFEBS JOURNAL, Issue 3 2007Markus Bach We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein,Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3,12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on/off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on/off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on- and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable. [source] A new molecular tool for transgenic diatomsFEBS JOURNAL, Issue 13 2005Control of mRNA, protein biosynthesis by an inducible promoter, terminator cassette Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen-free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms. [source] Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulatorFEBS JOURNAL, Issue 17 2000Production of a suitable protein for structural studies Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure,function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation ,F508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N- 1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1. [source] The Helicobacter pylori plasticity region locus jhp0947,jhp0949 is associated with duodenal ulcer disease and interleukin-12 production in monocyte cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2004Ramon De Jonge Abstract Colonization with Helicobacter pylori always results in chronic gastritis, which is controlled by infiltration of mononuclear cells and the subsequent release of cytokines like interleukin (IL)-12. To identify H. pylori factors involved in inducing cytokine production in mononuclear cells, a random H. pylori mutant library was screened for the inability to induce IL-12 production in monocyte THP-1 cells. Of the 231 random mutants screened, one mutant (M1) showed a consistent twofold decrease in the amount of IL-12 induction compared to the parental strain 1061 (P<0.01). Further characterization of mutant M1 revealed that the kanamycin resistance cassette had integrated in the jhp0945 gene, which is situated in an H. pylori strain-specific plasticity region. Three reference strains possessing this plasticity region induced significantly higher amounts of IL-12 when compared to the H. pylori 26695 reference strain, which does not possess this plasticity region. The role in disease outcome of jhp0945 as well as the neighbouring plasticity region genes jhp0947 and jhp049 was assessed in a Dutch population cohort. Firstly, the presence of jhp0947 was completely linked with that of jhp0949 and was roughly associated with jhp0945 (P=0.072), but not with the cag pathogenicity island (PAI) (P=0.464). The presence of the jhp0947 and jhp0949 genes, but not of jhp0945, was significantly associated with duodenal ulcer disease when compared to gastritis (P=0.027). Therefore, the jhp0947,jhp0949 locus may be a novel putative H. pylori marker for disease outcome independent of the cag PAI. [source] Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the fieldFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Lotta Jäderlund Abstract Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25,tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25,tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25,tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions. [source] The F1FO ATP synthase genes in Methanosarcina acetivorans are dispensable for growth and ATP synthesisFEMS MICROBIOLOGY LETTERS, Issue 2 2009Regina Saum Abstract There is a long-standing discussion in the literature, based on biochemical and genomic data, whether some archaeal species may have two structurally and functionally distinct ATP synthases in one cell: the archaeal A1AO together with the bacterial F1FO ATP synthase. To address a potential role of the bacterial F1FO ATP synthase, we have exchanged the F1FO ATPase gene cluster in Methanosarcina acetivorans against a puromycin resistance cassette. Interestingly, the mutant was able to grow with no difference in growth kinetics to the wild type, and cellular ATP contents were identical in the wild type and the mutant. These data demonstrate that the F1FO ATP synthase is dispensable for the growth of M. acetivorans. [source] A novel neisserial shuttle plasmid: A useful new tool for meningococcal researchFEMS MICROBIOLOGY LETTERS, Issue 1 2005Clíona A. O' Dwyer Abstract We report the identification and nucleotide sequence analysis of a cryptic plasmid pMIDG2830 from the Gram-negative bacterium Neisseria flavescens. The largest open reading frame encodes a protein similar to the replication protein, RepA, found in pAB49 from Acinetobacter baumannii and pNI10 from Pseudomonas. Modified by the incorporation of a kanamycin resistance cassette, the plasmid can be stably maintained in Escherichia coli and Neisseria meningitidis, and can be used as a shuttle plasmid in meningococcal research. [source] Deprogrammed sporulation in StreptomycesFEMS MICROBIOLOGY LETTERS, Issue 1 2002Yasuo Ohnishi Abstract The bacterial genus Streptomyces forms chains of spores by septation at intervals in aerial hyphae and subsequent maturation on solid medium. Substrate hyphae undergo extensive lysis, liberating nutrients on which aerial hyphae develop. Some mutant strains, however, ectopically form spores by septation in substrate hyphae on solid medium or in vegetative hyphae in liquid medium, which suggests that all hyphae have the potential to differentiate into spores. A Streptomyces griseus mutant strain NP4, which has a mutation in the regulatory system for an ATP-binding cassette (ABC) transporter gene, forms ectopic spores in substrate hyphae only on glucose-containing medium. In addition, overexpression of a substrate-binding protein of the ABC transporter in the wild-type strain causes ectopic septation in very young substrate hyphae and subsequent sporulation in response to glucose. These ectopic spores germinate normally. The ectopic sporulation is independent of A-factor, a microbial hormone that determines the timing of aerial mycelium formation during normal development. Thus, substrate hyphae of Streptomyces have a potential to develop into spores without formation of aerial hyphae. For programmed development, therefore, the strict repression of septum formation in substrate mycelium should be necessary, as well as the positive signal relay leading to aerial mycelium formation followed by septation and sporulation. [source] Super-channel in bacteria: function and structure of the macromolecule import system mediated by a pit-dependent ABC transporterFEMS MICROBIOLOGY LETTERS, Issue 2 2001Yumiko Mishima Abstract In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on a pit, a mouth-like organ formed on the cell surface only when cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated the ,super-channel', and in this review, we discuss the three-dimensional structure and specific function of the ,super-channel' for macromolecule import found for the first time in a bacterium. [source] Tools for the genetic manipulation of Zygosaccharomyces rouxiiFEMS YEAST RESEARCH, Issue 8 2007Lenka Pribylova Abstract A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP,kanMX,loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase. [source] Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cellsGENES TO CELLS, Issue 9 2006Satoshi Watanabe An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis -acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars -element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars -element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars -element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars -element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars -element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars -element has also a barrier activity. These results indicated that the Ars -element act as an insulator in mammalian cells. [source] | ||||||||||||||||||||||||||||||||||||||||