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Sample records for bacterial atp-binding cassette

  1. Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

    Science.gov (United States)

    Heuveling, Johanna; Frochaux, Violette; Ziomkowska, Joanna; Wawrzinek, Robert; Wessig, Pablo; Herrmann, Andreas; Schneider, Erwin

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

  2. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

    Full Text Available Abstract There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx or out (efflux of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]. ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders.

  3. ATP-binding cassette (ABC) transporters in normal and pathological lung

    NARCIS (Netherlands)

    van der Deen, M; de Vries, EGE; Timens, W; Scheper, RJ; Timmer-Bosscha, H; Postma, DS

    2005-01-01

    ATP-binding cassette ( ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein ( P-gp), multidrug resistance-associated protein 1 ( MRP1) and breas

  4. Atovaquone and quinine anti-malarials inhibit ATP binding cassette transporter activity

    OpenAIRE

    Rijpma, S.R.; Heuvel, J. J.; van de Velden, M.; Sauerwein, R. W.; Russel, F. G.; Koenderink, J.B.

    2014-01-01

    BACKGROUND: Therapeutic blood plasma concentrations of anti-malarial drugs are essential for successful treatment. Pharmacokinetics of pharmaceutical compounds are dependent of adsorption, distribution, metabolism, and excretion. ATP binding cassette (ABC) transport proteins are particularly involved in drug deposition, as they are located at membranes of many uptake and excretory organs and at protective barriers, where they export endogenous and xenobiotic compounds, including pharmaceutica...

  5. Multidrug transport by ATP binding cassette transporters : a proposed two-cylinder engine mechanism

    NARCIS (Netherlands)

    van Veen, HW; Higgins, CF; Konings, WN

    2001-01-01

    The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. This review summarizes current insi

  6. The homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism

    NARCIS (Netherlands)

    van Veen, HW; Margolles, A; Muller, M; Higgins, CF; Konings, WN

    2000-01-01

    The bacterial LmrA protein and the mammalian multidrug resistance P-glycoprotein are closely related ATP-binding cassette (ABC) transporters that confer multidrug resistance on cells by mediating the extrusion of drugs at the expense of ATP hydrolysis. The mechanisms by which transport is mediated,

  7. Blood-Brain Barrier Active Efflux Transporters: ATP-Binding Cassette Gene Family

    OpenAIRE

    Löscher, Wolfgang; Potschka, Heidrun

    2005-01-01

    Summary: The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lip...

  8. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus

    OpenAIRE

    Yu, Fang; De Luca, Vincenzo

    2013-01-01

    The presence of biologically active monoterpenoid indole alkaloids (MIAs) on the leaf surfaces of medicinally important Catharanthus roseus has led to questions about the secretion processes involved and their prevalence within MIA-producing species of plants. This report shows that a transporter closely related to those involved in cuticle assembly in plants and belonging to the pleiotropic drug resistance family of ATP-binding cassette transporters is specialized for transport of the MIA ca...

  9. Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia

    OpenAIRE

    Heemskerk, Suzanne; van Koppen, Arianne; van den Broek, Luc; Poelen, Geert J. M.; Wouterse, Alfons C; Dijkman, Henry B. P. M.; Russel, Frans G. M.; Masereeuw, Rosalinde

    2007-01-01

    Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar–Hannover rats were injected with lipopolysaccharide (LPS+) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS−). After LPS+, proximal tubular damage and a reduction in renal...

  10. New ATP-binding cassette A3 mutation causing surfactant metabolism dysfunction pulmonary type 3.

    Science.gov (United States)

    Piersigilli, Fiammetta; Peca, Donatella; Campi, Francesca; Corsello, Mirta; Landolfo, Francesca; Boldrini, Renata; Danhaive, Olivier; Dotta, Andrea

    2015-10-01

    Respiratory distress syndrome (RDS) may occur in term and near-term infants because of mutations in surfactant-related genes. ATP-binding cassette A3 (ABCA3), a phospholipid carrier specifically expressed in the alveolar epithelium, is the most frequently involved protein. We report the case of a couple of late-preterm fraternal twin infants of opposite sex carrying the same compound heterozygous ABCA3 mutations, one of which has never been previously reported, with different disease severity, suggesting variable penetrance or sex-related differences. ABCA3 deficiency should be considered in term or near-term babies who develop unexplained RDS. PMID:26508177

  11. Characterisation of single domain ATP-binding cassette protien homologues of Theileria parva.

    Science.gov (United States)

    Kibe, M K; Macklin, M; Gobright, E; Bishop, R; Urakawa, T; ole-MoiYoi, O K

    2001-09-01

    Two distinct genes encoding single domain, ATP-binding cassette transport protein homologues of Theileria parva were cloned and sequenced. Neither of the genes is tandemly duplicated. One gene, TpABC1, encodes a predicted protein of 593 amino acids with an N-terminal hydrophobic domain containing six potential membrane-spanning segments. A single discontinuous ATP-binding element was located in the C-terminal region of TpABC1. The second gene, TpABC2, also contains a single C-terminal ATP-binding motif. Copies of TpABC2 were present at four loci in the T. parva genome on three different chromosomes. TpABC1 exhibited allelic polymorphism between stocks of the parasite. Comparison of cDNA and genomic sequences revealed that TpABC1 contained seven short introns, between 29 and 84 bp in length. The full-length TpABC1 protein was expressed in insect cells using the baculovirus system. Application of antibodies raised against the recombinant antigen to western blots of T. parva piroplasm lysates detected an 85 kDa protein in this life-cycle stage.

  12. Identification and Characterization of a Brucella abortus ATP-Binding Cassette Transporter Homolog to Rhizobium meliloti ExsA and Its Role in Virulence and Protection in Mice

    OpenAIRE

    G.M.S. Rosinha; Freitas, Daniela A.; Miyoshi, Anderson; Azevedo, Vasco; Campos, Eleonora; Cravero, Silvio L; Rossetti, Osvaldo; Splitter, Gary; S.C. Oliveira

    2002-01-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The ded...

  13. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    Science.gov (United States)

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.

  14. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    Science.gov (United States)

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients.

  15. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    Science.gov (United States)

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients. PMID:24100054

  16. TSH increases synthesis of hepatic ATP-binding cassette subfamily A member 1 in hypercholesterolemia.

    Science.gov (United States)

    Zhang, Tiantian; Zhou, Lingyan; Li, Cong Cong; Shi, Hong; Zhou, Xinli

    2016-07-22

    Epidemiological evidence suggests that thyrotropin (TSH) levels are closely correlated with the severity of hypercholesterolemia. Reverse cholesterol transfer (RCT) plays an important role in regulating bloodcholesterol. However, the molecular mechanism of hypercholesterolemia in subclinical hypothyroidism (SCH) has not been fully clarified. The SCH mouse model, which is characterized by elevated serum TSH but not thyroid hormone levels, demonstrated a significant increase in plasma cholesterol compared with controls. Interestingly, Tshr KO mice, with normal thyroid hormone levels after thyroid hormone supplementation, showed lower plasma cholesterol levels compared with their wild-type littermates. ATP binding cassette subfamily A member 1(ABCA1) is a member of the ABC superfamily, which induces transfer of intracellular cholesterol to extracellular apolipoprotein. TSH upregulated hepatic ABCA1 to promote the efflux of intercellular cumulative cholesterol, resulting in increased plasma cholesterol. These data might partially explain the pathogenesis of hypercholesterolemia in SCH. PMID:27179782

  17. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    Science.gov (United States)

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium. PMID:21561685

  18. Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

    DEFF Research Database (Denmark)

    Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.;

    2014-01-01

    Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been...... implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found...... that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S...

  19. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    Science.gov (United States)

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  20. Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9)

    NARCIS (Netherlands)

    Wolters, Justina Clarinda; Abele, Rupert; Tampé, Robert

    2005-01-01

    The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a h

  1. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter

    NARCIS (Netherlands)

    Yang, Nuan; Driessen, Arnold J. M.

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporte

  2. Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus.

    Directory of Open Access Journals (Sweden)

    Akifumi Sugiyama

    Full Text Available LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.

  3. The pharmacological impact of ATP-binding cassette drug transporters on vemurafenib-based therapy

    Directory of Open Access Journals (Sweden)

    Chung-Pu Wu

    2014-04-01

    Full Text Available Melanoma is the most serious type of skin cancer and one of the most common cancers in the world. Advanced melanoma is often resistant to conventional therapies and has high potential for metastasis and low survival rates. Vemurafenib is a small molecule inhibitor of the BRAF serine-threonine kinase recently approved by the United States Food and Drug Administration to treat patients with metastatic and unresectable melanomas that carry an activating BRAF (V600E mutation. Many clinical trials evaluating other therapeutic uses of vemurafenib are still ongoing. The ATP-binding cassette (ABC transporters are membrane proteins with important physiological and pharmacological roles. Collectively, they transport and regulate levels of physiological substrates such as lipids, porphyrins and sterols. Some of them also remove xenobiotics and limit the oral bioavailability and distribution of many chemotherapeutics. The overexpression of three major ABC drug transporters is the most common mechanism for acquired resistance to anticancer drugs. In this review, we highlight some of the recent findings related to the effect of ABC drug transporters such as ABCB1 and ABCG2 on the oral bioavailability of vemurafenib, problems associated with treating melanoma brain metastases and the development of acquired resistance to vemurafenib in cancers harboring the BRAF (V600E mutation.

  4. Functional analysis of the ATP-binding cassette (ABC transporter gene family of Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Broehan Gunnar

    2013-01-01

    Full Text Available Abstract Background The ATP-binding cassette (ABC transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. Results We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H. This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. Conclusions The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

  5. Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

    Science.gov (United States)

    Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D; Andersen, Tonni G; Pomorski, Thomas G

    2014-12-01

    Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

  6. A novel ATP-binding cassette transporter, ABCG6 is involved in chemoresistance of Leishmania.

    Science.gov (United States)

    BoseDasgupta, Somdeb; Ganguly, Agneyo; Roy, Amit; Mukherjee, Tanmoy; Majumder, Hemanta K

    2008-04-01

    ATP-binding cassette (ABC) transporters constitute the biggest family of membrane proteins involved in drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted camptothecin (CPT)-resistant parasites show overexpression of a novel ABC transporter, which was classified as ABCG6. Transfection and overexpression of LdABCG6 in wild type parasites, shows its localization primarily in the plasma membrane and flagellar pocket region. Overexpressed LdABCG6 confers substantial CPT resistance to the parasites by rapid drug efflux. Various inhibitors have been tested for their ability to revert the CPT-resistant phenotype to specifically understand the inhibition of LdABCG6 transporter. Transport experiments using everted membrane vesicles were carried out to gain an insight into the kinetics of drug transport. This study provides further knowledge of specific membrane traffic ATPase and its involvement in the chemoresistance of Leishmania. PMID:18243364

  7. Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori

    OpenAIRE

    Liu, Shumin; Zhou, Shun; Tian, Ling; Guo, Enen; Luan, Yunxia; Zhang, Jianzhen; Li, Sheng

    2011-01-01

    Background The ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development. Results From the genome of the silkworm, Bombyx mori, we have identified 51 putative ABC genes which are classified into eight subfamil...

  8. AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans.

    Science.gov (United States)

    Handali, Melody; Roychowdhury, Hridindu; Neupane, Durga P; Yukl, Erik T

    2015-12-11

    Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC). Transcription of the aztD gene is significantly up-regulated under conditions of zinc starvation. Recombinantly expressed AztD purifies with slightly substoichiometric zinc from the periplasm of Escherichia coli and is capable of binding up to three zinc ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound zinc nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a zinc chaperone to AztC and likely functions to maintain zinc homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic zinc trafficking and the function of chaperones in this process.

  9. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    Science.gov (United States)

    Lara, Flavio Alves; Pohl, Paula C; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H F; Almeida, Igor C; Vaz, Itabajara da Silva; Oliveira, Pedro L

    2015-01-01

    In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new

  10. Endothelial ATP-binding cassette G1 in mouse endothelium protects against hemodynamic-induced atherosclerosis.

    Science.gov (United States)

    Xue, Shanshan; Wang, Jiaxing; Zhang, Xu; Shi, Ying; Li, Bochuan; Bao, Qiankun; Pang, Wei; Ai, Ding; Zhu, Yi; He, Jinlong

    2016-08-19

    Activated vascular endothelium inflammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid homeostasis by transporting cholesterol efflux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inflammation activation during early-stage atherogenesis in mice and the underlying mechanisms. Endothelial cell (EC)-specific ABCG1 transgenic (EC-ABCG1-Tg) mice were generated and cross-bred with low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. After a 4-week Western-type diet, the mice were sacrificed for assessing atherosclerosis. Human umbilical vein ECs were treated with different flows, and ABCG1 was adenovirally overexpressed to investigate the mechanism in vitro. Compared with Ldlr(-/-) mouse aortas, EC-ABCG1-Tg/Ldlr(-/-) aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr(-/-) mouse aortic arch but not thoracic aorta was significantly reduced, which suggests a protective role of ABCG1 under atheroprone flow. In vitro, overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. Overexpression of ABCG1 blunted cholesterol-activated ECs in vitro. In exploring the mechanisms of ABCG1 attenuating endothelial inflammation, we found that ABCG1 inhibited oscillatory flow-activated nuclear factor kappa B and NLRP3 inflammasome in ECs. ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory shear stress via suppressing the inflammatory response. PMID:27297110

  11. Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages

    Institute of Scientific and Technical Information of China (English)

    Chao-ke TANG; Guang-hui YI; Jun-hao YANG; Lu-shan LIU; Zuo WANG; Chang-geng RUAN; Yong-zong YANG

    2004-01-01

    AIM: To study the effect of oxidized low density lipoprotein (ox-LDL) on ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages. METHODS: After exposing the cultured THP-1 macrophages to ox-LDL for different periods, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively.The cholesterol level in THP-1 macrophage foam cells was detected by high performance liquid chromatography.RESULTS: ox-LDL elevated AB CA1 in both protein and mRNA levels and increased apolipoprotein (apo) A-I-mediated cholesterol efflux in a time- and dose-dependent manner. 22(R)-hydroxyeholesterol and 9-cis-retinoic acid did significantly increase cholesterol efflux in THP-1 macrophage foam cells (P<0.05), respectively. Both of them further promoted cholesterol efflux (P<0.01). As expected, liver X receptor (LXR) agonist decreased content of esterified cholesterol in the macrophage foam cells compared with control, whereas only a slight decrease of free cholesterol was observed. LXR activity was slightly increased by oxidized LDL by 12 % at 12 h compared with 6 h.However, LXR activity was increased about 1.8 times at 24 h, and oxidized LDL further increased LXR activity by about 2.6 times at 48 h. CONCLUSION: ABCA1 gene expression was markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 plays an important role in the homeostasis of cholesterol in the macrophages.

  12. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

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    Flavio Alves Lara

    Full Text Available In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA, a well-known inhibitor of ATP binding cassette (ABC transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may

  13. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

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    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  14. Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

    OpenAIRE

    Taniguchi, Naohiro; Tokuda, Hajime

    2008-01-01

    An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

  15. Computational characterization of TTHA0379: A potential glycerophosphocholine binding protein of Ugp ATP-binding cassette transporter.

    Science.gov (United States)

    Chandravanshi, Monika; Gogoi, Prerana; Kanaujia, Shankar Prasad

    2016-11-01

    For the de novo biosynthesis of phospholipids, byproducts such as sn-glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) of glycerophospholipid metabolic pathway are imported inside the cell by an ATP-binding cassette (ABC) transporter known as UgpABCE. Of which, UgpA and UgpE constitutes the transmembrane domains (TMDs), UgpC forms the dimer of ATP-hydrolyzing component and UgpB is the periplasmic substrate binding protein. Structurally, UgpABCE transporter displays similarity to the maltose ABC transporter of Escherichia coli; thus, has been grouped into the CUT1 (Carbohydrate Uptake Transporter-1) family of bacterial ABC transporters. Being a member of CUT1 family, several Ugp (Uptake glycerol phosphate) protein sequences in biological database(s) exhibit sequence and structure similarity to sugar ABC transporters and have been annotated as sugar binding proteins; one of such proteins is TTHA0379 from Thermus thermophilus HB8. Here, in this study, we used computational method(s) to distinguish UgpB and sugar binding proteins based on their primary and tertiary structure features. A comprehensive analysis of these proteins indicates that they are evolutionarily related to each other having common conserved features at their primary and tertiary structure levels. However, they display differences at their active sites owing to the dissimilarity in their ligand preferences. In addition, phylogenetic analysis of TTHA0379 along with UgpB and sugar binding proteins reveals that both the groups of proteins forms two distinct clades and TTHA0379 groups with UgpB proteins. Furthermore, analysis of the ligand binding pocket shows that all the essential features of glycerophosphocholine binding protein i.e. UgpB, are conserved in TTHA0379 as well. Combining these features, here, we designate TTHA0379 to be a GPC binding protein.

  16. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  17. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    OpenAIRE

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, ...

  18. Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance

    Directory of Open Access Journals (Sweden)

    Yi-Jun Wang

    2014-09-01

    Full Text Available The phenomenon of multidrug resistance (MDR has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs, such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance.

  19. In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

    Science.gov (United States)

    Bakhsheshian, Joshua; Wei, Bih-Rong; Hall, Matthew D; Simpson, R Mark; Gottesman, Michael M

    2016-01-01

    We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter. PMID:27424909

  20. Substrate Specificity and Ionic Regulation of GlnPQ from Lactococcus lactis. An ATP-Binding Cassette Transporter with Four Extracytoplasmic Substrate-Binding Domains

    NARCIS (Netherlands)

    Schuurman-Wolters, Gea K.; Poolman, Bert

    2005-01-01

    We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmi

  1. Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

    Science.gov (United States)

    Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

  2. Secretion of natural and synthetic toxic compounds from filamentous fungi by membrane transporters of the ATP-binding cassette and major facilitator superfamily

    NARCIS (Netherlands)

    Stergiopoulos, I.; Zwiers, L.H.; Waard, De M.A.

    2002-01-01

    This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environme

  3. LrABCF1, a GCN-type ATP-binding cassette transporter from Lilium regale, is involved in defense responses against viral and fungal pathogens

    Science.gov (United States)

    ATP-binding cassette (ABC) transporters are essential for membrane translocation in diverse biological processes, such as plant development and defense response. Here, a general control non-derepressible (GCN)-type ABC transporter gene, designated LrABCF1, was identified from Cucumber mosaic virus (...

  4. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    Science.gov (United States)

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

  5. The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

    Institute of Scientific and Technical Information of China (English)

    Bai-Xiao Niu; Fu-Rong He; Ming He; Ding Ren; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    Plant male reproductive development is a complex biological process,but the underlying mechanism is not well understood.Here,we characterized a rice (Oryza sativa L.) male sterile mutant.Based on mapbased cloning and sequence analysis,we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene,OsABCG15,causing abnormal anthers and male sterility.Therefore,we named this mutant osabcg15.Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis Ⅱ stage) to stage 10 (late microspore stage).Two genes CYP704B2 and WDA1,involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine,were downregulated in osabcg15 mutant,suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules.Consistently,histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration,leading to rapid degredation of young microspores.The results suggest that OsABCG15 plays a critical role in exine formation and pollen development,similar to the homologous gene of AtABCG26 in Arabidopsis.This work is helpful to understand the regulatory network in rice anther development.

  6. HG-829 is a potent noncompetitive inhibitor of the ATP-binding cassette multidrug resistance transporter ABCB1.

    Science.gov (United States)

    Caceres, Gisela; Robey, Robert W; Sokol, Lubomir; McGraw, Kathy L; Clark, Justine; Lawrence, Nicholas J; Sebti, Said M; Wiese, Michael; List, Alan F

    2012-08-15

    Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies. PMID:22761337

  7. Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

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    Polin Haghvirdizadeh

    2015-01-01

    Full Text Available Background. Type 2 diabetes mellitus (T2DM is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1 gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs2230806 (R219K, rs1800977 (C69T, and rs9282541 (R230C polymorphisms Malaysian subjects. Methods. A total of 164 T2DM and 165 controls were recruited and their genotypes for ABCA1 gene polymorphisms were determined based on the real time high resolution melting analysis. Results. There was a significant difference between the subjects in terms of age, BMI, FPG, HbA1c, HDL, LDL, and TG P<0.05. There was a significant association between HOM of R219K P=0.005, among Malaysian subjects; moreover, allele frequency revealed the significant difference in A allele of R219K P=0.003. But, there was no significant difference in genotypic and allelic frequencies of C69T and R230C polymorphism. Conclusion. R219K polymorphism of ABCA1 gene can be considered as a genetic risk factor for T2DM subjects among Malaysians.

  8. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    Science.gov (United States)

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-01

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. PMID:26708605

  9. High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Jiahong Xue; Zuyi Yuan; Yue Wu; Yan Zhao; Zhaofei Wan

    2008-01-01

    Objective:ATP-binding cassette transporters(ABC) A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods:Incubation of human VSMCs with D-ghicose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-kB inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF-kB inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

  10. Stickleback embryos use ATP-binding cassette transporters as a buffer against exposure to maternally derived cortisol.

    Science.gov (United States)

    Paitz, Ryan T; Bukhari, Syed Abbas; Bell, Alison M

    2016-03-16

    Offspring from females that experience stressful conditions during reproduction often exhibit altered phenotypes and many of these effects are thought to arise owing to increased exposure to maternal glucocorticoids. While embryos of placental vertebrates are known to regulate exposure to maternal glucocorticoids via placental steroid metabolism, much less is known about how and whether egg-laying vertebrates can control their steroid environment during embryonic development. We tested the hypothesis that threespine stickleback (Gasterosteus aculeatus) embryos can regulate exposure to maternal steroids via active efflux of maternal steroids from the egg. Embryos rapidly (within 72 h) cleared intact steroids, but blocking ATP-binding cassette (ABC) transporters inhibited cortisol clearance. Remarkably, this efflux of cortisol was sufficient to prevent a transcriptional response of embryos to exogenous cortisol. Taken together, these findings suggest that, much like their placental counterparts, developing fish embryos can actively regulate their exposure to maternal cortisol. These findings highlight the fact that even in egg-laying vertebrates, the realized exposure to maternal steroids is mediated by both maternal and embryonic processes and this has important implications for understanding how maternal stress influences offspring development. PMID:26984623

  11. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  12. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    Science.gov (United States)

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins. PMID:19961540

  13. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    Science.gov (United States)

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

  14. Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain

    OpenAIRE

    Zhou, Chunyan; Chen, Jing; Zhang, Xiaolu; Costa, Lucio G.; Guizzetti, Marina

    2014-01-01

    Aims: Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cho...

  15. Disruption of lolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal for Escherichia coli and Prevents Release of Lipoproteins from the Inner Membrane

    OpenAIRE

    Narita, Shin-ichiro; Tanaka, Kimie; Matsuyama, Shin-ichi; Tokuda, Hajime

    2002-01-01

    ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstituti...

  16. A Selective ATP-binding Cassette Sub-family G Member 2 Efflux Inhibitor Revealed Via High-Throughput Flow Cytometry

    OpenAIRE

    Strouse, J. Jacob; Ivnitski-Steele, Irena; Khawaja, Hadya M.; Perez, Dominique; Ricci, Jerec; Yao, Tuanli; Weiner, Warren S.; Schroeder, Chad E.; Simpson, Denise S.; Maki, Brooks E.; Li, Kelin; Golden, Jennifer E.; Foutz, Terry D.; Waller, Anna; Evangelisti, Annette M.

    2012-01-01

    Chemotherapeutics tumor resistance is a principal reason for treatment failure and clinical and experimental data indicate that multidrug transporters such as ATP-binding Cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is ...

  17. Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

    OpenAIRE

    Falcón-Pérez, Juan M.; Martínez-Burgos, Mónica; Molano, Jesús; Mazón, María J.; Eraso, Pilar

    2001-01-01

    The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate ener...

  18. Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori

    OpenAIRE

    Zhang Jianzhen; Luan Yunxia; Guo Enen; Tian Ling; Zhou Shun; Liu Shumin; Li Sheng

    2011-01-01

    Abstract Background The ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development. Results From the genome of the silkworm, Bombyx mori, we have identified 51 putative ABC genes which are classified into eight...

  19. Influence of ATP-Binding Cassette Transporter 1 R219K and M883I Polymorphisms on Development of Atherosclerosis: A Meta-Analysis of 58 Studies

    OpenAIRE

    Yin, Yan-Wei; Li, Jing-Cheng; Gao, Dong; Chen, Yan-Xiu; Li, Bing-Hu; Wang, Jing-Zhou; Liu, Yun; Liao, Shao-Qiong; Zhang, Ming-Jie; Chang-yue GAO; Zhang, Li-li

    2014-01-01

    Background Numerous epidemiological studies have evaluated the associations between ATP-binding cassette transporter 1 (ABCA1) R219K (rs2230806) and M883I (rs4149313) polymorphisms and atherosclerosis (AS), but results remain controversial. The purpose of the present study is to investigate whether these two polymorphisms facilitate the susceptibility to AS using a meta-analysis. Methods PubMed, Embase, Web of Science, Medline, Cochrane database, Clinicaltrials.gov, Current Controlled Trials,...

  20. Overexpression of the ATP binding cassette gene ABCA1 determines resistance to Curcumin in M14 melanoma cells

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    Angelini Giovanna

    2009-12-01

    Full Text Available Abstract Background Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. Results We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NFκB signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NFκB dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NFκB. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Conclusion Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

  1. ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence.

    Science.gov (United States)

    Murphy, Timothy F; Brauer, Aimee L; Johnson, Antoinette; Kirkham, Charmaine

    2016-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. PMID:27391026

  2. Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

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    Yu Lan

    2012-08-01

    Full Text Available Abstract Background The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. Results In order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877 and 1.4-fold in SR16 (P = 0.00973 duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively. Conclusions The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

  3. Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori

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    Zhang Jianzhen

    2011-10-01

    Full Text Available Abstract Background The ATP-binding cassette (ABC transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development. Results From the genome of the silkworm, Bombyx mori, we have identified 51 putative ABC genes which are classified into eight subfamilies (A-H by phylogenetic analysis. Gene duplication is very evident in the ABCC and ABCG subfamilies, whereas gene numbers and structures are well conserved in the ABCD, ABCE, ABCF, and ABCH subfamilies. Microarray analysis revealed that expression of 32 silkworm ABC genes can be detected in at least one tissue during different developmental stages, and the expression patterns of some of them were confirmed by quantitative real-time PCR. A large number of ABC genes were highly expressed in the testis compared to other tissues. One of the ABCG genes, BmABC002712, was exclusively and abundantly expressed in the Malpighian tubule implying that BmABC002712 plays a tissue-specific role. At least 5 ABCG genes, including BmABC005226, BmABC005203, BmABC005202, BmABC010555, and BmABC010557, were preferentially expressed in the midgut, showing similar developmental expression profiles to those of 20-hydroxyecdysone (20E-response genes. 20E treatment induced the expression of these ABCG genes in the midgut and RNA interference-mediated knockdown of USP, a component of the 20E receptor, decreased their expression, indicating that these midgut-specific ABCG genes are 20E-responsive. Conclusion In this study, a genome-wide analysis of the silkworm ABC transporters has been conducted. A comparison of ABC transporters from 5 insect species provides an overview of this vital gene superfamily in insects. Moreover, tissue- and stage-specific expression data of the

  4. Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

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    Simone Bocchetta

    Full Text Available Hepatitis C virus (HCV establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1 mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts. The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis

  5. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

    OpenAIRE

    Estelita Pereira Lima; Marília Oliveira Fonseca Goulart; Modesto Leite Rolim Neto

    2014-01-01

    The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in t...

  6. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti.

    Science.gov (United States)

    Lima, Estelita Pereira; Goulart, Marília Oliveira Fonseca; Rolim Neto, Modesto Leite

    2014-11-01

    The role of ATP-binding cassette (ABC) transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM). The best result in the series was obtained with the addition of verapamil (40 μM), which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated. PMID:25411004

  7. Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

    OpenAIRE

    Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

    2006-01-01

    The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

  8. Cloning and Characterization of the Pseudomonas fluorescens ATP-Binding Cassette Exporter, HasDEF, for the Heme Acquisition Protein HasA

    OpenAIRE

    Idei, Akiko; Kawai, Eri; Akatsuka, Hiroyuki; Omori, Kenji

    1999-01-01

    Two ATP-binding cassette (ABC) exporters are present in Pseudomonas fluorescens no. 33; one is the recently reported AprDEF system and the other is HasDEF, which exports a heme acquisition protein, HasA. The hasDEF genes were cloned by DNA hybridization with a DNA probe coding for the LipB protein, one of the components of the Serratia marcescens ABC exporter Lip system. P. fluorescens HasA showed sequence identity of 40 to 49% with HasA proteins from Pseudomonas aeruginosa and Serratia marce...

  9. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Estelita Pereira Lima

    2014-11-01

    Full Text Available The role of ATP-binding cassette (ABC transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM. The best result in the series was obtained with the addition of verapamil (40 μM, which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

  10. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption

    NARCIS (Netherlands)

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W.

    2013-01-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding

  11. ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10 (ABCC10) are not primary resistance factors for cabazitaxel

    Institute of Scientific and Technical Information of China (English)

    Rishil J Kathawala; Yi-Jun Wang; Suneet Shukla; Yun-Kai Zhang; Saeed Alqahtani; Amal Kaddoumi; Suresh V Ambudkar; Charles R Ashby Jr; Zhe-Sheng Chen

    2015-01-01

    Introduction:ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10 (ABCC10) proteins are efflux transporters that couple the energy derived from ATP hydrolysis to the translocation of toxic substances and chemotherapeutic drugs out of cells. Cabazitaxel is a novel taxane that differs from paclitaxel by its lower affinity for ATP-binding cassette (ABC) transporters. Methods:We determined the effects of cabazitaxel, a novel tubulin-binding taxane, and paclitaxel on paclitaxel-resistant, ABCB1-overexpressing KB-C2 and LLC-MDR1-WT cells and paclitaxel-resistant, ABCC10-overexpressing HEK293/ABCC10 cells by calculating the degree of drug resistance and measuring ATPase activity of the ABCB1 transporter. Results:Decreased resistance to cabazitaxel compared with paclitaxel was observed in KB-C2, LLC-MDR1-WT, and HEK293/ABCC10 cells. Moreover, cabazitaxel had low efficacy, whereas paclitaxel had high efficacy in stimulating the ATPase activity of ABCB1, indicating a direct interaction of both drugs with the transporter. Conclusion:ABCB1 and ABCC10 are not primary resistance factors for cabazitaxel compared with paclitaxel, suggesting that cabazitaxel may have a low affinity for these efflux transporters.

  12. Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette transporters gene enrichment in typhoid fever-infected Nigerian children

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    Resau James H

    2011-09-01

    bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. Conclusions We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

  13. Cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1) and cholesterol uptake via the LDL receptor influences cholesterol-induced impairment of beta cell function in mice

    NARCIS (Netherlands)

    Kruit, J. K.; Kremer, P. H. C.; Dai, L.; Tang, R.; Ruddle, P.; de Haan, W.; Brunham, L. R.; Verchere, C. B.; Hayden, M. R.

    2010-01-01

    Cellular cholesterol accumulation is an emerging mechanism for beta cell dysfunction in type 2 diabetes. Absence of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) results in increased islet cholesterol and impaired insulin secretion, indicating that impaired cholesterol effl

  14. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Kostsin, Dzmitry G. [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya Str. 27, Minsk 220072 (Belarus); Kashiwayama, Yoshinori [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Takanashi, Kojiro; Yazaki, Kazufumi [Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoko University, Uji, Kyoto 611-0011 (Japan); Imanaka, Tsuneo, E-mail: imanaka@pha.u-toyama.ac.jp [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Morita, Masashi [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2014-10-24

    Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

  15. Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

    Science.gov (United States)

    Garroum, Imène; Bidzinski, Przemyslaw; Daraspe, Jean; Mucciolo, Antonio; Humbel, Bruno M; Morel, Jean-Benoit; Nawrath, Christiane

    2016-06-01

    The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense. PMID:27121976

  16. Distinct alterations in ATP-binding cassette transporter expression in liver, kidney, small intestine, and brain in adjuvant-induced arthritic rats.

    Science.gov (United States)

    Kawase, Atsushi; Norikane, Sari; Okada, Ayaka; Adachi, Mamiko; Kato, Yukio; Iwaki, Masahiro

    2014-08-01

    Pathophysiological changes of infection or inflammation are associated with alterations in the production of numerous absorption, distribution, metabolism and excretion-related proteins. However, little information is available on the effects of inflammation on the expression levels and activities of ATP-binding cassette (ABC) transporters. We examined the effect of acute (on day 7) and chronic (on day 21) inflammation on the expression of ABC transporters in some major tissues in rat. Adjuvant-induced arthritis (AA) in rats was used as an animal model for inflammation. The mRNA levels of mdr1a and mdr1b encoding P-glycoprotein (P-gp) decreased significantly in livers of AA rats on day 21. Hepatic protein levels of P-gp, Mrp2, and Bcrp decreased significantly in membranes but not homogenates of AA rats after 7 days and after 21 days of treatment with adjuvant. Contrary to liver, protein levels of P-gp and Mrp2, but not Bcrp in kidney, increased significantly in membranes. The biliary excretion of rhodamine 123 was decreased in rats with chronic inflammation owing to decreases in efflux activities of P-gp. Our results showed that the expression of transporters in response to inflammation was organ dependent. In particular, hepatic and renal P-gp and Mrp2 exhibited opposite changes in membrane protein levels.

  17. Role of NH2-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    International Nuclear Information System (INIS)

    Highlights: • ABCD proteins classifies based on with or without NH2-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH2-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH2-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH2-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH2-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH2-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH2-terminal H0 motif in organelle targeting is widely conserved in living organisms

  18. Hernandezine, a Bisbenzylisoquinoline Alkaloid with Selective Inhibitory Activity against Multidrug-Resistance-Linked ATP-Binding Cassette Drug Transporter ABCB1.

    Science.gov (United States)

    Hsiao, Sung-Han; Lu, Yu-Jen; Yang, Chun-Chiao; Tuo, Wei-Cherng; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung; Hung, Tai-Ho; Wu, Chung-Pu

    2016-08-26

    The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, MDR1) is the most studied mechanism of multidrug resistance (MDR), which remains a major obstacle in clinical cancer chemotherapy. Consequently, resensitizing MDR cancer cells by inhibiting the efflux function of ABCB1 has been considered as a potential strategy to overcome ABCB1-mediated MDR in cancer patients. However, the task of developing a suitable modulator of ABCB1 has been hindered mostly by the lack of selectivity and high intrinsic toxicity of candidate compounds. Considering the wide range of diversity and relatively nontoxic nature of natural products, developing a potential modulator of ABCB1 from natural sources is particularly valuable. Through screening of a large collection of purified bioactive natural products, hernandezine was identified as a potent and selective reversing agent for ABCB1-mediated MDR in cancer cells. Experimental data demonstrated that the bisbenzylisoquinoline alkaloid hernandezine is selective for ABCB1, effectively inhibits the transport function of ABCB1, and enhances drug-induced apoptosis in cancer cells. More importantly, hernandezine significantly resensitizes ABCB1-overexpressing cancer cells to multiple chemotherapeutic drugs at nontoxic, nanomolar concentrations. Collectively, these findings reveal that hernandezine has great potential to be further developed into a novel reversal agent for combination therapy in MDR cancer patients. PMID:27504669

  19. Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease.

    Science.gov (United States)

    Sabirzhanova, Inna; Lopes Pacheco, Miquéias; Rapino, Daniele; Grover, Rahul; Handa, James T; Guggino, William B; Cebotaru, Liudmila

    2015-08-01

    Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment. PMID:26092729

  20. Whole-transcriptome survey of the putative ATP-binding cassette (ABC transporter family genes in the latex-producing laticifers of Hevea brasiliensis.

    Directory of Open Access Journals (Sweden)

    Nie Zhiyi

    Full Text Available The ATP-binding cassette (ABC proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree. A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis.

  1. Cooperative transcriptional activation of ATP-binding cassette sterol transporters ABCG5 and ABCG8 genes by nuclear receptors including Liver-X-Receptor

    Directory of Open Access Journals (Sweden)

    Su Sun Back

    2013-06-01

    Full Text Available The ATP-binding cassette transporters ABCG5 and ABCG8 formheterodimers that limit absorption of dietary sterols in theintestine and promote cholesterol elimination from the bodythrough hepatobiliary secretion. To identify cis-regulatoryelements of the two genes, we have cloned and analyzedtwenty-three evolutionary conserved region (ECR fragmentsusing the CMV-luciferase reporter system in HepG2 cells. TwoECRs were found to be responsive to the Liver-X-Receptor (LXR.Through elaborate deletion studies, regions containing putativeLXREs were identified and the binding of LXRα wasdemonstrated by EMSA and ChIP assay. When the LXREs wereinserted upstream of the intergenic promoter, synergisticactivation by LXRα/RXRα in combination with GATA4, HNF4α,and LRH-1, which had been shown to bind to the intergenicregion, was observed. In conclusion, we have identified twoLXREs in ABCG5/ABCG8 genes for the first time and proposethat these LXREs, especially in the ECR20, play major roles inregulating these genes. [BMB Reports 2013; 46(6: 322-327

  2. The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2014-02-01

    Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

  3. The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type

    Science.gov (United States)

    Ceruso, Marina; Fratamico, Pina; Chirollo, Claudia; Taglialatela, Rosanna; Cortesi, Maria Luisa

    2014-01-01

    Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment. PMID:27800311

  4. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    Science.gov (United States)

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-01

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  5. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  6. Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

    Science.gov (United States)

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V; Schuetz, John D

    2013-09-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV-associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  7. Prognostic significance and molecular mechanism of ATP-binding cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Zhiqi Yu

    Full Text Available BACKGROUND: Mechanism of radioresistance in rectal carcinoma remains largely unknown. We aimed to evaluate the predictive role of ATP-binding cassette subfamily C member 4 (ABCC4 in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy. METHODS: The expression of ABCC4 and P53 mutant in biopsy tissue specimens from 121 locally advanced rectal carcinoma patients was examined using immunohistochemistry. The factors contributing to 3-year overall survival and disease-free survival were evaluated using the Kaplan-Meier method and Cox proportional hazard model. Lentivirus-mediated small hairpin RNA was applied to inhibit ABCC4 expression in colorectal carcinoma cell line RKO, and investigate the radiosensitivity in xenograft model. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were detected. RESULTS: High expression of ABCC4 and p53 mutant in pretreated tumors, poor pathological response, and high final tumor staging were significant factors independently predicted an unfavorable prognosis of locally advanced rectal carcinoma patients after neoadjuvant radiotherapy. Down-regulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model. Furthermore, down-regulation of ABCC4 expression enhanced intracellular cyclic adenosine monophosphate production and noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation. CONCLUSIONS: Our study suggests that ABCC4 serves as a novel predictive biomarker that is responsible for the radioresistance and predicts a poor prognosis for locally advanced rectal carcinoma after neoadjuvant radiotherapy.

  8. An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates.

    Science.gov (United States)

    Ejby, Morten; Fredslund, Folmer; Andersen, Joakim Mark; Vujičić Žagar, Andreja; Henriksen, Jonas Rosager; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan; Abou Hachem, Maher

    2016-09-16

    The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

  9. Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.

    Science.gov (United States)

    Weidlich, Daniela; Wiesemann, Nicole; Heuveling, Johanna; Wardelmann, Kristina; Landmesser, Heidi; Sani, Katayoun Behnam; Worth, Catherine L; Preissner, Robert; Schneider, Erwin

    2013-09-01

    The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function. PMID:23747295

  10. Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: implications for their therapeutic and teratogenic effects.

    Science.gov (United States)

    Chen, Jing; Costa, Lucio G; Guizzetti, Marina

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

  11. Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: implications for their therapeutic and teratogenic effects.

    Science.gov (United States)

    Chen, Jing; Costa, Lucio G; Guizzetti, Marina

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions.

  12. Pharmacophore Modeling of Nilotinib as an Inhibitor of ATP-Binding Cassette Drug Transporters and BCR-ABL Kinase Using a Three-Dimensional Quantitative Structure–Activity Relationship Approach

    OpenAIRE

    Shukla, Suneet; Kouanda, Abdul; Silverton, Latoya; Talele, Tanaji T.; Suresh V Ambudkar

    2014-01-01

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with mi...

  13. Advances in research of ATP-binding cassette transporters in drug resistance mechanisms of intractable epilepsy%ATP结合盒式蛋白在难治性癫(痫)耐药性机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    付帅

    2014-01-01

    Epilepsy is one of the common diseases in the nervous system with its complicated pathogenesis still remains unknown.The drug resistance mechanism of intractable epilepsy has always been a key point in the research of neuroscience.A possible cause for the drug resistance is the over expression of efflux drug transporters,e.g.ATP-binding cassette transporters,which may decrease extracellular antiepileptic drugs levels in brains of intractable epilepsy patients.ATP-binding cassette transporters are super family of transporter proteins that require ATP hydrolysis for the transport of substrates across membranes,including P-glycoprotein,multidrug resistance-associated protein,major vault protein and breast cancer resistance associated protein.They are major impediment for the AED successful treatment of many forms of refractory epilepsy in human.This paper reviews the research progress on over-expression of ATP-binding cassette transporters and mechanism of drug resistance in intractable epilepsy.%难治性癫(痫)因其耐药机制的复杂性,迄今尚未清楚,目前探究其对抗癫(痫)药物的多重耐药性的一大热点是外流性药物转运蛋白.ATP结合盒式蛋白是外流性药物转运蛋白的代表,其中包括P糖蛋白、多药耐药蛋白、穹窿体主蛋白、乳腺癌耐药蛋白等,它们可以决定抗癫(痫)药物能否有效地作用于癫(痫)部位,而难治性癫(痫)患者对这些蛋白的高表达普遍存在,但是否与疾病耐药性相关仍需进一步探讨.该文从癫(痫)患者的ATP结合盒式蛋白高表达原因和蛋白对药物转运的作用机制方面对患者耐药性影响方面作一综述.

  14. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhaojing [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Xu, Yonghong [Institute of Ophthalmological Research, Department of Ophthalmology, Renmin Hospital of Wuhan University, 430060 Wuhan (China); Meng, Xiangning [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Watari, Fumio [Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586 (Japan); Liu, Hudan, E-mail: hudanliu@hust.edu.cn [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Chen, Xiao, E-mail: mornsmile@yahoo.com [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China)

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  15. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

    International Nuclear Information System (INIS)

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance

  16. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    Science.gov (United States)

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

  17. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    Science.gov (United States)

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  18. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    Science.gov (United States)

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  19. Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica.

    Science.gov (United States)

    Osborne, Suzanne E; Tuinema, Brian R; Mok, Mac C Y; Lau, Pui Sai; Bui, Nhat Khai; Tomljenovic-Berube, Ana M; Vollmer, Waldemar; Zhang, Kun; Junop, Murray; Coombes, Brian K

    2012-05-01

    Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome. PMID:22418438

  20. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  1. ATP-Binding Cassette Systems of Brucella

    Directory of Open Access Journals (Sweden)

    Dominic C. Jenner

    2009-01-01

    Full Text Available Brucellosis is a prevalent zoonotic disease and is endemic in the Middle East, South America, and other areas of the world. In this study, complete inventories of putative functional ABC systems of five Brucella species have been compiled and compared. ABC systems of Brucella melitensis 16M, Brucella abortus 9-941, Brucella canis RM6/66, Brucella suis 1330, and Brucella ovis 63/290 were identified and aligned. High numbers of ABC systems, particularly nutrient importers, were found in all Brucella species. However, differences in the total numbers of ABC systems were identified (B. melitensis, 79; B. suis, 72; B. abortus 64; B. canis, 74; B. ovis, 59 as well as specific differences in the functional ABC systems of the Brucella species. Since B. ovis is not known to cause human brucellosis, functional ABC systems absent in the B. ovis genome may represent virulence factors in human brucellosis.

  2. The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

    Science.gov (United States)

    Rioult, Damien; Pasquier, Jennifer; Boulangé-Lecomte, Céline; Poret, Agnès; Abbas, Imane; Marin, Matthieu; Minier, Christophe; Le Foll, Frank

    2014-08-01

    In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK

  3. The multi-xenobiotic resistance (MXR) efflux activity in hemocytes of Mytilus edulis is mediated by an ATP binding cassette transporter of class C (ABCC) principally inducible in eosinophilic granulocytes.

    Science.gov (United States)

    Rioult, Damien; Pasquier, Jennifer; Boulangé-Lecomte, Céline; Poret, Agnès; Abbas, Imane; Marin, Matthieu; Minier, Christophe; Le Foll, Frank

    2014-08-01

    In marine and estuarine species, immunotoxic and/or immunomodulatory mechanisms are the crossroad of interactions between xenobiotics, microorganisms and physicochemical variations of the environment. In mussels, immunity relies exclusively on innate responses carried out by cells collectively called hemocytes and found in the open hemolymphatic circulatory system of these organisms. However, hemocytes do not form a homogenous population of immune cells since distinct subtypes of mussel blood cells can be distinguished by cytochemistry, flow cytometry or cell motility analysis. Previous studies have also shown that these cells are able to efflux xenobiotics by means of ATP binding cassette (ABC) transporter activities conferring a multixenobiotic resistance (MXR) phenotype. ABC transporters corresponding to vertebrate class B/P-glycoprotein (P-gp) and to class C/multidrug resistance related protein (MRP) are characterized in Mytilidae. Herein, we have investigated the relative contributions of ABCB- and ABCC-mediated efflux within the different hemocyte subpopulations of Mytilus edulis mussels, collected from areas differentially impacted by chemical contaminants in Normandy (France). RT-PCR analyses provide evidence for the presence of ABCB and ABCC transporters transcripts in hemocytes. Immunodetection of ABCB/P-gp with the monoclonal antibody UIC2 in living hemocytes revealed that expression was restricted to granular structures of spread cells. Efflux transporter activities, with calcein-AM as fluorescent probe, were measured by combining flow cytometry to accurate Coulter cell size measurements in order to get a cell-volume normalized fluorescence concentration. In these conditions, basal fluorescence levels were higher in hemocytes originating from Yport (control site) than in cells collected from the harbor of Le Havre, where mussels are more exposed to with persistent pollutants. By using specific ABCB/P-gp (verapamil, PSC833, zosuquidar) and ABCC/MRP (MK

  4. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

    OpenAIRE

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in...

  5. Localization of the ATP-binding cassette (ABC) transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane.

    NARCIS (Netherlands)

    Kavishe, R.A.; Heuvel, J.M.W. van den; Vegte-Bolmer, M. van de; Luty, A.J.; Russel, F.G.M.; Koenderink, J.B.

    2009-01-01

    BACKGROUND: The spread of drug resistance has been a major obstacle to the control of malaria. The mechanisms underlying drug resistance in malaria seem to be complex and multigenic. The current literature on multiple drug resistance against anti-malarials has documented PfMDR1, an ATP-binding casse

  6. 三磷酸腺苷结合盒转运体A1在巨噬细胞胆固醇流出中的作用%Effects of ATP binding cassette transporter A1 on cholesterol efflux in macrophages

    Institute of Scientific and Technical Information of China (English)

    唐朝克; 严鹏科; 杨永宗

    2003-01-01

    Tangier disease is caused by mutations in ATP binding cassette transporter AI( ABCA1).ABCA1 interacts with lipid-free apolipoproteins, promoting phospholipid and cholesterol ettlux fzom cells and giving rise to HDL particles. ABCA1 may act as a phospholipid translocase facilitating phospholipid binding to apoA-Ⅰ. ABCA1 gene expression is upregulated in cholesterol-loaded cells as a result of activation of IXR/RXR- mediated gene transcription. LXR and RXR coordinately induce a battery of genes mediating cellular cholesterol efllux, centripetal cholesterol tramport, and cholesterol excretion in bile. Small- molecule activators of LXR/RXR or other stimulators of macrophage or intestinal cholesterol efl]ux hold great promise as future treat-ments for atherosclerosis.

  7. Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

    Science.gov (United States)

    Manjula, M; Pampa, K J; Kumar, S M; Mukherjee, S; Kunishima, N; Rangappa, K S; Lokanath, N K

    2015-03-27

    The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

  8. Lipid raft involved in drug resistance: relationship between multidrug resistance ATP-binding cassette transporters and lipid raft%脂筏参与耐药: 多药耐药相关ABC转运蛋白与脂筏的关系

    Institute of Scientific and Technical Information of China (English)

    王琳; 贾宇; 姜远英

    2011-01-01

    Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. Recently ATP-binding cassette (ABC) transporters, which are associated with multidrug resistance, have been found in lipid rafts; therefore they might be related to drug resistance. Here we introduce the relationship between the localization and functions of three multi-drug related ABC transporters, including two relevant to multidrug resistance in tumor cells(Pgp/ABCB1 and MRP1/ABCC1) and one relevant to multidrug resistance in Candida albicans (Cdrlp). We also discuss the influence of sphingolipids and cholesterol, two major components of lipid rafts, on the localization and function of the above three ABC transporters.%脂筏(lipid raft)和细胞的许多功能,如信号转导、蛋白质和脂类的转运等都相关.近来有研究发现,与多药耐药密切相关的ABC转运蛋白(ATP-binding cassette transporter)定位于脂筏中,因此推测脂筏可能与耐药性有一定关系.本文综述了3种和耐药相关的ABC转运蛋白的定位与其功能之间的联系,分别是和肿瘤细胞多药耐药相关的ABC转运蛋白Pgp/ABCB1、MRP1/ABCC1以及与白假丝酵母菌(白念珠菌)多药耐药相关的ABC转运蛋白Cdr1p;并进一步讨论了脂筏的重要组成成分胆固醇和鞘脂对上述3种ABC转运蛋白的定位和功能的影响.

  9. Mutant cycles at CFTR’s non-canonical ATP-binding site support little interface separation during gating

    OpenAIRE

    Szollosi, A; Muallem, D. R.; Csanady, L.; P.; Vergani

    2011-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is tr...

  10. Expression and significance of liver X receptor -β and ATP binding cassette transport protein A1 in human glioblastoma%肝X受体-β和三磷酸腺苷结合盒转运子A1在人脑胶质母细胞瘤中的表达和意义

    Institute of Scientific and Technical Information of China (English)

    滕志朋; 王晨; 刘斌; 李昱

    2012-01-01

    目的 研究肝X受体-β(Liver X receptor-β,LXR-β)和三磷酸腺苷结合盒转运子A1( ATP binding cassette transport protein A1,ABCA1)蛋白在人脑胶质母细胞瘤(Glioblastoma,GBM)中的表达及相关性,并探讨其对GBM的意义.方法 分析48例GBM患者的资料,并取患者肿瘤组织石蜡切片,另取19份瘤旁正常脑组织石蜡切片为对照组,采用SP免疫组化法检测LXR-β和ABCA1蛋白的表达,并分析LXR-β与ABCA1表达的相关性.结果 LXR-β和ABCA1蛋白在GBM中的表达率分别为81.3%和77.1%,与对照组(15.8%和26.3%)相比,差异有统计学意义(P<0.001),且LXR-β与ABCA1的表达呈正相关(rs=0.500,P<0.05).患者性别、年龄、胶质瘤复发、手术切除范围、肿瘤最大直径、术后放化疗情况与LXR-β、ABCA1蛋白的表达差异无统计学意义(P>0.05).结论 LXR-3和ABCA1蛋白与GBM的形成和进展有一定关系,有可能成为临床诊断及治疗GBM的生物学指标.%Objective To investigate the expressions of liver X receptor-β (LXR-β) and ATP binding cassette transport protein Al (ABCA1) in human glioblastoma (GBM) and evaluate their relationship as well as their roles in GBM. Methods The clinical data on 48 cases of GBM were analyzed. The tumor tissues of patients were prepared into paraffin sections and determined for expressions of LXR-β and ABCAl by immunohistochemical assay with SP staining, using 19 sections of normal brain tissue as control, based on which the relation ship between expressions of LXR-β and ABCAl was analyzed. Results The expression rates of LXR-β and ABCAl GBM were 81- 3% and 77. 1% respectively, which showed no significant difference with those in control group (15. 8% and 26. 3% respectively )(P 0. 05). Conclusion LXR-β and ABCAl showed a certain relationship to the onset and progress of GBM, which might be used as a biological index for clinical diagnosis and treatment of GBM.

  11. Correlation between R219K polymorphism in the ATP-binding cassette transporter A1 gene and ischemic stroke in young adults%ABCA1基因R219K多态性与青年缺血性脑卒中的相关性研究

    Institute of Scientific and Technical Information of China (English)

    刘竞丽; 李劲频; 汪晓玲

    2009-01-01

    Objective To investigate the correlation between the R219K polymorphism in the ATP-binding cassette transporter A1 (ABCA1) gene and arterial ischemic stroke in young adults. Methods The R219K polymorphism in the ABCA1 from 131 young patients with ischemic stroke and 135 age- and gender-matched controls was explored using polymerase chain reaction technique. The level of plasma lipids in the two groups was determined and carotid ultrasonography was employed to measure the carotid intima-media thickness (IMT). Results The frequency distributions of R219K genotype was significantly different between young patients and controls, and the patients held fewer KK genotypes than the the controls (P0.05); the level of HDL-C in the K allele carriers was significantly higher as compared with that in the non-K allele carriers (P0.05);而携带者HDL-C水平较非携带者增高,差异有统计学意义(P0.05),而携带者的颈动脉内膜、中膜厚度较非携带者明显减少,差异有统计学意义(P<0.05). 结论 ABCA1基因R219K多态性与青年缺血性脑卒中的遗传易感性相关,其中K等位基因可能对缺血性脑卒中有保护作用.推测其保护机制为通过升高血浆HDL-C水平起到减缓颈动脉内膜、中膜增厚,避免动脉粥样硬化的作用.

  12. 中国人内源性高甘油三酯血症患者ATP结合盒转运子A1基因R219K多态性研究%Analysis of ATP binding cassette A1 gene R219K polymorphism in patients with endogenous hypertriglyceridemia in Chinese population

    Institute of Scientific and Technical Information of China (English)

    吴银; 白怀; 刘瑞; 刘宇; 刘秉文

    2007-01-01

    目的 研究ATP结合盒转运子A1(ATP binding cassette A1, ABCA1)基因R219K多态性是否与中国人内源性高甘油三酯血症(hypertriglyceridemia, HTG)有关联,为探讨本病的分子遗传基础提供依据.方法 应用聚合酶链反应-限制性片段长度多态性分析法,对成都地区309名汉族人(200名正常人和109例内源性高甘油三酯血症患者)ABCA1基因R219K多态性位点进行分析.结果 中国人ABCA1基因R219K多态位点K等位基因频率在对照组和HTG组分别为0.472与0.436; HTG组和对照组R219K位点之间基因型和等位基因的频率差异无统计学意义.对照组和HTG组KK基因型携带者血清高密度脂蛋白胆固醇(high density lipoprotein-cholesterol, HDL-C)水平均较相应组RR基因型携带者显著升高[(1.48±0.45) mmol/L vs (1.27±0.29) mmol/L, P<0.05;(1.07±0.30) mmol/L vs (0.87±0.19) mmol/L, P<0.05];对照组RK型携带者血清甘油三酯水平较RR型携带者显著降低[(1.22±0.37) mmol/L vs (1.41±0.84) mmol/L, P<0.05],HTG组血清甘油三酯在RR、RK、KK型之间有逐渐降低的趋势[(3.82±2.02) mmol/L vs (3.42±1.67) mmol/L vs (3.33±1.43) mmol/L, P>0.05]; HTG组K等位基因携带者(RK或KK型者)总胆固醇(total cholesterol, TC)/HDL-C比值均较RR型携带者显著降低(KK vs RK vs RR:4.82±1.28 vs 5.42±1.62 vs 6.33±1.70, P<0.05).结论 ABCA1基因R219K多态性不仅与中国成都地区正常汉族人血清HDL-C、甘油三酯含量有关,而且还与内源性高甘油三酯血症患者血清HDL-C水平、TC/HDL-C比值相关联.

  13. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ

    2002-01-01

    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural a

  14. Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Taras Gout

    2012-01-01

    Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

  15. Reconstruction of a Bacterial Genome from DNA Cassettes

    Energy Technology Data Exchange (ETDEWEB)

    Christopher Dupont; John Glass; Laura Sheahan; Shibu Yooseph; Lisa Zeigler Allen; Mathangi Thiagarajan; Andrew Allen; Robert Friedman; J. Craig Venter

    2011-12-31

    This basic research program comprised two major areas: (1) acquisition and analysis of marine microbial metagenomic data and development of genomic analysis tools for broad, external community use; (2) development of a minimal bacterial genome. Our Marine Metagenomic Diversity effort generated and analyzed shotgun sequencing data from microbial communities sampled from over 250 sites around the world. About 40% of the 26 Gbp of sequence data has been made publicly available to date with a complete release anticipated in six months. Our results and those mining the deposited data have revealed a vast diversity of genes coding for critical metabolic processes whose phylogenetic and geographic distributions will enable a deeper understanding of carbon and nutrient cycling, microbial ecology, and rapid rate evolutionary processes such as horizontal gene transfer by viruses and plasmids. A global assembly of the generated dataset resulted in a massive set (5Gbp) of genome fragments that provide context to the majority of the generated data that originated from uncultivated organisms. Our Synthetic Biology team has made significant progress towards the goal of synthesizing a minimal mycoplasma genome that will have all of the machinery for independent life. This project, once completed, will provide fundamentally new knowledge about requirements for microbial life and help to lay a basic research foundation for developing microbiological approaches to bioenergy.

  16. The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations.

    Science.gov (United States)

    Furukawa-Hagiya, Tomoka; Furuta, Tadaomi; Chiba, Shuntaro; Sohma, Yoshiro; Sakurai, Minoru

    2013-01-10

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions. PMID:23214920

  17. Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux in a mammalian cell line.

    Directory of Open Access Journals (Sweden)

    Dan M Close

    Full Text Available The bacterial luciferase (lux gene cassette consists of five genes (luxCDABE whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2 was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp from Vibrio harveyi. FMNH(2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

  18. Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

    OpenAIRE

    Szollosi, A; P.; Vergani; Csanady, L.

    2010-01-01

    The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dim...

  19. Association between two common polymorphisms in ATP-binding cassette A1 gene and coronary heart disease complicated with diabetes in Chinese Han people%三磷酸腺苷结合盒转运子A1启动子区及7外显子基因突变与合并糖尿病的冠心病关联研究

    Institute of Scientific and Technical Information of China (English)

    祁莉萍; 严晓伟; 叶平; 党爱民

    2010-01-01

    Objective The promoter-565C/T variant and the 7exon R219K variant are associated with risk of Coronary heart disease (CAD), but the association also remains controversial. At present, there are few studies focusing on the associations between ATP-binding cassette A1 (ABCA1), and CAD with Diabetes mellitus (DM) in Chinese population. Since decreased serum level of HDL-C is often observed in DM,it is natural to hypothesize that polymorphisms of the ABCA1 gene might be related to CAD complicated with DM. Objective To study the mutations and genetic characteristics of ABCA1 promoter -565C/T and 7Exon R219K in CAD with DM patients in Chinese Han people. Methods One hundred and seventy-three patients of CAD with DM and 389 controls were genotyped for-565C/T, R219K used with LDR. Genetic association analysis was performed. Results The frequencies of the CC, CT, and TT genotypes in CAD with DM were 0.360(n=63), 0.482 (n=83) and 0.157 (n=27), respectively. The frequency of the TT genotype and T allele at the-565C/T locus had no significant alterations between CAD with DM patients and Controls (0.157 vs 0.163; 0.398 vs 0.409,P>0.05). The frequency of the AA and GA geno-type at the R219K locus was lower in CAD patients compared with diabetes (0.65 vs 0.73,P=0.079). Logistic re-gression model were performed, revealed no interaction between 2 SNPs and traditional risk factors, but R219K had a protection effect, OR=0.428 (95%CI 0.227-0.603), P=0.009. Conclusions ABCA1 the T allele of-565 C/T SNP has no significant association with CAD with DM. R219K SNP predicts differences in CAD with diabetes. The AA genotype may protect against subclinical cardiovascular disease.%目的 首次研究汉族人群冠心病合并糖尿病与三磷酸腺苷结合盒转运子A1(ABCA1)基因启动子区-565C/T及7外显子R219K基因多态性关联分析.方法 应用连接酶检测反应法对172例合并糖尿病冠心病患者及393例对照组测试-565C/T及R219K基因型.结果

  20. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    OpenAIRE

    Villamor, J. G.; Kaschani, F.; Colby, T; Oeljeklaus, J.; Zhao, D; Kaiser, M.; Patricelli, M. P.; R. A. L. van der Hoorn

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding prot...

  1. Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

    Science.gov (United States)

    Ayaori, Makoto; Yakushiji, Emi; Ogura, Masatsune; Nakaya, Kazuhiro; Hisada, Tetsuya; Uto-Kondo, Harumi; Takiguchi, Shunichi; Terao, Yoshio; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Yogo, Makiko; Uehara, Yoshinari; Kagechika, Hiroyuki; Nakanishi, Tsuyoshi; Ikewaki, Katsunori

    2012-04-01

    ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

  2. The ABCC6 transporter: what lessons can be learnt from other ATP-binding cassette transporters?

    Directory of Open Access Journals (Sweden)

    Olivier M. Vanakker

    2013-10-01

    Full Text Available ABC transporters represent a large family of ATP-driven transmembrane transporters involved in uni- or bidirectional transfer of a variety of substrates. Divided in 7 families, they represent 48 transporter proteins, several of which are associated with human disease. Among the latter is ABCC6, a unidirectional exporter protein primarily expressed in liver and kidney. ABCC6 deficiency causes pseudoxanthoma elasticum (PXE, characterised by calcification and fragmentation of elastic fibres, resulting in oculocutaneous and cardiovascular symptoms. Unique among connective tissue disorders, the relation between the ABCC6 transporter and ectopic mineralization in PXE remains enigmatic, not in the least because of lack of knowledge on the substrate(s of ABCC6 and its unusual expression pattern. Because many features, including structure and transport mechanism, are shared by ABC transporters, it is worthwhile to evaluate if and to what extent the knowledge on the (pathophysiology of other transporters may provide useful clues towards understanding the (pathophysiological role of ABCC6. In this paper, we summarize relevant knowledge and methods for analysis on ABC transporters which may be useful for the further study of ABCC6.

  3. ATP binding cassette transporters modulate both coelenterazine- and D-luciferin- based bioluminescence imaging

    OpenAIRE

    Huang, Ruimin; Vider, Jelena; Serganova, Inna; Blasberg, Ronald G.

    2011-01-01

    Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI-readout-intensity from intact living cells.

  4. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus.

    Science.gov (United States)

    Yu, Fang; De Luca, Vincenzo

    2013-09-24

    The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface. PMID:24019465

  5. Masitinib Antagonizes ATP-Binding Cassette Subfamily C Member 10-Mediated Paclitaxel Resistance: A Preclinical Study

    OpenAIRE

    Kathawala, Rishil J; Sodani, Kamlesh; Chen, Kang; PATEL, ATISH; Abuznait, Alaa H.; Anreddy, Nagaraju; Sun, Yue-Li; Kaddoumi, Amal; Ashby, Charles R.; Chen, Zhe-Sheng

    2014-01-01

    Paclitaxel displays clinical activity against a wide variety of solid tumors. However, resistance to paclitaxel significantly attenuates the response to chemotherapy. The ABC transporter subfamily C member 10 (ABCC10), also known as multi-drug resistance protein 7 (MRP7) efflux transporter, is a major mediator of paclitaxel resistance. In this study, we show that masitinib, a small molecule stem-cell growth factor receptor (c-Kit) tyrosine kinase inhibitor, at non-toxic concentrations, signif...

  6. ATP-Binding-Cassette Transporters in Biliary Efflux and Drug-Induced Liver Injury

    OpenAIRE

    Pedersen, Jenny M.

    2013-01-01

    Membrane transport proteins are known to influence the absorption, distribution, metabolism, excretion and toxicity (ADMET) of drugs. At the onset of this thesis work, only a few structure-activity models, in general describing P-glycoprotein (Pgp/ABCB1) interactions, were developed using small datasets with little structural diversity. In this thesis, drug-transport protein interactions were explored using large, diverse datasets representing the chemical space of orally administered registe...

  7. Isotope-coded ATP Probe for Quantitative Affinity Profiling of ATP-binding Proteins

    OpenAIRE

    Xiao, Yongsheng; Guo, Lei; Wang, Yinsheng

    2013-01-01

    ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding ...

  8. Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion.

    Science.gov (United States)

    Hodges, Larry D; Vergunst, Annette C; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M; Hooykaas, Paul J J; Ream, Walt

    2006-12-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  9. Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

    Science.gov (United States)

    Close, Dan; Webb, James; Ripp, Steven; Patterson, Stacey; Sayler, Gary

    2012-06-01

    Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

  10. Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

    Science.gov (United States)

    Szollosi, Andras; Muallem, Daniella R; Csanády, László; Vergani, Paola

    2011-06-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains

  11. ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation.

    Science.gov (United States)

    Hammarén, Henrik M; Ungureanu, Daniela; Grisouard, Jean; Skoda, Radek C; Hubbard, Stevan R; Silvennoinen, Olli

    2015-04-14

    Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches. PMID:25825724

  12. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  13. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

    Directory of Open Access Journals (Sweden)

    Joshua E Drumm

    2009-05-01

    Full Text Available Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  14. Regulation of Yeast Nutrient Permease Endocytosis by ATP-binding Cassette Transporters and a Seven-transmembrane Protein, RSB1*

    OpenAIRE

    Johnson, Soraya S.; Hanson, Pamela K.; Manoharlal, Raman; Brice, Sarah E.; Cowart, L. Ashley; Moye-Rowley, W. Scott

    2010-01-01

    Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivi...

  15. Linoleic acid suppresses cholesterol efflux and ATP-binding cassette transporters in murine bone marrow-derived macrophages

    Science.gov (United States)

    Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease (CVD), possibly associated with elevated plasma free fatty acid concentrations. Paradoxically, evidence suggests that unsaturated, compared to saturated fatty acids, suppress macrophage chole...

  16. Heterocyclic cyclohexanone monocarbonyl analogs of curcumin can inhibit the activity of ATP-binding cassette transporters in cancer multidrug resistance.

    Science.gov (United States)

    Revalde, Jezrael L; Li, Yan; Hawkins, Bill C; Rosengren, Rhonda J; Paxton, James W

    2015-02-01

    Curcumin (CUR) is a phytochemical that inhibits the xenobiotic ABC efflux transporters implicated in cancer multidrug resistance (MDR), such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins 1 and 5 (MRP1 and MRP5). The use of CUR in the clinic however, is complicated by its instability and poor pharmacokinetic profile. Monocarbonyl analogs of CUR (MACs) are compounds without CUR's unstable β-diketone moiety and were reported to have improved stability and in vivo disposition. Whether the MACs can be used as MDR reversal agents is less clear, as the absence of a β-diketone may negatively impact transporter inhibition. In this study, we investigated 23 heterocyclic cyclohexanone MACs for inhibitory effects against P-gp, BCRP, MRP1 and MRP5. Using flow cytometry and resistance reversal assays, we found that many of these compounds inhibited the transport activity of the ABC transporters investigated, often with much greater potency than CUR. Overall the analogs were most effective at inhibiting BCRP and we identified three compounds, A12 (2,6-bis((E)-2,5-dimethoxy-benzylidene)cyclohexanone), A13 (2,6-bis((E)-4-hydroxyl-3-methoxybenzylidene)-cyclohexanone) and B11 (3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one), as the most promising BCRP inhibitors. These compounds inhibited BCRP activity in a non-cell line, non-substrate-specific manner. Their inhibition occurred by direct transporter interaction rather than modulating protein or cell surface expression. From these results, we concluded that MACs, such as the heterocyclic cyclohexanone analogs in this study, also have potential as MDR reversal agents and may be superior alternatives to the unstable parent compound, CUR.

  17. Conformational changes of the histidine ATP-binding cassette transporter studied by double electron-electron resonance spectroscopy.

    Science.gov (United States)

    Sippach, Michael; Weidlich, Daniela; Klose, Daniel; Abé, Christoph; Klare, Johann; Schneider, Erwin; Steinhoff, Heinz-Jürgen

    2014-07-01

    The conformational dynamics of the histidine ABC transporter HisQMP2 from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron-electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP2 in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP2 as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP2. Our results are in line with a rearrangement of transmembrane helices 4 and 4' of HisQM during the closed to the semi-open transition of HisP2 driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis. PMID:24583084

  18. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    OpenAIRE

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support cha...

  19. Structural models of zebrafish (Danio rerio NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Jitendra Maharana

    Full Text Available Nucleotide-binding oligomerization domain-containing protein 1 (NOD1 and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds and Aspartic acid (Walker B formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2 interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

  20. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    OpenAIRE

    Jun Adachi; Marina Kishida; Shio Watanabe; Yuuki Hashimoto; Kazuna Fukamizu; Takeshi Tomonaga

    2015-01-01

    Interactions between ATP and ATP-binding proteins (ATPome) are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes) that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014) [1]. As a result, we identified ...

  1. Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.

    Science.gov (United States)

    Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-02-14

    Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 μM for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

  2. Identification of a MAP 2-like ATP-binding protein associated with axoplasmic vesicles that translocate on isolated microtubules

    OpenAIRE

    1986-01-01

    Axoplasmic vesicles were purified and observed to translocate on isolated microtubules in an ATP-dependent, trypsin-sensitive manner, implying that ATP-binding polypeptides essential for force generation were present on the vesicle surface. To identify these proteins [alpha 32P]8-azidoadenosine 5'-triphosphate ([alpha 32P]8-N3ATP), a photoaffinity analogue of ATP, was used. The results presented here identify and characterize a vesicle-associated polypeptide having a relative molecular mass o...

  3. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    Directory of Open Access Journals (Sweden)

    Jun Adachi

    2015-12-01

    Full Text Available Interactions between ATP and ATP-binding proteins (ATPome are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014 [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014 [1] have been deposited to the ProteomeXchange with identifier PXD001200.

  4. Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

    Science.gov (United States)

    Ghauri, Muhammad A; Khalid, Ahmad M; Grant, Susan; Grant, William D; Heaphy, Shaun

    2006-06-01

    Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated. PMID:16732461

  5. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    2016-04-01

    Full Text Available One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  6. Cystathionine beta-Synthase (CBS) Domains 1 and 2 Fulfill Different Roles in Ionic Strength Sensing of the ATP-binding Cassette (ABC) Transporter OpuA

    NARCIS (Netherlands)

    Karasawa, Akira; Erkens, Guus B.; Berntsson, Ronnie P. -A.; Otten, Renee; Schuurman-Wolters, Gea K.; Mulder, Frans A. A.; Poolman, Bert

    2011-01-01

    The cystathionine beta-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are crit

  7. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells

    DEFF Research Database (Denmark)

    Robey, R W; Medina-Pérez, W Y; Nishiyama, K;

    2001-01-01

    microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold...

  8. Tiaozhi Tongmai Granules reduce atherogenesis and promote the expression of ATP-binding cassette transporter A1 in rabbit atherosclerotic plaque macrophages and the liver

    Directory of Open Access Journals (Sweden)

    Qing Sun

    2014-07-01

    Conclusions: Tiaozhi Tongmai Granules appear to have an anti-atherogenic effect that is most likely mediated by simultaneously upregulating the protein expression of ABCA1 in rabbit atherosclerotic plaque macrophages and in the liver.

  9. Tandutinib (MLN518/CT53518) targeted to stem-like cells by inhibiting the function of ATP-binding cassette subfamily G member 2.

    Science.gov (United States)

    Zhao, Xiao-qin; Dai, Chun-ling; Ohnuma, Shinobu; Liang, Yong-ju; Deng, Wen; Chen, Jun-Jiang; Zeng, Mu-Sheng; Ambudkar, Suresh V; Chen, Zhe-Sheng; Fu, Li-Wu

    2013-06-14

    Tandutinib is a novel inhibitor of tyrosine kinases FLT3, PDGFR and KIT. Our study was to explore the capability of tandutinib to reverse ABC transporter-mediated multidrug resistance. Tandutinib reversed ABCG2-mediated drug resistance in ABCG2-482-R2, ABCG2-482-G2, ABCG2-482-T7 and S1-M1-80 cells and increased the accumulation of doxorubicin, rhodamine 123 and [H(3)] mitoxantrone in ABCG2-overexpressing cells. Importantly, tandutinib selectively sensitized side population cells to mitoxantrone. Taken together, our results advocate the potency of tandutinib as an ABCG2 modulator and stem-like cells targeted agent to increase efficiency of anticancer drugs. PMID:23619284

  10. Association of three common single nucleotide polymorphisms of ATP binding cassette G8 gene with gallstone disease: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Zhao-Yan Jiang

    Full Text Available BACKGROUND: In this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C. METHODS: Data were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well. RESULTS: Ten papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H (OR = 2.43, 95%CI: 2.23-2.64, P<0.001, and for Y54C (OR = 1.36, 95%CI: 1.01-1.83, P = 0.044, or T400K (OR = 1.17, 95%CI: 0.96-1.43. P = 0.110. In allele model, minor alleles of D19H polymorphism (allele D: OR = 2.25, 95%CI: 2.10-2.42, P<0.001 and of T400K polymorphism (allele K: OR = 1.18, 95%CI: 1.06-1.31, P<0.001 were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism (allele Y, OR = 1.08, 95%CI: 0.96-1.21, P = 0.146 was not related with gallstone disease. I(2 statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger's test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found. CONCLUSIONS: Our study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease.

  11. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Directory of Open Access Journals (Sweden)

    Shabeesh Balan

    Full Text Available Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS (prototype for AED-resistant epilepsy; juvenile myoclonic epilepsy (JME (prototype for AED-responsive epilepsy; and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004. This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004 and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05 cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE-HS in south Indian ancestry from Kerala.

  12. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Science.gov (United States)

    Balan, Shabeesh; Bharathan, Sumitha Prameela; Vellichiramal, Neetha Nanoth; Sathyan, Sanish; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE-HS in south Indian ancestry from Kerala.

  13. Genome-wide identification and evolution of ATP-binding cassette transporters in the ciliate Tetrahymena thermophila: A case of functional divergence in a multigene family

    Directory of Open Access Journals (Sweden)

    Yuan Dongxia

    2010-10-01

    Full Text Available Abstract Background In eukaryotes, ABC transporters that utilize the energy of ATP hydrolysis to expel cellular substrates into the environment are responsible for most of the efflux from cells. Many members of the superfamily of ABC transporters have been linked with resistance to multiple drugs or toxins. Owing to their medical and toxicological importance, members of the ABC superfamily have been studied in several model organisms and warrant examination in newly sequenced genomes. Results A total of 165 ABC transporter genes, constituting a highly expanded superfamily relative to its size in other eukaryotes, were identified in the macronuclear genome of the ciliate Tetrahymena thermophila. Based on ortholog comparisons, phylogenetic topologies and intron characterizations, each highly expanded ABC transporter family of T. thermophila was classified into several distinct groups, and hypotheses about their evolutionary relationships are presented. A comprehensive microarray analysis revealed divergent expression patterns among the members of the ABC transporter superfamily during different states of physiology and development. Many of the relatively recently formed duplicate pairs within individual ABC transporter families exhibit significantly different expression patterns. Further analysis showed that multiple mechanisms have led to functional divergence that is responsible for the preservation of duplicated genes. Conclusion Gene duplications have resulted in an extensive expansion of the superfamily of ABC transporters in the Tetrahymena genome, making it the largest example of its kind reported in any organism to date. Multiple independent duplications and subsequent divergence contributed to the formation of different families of ABC transporter genes. Many of the members within a gene family exhibit different expression patterns. The combination of gene duplication followed by both sequence divergence and acquisition of new patterns of expression likely plays a role in the adaptation of Tetrahymen a to its environment.

  14. Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette Transporter (ΔabcBA) Protects against Wild Type Brucella ovis in Rams.

    Science.gov (United States)

    Silva, Ana Patrícia C; Macêdo, Auricélio A; Costa, Luciana F; Rocha, Cláudia E; Garcia, Luize N N; Farias, Jade R D; Gomes, Priscilla P R; Teixeira, Gustavo C; Fonseca, Kessler W J; Maia, Andréa R F; Neves, Gabriela G; Romão, Everton L; Silva, Teane M A; Mol, Juliana P S; Oliveira, Renata M; Araújo, Márcio S S; Nascimento, Ernane F; Martins-Filho, Olindo A; Brandão, Humberto M; Paixão, Tatiane A; Santos, Renato L

    2015-01-01

    This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams. PMID:26317399

  15. Polymorphisms of ATP binding cassette G5 and G8 transporters: their effect on cholesterol metabolism after moderate weight loss in overweight and obese hyperlipidemic women

    Science.gov (United States)

    To determine the effect of polymorphisms ABCG5 and ABCG8 transporters on changes in lipid levels, cholesterol absorption rate (ABS), fractional synthesis rate (FSR), and turnover (TO) after moderate weight loss (WtL) in women. Cholesterol metabolism was measured pre and post WtL in 35 hyperlipidemic...

  16. Inhibitory Potential of Antifungal Drugs on ATP-Binding Cassette Transporters P-Glycoprotein, MRP1 to MRP5, BCRP, and BSEP.

    Science.gov (United States)

    Lempers, Vincent J C; van den Heuvel, Jeroen J M W; Russel, Frans G M; Aarnoutse, Rob E; Burger, David M; Brüggemann, Roger J; Koenderink, Jan B

    2016-06-01

    Inhibition of ABC transporters is a common mechanism underlying drug-drug interactions (DDIs). We determined the inhibitory potential of antifungal drugs currently used for invasive fungal infections on ABC transporters P-glycoprotein (P-gp), MRP1 to MRP5, BCRP, and BSEP in vitro Membrane vesicles isolated from transporter-overexpressing HEK 293 cells were used to investigate the inhibitory potential of antifungal drugs (250 μM) on transport of model substrates. Concentration-inhibition curves were determined if transport inhibition was >60%. Fifty percent inhibitory concentrations (IC50s) for P-gp and BCRP were both 2 μM for itraconazole, 5 and 12 μM for hydroxyitraconazole, 3 and 6 μM for posaconazole, and 3 and 11 μM for isavuconazole, respectively. BSEP was strongly inhibited by itraconazole and hydroxyitraconazole (3 and 17 μM, respectively). Fluconazole and voriconazole did not inhibit any transport for >60%. Micafungin uniquely inhibited all transporters, with strong inhibition of MRP4 (4 μM). Anidulafungin and caspofungin showed strong inhibition of BCRP (7 and 6 μM, respectively). Amphotericin B only weakly inhibited BCRP-mediated transport (127 μM). Despite their wide range of DDIs, azole antifungals exhibit selective inhibition on efflux transporters. Although echinocandins display low potential for clinically relevant DDIs, they demonstrate potent in vitro inhibitory activity. This suggests that inhibition of ABC transporters plays a crucial role in the inexplicable (non-cytochrome P450-mediated) DDIs with antifungal drugs. PMID:27001813

  17. Variants in the ATP-Binding Cassette Transporter (ABCA7), Apolipoprotein E ε4, and the Risk of Late-Onset Alzheimer Disease in African Americans

    Science.gov (United States)

    Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B.; Graff-Radford, Neill R.; Evans, Denis; De Jager, Philip L.; Crane, Paul K.; Buxbaum, Joseph D.; Murrell, Jill R.; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T.; Green, Robert C.; Barnes, Lisa L.; Cantwell, Laura B.; Fallin, M. Daniele; Go, Rodney C. P.; Griffith, Patrick; Obisesan, Thomas O.; Manly, Jennifer J.; Lunetta, Kathryn L.; Kamboh, M. Ilyas; Lopez, Oscar L.; Bennett, David A.; Hendrie, Hugh; Hall, Kathleen S.; Goate, Alison M.; Byrd, Goldie S.; Kukull, Walter A.; Foroud, Tatiana M.; Haines, Jonathan L.; Farrer, Lindsay A.; Pericak-Vance, Margaret A.; Schellenberg, Gerard D.; Mayeux, Richard

    2013-01-01

    Importance Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. Objective To identify genetic loci associated with late-onset Alzheimer disease in African Americans. Design, Setting, and Participants The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance–weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Main Outcomes and Measures Presence of Alzheimer disease according to standardized criteria. Results Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10–9), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8

  18. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis%囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

    Institute of Scientific and Technical Information of China (English)

    BOMPADRE; Silvia; G; HWANG; Tzyh-Chang

    2007-01-01

    囊性纤维化跨膜电导调节体(cystic fibrosis transmembrane conductance regulator,CFTR)是一种Cl-通道,属于ATP结合(ATP-binding cassette,ABC)转运体超家族.CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化(cystic fibrosis,CF)发生的主要原因.这种疾病患者各组织上皮细胞内Cl-转运失调.目前,与CF相关的不同突变超过1 400种.CFTR调节(regulatory,R)域负责调控,核苷酸结合域(nucleotide-binding domains,NBDs)NBD1和NBD2负责ATP结合和水解门控.近期研究发现CFTR的NBDs与其它ABC蛋白一样可以二聚化.二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列(signature sequence)形成ATP结合袋(ATP-binding pockets,ABPs)ABP1和ABP2.ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用.ABP2由NBD2上的Walk A和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象.有一些CFTR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路.%The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1

  19. Radiographic film cassette unloading apparatus

    International Nuclear Information System (INIS)

    Apparatus for unloading cassettes, containing exposed radiographic films, has means for unfastening the cassettes, an inclined pathway for gravity feeding and rotating feed members (rollers or belts) to propel the films into the processor. (UK)

  20. Automated cassette-to-cassette substrate handling system

    Science.gov (United States)

    Kraus, Joseph Arthur; Boyer, Jeremy James; Mack, Joseph; DeChellis, Michael; Koo, Michael

    2014-03-18

    An automated cassette-to-cassette substrate handling system includes a cassette storage module for storing a plurality of substrates in cassettes before and after processing. A substrate carrier storage module stores a plurality of substrate carriers. A substrate carrier loading/unloading module loads substrates from the cassette storage module onto the plurality of substrate carriers and unloads substrates from the plurality of substrate carriers to the cassette storage module. A transport mechanism transports the plurality of substrates between the cassette storage module and the plurality of substrate carriers and transports the plurality of substrate carriers between the substrate carrier loading/unloading module and a processing chamber. A vision system recognizes recesses in the plurality of substrate carriers corresponding to empty substrate positions in the substrate carrier. A processor receives data from the vision system and instructs the transport mechanism to transport substrates to positions on the substrate carrier in response to the received data.

  1. Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    OpenAIRE

    Pougach, Ksenia; Semenova, Ekaterina; Bogdanova, Ekaterina; Datsenko, Kirill A.; Djordjevic, Marko; Wanner, Barry L.; Severinov, Konstantin

    2010-01-01

    CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laborato...

  2. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene

    International Nuclear Information System (INIS)

    Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation (δF508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-δF508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward

  3. Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.

    Science.gov (United States)

    Chuang, W J; Abeygunawardana, C; Gittis, A G; Pedersen, P L; Mildvan, A S

    1995-05-10

    PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of

  4. Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate

    Science.gov (United States)

    Sung, Nuri; Lee, Jungsoon; Kim, Ji-Hyun; Chang, Changsoo; Joachimiak, Andrzej; Lee, Sukyeong; Tsai, Francis T. F.

    2016-01-01

    Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding. PMID:26929380

  5. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    Science.gov (United States)

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  6. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    Science.gov (United States)

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  7. The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

    Directory of Open Access Journals (Sweden)

    Emilia Herrera-Moyano

    2014-12-01

    Full Text Available The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes. We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS, we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

  8. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  9. An investigation of infection control for x-ray cassettes in a diagnostic imaging department

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Matthew [School of Allied Health Professions and Science, Faculty of Health, Wellbeing and Science, University Campus Suffolk, Rope Walk, Ipswich, Suffolk, IP4 1LT (United Kingdom); Harvey, Jane M. [School of Allied Health Professions and Science, Faculty of Health, Wellbeing and Science, University Campus Suffolk, Rope Walk, Ipswich, Suffolk, IP4 1LT (United Kingdom)], E-mail: j.harvey@ucs.ac.uk

    2008-11-15

    Introduction: This research was conducted to investigate if X-ray cassettes could be a possible source of pathogens capable of causing nosocomial infections, and if they could be a possible vector for cross infection within the hospital environment. Method: The research involved the swabbing of X-ray cassettes in a Diagnostic Imaging Department of a large hospital in the east of England. Two areas of the Diagnostic Imaging Department were included in the study. Research concentrated on X-ray cassettes used for mobile radiography, accident and emergency and inpatient use. Forty cassettes were swabbed in total specifically for general levels of bacterial contamination, also for the presence or absence of methicillin-resistant Staphylococcus aureus (MRSA). A mapping exercise was completed following the location of an X-ray cassette typically used in mobile radiography. The exercise noted the level of direct contact with patient's skin and other possible routes of infection. Results: The results demonstrated that there were large levels of growth of samples taken from cassettes and developed in the Microbiology Department. Coagulase-negative Staphylococcus, Micrococci, Diptheroids and species of Bacillus were all identified. The mapping exercise in which the journey of a 35/43 cm cassette used for mobile radiography was tracked found that contact with patient's skin and potential pathogens or routes of cross infection was a common occurrence whilst undertaking mobile radiography. Conclusion: The research has identified the presence of bacterial contamination on cassettes. The research established that X-ray cassettes/imaging plates are often exposed to pathogens and possible routes of cross infection; also that patient's skin often comes directly in contact with the X-ray cassette/imaging plate. The research also shows that as cassettes/imaging plates are a potential source of cross infection, the Diagnostic Imaging Department may be partly responsible

  10. An investigation of infection control for x-ray cassettes in a diagnostic imaging department

    International Nuclear Information System (INIS)

    Introduction: This research was conducted to investigate if X-ray cassettes could be a possible source of pathogens capable of causing nosocomial infections, and if they could be a possible vector for cross infection within the hospital environment. Method: The research involved the swabbing of X-ray cassettes in a Diagnostic Imaging Department of a large hospital in the east of England. Two areas of the Diagnostic Imaging Department were included in the study. Research concentrated on X-ray cassettes used for mobile radiography, accident and emergency and inpatient use. Forty cassettes were swabbed in total specifically for general levels of bacterial contamination, also for the presence or absence of methicillin-resistant Staphylococcus aureus (MRSA). A mapping exercise was completed following the location of an X-ray cassette typically used in mobile radiography. The exercise noted the level of direct contact with patient's skin and other possible routes of infection. Results: The results demonstrated that there were large levels of growth of samples taken from cassettes and developed in the Microbiology Department. Coagulase-negative Staphylococcus, Micrococci, Diptheroids and species of Bacillus were all identified. The mapping exercise in which the journey of a 35/43 cm cassette used for mobile radiography was tracked found that contact with patient's skin and potential pathogens or routes of cross infection was a common occurrence whilst undertaking mobile radiography. Conclusion: The research has identified the presence of bacterial contamination on cassettes. The research established that X-ray cassettes/imaging plates are often exposed to pathogens and possible routes of cross infection; also that patient's skin often comes directly in contact with the X-ray cassette/imaging plate. The research also shows that as cassettes/imaging plates are a potential source of cross infection, the Diagnostic Imaging Department may be partly responsible for adding to

  11. Molecular dynamics simulations of the bacterial ABC transporter SAV1866 in the closed form.

    Science.gov (United States)

    St-Pierre, Jean-François; Bunker, Alex; Róg, Tomasz; Karttunen, Mikko; Mousseau, Normand

    2012-03-01

    The ATP binding cassette (ABC) transporter family of proteins contains members involved in ATP-mediated import or export of ligands at the cell membrane. For the case of exporters, the translocation mechanism involves a large-scale conformational change that involves a clothespin-like motion from an inward-facing open state, able to bind ligands and adenosine triphosphate (ATP), to an outward-facing closed state. Our work focuses on SAV1866, a bacterial member of the ABC transporter family for which the structure is known for the closed state. To evaluate the ability of this protein to undergo conformational changes at physiological temperature, we first performed conventional molecular dynamics (MD) on the cocrystallized adenosine diphosphate (ADP)-bound structure and on a nucleotide-free structure. With this assessment of SAV1866's stability, conformational changes were induced by steered molecular dynamics (SMD), in which the nucleotide binding domains (NBD) were pushed apart, simulating the ATP hydrolysis energy expenditure. We found that the transmembrane domain is not easily perturbed by large-scale motions of the NBDs. PMID:22339391

  12. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  13. Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity

    International Nuclear Information System (INIS)

    To test whether the tyrosine kinase activity of the insulin receptor is crucial for insulin action, the authors have constructed mutations of the human insulin receptor at Lys-1030, which is in the presumed ATP-binding region. By using oligonucleotide-directed mutagenesis, this lysine residue was replaced with either methionine, arginine, or alanine. Chinese hamster ovary cells were transfected by mutant cDNAs and the expressed insulin receptors were characterized. They show here that none of these mutants exhibited insulin-activated autophosphorylation and kinase activity in vitro. They also do not mediate insulin- and antibody-stimulated uptake of 2-deoxyglucose. The tyrosine kinase activity is thus required for a key physiological response of insulin

  14. Magnetic cassette for radiographic film material

    International Nuclear Information System (INIS)

    A radiographic film cassette having a plurality of magnet components integral with the cassette holder for adhering the cassette to ferrous material in X-raying for defects in welds or fissures in shipyards, pipe lines, or the like. What is provided is a substantially flexible cassette envelope comprising first and second layers of radiographic intensifying screens with a sheet of radiographic film positioned therebetween. The cassette would be a cassette envelope constructed of waterproof fabric or other suitable material providing a light-free environment, and having the ability to flex around the curvature of the surface of a pipe or the like to be x-rayed. There is further provided a plurality of magnet components, preferably situated in each corner of the cassette envelope and flexibly attached thereto for overall adherence of the envelope to the surface of the pipe or the like to be x-rayed during the process

  15. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

    OpenAIRE

    Okuda, Suguru; Tokuda, Hajime

    2009-01-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

  16. Heart ATP-Binding Cassette Protein A1 and G1, Peroxisome Proliferator-Activated Receptor-α and Liver X Receptors Genes Expression in Response to Intensive Treadmill Running and Red Crataegus pentaegyna (Sorkh valik) in Male Rats

    OpenAIRE

    Abbass Ghanbari Niaki; Safieyh Ghanbari Abarghooi; Monireh Gholizadeh

    2015-01-01

    Background: The heart has a very high energy demand and to sustain sufficient ATP generation, can use a variety of different carbon substrates as energy sources if available. Objectives: The purpose of the current study was to investigate the effect of a high intensity treadmill running training (8 weeks) with or without aqueous extraction of Crataegus pentaegyna (Sorkh valik) on heart ABCA1, ABCG1, PPARα and LXR genes expression. Materials and Methods: In this experimental study, 20 Wi...

  17. Polymorphisms in ATP-binding cassette transporter genes and interaction with diet and life style factors in relation to colorectal cancer in a Danish prospective case-cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Andersen, Vibeke; Tjonneland, Anne;

    2015-01-01

    to assess whether polymorphisms in ABCB1, ABCC2 and ABCG2 were associated with risk of colorectal cancer (CRC) and to investigate gene-environment (dietary factors, smoking and use of non-steroidal anti-inflammatory drugs) and gene-gene interactions between previously studied polymorphisms in IL1B and IL10...... of the polymorphisms were associated with CRC, but ABCB1 and ABCG2 haplotypes were associated with risk of CRC. ABCB1/rs1045642 interacted with intake of cereals and fiber (p-Value for interaction (Pint) = 0.001 and 0.01, respectively). In a three-way analysis, both ABCB1/rs1045642 and ABCG2/rs2231137 in combination...... with IL10/rs3024505 interacted with fiber intake in relation to risk of CRC (Pint = 0.0007 and 0.009). Our results suggest that the ABC transporters P-glycoprotein/multidrug resistance 1 and BRCP, in cooperation with IL-10, are involved in the biological mechanism underlying the protective effect of fiber...

  18. EFFECTS OF ANTHOCYANIN ON ATP BINDING CASSETTE TRANSPORTER G1 EXPRESSION AND ANALYSIS OF THE MECHANISMS%花色苷对THP-1细胞中ABCG1表达的影响及机制分析

    Institute of Scientific and Technical Information of China (English)

    张英慧; 董华强; 钟希琼; 黄剑波

    2011-01-01

    目的 研究花色苷矢车菊素-3-O-β-葡萄糖苷(C3G)对人巨噬细胞THP-1中ABCG1表达的影响,并分析相关作用机制.方法 以C3G干预培养诱导分化的THP-1细胞,荧光实时定量RT-PCR检测ATP结合盒式转运蛋白G1(ABCG1)以及其直接调节因子肝脏X受体α(liver X receptotα,LXRα)的mRNA表达,时间分辨荧光共振能量转移(time-resolved fluorescence resonance energy transfer,TR-FRET)方法检测C3G与LXRα配体结合域的结合能力.结果 100μ mol/L C3G处理THP-1细胞16 h显著增加了ABCG1 mRNA的表达(为对照组的1.98倍,P<0.05);尽管在TR-FRET试验中,C3G并未呈现典型的配体结合曲线,而50 μ mol/L和100μmol/L C3G显著增加了THP-1细胞中LXRαmRNA的表达(分别为对照组的2.21倍和2.83倍,P<0.05).结论 C3G促进了ABCG1表达,该机制可能通过增加核受体LXRαmRNA表达来实现.

  19. Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

    Science.gov (United States)

    Eom, Gyeong Tae; Oh, Joon Young; Park, Ji Hyun; Lim, Hye Jin; Lee, So Jeong; Kim, Eun Young; Choi, Ji-Eun; Jegal, Jonggeon; Song, Bong Keun; Yu, Ju-Hyun; Song, Jae Kwang

    2016-09-01

    An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. PMID:27033673

  20. Inhibiting NF-K B increases cholesterol efflux from THP-1 derived- foam cells treated with Angll via up-regulating the expression of ATP-binding cassette transporter A1

    Institute of Scientific and Technical Information of China (English)

    Kun Liu; Yanfu Wang; Zhijian Chen; Yuhua Liao; Xiang Gao; Jian Chen

    2008-01-01

    Objective:To study the role of nuclear factor-kappa B(NF- K B) in cholesterol efflux from THP-I derived-foam cells treated with Angiotensin Ⅱ (Ang Ⅱ ). Methods:Cultured THP-l derived-foam cells were treated with Ang Ⅱ or preincubated with tosyl-phenylalan inechloromethyl-ketone(TPCK) NF-K B inhibitor. The levels of activated NF-K B in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCAI mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before Ang Ⅱ stimulation attenuated the response of NF- K B p65 nuclear translocation induced by Ang Ⅱ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol effiux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively, when compared with Ang Ⅱ group. In accordance, the ABCAl mRNA and protein were increased by 30% and 19%(P< 0.05) respectively, when compared with Ang Ⅱ group. Conclusion:Ang Ⅱ can down- regulate ABCAI in THP-l derived-foam cells via NF- K B, which leads to less cholesterol effiux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.

  1. The Second Extracellular Loop of Pore-Forming Subunits of ATP-Binding Cassette Transporters for Basic Amino Acids Plays a Crucial Role in Interaction with the Cognate Solute Binding Protein(s)▿

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-01-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)2 complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP2 of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P2 variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  2. The second extracellular loop of pore-forming subunits of ATP-binding cassette transporters for basic amino acids plays a crucial role in interaction with the cognate solute binding protein(s).

    Science.gov (United States)

    Eckey, Viola; Weidlich, Daniela; Landmesser, Heidi; Bergmann, Ulf; Schneider, Erwin

    2010-04-01

    In the thermophile Geobacillus stearothermophilus, the uptake of basic amino acids is mediated by an ABC transporter composed of the substrate binding protein (receptor) ArtJ and a homodimer each of the pore-forming subunit, ArtM, and the nucleotide-binding subunit, ArtP. We recently identified two putative binding sites in ArtJ that might interact with the Art(MP)(2) complex, thereby initiating the transport cycle (A. Vahedi-Faridi et al., J. Mol. Biol. 375:448-459, 2008). Here we investigated the contribution of charged amino acid residues in the second extracellular loop of ArtM to contact with ArtJ. Our results demonstrate a crucial role for residues K177, R185, and E188, since mutations to oppositely charged amino acids or glutamine led to a complete loss of ArtJ-stimulated ATPase activity of the complex variants in proteoliposomes. The defects could not be suppressed by ArtJ variants carrying mutations in site I (K39E and K152E) or II (E163K and D170K), suggesting a more complex interplay than that by a single salt bridge. These findings were supported by cross-linking assays demonstrating physical proximity between ArtJ(N166C) and ArtM(E182C). The importance of positively charged residues for receptor-transporter interaction was underscored by mutational analysis of the closely related transporter HisJ/LAO-HisQMP(2) of Salmonella enterica serovar Typhimurium. While transporter variants with mutated positively charged residues in HisQ displayed residual ATPase activities, corresponding mutants of HisM could no longer be stimulated by HisJ/LAO. Interestingly, the ATPase activity of the HisQM(K187E)P(2) variant was inhibited by l- and d-histidine in detergent, suggesting a role of the residue in preventing free histidine from gaining access to the substrate binding site within HisQM. PMID:20154136

  3. A Previously Unknown Unique Challenge for Inhibitors of SYK ATP-Binding Site: Role of SYK as A Cell Cycle Checkpoint Regulator

    Directory of Open Access Journals (Sweden)

    Fatih M. Uckun

    2014-11-01

    Full Text Available The identification of SYK as a molecular target in B-lineage leukemia/lymphoma cells prompted the development of SYK inhibitors as a new class of anti-cancer drug candidates. Here we report that induction of the SYK gene expression in human cells causes a significant down-regulation of evolutionarily conserved genes associated with mitosis and cell cycle progression providing unprecedented evidence that SYK is a master regulator of cell cycle regulatory checkpoint genes in human cells. We further show that SYK regulates the G2 checkpoint by physically associating with and inhibiting the dual-specificity phosphatase CDC25C via phosphorylation of its S216 residue. SYK depletion by RNA interference or treatment with the chemical SYK inhibitor prevented nocodazole-treated human cell lines from activating the G2 checkpoint via CDC25C S216-phosphorylation and resulted in polyploidy. Our study provides genetic and biochemical evidence that spleen tyrosine kinase (SYK has a unique role in the activation of the G2 checkpoint in both non-lymphohematopoietic and B-lineage lymphoid cells. This previously unknown role of SYK as a cell cycle checkpoint regulator represents an unforeseen and significant challenge for inhibitors of SYK ATP binding site.

  4. Two different point mutations in ABL gene ATP-binding domain conferring Primary Imatinib resistance in a Chronic Myeloid Leukemia (CML patient: A case report

    Directory of Open Access Journals (Sweden)

    Iqbal Zafar

    2004-01-01

    Full Text Available Imatinib (Gleevec is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance.

  5. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis.

    Science.gov (United States)

    Crawford, Matthew A; Lowe, David E; Fisher, Debra J; Stibitz, Scott; Plaut, Roger D; Beaber, John W; Zemansky, Jason; Mehrad, Borna; Glomski, Ian J; Strieter, Robert M; Hughes, Molly A

    2011-10-11

    Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Here, we report that disruption of the gene ftsX, which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms.

  6. Cellular pathways controlling integron cassette site folding.

    Science.gov (United States)

    Loot, Céline; Bikard, David; Rachlin, Anna; Mazel, Didier

    2010-08-01

    By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single-stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double-stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non-recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination. PMID:20628355

  7. ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

    Science.gov (United States)

    Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Chen, Zhongzhou; Lam, Hon-Ming

    2016-03-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

  8. ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

    Science.gov (United States)

    Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Chen, Zhongzhou; Lam, Hon-Ming

    2016-03-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life.

  9. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    Science.gov (United States)

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  10. Solution structure of the 45-residue ATP-binding peptide of adenylate kinase as determined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, E.M.; Kuby, S.A.; Mildyan, A.S.

    1986-05-01

    In the X-ray structure of adenylate kinase residues 1-45 exist as 47% ..cap alpha..-helix, 29% ..beta..-structure (strands and turns) and 24% coil. The solution structure of a synthetic peptide corresponding to residues 1-45, which constitutes the MgATP binding site was studied by 3 independent spectroscopic methods. Globularity of the peptide was shown by its broad NMR resonances which narrow upon denaturation, and by its ability to bind MgATP with similar affinity and conformation as the intact enzyme does. COSY and NOESY NMR methods at 250 and 500 MHz reveal proximities among NH, C..cap alpha.., and C..beta.. protons indicative of >20% ..cap alpha..-helix, and >20% ..beta..-structure. Correlation of regions of secondary structure with the primary sequence by 2D NMR indicates at least one ..cap alpha..-helix (res. 23 to 29) and two ..beta..-strands (res. 12 to 15 and 34 to 38). The broad amide I band in the deconvoluted FTIR spectrum could be fit as the sum of 4 peaks due to specific secondary structures, yielding less than or equal to=45% ..cap alpha..-helix, less than or equal to=40% ..beta..-structure and greater than or equal to=15% coil. The CD spectrum, from 185-250 nm, interpreted with a 3-parameter basis set, yielded 20 +/- 5% ..cap alpha..=helix, and less than or equal to=20% ..beta..-structure. The solution structure of peptide 1-45 thus approximates that of residues 1-45 in the crystal.

  11. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, M.; Kuby, S.A.; Mildvan A.S.

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme, appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase, with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of ..beta..-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% ..cap alpha..-helix, 38% ..beta..-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possible due to disorder, it can be fit by using methods developed on well-characterized globular proteins. The CD spectrum is best fit by assuming the presence of at most 13% ..cap alpha..-helix in the peptide, 24 +/- 2% ..beta..-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformation changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assess by CD.

  12. Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.

    Directory of Open Access Journals (Sweden)

    Visaahini Sureshan

    Full Text Available Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites, as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.

  13. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    Science.gov (United States)

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  14. Involvement of a bacterial microcompartment in the metabolism of fucose and rhamnose by Clostridium phytofermentans.

    Directory of Open Access Journals (Sweden)

    Elsa Petit

    Full Text Available BACKGROUND: Clostridium phytofermentans, an anaerobic soil bacterium, can directly convert plant biomass into biofuels. The genome of C. phytofermentans contains three loci with genes encoding shell proteins of bacterial microcompartments (BMC, organelles composed entirely of proteins. METHODOLOGY AND PRINCIPAL FINDINGS: One of the BMC loci has homology to a BMC-encoding locus implicated in the conversion of fucose to propanol and propionate in a human gut commensal, Roseburia inulinivorans. We hypothesized that it had a similar role in C. phytofermentans. When C. phytofermentans was grown on fucose, the major products identified were ethanol, propanol and propionate. Transmission electron microscopy of fucose- and rhamnose-grown cultures revealed polyhedral structures, presumably BMCs. Microarray analysis indicated that during growth on fucose, operons coding for the BMC locus, fucose dissimilatory enzymes, and an ATP-binding cassette transporter became the dominant transcripts. These data are consistent with fucose fermentation producing a 1,2-propanediol intermediate that is further metabolized in the microcompartment encoded in the BMC locus. Growth on another deoxyhexose sugar, rhamnose, resulted in the expression of the same BMC locus and similar fermentation products. However, a different set of dissimilatory enzymes and transport system genes were induced. Quite surprisingly, growth on fucose or rhamnose also led to the expression of a diverse array of complex plant polysaccharide-degrading enzymes. CONCLUSIONS/SIGNIFICANCE: Based on physiological, genomic, and microarray analyses, we propose a model for the fermentation of fucose and rhamnose in C. phytofermentans that includes enzymes encoded in the same BMC locus. Comparative genomic analysis suggests that this BMC may be present in other clostridial species.

  15. An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification.

    Science.gov (United States)

    Ouyang, Mengxing; Mohan, Ruchika; Lu, Yi; Liu, Tingting; Mach, Kathleen E; Sin, Mandy L Y; McComb, Mason; Joshi, Janhvi; Gau, Vincent; Wong, Pak Kin; Liao, Joseph C

    2013-07-01

    To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.

  16. Books (on Cassette) Are Better Than Ever.

    Science.gov (United States)

    Davis, Bryan

    1984-01-01

    Describes introduction of books on tape at Oskaloosa (Iowa) Public Library, highlighting determination of audience and use, display of recorded books, packaging of tapes, cataloging, and quality of tapes. A list of 19 production companies and six distributors noting address, telephone number, type of cassettes, and price range is included. (EJS)

  17. 21 CFR 892.1850 - Radiographic film cassette.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Radiographic film cassette. 892.1850 Section 892...) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A radiographic film cassette is a device intended for use during diagnostic x-ray procedures...

  18. CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

    Energy Technology Data Exchange (ETDEWEB)

    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L. [Case Western; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

  19. Discovery of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazoles as Novel DNA Gyrase Inhibitors Targeting the ATP-Binding Site.

    Science.gov (United States)

    Tomašič, Tihomir; Katsamakas, Sotirios; Hodnik, Žiga; Ilaš, Janez; Brvar, Matjaž; Solmajer, Tom; Montalvão, Sofia; Tammela, Päivi; Banjanac, Mihailo; Ergović, Gabrijela; Anderluh, Marko; Peterlin Mašič, Lucija; Kikelj, Danijel

    2015-07-23

    Bacterial DNA gyrase and topoisomerase IV are essential enzymes that control the topological state of DNA during replication and validated antibacterial drug targets. Starting from a library of marine alkaloid oroidin analogues, we identified low micromolar inhibitors of Escherichia coli DNA gyrase based on the 5,6,7,8-tetrahydroquinazoline and 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole scaffolds. Structure-based optimization of the initial hits resulted in low nanomolar E. coli DNA gyrase inhibitors, some of which exhibited micromolar inhibition of E. coli topoisomerase IV and of Staphylococcus aureus homologues. Some of the compounds possessed modest antibacterial activity against Gram positive bacterial strains, while their evaluation against wild-type, impA and ΔtolC E. coli strains suggests that they are efflux pump substrates and/or do not possess the physicochemical properties necessary for cell wall penetration. Our study provides a rationale for optimization of this class of compounds toward balanced dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

  20. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Cellular Slime Mold, Dictyostelium Discoideum

    Science.gov (United States)

    Hanely, Julia C.; Reinsch, Sigrid; Myers, Zachary A.; Freeman, John; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David G.

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. To expand the use of flight verified hardware for various model organisms, we performed ground experiments to determine whether ARC EMCS Seed Cassettes could be adapted for use with cellular slime mold for future space flight experiments. Dictyostelium is a cellular slime mold that can exist both as a single-celled independent organism and as a part of a multicellular colony which functions as a unit (pseudoplasmodium). Under certain stress conditions, individual amoebae will aggregate to form multicellular structures. Developmental pathways are very similar to those found in Eukaryotic organisms, making this a uniquely interesting organism for use in genetic studies. Dictyostelium has been used as a genetic model organism for prior space flight experiments. Due to the formation of spores that are resistant to unfavorable conditions such as desiccation, Dictyostelium is also a good candidate for use in the EMCS Seed Cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membranes contains dried growth medium. The goals of this study were to (1) verify that Dictyostelium are capable of normal growth and development on PES membranes, (2) develop a method for dehydration of Dictyostelium spores with successful recovery and development after rehydration, and (3) successful mock rehydration experiments in cassettes. Our results show normal developmental progression in two strains of Dictyostelium discoideum on PES membranes with a bacterial food source. We have successfully performed a mock rehydration of spores with developmental progression from aggregation to slug formation, and production of morphologically normal spores within 9 days of rehydration. Our results indicate that experiments on the ISS using the slime mold, Dictyostelium discoideum could potentially be performed in the flight verified hardware of

  1. Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains

    DEFF Research Database (Denmark)

    Tanabe, Mikio; Mirza, Osman; Bertrand, Thomas;

    2007-01-01

    Bacterial ATP-binding cassette (ABC) transport systems couple ATP hydrolysis with the uptake and efflux of a wide range of substances across bacterial membranes. These systems are comprised of transmembrane domains, nucleotide binding domains and, in the case of uptake systems, periplasmic bindin...

  2. A Cassette Based System for Hydrogen Storage and Delivery

    Energy Technology Data Exchange (ETDEWEB)

    Britton Wayne E.

    2006-11-29

    A hydrogen storage system is described and evaluated. This is based upon a cassette, that is a container for managing hydrogen storage materials. The container is designed to be safe, modular, adaptable to different chemistries, inexpensive, and transportable. A second module receives the cassette and provides the necessary infrastructure to deliver hydrogen from the cassette according to enduser requirements. The modular concept has a number of advantages over approaches that are all in one stand alone systems. The advantages of a cassette based system are discussed, along with results from model and laboratory testing.

  3. Sub classification and targeted characterization of prophage-encoded two-component cell lysis cassette

    Indian Academy of Sciences (India)

    K V Srividhya; S Krishnaswamy

    2007-08-01

    Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies. The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E. coli and the purified protein was functional, exhibiting lytic activity against E. coli and Salmonella typhi cell wall substrate. Such targeted sequence-structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

  4. Kinetics, in silico docking, molecular dynamics, and MM-GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined.

    Science.gov (United States)

    Hayes, Joseph M; Skamnaki, Vicky T; Archontis, Georgios; Lamprakis, Christos; Sarrou, Josephine; Bischler, Nicolas; Skaltsounis, Alexios-Leandros; Zographos, Spyros E; Oikonomakos, Nikos G

    2011-03-01

    With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC₅₀ > 50 μM), indirubin-3'-oxime (IC₅₀ = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts. PMID:21287607

  5. Observing cassette culture: user interface implications for digital music libraries

    OpenAIRE

    Toal, Jason

    2007-01-01

    Many people keep their collections of music on cassette tape even if they rarely listen to them. Images of these collections can be found online on photo sharing websites. What can we learn from such collections and what might they tell us about designing interfaces for new digital music libraries? The author conducts an online ethnographic study of over two hundred cassette tape collections, and over sixty participants with the aim of guiding future design of music collections. The author pr...

  6. Detection of oral Helicobacter Pylori infection using saliva test cassette

    OpenAIRE

    Yu, Min; Zhang, Xue-Yan; Yu, Qing

    2015-01-01

    Objective: To investigate the incidence of oral infection with Helicobacter pylori (H. pylori) and identify related epidemiological factors among freshmen of four colleges in Yancheng. Methods: The data, scored positive or negative, were collected on 160 individuals who had been diagnosed by H. pylori Saliva Test Cassette (HPS) during October 2013 to October 2014. H. pylori Saliva Test Cassette (HPS) is to use colloidal gold technique to specifically identify urease in saliva. A standard ques...

  7. Staphylococcal Cassette Chromosome mec Types Among Methicillin-Resistant Staphylococcus aureus in Northern Iran

    Science.gov (United States)

    Taherirad, Akram; Jahanbakhsh, Roghayeh; Shakeri, Fatemeh; Anvary, Shaghayegh; Ghaemi, Ezzat Allah

    2016-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of nosocomial and community-acquired infections around the world. Staphylococcal cassette chromosome mec (SCCmec) typing methods are often used to study MRSA molecular epidemiology. Objectives The current study was designed to explore the distribution profiles of different SCCmec types among methicillin-resistant S. aureus strains isolated from hospitals in Gorgan, in northern Iran, and to correlate the types into observed bacterial virulence factors. Materials and Methods Staphylococcal cassette chromosome mec typing of 62 MRSA strains isolated from patients and health-care workers in Gorgan was performed using multiplex polymerase chain reaction (PCR) assay. The prevalence of the strains was then compared according to isolation source, antibiotic susceptibility profiles, biofilm production, and the presence of the Panton-Valentine gene in isolates. Results The most common SCCmec type was type III, with a frequency rate of 76%, followed by types IV, I, and V, with frequency rates of 11.2%, 4.8%, and 3.2%, respectively; three isolates (4.8%) were not typeable by this method. SCCmec type I was only isolated from blood culture, and types IV and V were mainly isolated from wounds and urine samples; SCCmec type III was isolated from all of the clinically samples. All of the MRSA strains that were isolated from healthy carriers were type III. Multidrug resistance in the type III strains was higher compared to the other types. The frequencies of Panton-Valentine and biofilm production were significantly lower in the type III strains compared to the other SCCmec types (P < 0.05). Conclusions Similarly to other geographical regions of Iran, the SCCmec type III MRSA strain was the most frequently isolated strain from patients in Gorgan. Staphylococcal cassette chromosome mec type III showed fewer virulence factors compared to other SCCmec types. PMID:27800133

  8. ABCA1基因启动子区-191G/C单核苷酸多态性在冠心病中的意义%Significance of - 191G/C single nucleotide polymorphisms in the promoter region of ATP-binding cassette transporter gene in coronary artery disease

    Institute of Scientific and Technical Information of China (English)

    刘凌; 郭志刚; 王琦光; 刘胜林; 赖文岩; 屠燕

    2005-01-01

    目的研究ABCA1基因启动子区-191G/C单核苷酸多态性(SNP)对血脂的影响及其在冠心病中的意义.方法 用聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)探讨204例冠心病患者和114例正常人的ABCA1基因启动子区-191位点G/C SNP在两组间、冠心病组不同临床表现型之间分布的差异及3种等位基因型与冠心病相关临床指标的关系.结果冠心病组与正常人组比较,3种等位基因型GG、GC、CC分布频率差异具有显著性,CC基因型在冠心病组中的分布频率明显高于正常人组(P<0.05),C等位基因在冠心病组中的分布频率明显高于正常人组(P<0.01).在稳定性心绞痛组(SAP)和急性冠脉综合征组(ACS),3种等位基因型GG、GC、CC分布频率差异也具有显著性,ACS组CC基因型明显高于SAP组(P<0.05),C等位基因在ACS组中分布频率明显高于SAP组(P<0.01).在冠心病组中,3种等位基因型间体重指数、总胆固醇、总甘油三脂、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、极低密度脂蛋白胆固醇等临床指标的比较无显著性差异.结论 ABCA1基因启动子区-191G/C SNP在不影响中国冠心病人群血脂水平的情况下增加冠心病的发生,C等位基因与冠心病的稳定性相关.

  9. ω-carboxyl Like Linoleic Acid Oxides Promoting the Expression of ATP-binding Cassette Transporter A1 (ABCA1)%ω-羧基样亚油酸氧化物促进ATP结合盒转运子A1(ABCA1)表达

    Institute of Scientific and Technical Information of China (English)

    马艳华; 刘庆平; 娜琴; 苑红; 扈瑞平

    2015-01-01

    ω-羧基样亚油酸氧化物(7-KC-9-COOH)是由Cu2+氧化低密度脂蛋白(oxLDL)中得到的负性胆固醇氧化物.研究其对ATP结合盒(ABC)转运子超家族成员ABCA1表达的调控作用,并探讨其对高密度脂蛋白(HDL)介导的胆固醇流出的影响.THP-1单核细胞经佛波酯(PMA)诱导分化为巨噬细胞,化学合成的7-KC-9-COOH以时间梯度孵育细胞,随后采用RTPCR,western blot研究其对ABCA1基因和蛋白水平表达的影响.7-KC-9-COOH显著促进了ABCAl mRNA和蛋白水平表达,其中作用2h效果最为显著,基因水平增加48.6%(*P<0.05),蛋白水平增加285.3%,较空白组升高近3倍(**P<0.01).抑制其上游核转录因子PPARγ与LXRα,其蛋白表达量下降51.2%.ω-COOH亚油酸氧化物7-KC-9-COOH经由PPARγ-LXRα信号通路提高了ABCA1的表达,促进了胆固醇逆转运(RCT)和HDL介导的胆固醇流出.

  10. ATP结合盒转运子1R219K基因多态性与阿尔茨海默病相关性研究%Research on association between polymorphism of ATP-binding cassette transporter A1 and Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    王娟; 陈卫蓉; 肖志杰

    2013-01-01

    Objective To assess the effect of single-nucleotide polymorphisms (SNPs) in the cholesterol-related genes ABCA1 exon R219K variation on the risk of AD and cognitive function.Methods Using case-control study,a total of sporadic 104 AD patients and 104 individuals above 60 years without cognitive impairment from a population of Chinese Han nationality in Changsha area were studied to determine the polymorphisms of ABCA1 R219K.Adopt SPSS 13.0 for statistical analysis.Results There were significant differences in the frequencies of three genotypes (RR、RK and KK) between the AD patients and controls (x2 =8.777,P =0.012).The frequency of KK genotype in AD group is obviously lower than that of controls (x2 =5.261,P =0.022).The frequency of KK+RK genotype in AD patients was 54.8%,was significantly lower than that of controls (70.2%,P =0.022).There was significantly higher levels of HDL-C,apoA-I and cognitive scores in the carriers of KK genotype and K allele (P < 0.05).There was significantly higher levels of MMSE and WMS scores in the carriers of K K genotype and K allele (P < 0.05).Further in accordance with memory types including long-term memory,short-term memory,transient memory,compared with RR genotype,the scores of short-term memory,transient memory of RK,KK and RK + KK genotypes were higher (P < 0.05),but there was no significant difference in the score of long-term memory (P > 0.05).Multifactor logistic regression analysis showed that carrying K allele was a single protective factor for AD.Conclusions The polymorphism of ABCA1R219K is associated with AD.Carrying K allele may play a positive role in serum lipids and cognition,and produce a protective effect on risk of AD.%目的 探讨胆固醇相关基因ABCA1外显子R219K遗传变异对阿尔茨海默病(AD)发病风险及认知功能的影响.方法 采用病例对照研究方法测定湖南长沙地区汉族人群104例散发性AD患者以及104名60岁以上认知功能正常者的ABCA1R219K基因多态性.采用SPSS 13.0软件进行统计分析.结果 两组间RR、RK及KK 3种基因型分布差异有统计学意义(x2=8.777,P=0.012),AD组KK型频率显著低于对照组(x2=5.261,P=0.022).AD组(RK+KK)型频率较对照组显著降低(54.8% & 70.2%,P=0.022).KK型及K等位基因携带者血浆HDL—C、apoA-I水平增高,差异有统计学意义(P<0.05).在总体研究人群中,KK型及RK+ KK型携带者的MMSE评分以及WMS评分显著增高(P<0.05);进一步按照长时记忆、短时记忆和瞬时记忆3种记忆类型分析,与RR型相比,RK、KK型及RK+KK型携带者的短时记忆及瞬时记忆得分显著增高(P<0.05),而长时记忆得分的比较差异无统计学意义(P>0.05).Logistic回归结果表明K等位基因携带是AD发病的独立保护因素.结论 ABCA1R219K变异与AD的发病有关,K等住基因携带对血脂及认知功能可能产生有益作用,并对AD的发生可能有一定保护作用.

  11. 姜黄素对脂变肝细胞胆固醇流出及ABCA1表达的影响%Effects of Curcumin on ATP Binding Cassette Transporter A1 Expression and Cholesterol Effiux in Human L-02 Hepatocyte

    Institute of Scientific and Technical Information of China (English)

    余其林; 阳学风

    2009-01-01

    目的 研究姜黄素对脂变肝细胞胆固醇的转运机制. 方法 取正常人肝L-02细胞作为研究对象.用10%(V/V)医用脂肪乳注射液和10%(V/V)胎牛血清的RPMI-1640完全培养基孵育24 h建立的脂肪变性肝细胞模型.实验分为为6个组,即正常肝细胞组、人肝L-02细胞脂肪变性模型组、姜黄素处理组(脂变细胞模型建立后,分别加入不等量姜黄素,使其终浓度分别为12.5、25、50、100 μmol/L).RT-PCR检测细胞ABCA1表达;高效液相色谱法定量检测细胞内外胆固醇含量. 结果 姜黄素呈浓度依赖性地减少细胞内脂变肝细胞内总胆固醇(TC)、胆固醇酯(CE)含量,增加培养基中游离胆固醇(FC)含量,同时增加ABCA1的表达. 结论 姜黄素能降低脂变人肝L-02细胞TC及CE含量,增加FC的外流;姜黄素增加脂变肝细胞胆固醇转运可能与ABCA1的上调有关.

  12. Effects of ATP-binding cassette exporters on virulence factors in Streptococcus mutans%三磷酸腺苷结合盒外排子对变异链球菌毒力因子影响的研究进展

    Institute of Scientific and Technical Information of China (English)

    曾荟荟; 凌均棨

    2015-01-01

    ABC transporters have been proved to be integral membrane proteins that actively transported a diverse range of substrates across cell membranes. ABC transporters had varied functions, and took part in gene competence, (p)ppGpp accumulation, bacteriocin secretion and immunity in Streptococcus mutans. The structures, functions, mechanisms and inhibitors of the known ABC exporters in Streptococcus mutans were summarized.%三磷酸腺苷结合盒(ABC)转运子是膜蛋白的一部分,透过细胞膜转运各种生物分子,参与多种生理功能。在变异链球菌中,ABC外排子与基因感受态、四(五)磷酸鸟苷[(p)ppGpp]累积、细菌素分泌与免疫密切相关。本文就变异链球菌ABC外排子的结构、生理功能、作用机制和抑制剂作一综述。

  13. Fatigue analysis of steam generator cassette parts based on CAE

    International Nuclear Information System (INIS)

    Fatigue analysis has been performed for steam generator nozzle header and tube based on CAE. Three dimensional model was produced using the commercial CAD program, IDEAS and the geometry and boundary condition information have been transformed into input format of ABAQUS for thermal analysis, stress analysis, and fatigue analysis. Cassette nozzle, which has a complex geometry, has been analysed by using the three dimensional model. But steam generator tube has been analysed according to ASME procedure since it can be modelled as a two dimensional finite element model. S-N curve for the titanium alloy of the steam generator tube material was obtained from the material tests. From the analysis, it has been confirmed that these parts of the steam generator cassette satisfy the lifetime of the steam generator cassette. Three dimensional modelling strategy from the thermal analysis to fatigue analysis should be implemented into the design of reactor major components to enhance the efficiency of design procedure

  14. Construction of deletion mutants in the phosphotransferase transport system and adenosine triphosphate-binding cassette transporters in Listeria monocytogenes and analysis of their growth under different stress conditions

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2013-10-01

    Full Text Available Functional genomics approaches enable us to investigate the biochemical, cellular, and physiological properties of each gene product and are nowadays applied to enhance food safety by understanding microbial stress responses in food and host-pathogen interactions. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to eliminate this pathogen since it can survive under multiple stress conditions such as low pH and low temperature. Detailed studies are needed to determine its mode of action and to understand the mechanisms that protect the pathogen when it is subjected to stress. In this study, deletion mutants of phosphotransferase transport system genes (PTS and adenosine triphosphate(ATP-binding cassette transporters (ABC of Listeria monocytogenes F2365 were created using molecular techniques. These mutants and the wild-type were tested under different stress conditions, such as in solutions with different NaCl concentration, pH value and for nisin resistance. Results demonstrate that the behaviour of these deletion mutants is different from the wild type. In particular, deleted genes may be involved in L. monocytogenes resistance to nisin and to acid and salt concentrations. Functional genomics research on L. monocytogenes allows a better understanding of the genes related to stress responses and this knowledge may help in intervention strategies to control this food-borne pathogen. Furthermore, specific gene markers can be used to identify and subtype L. monocytogenes. Thus, future development of this study will focus on additional functional analyses of important stress response-related genes, as well as on methods for rapid and sensitive detection of L. monocytogenes such as using DNA microarrays.

  15. Comparative structural and functional analysis of staphylococcus aureus glucokinase with other bacterial glucokinases

    Directory of Open Access Journals (Sweden)

    P S Kumar

    2014-01-01

    Full Text Available Glucokinase is classified in bacteria based upon having ATP binding site and ′repressor/open reading frames of unknown function/sugar kinases′ motif, the sequence of glucokinase gene (JN645812 of Staphylococcus aureus ATCC12600 showed presence of ATP binding site and ′repressor/open reading frames of unknown function/sugar kinases′ motif. We have earlier observed glucokinase of S. aureus has higher affinity towards the substrate compared to other bacterial glucokinase and under anaerobic condition with increased glucose concentration S. aureus exhibited higher rate of biofilm formation. To establish this, 3D structure of glucokinase was built using homology modeling method, the PROCHECK and ProSA-Web analysis indicated this built glucokinase structure was close to the crystal structure. This structure was superimposed with different bacterial glucokinase structures and from the root-mean-square deviation values, it is concluded that S. aureus glucokinase exhibited very close homology with Enterococcus faecalis and Clostridium difficle while with other bacteria it showed high degree of variations both in domain and nondomain regions. Glucose docking results indicated -12.3697 kcal/mol for S. aureus glucokinase compared with other bacterial glucokinase suggesting higher affinity of glucose which correlates with enzyme kinetics and higher rate of biofilm formation.

  16. DIY series of genetic cassettes useful in construction of versatile vectors specific for Alphaproteobacteria.

    Science.gov (United States)

    Dziewit, Lukasz; Adamczuk, Marcin; Szuplewska, Magdalena; Bartosik, Dariusz

    2011-08-01

    We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria. PMID:21569803

  17. Co-assortment in integron-associated gene cassette assemblages in environmental DNA samples

    Directory of Open Access Journals (Sweden)

    Michael Carolyn A

    2010-08-01

    Full Text Available Abstract Background It has been shown that integron-associated gene cassettes exist largely in tandem arrays of variable size, ranging from antibiotic resistance arrays of three to five cassettes up to arrays of more than 100 cassettes associated with the vibrios. Further, the ecology of the integron/gene cassette system has been investigated by showing that very many different cassettes are present in even small environmental samples. In this study, we seek to extend the ecological perspective on the integron/gene cassette system by investigating the way in which this diverse cassette metagenome is apportioned amongst prokaryote lineages in a natural environment. Results We used a combination of PCR-based techniques applied to environmental DNA samples and ecological analytical techniques to establish co-assortment within cassette populations, then establishing the relationship between this co-assortment and genomic structures. We then assessed the distribution of gene cassettes within the environment and found that the majority of gene cassettes existed in large co-assorting groups. Conclusions Our results suggested that the gene cassette diversity of a relatively pristine sampling environment was structured into co-assorting groups, predominantly containing large numbers of cassettes per group. These co-assorting groups consisted of different gene cassettes in stoichiometric relationship. Conservatively, we then attributed co-assorting cassettes to the gene cassette complements of single prokaryote lineages and by implication, to large integron-associated arrays. The prevalence of large arrays in the environment raises new questions about the assembly, maintenance and utility of large cassette arrays in prokaryote populations.

  18. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... radiographic cassette holder. (a) Identification. A wall-mounted radiographic cassette holder is a device...

  19. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    Directory of Open Access Journals (Sweden)

    Vera Kozjak-Pavlovic

    2009-10-01

    Full Text Available The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m. Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m loss and apoptosis, demonstrating that dissipation of DeltaPsi(m is a requirement for cell death caused by neisserial infection.

  20. Is this charred material from a VHS video cassette?

    Science.gov (United States)

    Fruchtenicht, Tara; Blackledge, Robert D.; Williams, Teresa R.

    2010-06-01

    At his residence, a victim in a double homicide had installed a home-built video surveillance system. The suspects either knew of or discovered this system and removed it. In a backyard at a location associated with the suspects was a barrel used for burning trash. Could charred debris recovered from a metal bowl found among the contents of the barrel be the remains of a VHS video cassette? A positive answer to the question was obtained through a combination of optical microscopy, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Energy Dispersive Spectroscopy (EDS).

  1. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to. the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

  2. A two-cassette reporter system for assessing target gene translation and target gene product inclusion body formation

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention relates to a dual cassette reporter system capable of assessing target gene translation and target gene product folding. The present invention further relates to vectors and host cells comprising the dual cassette reporter system. In addition the invention relates to the use...... of the dual cassette reporter system for assessing target gene translation and target gene product folding....

  3. A clinical trial of a rare earth screen/film system in a periapical cassette

    International Nuclear Information System (INIS)

    In a clinical trial, a slow rare earth screen/film system (Siemens Titan 2D/Kodak XG) was used to obtain intraoral radiographs at conventional monitoring stages in endodontic treatment. The screen film image proved to be an effective substitute for the direct-exposure Ultraspeed periapical film. The intraoral cassettes, designed and fabricated for the study, were an adaptation of the flexible, vacuum-sealed cassettes used in mammography. It is believed that when a practicable periapical cassette is manufactured, many additional indications for the system are probable. Major reductions in patient exposure of at least 85% to 90% per periapical film would be effected

  4. Genetic Variation of the Borrelia burgdorferi Gene vlsE Involves Cassette-Specific, Segmental Gene Conversion

    OpenAIRE

    Zhang, Jing-Ren; Norris, Steven J

    1998-01-01

    The Lyme disease spirochete Borrelia burgdorferi possesses 15 silent vls cassettes and a vls expression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5′ and 3′ coding sequences of vlsE that flank the cassette region are not subject to sequence variation...

  5. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB.

    Science.gov (United States)

    Okuda, Suguru; Tokuda, Hajime

    2009-04-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detail. However, it remained unclear how Lol factors interact with each other to conduct very efficient lipoprotein transfer in the periplasm where ATP is not available. To address this issue, a photo-reactive phenylalanine analogue, p-benzoyl-phenylalanine, was introduced at various positions of LolA and LolB, of which the overall structures are very similar and comprise an incomplete beta-barrel with a hydrophobic cavity inside. Cells expressing LolA or LolB derivatives containing the above analogue were irradiated with UV for in vivo photo-cross-linking. These analyses revealed a hot area in the same region of LolA and LolB, through which LolA and LolB interact with each other. This area is located at the entrance of the hydrophobic cavity. Moreover, this area in LolA is involved in the interaction with a membrane subunit, LolC, whereas no cross-linking occurs between LolA and the other membrane subunit, LolE, or ATP-binding subunit LolD, despite the structural similarity between LolC and LolE. The hydrophobic cavities of LolA and LolB were both found to bind lipoproteins inside. These results indicate that the transfer of lipoproteins through Lol proteins occurs in a mouth-to-mouth manner. PMID:19307584

  6. Influence of cassette design on three-dimensional perfusion culture of artificial bone.

    Science.gov (United States)

    Du, Dajiang; Ushida, Takashi; Furukawa, Katsuko S

    2015-01-01

    Media perfusion is often required to maintain cell viability within topographically complex 3-dimensional scaffold cultures. Osteoblast-seeded scaffolds for bone regeneration require robust cell proliferation and survival both within the scaffold and over the exterior for optimal osteogenic capacity. Conventional press-fitting cassettes ensure internal fluid flow through the scaffold but may restrict external flow around the scaffold, resulting in a barren (cell-free) external scaffold surface. In this study, we aimed to solve this problem by modifying the cassette structure to enhance external flow in an oscillatory perfusion culture system. Mouse osteoblast-like MC 3T3-E1 cells were seeded in porous ceramic scaffolds and incubated for 3 days either under static culture conditions or in an oscillatory perfusion bioreactor. Scaffolds were held in the bioreactor with either conventional press-fitting cassettes or cassettes with rings to separate the scaffold exterior from the internal cassette wall. The external surfaces of scaffolds maintained under static conditions were well seeded, but cells failed to grow deeply into the core, reflecting poor internal chemotransport. Alternatively, scaffolds cultured by perfusion with press-fitting cassettes had poor cell viability at the cassette-external scaffold surface interface, but cells were widely distributed within the scaffold core. Scaffolds cultured using the modified cassettes with 1 or 2 rings exhibited uniformly distributed living cells throughout the internal pores and over the entire external surface, possibly because of the improved medium flow over the scaffold surface. This modified oscillatory perfusion culture system may facilitate the production of engineered bone with superior osteogenic capacity for grafting.

  7. ATP binding to a multisubunit enzyme: statistical thermodynamics analysis

    CERN Document Server

    Zhang, Yunxin

    2012-01-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical thermodynamics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provided a new way to understand biophysical processes by statistical thermodynamics analysis.

  8. Effect of attC structure on cassette excision by integron integrases

    Directory of Open Access Journals (Sweden)

    Larouche André

    2011-02-01

    Full Text Available Abstract Background Integrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates. Results We compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G. We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical

  9. NCBI nr-aa BLAST: CBRC-TTRU-01-1060 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1060 ref|XP_002523691.1| ATP-binding cassette transporter, putative [Ricinus... communis] gb|EEF38631.1| ATP-binding cassette transporter, putative [Ricinus communis] XP_002523691.1 0.012 25% ...

  10. NCBI nr-aa BLAST: CBRC-MLUC-01-0864 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MLUC-01-0864 ref|XP_002522797.1| ATP-binding cassette transporter, putative [Ricinus... communis] gb|EEF39648.1| ATP-binding cassette transporter, putative [Ricinus communis] XP_002522797.1 1.3 24% ...

  11. Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection

    Science.gov (United States)

    BAKHTIARI, Ronak; AHMADIAN, Shahin; FALLAH MEHRABADI, Jalil

    2016-01-01

    Background: Uropathogenic Escherichia coli (UPEC) are major bacterial agent of Urinary Tract Infection (UTI). This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6–12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing. Methods: This survey was conducted during 2014–2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14). One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured. Results: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05). Conclusion: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI. PMID:27516995

  12. Film cassette for quality assurance of dental X-ray tubes

    International Nuclear Information System (INIS)

    In this brief technical note, the authors describe a film cassette to enable routine quality assurance checks of equipment to be made by dental staff without the use of an ion chamber. The film is sent to the Physics Department of Guys Hospital, London, for processing and densitometric analysis. (UK)

  13. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L;

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  14. Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

    Science.gov (United States)

    Oh, Young Hoon; Eom, Gyeong Tae; Kang, Kyoung Hee; Joo, Jeong Chan; Jang, Young-Ah; Choi, Jae Woo; Song, Bong Keun; Lee, Seung Hwan; Park, Si Jae

    2016-04-01

    Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources. PMID:26780375

  15. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

    Directory of Open Access Journals (Sweden)

    Khorchid Ahmad

    2003-07-01

    Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

  16. Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

    NARCIS (Netherlands)

    Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

    2007-01-01

    Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found t

  17. Temperature variations around medication cassette and carry bag in routine use of epoprostenol administration in healthy volunteers.

    Directory of Open Access Journals (Sweden)

    Yuichi Tamura

    Full Text Available BACKGROUND: According to several treatment guidelines, epoprostenol is an important treatment option for pulmonary arterial hypertension. However, the pharmacokinetic characteristics and poor stability of epoprostenol at room temperature make its administration challenging. We therefore studied temperature fluctuations between the drug administration cassette and atmosphere to promote the safe use of epoprostenol. METHODS AND FINDINGS: Five healthy volunteers carried a portable intravenous infusion pump attached to a medication cassette containing saline in a bag during their ordinary activities over 16 days during which the mean atmospheric temperature was 29.6 ± 1.5°C. The temperature around the medication cassette was not less than 25°C on any occasion, and the mean period over 24 h during which the temperature around the cassette exceeded 35°C and 40°C was 96.9 ± 156.4 min and 24.4 ± 77.3 min, respectively. Significant correlations were observed between the temperatures outside the bag and around the cassette, as well as between temperatures around the cassette and of the saline solution in the cassette (r = 0.9258 and 0.8276, respectively. There were no differences in the temperatures outside the bag or around the cassette with respect to the bag material. CONCLUSIONS: Temperatures around a medication cassette and outside the bag containing the medication increase with sunlight exposure. The temperature around cassettes used for administering epoprostenol must therefore be kept low for as long as possible during hot summer conditions to maintain the drug stability.

  18. Utilizing ARC EMCS Seedling Cassettes as Highly Versatile Miniature Growth Chambers for Model Organism Experiments

    Science.gov (United States)

    Freeman, John L.; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David; Reinsch, S.; DeSimone, Julia C.; Myers, Zachary A.

    2014-01-01

    The aim of our ground testing was to demonstrate the capability of safely putting specific model organisms into dehydrated stasis, and to later rehydrate and successfully grow them inside flight proven ARC EMCS seedling cassettes. The ARC EMCS seedling cassettes were originally developed to support seedling growth during space flight. The seeds are attached to a solid substrate, launched dry, and then rehydrated in a small volume of media on orbit to initiate the experiment. We hypothesized that the same seedling cassettes should be capable of acting as culture chambers for a wide range of organisms with minimal or no modification. The ability to safely preserve live organisms in a dehydrated state allows for on orbit experiments to be conducted at the best time for crew operations and more importantly provides a tightly controlled physiologically relevant growth experiment with specific environmental parameters. Thus, we performed a series of ground tests that involved growing the organisms, preparing them for dehydration on gridded Polyether Sulfone (PES) membranes, dry storage at ambient temperatures for varying periods of time, followed by rehydration. Inside the culture cassettes, the PES membranes were mounted above blotters containing dehydrated growth media. These were mounted on stainless steel bases and sealed with plastic covers that have permeable membrane covered ports for gas exchange. The results showed we were able to demonstrate acceptable normal growth of C.elegans (nematodes), E.coli (bacteria), S.cerevisiae (yeast), Polytrichum (moss) spores and protonemata, C.thalictroides (fern), D.discoideum (amoeba), and H.dujardini (tardigrades). All organisms showed acceptable growth and rehydration in both petri dishes and culture cassettes initially, and after various time lengths of dehydration. At the end of on orbit ISS European Modular Cultivation System experiments the cassettes could be frozen at ultra-low temperatures, refrigerated, or chemically

  19. Functional importance of different patterns of correlation between adjacent cassette exons in human and mouse

    Directory of Open Access Journals (Sweden)

    Zhang Xuegong

    2008-04-01

    Full Text Available Abstract Background Alternative splicing expands transcriptome diversity and plays an important role in regulation of gene expression. Previous studies focus on the regulation of a single cassette exon, but recent experiments indicate that multiple cassette exons within a gene may interact with each other. This interaction can increase the potential to generate various transcripts and adds an extra layer of complexity to gene regulation. Several cases of exon interaction have been discovered. However, the extent to which the cassette exons coordinate with each other remains unknown. Results Based on EST data, we employed a metric of correlation coefficients to describe the interaction between two adjacent cassette exons and then categorized these exon pairs into three different groups by their interaction (correlation patterns. Sequence analysis demonstrates that strongly-correlated groups are more conserved and contain a higher proportion of pairs with reading frame preservation in a combinatorial manner. Multiple genome comparison further indicates that different groups of correlated pairs have different evolutionary courses: (1 The vast majority of positively-correlated pairs are old, (2 most of the weakly-correlated pairs are relatively young, and (3 negatively-correlated pairs are a mixture of old and young events. Conclusion We performed a large-scale analysis of interactions between adjacent cassette exons. Compared with weakly-correlated pairs, the strongly-correlated pairs, including both the positively and negatively correlated ones, show more evidence that they are under delicate splicing control and tend to be functionally important. Additionally, the positively-correlated pairs bear strong resemblance to constitutive exons, which suggests that they may evolve from ancient constitutive exons, while negatively and weakly correlated pairs are more likely to contain newly emerging exons.

  20. Evaluating the accuracy of technicians and pharmacists in checking unit dose medication cassettes.

    Science.gov (United States)

    Ambrose, Peter J; Saya, Frank G; Lovett, Larry T; Tan, Sandy; Adams, Dale W; Shane, Rita

    2002-06-15

    The accuracy rates of board-registered pharmacy technicians and pharmacists in checking unit dose medication cassettes in the inpatient setting at two separate institutions were examined. Cedars-Sinai Medical Center and Long Beach Memorial Medical Center, both in Los Angeles county, petitioned the California State Board of Pharmacy to approve a waiver of the California Code of Regulations to conduct an experimental program to compare the accuracy of unit dose medication cassettes checked by pharmacists with that of cassettes checked by trained, certified pharmacy technicians. The study consisted of three parts: assessing pharmacist baseline checking accuracy (Phase I), developing a technician-training program and certifying technicians who completed the didactic and practical training (Phase II), and evaluating the accuracy of certified technicians checking unit dose medication cassettes as a daily function (Phase III). Twenty-nine pharmacists and 41 technicians (3 of whom were pharmacy interns) participated in the study. Of the technicians, all 41 successfully completed the didactic and practical training, 39 successfully completed the audits and became certified checkers, and 2 (including 1 of the interns) did not complete the certification audits because they were reassigned to another work area or had resigned. In Phase II, the observed accuracy rate and its lower confidence limit exceeded the predetermined minimum requirement of 99.8% for a certified checker. The mean accuracy rates for technicians were identical at the two institutions (p = 1.0). The difference in mean accuracy rates between pharmacists (99.52%; 95% confidence interval [CI] 99.44-99.58%) and technicians, (99.89%; 95% CI 99.87-99.90%) was significant (p medication cassettes filled by other technicians. PMID:12073859

  1. Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines

    Institute of Scientific and Technical Information of China (English)

    CHANG Jin-Soo; PEN Xianghao; KIM Kyoung-Woong

    2008-01-01

    Biogeochemical cyclic activity of the ars (arsenic resistance system) operon is arsB influx/efflux encoded by the ecological of Pseudomonas putida. This suggests that studying arsenite-oxidizing bacteria may lead to a better understanding of molecular geomicrobiology, which can be applied to the bioremediation of arsenic-contaminated mines. This is the first report in which multiple arsB-binding mechanisms have been used on indigenous bacteria. In ArsB (swains OS-5; ABB83931; OS-19; ABB04282 and RW-28;ABB88574), there are ten putative enzyme, Histidine (His) 131, His 133, His 137, Arginine (Arg) 135, Arg 137, Arg 161, Trptohan (Trp) 142, Trp 164, Trp 166, and Trp 171, which are each located in different regions of the partial sequence. The adenosine triphosphate (ATP)-binding cassette transports, binding affinities and associating ratable constants show that As-binding is comparatively insensitive to the location of the residues within the moderately stable a-helical structure. The α-helical structures in ArsB-permease and anion permease arsB have been shown to import/export arsenic in P. putida. We proposed that arsB residues, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171 are required for arsenic binding and activation ofarsA/arsB or arsAB.This arsB influx/efflux pum-ping is important, and the effect in arsenic species change and mobility in mine soil has got a significantly ecological role because it allows arsenic oxidizing/reducing bacteria to control biogeochemical cycle of abandoned mines.

  2. Biogeochemical cyclic activity of bacterial arsB in arsenic-contaminated mines.

    Science.gov (United States)

    Chang, Jin-Soo; Ren, Xianghao; Kim, Kyoung-Woong

    2008-01-01

    Biogeochemical cyclic activity of the ars (arsenic resistance system) operon is arsB influx/efflux encoded by the ecological of Pseudomonas putida. This suggests that studying arsenite-oxidizing bacteria may lead to a better understanding of molecular geomicrobiology, which can be applied to the bioremediation of arsenic-contaminated mines. This is the first report in which multiple arsB-binding mechanisms have been used on indigenous bacteria. In ArsB (strains OS-5; ABB83931; OS-19; ABB04282 and RW-28; ABB88574), there are ten putative enzyme, Histidine (His) 131, His 133, His 137, Arginine (Arg) 135, Arg 137, Arg 161, Trptohan (Trp) 142, Trp 164, Trp 166, and Trp 171, which are each located in different regions of the partial sequence. The adenosine triphosphate (ATP)-binding cassette transports, binding affinities and associating ratable constants show that As-binding is comparatively insensitive to the location of the residues within the moderately stable alpha-helical structure. The alpha-helical structures in ArsB-permease and anion permease arsB have been shown to import/export arsenic in P. putida. We proposed that arsB residues, His 131, His 133, His 137, Arg 135, Arg 137, Arg 161, Trp 142, Trp 164, Trp 166, and Trp 171 are required for arsenic binding and activation of arsA/arsB or arsAB. This arsB influx/efflux pum-ping is important, and the effect in arsenic species change and mobility in mine soil has got a significantly ecological role because it allows arsenic oxidizing/reducing bacteria to control biogeochemical cycle of abandoned mines. PMID:19202875

  3. Bacterial Vaginosis

    Science.gov (United States)

    ... 586. Related Content STDs during Pregnancy Fact Sheet Pregnancy and HIV, Viral Hepatitis, and STD Prevention Pelvic Inflammatory Disease ( ... Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes Hepatitis HIV/AIDS & STDs Human Papillomavirus ... STDs See Also Pregnancy Reproductive ...

  4. Bacterial Meningitis

    Science.gov (United States)

    ... Schedules Preteen & Teen Vaccines Meningococcal Disease Sepsis Bacterial Meningitis Recommend on Facebook Tweet Share Compartir On this ... serious disease. Laboratory Methods for the Diagnosis of Meningitis This manual summarizes laboratory methods used to isolate, ...

  5. Integration of multiple expression cassettes into mammalian genomes in a single step

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Andrijana Kriz, Katharina Schmid, Kurt Ballmer & Philipp Berger ### Abstract The modification of mammalian cells by the expression of multiple genes is a crucial technology in modern biological research. MultiLabel allows the modular assembly of independent expression units in a single plasmid which can be used for transient and stable modification of cells. In contrast to other methods, the assembly of the expression cassettes does not require restriction enzymes since i...

  6. Identification of a Novel Cassette Array in Integronbearing Helicobacter Pylori Strains Isolated from Iranian Patients.

    Science.gov (United States)

    Goudarzi, Mehdi; Seyedjavadi, Sima Sadat; Fazeli, Maryam; Roshani, Maryam; Azad, Mehdi; Heidary, Mohsen; Navidinia, Masoumeh; Goudarzi, Hossein

    2016-01-01

    Helicobacter pylori as the second most common cause of gastric cancer in the world infects approximately half of the developed countries population and 80% of the population living in developing countries. Integrons as genetic reservoirs play major roles in dissemination of antimicrobial resistance genes. To the best of our knowledge, this is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes in H. pylori isolates from Iran. This crosssectional study was conducted in Tehran among 110 patients with H. pylori infection. Antimicrobial susceptibility testing (AST) for H. pylori strains were assessed by the micro broth dilution method. Class 1 and 2 integrons were detected using PCR. In order to determine gene cassettes, amplified fragments were subjected to DNA sequencing of both amplicon strands. The prevalence of resistance to clarithromycin, metronidazole, clarithromycin, tetracycline, amoxicillin, rifampin, and levofloxacin were 68.2% (n=75), 25.5% (n=28), 24.5% (n=27), 19.1% (n=21), 18.2% (n=20) and 16.4% (n=18), respectively. Frequency of multidrug resistance among H. pylori isolates was 12.7%. Class 2 integron was detected in 50 (45.5%) and class 1 integron in 10 (9.1%) H. pylori isolates. The most predominant gene cassette arrays in class 2 integron bearing H. pylori were included sateraaadA1, dfrA1sat2aadA1, blaoxa2 and, aadB whereas common gene cassette arrays in class 1 integron were aadBaadA1cmlA6, aacA4, blaoxa2, and catB3. The high frequency of class 2 integron and multidrug resistance in the present study should be considered as a warning for clinicians that continuous surveillance is necessary to prevent the further spread of resistant isolates. PMID:27509968

  7. Methicillin-resistant Staphylococcus saprophyticus in Sweden carries various types of staphylococcal cassette chromosome mec (SCCmec).

    Science.gov (United States)

    Söderquist, B; Berglund, C

    2009-12-01

    Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections and is usually susceptible to the antimicrobial agents used for their treatment. However, S. saprophyticus resistant to beta-lactam antibiotics and carrying mecA has been reported. Eight Swedish isolates of mecA-positive S. saprophyticus with diverse origin carrying at least three different types of staphylococcal cassette chromosome mec (SCCmec) are described here.

  8. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy.

    Science.gov (United States)

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy. PMID:25636961

  9. Non-integrative lentivirus drives high-frequency cre-mediated cassette exchange in human cells.

    Directory of Open Access Journals (Sweden)

    Raul Torres

    Full Text Available Recombinase mediated cassette exchange (RMCE is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening.

  10. Linear friction weld process monitoring of fixture cassette deformations using empirical mode decomposition

    Science.gov (United States)

    Bakker, O. J.; Gibson, C.; Wilson, P.; Lohse, N.; Popov, A. A.

    2015-10-01

    Due to its inherent advantages, linear friction welding is a solid-state joining process of increasing importance to the aerospace, automotive, medical and power generation equipment industries. Tangential oscillations and forge stroke during the burn-off phase of the joining process introduce essential dynamic forces, which can also be detrimental to the welding process. Since burn-off is a critical phase in the manufacturing stage, process monitoring is fundamental for quality and stability control purposes. This study aims to improve workholding stability through the analysis of fixture cassette deformations. Methods and procedures for process monitoring are developed and implemented in a fail-or-pass assessment system for fixture cassette deformations during the burn-off phase. Additionally, the de-noised signals are compared to results from previous production runs. The observed deformations as a consequence of the forces acting on the fixture cassette are measured directly during the welding process. Data on the linear friction-welding machine are acquired and de-noised using empirical mode decomposition, before the burn-off phase is extracted. This approach enables a direct, objective comparison of the signal features with trends from previous successful welds. The capacity of the whole process monitoring system is validated and demonstrated through the analysis of a large number of signals obtained from welding experiments.

  11. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    Directory of Open Access Journals (Sweden)

    Ming Yan

    Full Text Available Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  12. Conservation of gene cassettes among diverse viruses of the human gut.

    Directory of Open Access Journals (Sweden)

    Samuel Minot

    Full Text Available Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.

  13. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    Science.gov (United States)

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research. PMID:19255730

  14. Extending the Glucosyl Ceramide Cassette Approach: Application in the Total Synthesis of Ganglioside GalNAc-GM1b

    Directory of Open Access Journals (Sweden)

    Miku Konishi

    2013-12-01

    Full Text Available The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of β-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b.

  15. Coping with depression: a pilot study to assess the efficacy of a self-help audio cassette.

    OpenAIRE

    Blenkiron, P

    2001-01-01

    BACKGROUND: The self-help audio cassette 'Coping with Depression' was produced and widely distributed as part of the national Defeat Depression Campaign. A central aim was to improve public understanding and encourage the use of cognitive-behavioural techniques. AIM: To formally assess the ability of the audio cassette to change attitudes to depression in primary care and the degree to which patients are motivated to practice its recommended coping strategies. DESIGN OF STUDY: Comparison of L...

  16. Bacterial carbonatogenesis

    International Nuclear Information System (INIS)

    Several series of experiments in the laboratory as well as in natural conditions teach that the production of carbonate particles by heterotrophic bacteria follows different ways. The 'passive' carbonatogenesis is generated by modifications of the medium that lead to the accumulation of carbonate and bicarbonate ions and to the precipitation of solid particles. The 'active' carbonatogenesis is independent of the metabolic pathways. The carbonate particles are produced by ionic exchanges through the cell membrane following still poorly known mechanisms. Carbonatogenesis appears to be the response of heterotrophic bacterial communities to an enrichment of the milieu in organic matter. The active carbonatogenesis seems to start first. It is followed by the passive one which induces the growth of initially produced particles. The yield of heterotrophic bacterial carbonatogenesis and the amounts of solid carbonates production by bacteria are potentially very high as compared to autotrophic or chemical sedimentation from marine, paralic or continental waters. Furthermore, the bacterial processes are environmentally very ubiquitous; they just require organic matter enrichment. Thus, apart from purely evaporite and autotrophic ones, all Ca and/or Mg carbonates must be considered as from heterotrophic bacterial origin. By the way, the carbon of carbonates comes from primary organic matter. Such considerations ask questions about some interpretations from isotopic data on carbonates. Finally, bacterial heterotrophic carbonatogenesis appears as a fundamental phase in the relationships between atmosphere and lithosphere and in the geo-biological evolution of Earth. (author)

  17. IncP-1ε plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes

    Directory of Open Access Journals (Sweden)

    Holger eHeuer

    2012-01-01

    Full Text Available The role of broad host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5’-nuclease assay for real time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridisation. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

  18. A GFP-based bacterial biosensor with chromosomally integrated sensing cassette for quantitative detection of Hg(II) in environment

    Institute of Scientific and Technical Information of China (English)

    Himanshu Priyadarshi; Absar Alam; Gireesh-Babu P; Rekha Das; Pankaj Kishore; Shivendra Kumar; Aparna Chaudhari

    2012-01-01

    A mercury biosensor was constructed by integrating biosensor genetic elements into E.coli JM109 chromosome in a single copy number,using the attP/attB recombination mechanism of λ phage.The genetic elements used include a regulatory protein gene (merR) along with operator/promoter (O/P) derived from the mercury resistance operon from pDU1358 plasmid of Serratia marcescens.The expression of reporter gene gfp is also controlled by merR/O/P.Integration of the construct into the chromosome was done to increase the stability and precision of the biosensor.This biosensor could detect Hg(Ⅱ) ions in the concentration range of 100-1700 mnol/L,and manifest the result as the expression of GFP.The GFP expression was significantly different (P ≤ 0.05) for each concentration of inducing Hg(Ⅱ) ions in the detection range,which reduces the chances of misinterpretation of results.A model using regression method was also derived for the quantification of the concentration of Hg(Ⅱ) in water samples.

  19. Bacterial Adhesion & Blocking Bacterial Adhesion

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk

    2008-01-01

    tract to the microbial flocs in waste water treatment facilities. Microbial biofilms may however also cause a wide range of industrial and medical problems, and have been implicated in a wide range of persistent infectious diseases, including implantassociated microbial infections. Bacterial adhesion...... is the first committing step in biofilm formation, and has therefore been intensely scrutinized. Much however, still remains elusive. Bacterial adhesion is a highly complex process, which is influenced by a variety of factors. In this thesis, a range of physico-chemical, molecular and environmental parameters......, which influence the transition from a planktonic lifestyle to a sessile lifestyle, have been studied. Protein conditioning film formation was found to influence bacterial adhesion and subsequent biofilm formation considerable, and an aqueous extract of fish muscle tissue was shown to significantly...

  20. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, mea

  1. Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Andrew Cameron

    Full Text Available Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.

  2. A new tocograph with cassette recording system and separate servo graphic recorder.

    Science.gov (United States)

    Zahn, V; Seitz, P

    1979-01-01

    In order to register contractional activity, especially in the case of high-risk pregnancies, a tocograph was develop by means of which the contractions are registered by a small cassette recorder, which the patient can carry by about with her. A separate graphic recorder is responsible for the playback and this recorder remains at the doctor's practice. The patient is able to register her contractions herself as the unit is so simple to use. The recording section weighs only 500 grams, including the specially developed pressure transducer with optical distance-meter. The tocograph is produced in series.

  3. Bacterial Ecology

    DEFF Research Database (Denmark)

    Fenchel, Tom

    2011-01-01

    Bacterial ecology is concerned with the interactions between bacteria and their biological and nonbiological environments and with the role of bacteria in biogeochemical element cycling. Many fundamental properties of bacteria are consequences of their small size. Thus, they can efficiently exploit...... biogeochemical processes are carried exclusively by bacteria. * Bacteria play an important role in all types of habitats including some that cannot support eukaryotic life....

  4. [Bacterial vaginosis].

    Science.gov (United States)

    Romero Herrero, Daniel; Andreu Domingo, Antonia

    2016-07-01

    Bacterial vaginosis (BV) is the main cause of vaginal dysbacteriosis in the women during the reproductive age. It is an entity in which many studies have focused for years and which is still open for discussion topics. This is due to the diversity of microorganisms that cause it and therefore, its difficult treatment. Bacterial vaginosis is probably the result of vaginal colonization by complex bacterial communities, many of them non-cultivable and with interdependent metabolism where anaerobic populations most likely play an important role in its pathogenesis. The main symptoms are an increase of vaginal discharge and the unpleasant smell of it. It can lead to serious consequences for women, such as an increased risk of contracting sexually transmitted infections including human immunodeficiency virus and upper genital tract and pregnancy complications. Gram stain is the gold standard for microbiological diagnosis of BV, but can also be diagnosed using the Amsel clinical criteria. It should not be considered a sexually transmitted disease but it is highly related to sex. Recurrence is the main problem of medical treatment. Apart from BV, there are other dysbacteriosis less characterized like aerobic vaginitis of which further studies are coming slowly but are achieving more attention and consensus among specialists. PMID:27474242

  5. Novel cassette array in a class 1 integron in clinical isolates of Acinetobacter baumannii from central Iran.

    Science.gov (United States)

    Japoni-Nejad, Alireza; Farshad, Shohreh; van Belkum, Alex; Ghaznavi-Rad, Ehsanollah

    2013-12-01

    Antibiotic resistance in Acinetobacter baumannii is a major problem in the hospital and outbreaks caused by this organism have been reported frequently. The present study aimed at determining the antibiotic susceptibility patterns, the prevalence of different classes of integrons and the characterization of integron class 1 gene cassettes in Iranian A. baumannii isolates. A total of 63 non-duplicate A. baumannii isolates were collected from clinical and environmental specimens in the Vali-Asr hospital in the central province of Iran (March to September, 2011). The antimicrobial susceptibility for 15 antibiotics which are used conventionally was determined by disk diffusion. The presence of different integron classes was investigated by PCR and the size of gene cassettes in class 1 integrons was then determined by PCR as well. Moreover, integron cassette arrays of isolates were delineated by RFLP and sequencing amplicons with different lengths. Of 63 isolates 62 (98.4%) carried a class 1 integron. The prevalence of IntI2 was 15.9% and the length of the amplicons ranged from 500 bp to 3 kb. Sequencing of integrons of class 1 revealed the presence of many resistance genes (aadA, aacA, aacC, dfrA, bla(GES) and bla(IMP)). We identified a completely new gene cassette which contained aacA7-qacF-aadA5-bla(IMP), this cassette has not been reported previously in A. baumannii. PMID:24161711

  6. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

    Science.gov (United States)

    Makhzoum, Abdullah; Benyammi, Roukia; Moustafa, Khaled; Trémouillaux-Guiller, Jocelyne

    2014-04-01

    Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing. PMID:23959796

  7. Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

    Science.gov (United States)

    Hiraki, Masahiko; Watanabe, Shokei; Chavas, Leonard M. G.; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Wakatsuki, Soichi; Fujihashi, Masahiro; Miki, Kunio; Baba, Seiki; Ueno, Go; Yamamoto, Masaki; Suzuki, Mamoru; Nakagawa, Atsushi; Watanabe, Nobuhisa; Tanaka, Isao

    2010-06-01

    To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

  8. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

    Science.gov (United States)

    Makhzoum, Abdullah; Benyammi, Roukia; Moustafa, Khaled; Trémouillaux-Guiller, Jocelyne

    2014-04-01

    Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

  9. Divertor cassette locking system remote handling trials with WHMAN at DTP2

    Energy Technology Data Exchange (ETDEWEB)

    Lyytikäinen, Ville; Kinnunen, Pasi; Koivumäki, Janne; Mattila, Jouni [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); Siuko, Mikko [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Esque, Salvador [F4E, Fusion for Energy, Torres Diagonal Litoral B3, Josep Pla2, 08019, Barcelona (Spain); Palmer, Jim, E-mail: ville.lyytikainen@tut.fi [ITER Organization, Route de Vinon sur Verdon, 13115 Saint Paul Lez Durance (France)

    2013-10-15

    Highlights: ► RH requirements were developed from operator feedback, potential problem analysis and task description. ► Tools were designed according to these RH specific requirements. ► Two RH capable were developed and their functionality was verified at DPT2. -- Abstract: A key ITER maintenance activity is the exchange of the divertor cassettes. The current major step in this programme involves the full scale physical test facility, namely divertor test platform 2 (DTP2), in Tampere, Finland. The objective of the DTP2 is the design and proof of concept studies of various remote handling (RH) device prototypes and their RH control systems, but is also important to define principles for standardizing control systems and methods around the ITER maintenance equipment. The development process of divertor cassette locking system (CLS) RH Tool prototypes is presented in this paper. The validation of the developed CLS Tool prototypes is accomplished in RH trials at DTP2. For this RH Trial, a CLS task description (TD) and tool prototypes were developed, manufactured and, finally, tested under remote operations. These tools, designed to be operated by water hydraulic manipulator (WHMAN), are water hydraulic jack (WHJ), pin tool (PT) and wrench tool (WT)

  10. THE REAL ISSUE WITH WALL DEPOSITS IN CLOSED FILTER CASSETTES - WHAT'S THE SAMPLE?

    Energy Technology Data Exchange (ETDEWEB)

    Brisson, M.

    2009-09-12

    The measurement of aerosol dusts has long been utilized to assess the exposure of workers to metals. Tools used to sample and measure aerosol dusts have gone through many transitions over the past century. In particular, there have been several different techniques used to sample for beryllium, not all of which might be expected to produce the same result. Today, beryllium samples are generally collected using filters housed in holders of several different designs, some of which are expected to produce a sample that mimics the human capacity for dust inhalation. The presence of dust on the interior walls of cassettes used to hold filters during metals sampling has been discussed in the literature for a number of metals, including beryllium, with widely varying data. It appears that even in the best designs, particulates can enter the sampling cassette and deposit on the interior walls rather than on the sampling medium. The causes are not well understood but are believed to include particle bounce, electrostatic forces, particle size, particle density, and airflow turbulence. Historically, the filter catch has been considered to be the sample, but the presence of wall deposits, and the potential that the filter catch is not representative of the exposure to the worker, puts that historical position into question. This leads to a fundamental question: What is the sample? This article reviews the background behind the issue, poses the above-mentioned question, and discusses options and a possible path forward for addressing that question.

  11. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

    Science.gov (United States)

    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

  12. Electricity generation from cattle manure slurry by cassette-electrode microbial fuel cells.

    Science.gov (United States)

    Inoue, Kengo; Ito, Toshihiro; Kawano, Yoshihiro; Iguchi, Atsushi; Miyahara, Morio; Suzuki, Yoshihiro; Watanabe, Kazuya

    2013-11-01

    Cassette-electrode microbial fuel cells (CE-MFCs) are efficient and scalable devices for electricity production from organic waste. Previous studies have demonstrated that CE-MFCs are capable of generating electricity from artificial wastewater at relatively high efficiencies. In this study, a single-cassette CE-MFC was constructed, and its capacity for electricity generation from cattle manure suspended in water (solid to water ratio of 1:50) was examined. The CE-MFC reactor was operated in batch mode for 49 days; electricity generation became stable 2 weeks after initiating the operation. The maximum power density was measured at 16.3 W m⁻³ on day 26. Sequencing analysis of PCR-amplified 16S rRNA gene fragments obtained from the original manure and from anode biofilms suggested that Chloroflexi and Geobacteraceae were abundant in the anode biofilm (29% and 18%, respectively), whereas no Geobacteraceae sequences were detected in the original manure sample. The results of this study suggest that CE-MFCs can be used to generate electricity from water-suspended cattle manure in a scalable MFC system.

  13. Bacterial Hydrodynamics

    Science.gov (United States)

    Lauga, Eric

    2016-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells, yet they represent the bulk of the world's biomass and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micrometer scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically complex environments. Using hydrodynamics as an organizing framework, I review the biomechanics of bacterial motility and look ahead to future challenges.

  14. Bacterial hydrodynamics

    CERN Document Server

    Lauga, Eric

    2015-01-01

    Bacteria predate plants and animals by billions of years. Today, they are the world's smallest cells yet they represent the bulk of the world's biomass, and the main reservoir of nutrients for higher organisms. Most bacteria can move on their own, and the majority of motile bacteria are able to swim in viscous fluids using slender helical appendages called flagella. Low-Reynolds-number hydrodynamics is at the heart of the ability of flagella to generate propulsion at the micron scale. In fact, fluid dynamic forces impact many aspects of bacteriology, ranging from the ability of cells to reorient and search their surroundings to their interactions within mechanically and chemically-complex environments. Using hydrodynamics as an organizing framework, we review the biomechanics of bacterial motility and look ahead to future challenges.

  15. NCBI nr-aa BLAST: CBRC-TTRU-01-0272 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0272 ref|ZP_03055167.1| cyclodextrin ABC superfamily ATP binding casse...tte transporter, membrane protein [Bacillus pumilus ATCC 7061] gb|EDW21594.1| cyclodextrin ABC superfamily A

  16. Genetic variation in ABCA1 predicts ischemic heart disease in the general population

    DEFF Research Database (Denmark)

    Frikke-Schmidt, Ruth; Nordestgaard, Børge G; Jensen, Gorm B;

    2008-01-01

    We tested the hypothesis that 6 nonsynonymous single nucleotide polymorphisms (SNPs) in ATP-Binding-Cassette transporter A1 (ABCA1) affect risk of ischemic heart disease (IHD) in the general population....

  17. The Actinobacterial mce4 Locus Encodes a Steroid Transporter

    NARCIS (Netherlands)

    Mohn, William W.; van der Geize, Robert; Stewart, Gordon R.; Okamoto, Sachi; Liu, Jie; Dijkhuizen, Lubbert; Eltis, Lindsay D.

    2008-01-01

    Bioinformatic analyses have suggested that Mce proteins in diverse actinobacteria are components of complex ATP-binding cassette transporter systems, comprising more than eight distinct proteins. In Mycobacterium tuberculosis, these proteins are implicated in interactions of this deadly pathogen wit

  18. Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy.

    Science.gov (United States)

    Ye, Zhisheng; Sun, Lei; Li, Rongbo; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2016-01-01

    Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes.

  19. On the integrity of a commercial cassette ultrafiltration membrane:implications for marine colloidal biogeochemistry

    Institute of Scientific and Technical Information of China (English)

    LIN Liangshi; CAI Yihua; SUN Xiuwu; CHEN Min

    2014-01-01

    The performance and integrity of a cassette cross-flow ultrafilter (Pellicon 2, Millipore) are examined with a suite of macromolecules of different molecular masses. The retention coefficient during the cross-flow ultrafiltration experiments increases with increasing molecular mass and reaches 90%with 10 kDa dextran in both milli-Q water and ultrafiltered seawater media. Based on a 90%retention coefficient, the molecular mass cut-off for the ultrafiltration membrane is defined at 10 kDa, which is ten times (1 kDa) that rated by the manufacturer. To further validate the accuracy of the laboratory calibration, the samples from the lower Zhujiang River and the Jiulong River Estuary are ultrafiltered with the cassette ultrafiltration membrane and the colloidal organic carbon abundances in these samples are quantified with the ultrafiltration per-meation model based on time series permeation subsamples. The colloidal organic carbon abundances are 5.8%-21.1%in the Jiulong River Estuary and 5.6%-11.0%in the lower Zhujiang River. These are consistent with the reported values for both estuaries as well as with the colloidal organic carbon abundances in ma-rine environments over the coastal and open oceans with 10 kDa cut-off membranes. Therefore, these field data support the laboratory calibration result and indicate the validity of the experimental and quantifica-tion procedure adopted. The discrepancy between the nominal molecular mass cut-off and the actual pore size of the ultrafiltration membrane should be of great concern for research in colloidal and nanoparticle biogeochemistry. Careful examination of the membrane integrity should be taken during ultrafiltration ex-periments in order to avoid misleading molecular mass cut-off information.

  20. Temperature Variations around Medication Cassette and Carry Bag in Routine Use of Epoprostenol Administration in Healthy Volunteers

    OpenAIRE

    Yuichi Tamura; Yasuo Nakajima; Yasushi Ozeki; Tomohiko Ono; Makoto Takei; Tsunehisa Yamamoto; Keiichi Fukuda

    2012-01-01

    BACKGROUND: According to several treatment guidelines, epoprostenol is an important treatment option for pulmonary arterial hypertension. However, the pharmacokinetic characteristics and poor stability of epoprostenol at room temperature make its administration challenging. We therefore studied temperature fluctuations between the drug administration cassette and atmosphere to promote the safe use of epoprostenol. METHODS AND FINDINGS: Five healthy volunteers carried a portable intravenous in...

  1. New film-cassette system to obtain wider field of craniocaudal view compared with conventional technique in screening mammography

    International Nuclear Information System (INIS)

    To evaluate the efficacy of a newly designed cassette and film system used to obtain a craniocaudal (CC) image during mammographic examination. We designed a film-cassette system for use in obtaining a CC image. The merit of this system is that the contact plane between the film and film cassette and the thoracic wall of the examinee changed from linear to concave, thus including more tissue on the image. Twenty women examined by screening mammography underwent conventional and new CC plane examinations. The distance from the nipple to the posterior margin of the included breast tissue, as seen on CC mammograms, was measured using the two techniques, and the difference between the respective results was analyzed by paired t-test. The distance from the nipple to the posterior margin was 12.9±1.7cm and 14.5±1.4cm at the lateral portion of the conventional and new CC image, respectively. This distance was thus significantly greater on the new than on the conventional image (p<0.001), but there was no significant difference between their medial portions. The newly designed cassette and film system used to obtain a craniocaudal image during mammography includes more breast tissue than the conventional system and may be helpful for the mammographic screening and diagnosis of peripheral breast lesions

  2. Integron gene cassettes and degradation of compounds associated with industrial waste: the case of the Sydney tar ponds.

    Directory of Open Access Journals (Sweden)

    Jeremy E Koenig

    Full Text Available Integrons are genetic platforms that accelerate lateral gene transfer (LGT among bacteria. They were first detected on plasmids bearing single and multiple drug resistance determinants in human pathogens, and it is abundantly clear that integrons have played a major role in the evolution of this public health menace. Similar genetic elements can be found in nonpathogenic environmental bacteria and in metagenomic environmental DNA samples, and it is reasonable to suppose that integrons have facilitated microbial adaptation through LGT in niches outside infectious disease wards. Here we show that a heavily impacted estuary, exposed for almost a century to products of coal and steel industries, has developed a rich and unique cassette metagenome, containing genes likely to aid in the catabolism of compounds associated with industrial waste found there. In addition, we report that the most abundant cassette recovered in this study is one that encodes a putative LysR protein. This autoregulatory transcriptional regulator is known to activate transcription of linked target genes or unlinked regulons encoding diverse functions including chlorocatechol and dichlorophenol catabolism. Finally, only class 1 integrase genes were amplified in this study despite using different primer sets, and it may be that the cassettes present in the Tar Ponds will prove to be associated with class 1 integrase genes. Nevertheless, our cassette library provides a snapshot of a complex evolutionary process involving integron-meditated LGT likely to be important in natural bioremediation.

  3. Integrase-mediated recombination of the veb1 gene cassette encoding an extended-spectrum β-lactamase.

    Directory of Open Access Journals (Sweden)

    Daniel Aubert

    Full Text Available The veb1 gene cassette encodes the extended spectrum β-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.

  4. Structural variations of staphylococcal cassette chromosome mec Type IVa in Staphylococcus aureus clonal complex 8 and unrelated lineages

    DEFF Research Database (Denmark)

    Damborg, Peter Panduro; Bartels, Mette Damkjær; Boye, Kit;

    2011-01-01

    PCR mapping of staphylococcal cassette chromosome mec type IVa and adjacent mobile elements in 94 methicillin-resistant Staphylococcus aureus (MRSA) strains identified two primary structures (A and B) that could be further classified into two (A1 and A2) and five (B1 to B5) variants, primarily...

  5. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    Science.gov (United States)

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption. PMID:27411901

  6. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    Science.gov (United States)

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption.

  7. A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish.

    Science.gov (United States)

    Fuentes, Fernando; Reynolds, Eric; Lewellis, Stephen W; Venkiteswaran, Gayatri; Knaut, Holger

    2016-01-01

    Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels. PMID:26818072

  8. EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.

    Science.gov (United States)

    Rice, Austin J; Alvarez, Frances J D; Schultz, Kathryn M; Klug, Candice S; Davidson, Amy L; Pinkett, Heather W

    2013-07-19

    In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

  9. Roles of the protruding loop of factor B essential for the localization of lipoproteins (LolB) in the anchoring of bacterial triacylated proteins to the outer membrane.

    Science.gov (United States)

    Hayashi, Yumi; Tsurumizu, Ryoji; Tsukahara, Jun; Takeda, Kazuki; Narita, Shin-ichiro; Mori, Makiko; Miki, Kunio; Tokuda, Hajime

    2014-04-11

    The Lol system comprising five Lol proteins, LolA through LolE, sorts Escherichia coli lipoproteins to outer membranes. The LolCDE complex, an ATP binding cassette transporter in inner membranes, releases outer membrane-specific lipoproteins in an ATP-dependent manner, causing formation of the LolA-lipoprotein complex in the periplasm. LolA transports lipoproteins through the periplasm to LolB on outer membranes. LolB is itself a lipoprotein anchored to outer membranes, although the membrane anchor is functionally dispensable. LolB then localizes lipoproteins to outer membranes through largely unknown mechanisms. The crystal structure of LolB is similar to that of LolA, and it possesses a hydrophobic cavity that accommodates acyl chains of lipoproteins. To elucidate the molecular function of LolB, a periplasmic version of LolB, mLolB, was mutagenized at various conserved residues. Despite the lack of acyl chains, most defective mutants were insoluble. However, a derivative with glutamate in place of leucine 68 was soluble and unable to localize lipoproteins to outer membranes. This leucine is present in a loop protruding from mLolB into an aqueous environment, and no analogous loop is present in LolA. Thus, leucine 68 was replaced with other residues. Replacement by acidic, but not hydrophobic, residues generated for the first time mLolB derivatives that can accept but cannot localize lipoproteins to outer membranes. Moreover, deletion of the leucine with neighboring residues impaired the lipoprotein receptor activity. Based on these observations, the roles of the protruding loop of LolB in the last step of lipoprotein sorting are discussed. PMID:24569999

  10. Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family.

    Science.gov (United States)

    Broussard, Tyler C; Miller, Darcie J; Jackson, Pamela; Nourse, Amanda; White, Stephen W; Rock, Charles O

    2016-03-18

    Fatty acid kinase (Fak) is a ubiquitous Gram-positive bacterial enzyme consisting of an ATP-binding protein (FakA) that phosphorylates the fatty acid bound to FakB. In Staphylococcus aureus, Fak is a global regulator of virulence factor transcription and is essential for the activation of exogenous fatty acids for incorporation into phospholipids. The 1.2-Å x-ray structure of S. aureus FakB2, activity assays, solution studies, site-directed mutagenesis, and in vivo complementation were used to define the functions of the five conserved residues that define the FakB protein family (Pfam02645). The fatty acid tail is buried within the protein, and the exposed carboxyl group is bound by a Ser-93-fatty acid carboxyl-Thr-61-His-266 hydrogen bond network. The guanidinium of the invariant Arg-170 is positioned to potentially interact with a bound acylphosphate. The reduced thermal denaturation temperatures of the T61A, S93A, and H266A FakB2 mutants illustrate the importance of the hydrogen bond network in protein stability. The FakB2 T61A, S93A, and H266A mutants are 1000-fold less active in the Fak assay, and the R170A mutant is completely inactive. All FakB2 mutants form FakA(FakB2)2 complexes except FakB2(R202A), which is deficient in FakA binding. Allelic replacement shows that strains expressing FakB2 mutants are defective in fatty acid incorporation into phospholipids and virulence gene transcription. These conserved residues are likely to perform the same critical functions in all bacterial fatty acid-binding proteins.

  11. pUNISHER: a high-level expression cassette for use with recombinant viral vectors for rapid and long term in vivo neuronal expression in the CNS.

    Science.gov (United States)

    Montesinos, Monica S; Chen, Zuxin; Young, Samuel M

    2011-12-01

    Fast onset and high-level neurospecific transgene expression in vivo is of importance for many areas in neuroscience, from basic to translational, and can significantly reduce the amount of vector load required to maintain transgene expression in vivo. In this study, we tested various cis elements to optimize transgene expression at transcriptional, posttranscriptional, and posttranslational levels and combined them together to create the high-level neuronal transgene expression cassette pUNISHER. Using a second-generation adenoviral vector system in combination with the pUNISHER cassette, we characterized its rate of onset of detectable expression and levels of expression compared with a neurospecific expression cassette driven by the 470-bp human synapsin promoter in vitro and in vivo. Our results demonstrate in primary neurons that the pUNISHER cassette, in a recombinant adenovirus type 5 background, led to a faster rate of onset of detectable transgene expression and higher level of transgene expression. More importantly, this cassette led to highly correlated neuronal expression in vivo and to stable transgene expression up to 30 days in the auditory brain stem with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held synapse. Thus the pUNISHER cassette is an ideal high-level neuronal expression cassette for use in vivo for neuroscience applications. PMID:21957229

  12. Analisis Tipe Staphylococcal Cassette Chromosome mec (SCCmec Isolat Methicillin Resistant Staphylococcus aureus (MRSA

    Directory of Open Access Journals (Sweden)

    Sunarjati Sudigdoadi

    2010-12-01

    Full Text Available Resistance of methicillin resistant Staphylococcus aureus (MRSA were based mainly on insertion of mobile genetic elements namely Staphylococcal cassette chromosome mec (SCCmec in the chromosome of Staphylococcus aureus. SCCmec consists of recombinase genes (ccr, mec genes complex, additional resistance genes, and insertion sequences. Recombinase genes structure mediates transfer of SCCmec from one bacteria to another. Identification of SCCmec is very important to know basic genetic resistance and to predict spreading of MRSA. The aim of this research was to analyze SCCmec type and antimicrobial susceptibility patterns. The design of this study was observational analytic study by typing SCCmec and antimicrobial susceptibility testing on July– December 2007. Isolation and identification of 45 MRSA isolates was performed in the Department of Microbiology, Faculty of Medicine, University of Padjadjaran, whereas identification of mecA gene and typing of SCCmec by multiplex PCR was performed in the Department of Microbiology, Faculty of Medicine, Sriwijaya University, Palembang. The result showed that all isolates contained mecA gene. Multiplex PCR revealed that 40 MRSA isolates had SCCmec type III and 5 isolates with type IV. All SCCmec type III isolates were multiresistant and all of the type IV were not multiresistant. In conclusion, MRSA isolates with SCCmec type III was associated with multiresistant whereas type IV was not.

  13. Product Variability of the ‘Cineole Cassette'Monoterpene Synthases of Related Nicotiana Species

    Institute of Scientific and Technical Information of China (English)

    Anke F(a)hnrich; Katrin Krause; Birgit Piechulla

    2011-01-01

    Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the ‘cineole cassere' comprising 1,8-cineole,limonene,myrcene,α-pinene,β-pinene,sabinene,and α-terpineol.We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes,which produced the characteristic monoterpenes of this ‘cineole cassette' with α-terpineol being most abundant in the volatile spectra.The amino acid sequences of both terpineol synthases were 99% identical.The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum.The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N.alata and N.langsdorfii were less efficient compared to the ‘cineole cassette′ monoterpene synthases of Arabidopsis thaliana,N.suaveolens,Salvia fruticosa,Salvia officinalis,and Citrus unshiu.The terpineol synthases of N.alata and N.langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals.The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N.alata was highest at the transition from day to night (diurnal rhythm).

  14. Energy use of televisions and video cassette recorders in the U.S.

    Energy Technology Data Exchange (ETDEWEB)

    Meier, Alan; Rosen, Karen

    1999-03-01

    In an effort to more accurately determine nationwide energy consumption, the U.S. Department of Energy has recently commissioned studies with the goal of improving its understanding of the energy use of appliances in the miscellaneous end-use category. This study presents an estimate of the residential energy consumption of two of the most common domestic appliances in the miscellaneous end-use category: color televisions (TVs) and video cassette recorders (VCRs). The authors used a bottom-up approach in estimating national TV and VCR energy consumption. First, they obtained estimates of stock and usage from national surveys, while TV and VCR power measurements and other data were recorded at repair and retail shops. Industry-supplied shipment and sales distributions were then used to minimize bias in the power measurement samples. To estimate national TV and VCR energy consumption values, ranges of power draw and mode usage were created to represent situations in homes with more than one unit. Average energy use values for homes with one unit, two units, etc. were calculated and summed to provide estimates of total national TV and VCR energy consumption.

  15. Human T24 Ha-ras cassette suitable for expression in eukaryotic cells

    Energy Technology Data Exchange (ETDEWEB)

    Bailleul, B.; Lang, J.; Wilkie, N.; Balmain, A.

    1988-01-11

    The T24 Ha-ras-1 oncogene is known to transform a wide range of eukaryotic cell types both in vivo and in vitro. Interpretation of the role played by ras genes in transformed cells would clearly by aided by employing a biological system capable of regulating ras p21 production. This can be achieved by substitution of the transcriptional control signals of Ha-ras with heterologous eukaryotic promoters. In this way, ras p21 may be expressed at high or low levels or inducibly expressed, depending on the promoter used. To facilitate construction of ras fusion genes, the authors have produced a T24 Ha-ras a cassette containing multiple cloning sites 5' to the Ha-ras coding exons. The polyadenylic site and the VTR region have been retained while the splice acceptor site upstream the ATG initiator codon has been deleted. The pR8-T24 construct should be useful for a variety of research applications, allowing insertion of any desired promoter into the 5' polylinker. In addition, potential regulatory elements could be inserted at the 3' end using the BamH1 site. This plasmid provides a great deal of flexibility in designing constructs which facilitate ras p21 expression in different cell types, either by transfection in vitro or in transgenic mice in vivo.

  16. Plug-and-Play Genetic Access to Drosophila Cell Types Using Exchangeable Exon Cassettes

    Science.gov (United States)

    Diao, Fengqiu; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C.; Ewer, John; Marr, Elizabeth; Potter, Christopher J.; Landgraf, Matthias; White, Benjamin H.

    2015-01-01

    Summary Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here we introduce a simple, versatile method for achieving cell type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e. introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system. PMID:25732830

  17. False positives observed on the Seratec® PSA SemiQuant Cassette Test with condom lubricants.

    Science.gov (United States)

    Bitner, Sara E

    2012-11-01

    In the course of the validation of a new component of the prostate-specific antigen (PSA) SemiQuant Cassette Test marketed by Seratec(®) , a false-positive reaction was observed when testing samples collected from the surface of unused, lubricated condoms. A variety of personal lubricants and condoms were tested to determine the frequency of the false positive, as well as its potential source. Samples were extracted in both water and the manufacturer-provided buffer, and the test was performed according to the manufacturer's suggested protocol. The false positive was observed intermittently, but occurred consistently with samples containing nonoxynol-9, a strong detergent utilized as a spermicide. The reaction may be attributable to the combination of latex and nonoxynol-9. Because of the unreliability of the test to confirm the presence of PSA in samples collected from condoms, the PSA cassette is an unsuitable method for confirming the presence of seminal fluid in condoms. PMID:22494324

  18. Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin

    DEFF Research Database (Denmark)

    Bertin, Gwladys I; Lavstsen, Thomas; Guillonneau, François;

    2013-01-01

    Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi......-conserved types defined by tandem runs of specific domains ("domain cassettes" (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection....

  19. A Novel Multiplex Real-Time PCR Assay for Rapid Typing of Major Staphylococcal Cassette Chromosome mec Elements

    Science.gov (United States)

    Francois, Patrice; Renzi, Gesuele; Pittet, Didier; Bento, Manuela; Lew, Daniel; Harbarth, Stephan; Vaudaux, Pierre; Schrenzel, Jacques

    2004-01-01

    We describe a novel procedure for rapid typing of the staphylococcal cassette chromosome mec element, a molecular marker allowing discrimination between community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains. Oligonucleotides targeting the recombinase genes were type specific and used to type a collection of 399 MRSA isolates recovered during patient screening at admission. This novel assay constitutes a valuable tool for evaluating the molecular epidemiology of MRSA and adjusting infection control strategies against MRSA. PMID:15243102

  20. A Novel Multiplex Real-Time PCR Assay for Rapid Typing of Major Staphylococcal Cassette Chromosome mec Elements

    OpenAIRE

    Francois, Patrice; Renzi, Gesuele; Pittet, Didier; Bento, Manuela; Lew, Daniel; Harbarth, Stephan; Vaudaux, Pierre; Schrenzel, Jacques

    2004-01-01

    We describe a novel procedure for rapid typing of the staphylococcal cassette chromosome mec element, a molecular marker allowing discrimination between community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains. Oligonucleotides targeting the recombinase genes were type specific and used to type a collection of 399 MRSA isolates recovered during patient screening at admission. This novel assay constitutes a valuable tool for evaluating the molecular epidemiol...

  1. Use of Cassette Dosing in Sandwich-Cultured Rat and Human Hepatocytes to Identify Drugs that Inhibit Bile Acid Transport

    OpenAIRE

    Kristina K Wolf; Vora, Sapana; Webster, Lindsey O.; Generaux, Grant T.; Polli, Joseph W; Brouwer, Kim L.R.

    2009-01-01

    Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid ...

  2. Staphylococcal Cassette Chromosome mec and Panton-Valentine Leukocidin Characterization of Methicillin-Resistant Staphylococcus aureus Clones▿

    OpenAIRE

    Moroney, Shannon M.; Heller, Loree C.; Arbuckle, Jesse; Talavera, Monica; Widen, Ray H.

    2006-01-01

    Staphylococcal cassette chromosome mec (SCCmec) types and Panton-Valentine leukocidin (PVL) gene carriage were compared among suspected community-associated methicillin-resistant Staphylococcus aureus MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) isolates. CA-MRSA isolates carried the SCCmec type IV complex, and most were PVL positive. The HA-MRSA isolates carried the SCCmec type II complex and did not harbor the PVL genes.

  3. Autoexcision of Bacterial Artificial Chromosome Facilitated by Terminal Repeat-Mediated Homologous Recombination: a Novel Approach for Generating Traceless Genetic Mutants of Herpesviruses ▿

    OpenAIRE

    Zhou, Fuchun; Li, Qiuhua; Wong, Scott W.; Gao, Shou-jiang

    2010-01-01

    Infectious bacterial artificial chromosomes (BACs) of herpesviruses are powerful tools for genetic manipulation. However, the presence of BAC vector sequence in the viral genomes often causes genetic and phenotypic alterations. While the excision of the BAC vector cassette can be achieved by homologous recombination between extra duplicate viral sequences or loxP site-mediated recombination, these methods either are inefficient or leave a loxP site mark in the viral genome. Here we describe t...

  4. Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis

    OpenAIRE

    Chang, W. L. William; Peter A Barry

    2003-01-01

    Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were re...

  5. Development of remote pipe cutting tool for divertor cassettes in JT-60SA

    International Nuclear Information System (INIS)

    Remote pipe cutting tool accessing from inside pipe has been newly developed for JT-60SA. The tool head equips a disk-shaped cutter blade and four rollers which are subjected to the reaction force. The tool pushes out the cutter blade by decreasing the distance between two cams. The tool cuts a cooling pipe by both pushing out the cutter blade and rotating the tool head itself. The roller holder is not pushed out anymore after touching the inner wall of the pipe. In other words, only cutter blade is pushed out after bringing the tool axis into the pipe axis. Outer diameter of the cutting tool head is 44 mm. The cutting tool is able to push out the cutter blade up to 32.5 mm in radius, i.e. 65 mm in diameter, which is enough to cut the pipe having an outer diameter of 59.8 mm. The thickness and material of the cooling pipe are 2.8 mm and SUS316L, respectively. The length of the cutting tool head is about 1 m. The tool is able to cut a pipe locates about 480 mm in depth from the mounting surface on the divertor cassette. The pipe cutting system equips two cutting heads and they are able to cut two pipes at the same time in order to remove the inner target plate. Reproducibility of the cross-sectional shape of the cut pipe is required for re-welding. The degree of reproducibility is inside 0.1 mm except for burr at outside of the pipe, which is enough to re-weld the cut pipe. Some swarf is generated during cutting the double-layered pipe assuming a plug located on the top of the pipe. The swarf is deposited on the bottom of the plug and collected by pulling out the plug in the actual equipment

  6. The Lophotrochozoan TGF-β signalling cassette - diversification and conservation in a key signalling pathway.

    Science.gov (United States)

    Kenny, Nathan J; Namigai, Erica K O; Dearden, Peter K; Hui, Jerome H L; Grande, Cristina; Shimeld, Sebastian M

    2014-01-01

    TGF-β signalling plays a key role in the patterning of metazoan body plans and growth. It is widely regarded as a 'module' capable of co-option into novel functions. The TGF-β pathway arose in the Metazoan lineage, and while it is generally regarded as well conserved across evolutionary time, its components have been largely studied in the Ecdysozoa and Deuterostomia. The recent discovery of the Nodal molecule in molluscs has underlined the necessity of untangling this signalling network in lophotrochozoans in order to truly comprehend the evolution, conservation and diversification of this key pathway. Three novel genome resources, the mollusc Patella vulgata, annelid Pomatoceros lamarcki and rotifer Brachionus plicatilis, along with other publicly available data, were searched for the presence of TGF-β pathway genes. Bayesian and Maximum Likelihood analyses, along with some consideration of conserved domain structure, was used to confirm gene identity. Analysis revealed conservation of key components within the canonical pathway, allied with extensive diversification of TGF-β ligands and partial loss of genes encoding pathway inhibitors in some lophotrochozoan lineages. We fully describe the TGF-β signalling cassette of a range of lophotrochozoans, allowing firm inference to be drawn as to the ancestral state of this pathway in this Superphylum. The TGF-β signalling cascade's reputation as being highly conserved across the Metazoa is reinforced. Diversification within the activin-like complement, as well as potential wide loss of regulatory steps in some Phyla, hint at specific evolutionary implications for aspects of this cascade's functionality in this Superphylum. PMID:25690968

  7. Comparative genome analysis: selection pressure on the Borrelia vls cassettes is essential for infectivity

    Directory of Open Access Journals (Sweden)

    Wilske Bettina

    2006-08-01

    Full Text Available Abstract Background At least three species of Borrelia burgdorferi sensu lato (Bbsl cause tick-borne Lyme disease. Previous work including the genome analysis of B. burgdorferi B31 and B. garinii PBi suggested a highly variable plasmid part. The frequent occurrence of duplicated sequence stretches, the observed plasmid redundancy, as well as the mainly unknown function and variability of plasmid encoded genes rendered the relationships between plasmids within and between species largely unresolvable. Results To gain further insight into Borreliae genome properties we completed the plasmid sequences of B. garinii PBi, added the genome of a further species, B. afzelii PKo, to our analysis, and compared for both species the genomes of pathogenic and apathogenic strains. The core of all Bbsl genomes consists of the chromosome and two plasmids collinear between all species. We also found additional groups of plasmids, which share large parts of their sequences. This makes it very likely that these plasmids are relatively stable and share common ancestors before the diversification of Borrelia species. The analysis of the differences between B. garinii PBi and B. afzelii PKo genomes of low and high passages revealed that the loss of infectivity is accompanied in both species by a loss of similar genetic material. Whereas B. garinii PBi suffered only from the break-off of a plasmid end, B. afzelii PKo lost more material, probably an entire plasmid. In both cases the vls gene locus encoding for variable surface proteins is affected. Conclusion The complete genome sequences of a B. garinii and a B. afzelii strain facilitate further comparative studies within the genus Borrellia. Our study shows that loss of infectivity can be traced back to only one single event in B. garinii PBi: the loss of the vls cassettes possibly due to error prone gene conversion. Similar albeit extended losses in B. afzelii PKo support the hypothesis that infectivity of Borrelia

  8. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    Management of bacterial infections is becoming increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens that are resistant to available antibiotics. Conventional antibiotics generally kill bacteria by interfering with vital cellular functions, an approach that ...

  9. Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

    Directory of Open Access Journals (Sweden)

    Quyen Thi Dinh

    2012-03-01

    Full Text Available Abstracts Background The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA and its chaperone (LipB from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems. Results To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB, six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein and only a small fraction of the overexpressed lipase was

  10. Prevention of bacterial adhesion

    DEFF Research Database (Denmark)

    Klemm, Per; Vejborg, Rebecca Munk; Hancock, Viktoria

    2010-01-01

    that imposes selection pressure for resistant bacteria. New approaches are urgently needed. Targeting bacterial virulence functions directly is an attractive alternative. An obvious target is bacterial adhesion. Bacterial adhesion to surfaces is the first step in colonization, invasion, and biofilm formation....... As such, adhesion represents the Achilles heel of crucial pathogenic functions. It follows that interference with adhesion can reduce bacterial virulence. Here, we illustrate this important topic with examples of techniques being developed that can inhibit bacterial adhesion. Some of these will become...

  11. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    Energy Technology Data Exchange (ETDEWEB)

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C. (MIT); (UT-Australia); (Macquarie); (Toronto); (New South)

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  12. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.

    Directory of Open Access Journals (Sweden)

    Chandrika N Deshpande

    Full Text Available The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes.We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators.Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  13. Investigation of integrons/cassettes in antimicrobial-resistant Escherichia coli isolated from food animals in China

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    In this study,326 Escherichia coli isolates from food animals collected during the last four decades in China were characterized using antimicrobial susceptibility testing and screening for integrons/cassettes.Minimum inhibitory concentration(MIC) testing indicated that the antimicrobial resistance of E.coli has increased since the 1970s.The findings of this study present a warning to veterinary practitioners about the excessive use of antimicrobials,and suggest the necessity for surveillance and control of antimicrobial resistance in veterinary clinical medicine in China.

  14. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes

    OpenAIRE

    Fine, Eli J; Appleton, Caleb M.; Douglas E. White; Brown, Matthew T.; Harshavardhan Deshmukh; Kemp, Melissa L.; Gang Bao

    2015-01-01

    CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in c...

  15. Concept design of the DEMO divertor cassette-to-vacuum vessel locking system adopting a systems engineering approach

    Energy Technology Data Exchange (ETDEWEB)

    Di Gironimo, G., E-mail: giuseppe.digironimo@unina.it [Università degli Studi di Napoli “Federico II”, Dipartimento di Ingegneria Industriale, Piazzale Tecchio 80, 80135 Napoli (Italy); Carfora, D. [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); VTT Technical Research Centre of Finland, Tekniikankatu 1, PO Box 1300, FI-33101 Tampere (Finland); Università degli Studi di Napoli “Federico II”, Dipartimento di Ingegneria Industriale, Piazzale Tecchio 80, 80135 Napoli (Italy); Esposito, G.; Lanzotti, A.; Marzullo, D. [Università degli Studi di Napoli “Federico II”, Dipartimento di Ingegneria Industriale, Piazzale Tecchio 80, 80135 Napoli (Italy); Siuko, M. [VTT Technical Research Centre of Finland, Tekniikankatu 1, PO Box 1300, FI-33101 Tampere (Finland)

    2015-05-15

    Highlights: • An iterative and incremental design process for cassette-to-VV locking system of DEMO divertor is presented. • Three different concepts have been developed with a systematic design approach. • The final concept has been selected with Fuzzy-Analytic Hierarchy Process in virtual reality. - Abstract: This paper deals with pre-concept studies of DEMO divertor cassette-to-vacuum vessel locking system under the work program WP13-DAS-07-T06: Divertor Remote Maintenance System pre-concept study. An iterative design process, consistent with Systems Engineering guidelines and named Iterative and Participative Axiomatic Design Process (IPADeP), is used in this paper to propose new innovative solutions for divertor locking system, which can overcome the difficulties in applying the ITER principles to DEMO. The solutions conceived have been analysed from the structural point of view using the software Ansys and, eventually, evaluated using the methodology known as Fuzzy-Analytic Hierarchy Process. Due to the lack and the uncertainty of the requirements in this early conceptual design stage, the aim is to cover a first iteration of an iterative and incremental process to propose an innovative design concept to be developed in more details as the information will be completed.

  16. Concept design of the DEMO divertor cassette-to-vacuum vessel locking system adopting a systems engineering approach

    International Nuclear Information System (INIS)

    Highlights: • An iterative and incremental design process for cassette-to-VV locking system of DEMO divertor is presented. • Three different concepts have been developed with a systematic design approach. • The final concept has been selected with Fuzzy-Analytic Hierarchy Process in virtual reality. - Abstract: This paper deals with pre-concept studies of DEMO divertor cassette-to-vacuum vessel locking system under the work program WP13-DAS-07-T06: Divertor Remote Maintenance System pre-concept study. An iterative design process, consistent with Systems Engineering guidelines and named Iterative and Participative Axiomatic Design Process (IPADeP), is used in this paper to propose new innovative solutions for divertor locking system, which can overcome the difficulties in applying the ITER principles to DEMO. The solutions conceived have been analysed from the structural point of view using the software Ansys and, eventually, evaluated using the methodology known as Fuzzy-Analytic Hierarchy Process. Due to the lack and the uncertainty of the requirements in this early conceptual design stage, the aim is to cover a first iteration of an iterative and incremental process to propose an innovative design concept to be developed in more details as the information will be completed

  17. Vimentin in Bacterial Infections

    Directory of Open Access Journals (Sweden)

    Tim N. Mak

    2016-04-01

    Full Text Available Despite well-studied bacterial strategies to target actin to subvert the host cell cytoskeleton, thus promoting bacterial survival, replication, and dissemination, relatively little is known about the bacterial interaction with other components of the host cell cytoskeleton, including intermediate filaments (IFs. IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge about the role of IFs in bacterial infections, focusing on the type III IF protein vimentin. Recent studies have revealed the involvement of vimentin in host cell defenses, acting as ligand for several pattern recognition receptors of the innate immune system. Two main aspects of bacteria-vimentin interactions are presented in this review: the role of vimentin in pathogen-binding on the cell surface and subsequent bacterial invasion and the interaction of cytosolic vimentin and intracellular pathogens with regards to innate immune signaling. Mechanistic insight is presented involving distinct bacterial virulence factors that target vimentin to subvert its function in order to change the host cell fate in the course of a bacterial infection.

  18. Diverse gene cassettes in class 1 integrons of facultative oligotrophic bacteria of River Mahananda,West Bengal, India.

    Directory of Open Access Journals (Sweden)

    Ranadhir Chakraborty

    Full Text Available BACKGROUND: In this study a large random collection (n=2188 of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007-2009 from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products. METHODOLOGY/PRINCIPAL FINDINGS: Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19% were antibiotic-resistant comprising of both single-antibiotic resistance (SAR and multiple-antibiotic resistant (MAR, and 521 (23.81% were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s of varying sizes from 0.15 to 3.45 KB. Chi-square (χ(2 test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6'-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families

  19. A Study on the Measurement of the Pollution Level of Bacteria and Disinfection of Table and IP Cassette

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Seok Hwan; Lee, Moo Sik; Lim, Chang Seon; Kim, Gha Jung [Koyang University, Koyang (Korea, Republic of)

    2008-09-15

    For the number of microbes and the pollution level of bacteriology of IP Cassette and Table by laboratories, after identifying the bacteria before and after using alcohol and tissue including disinfectant and statistically testing, this research was intended to provide the basic data for the prevention and the right disinfection guideline for infection management of hospitals in diagnostic radiology. The subject of this research was the general room of diagnostic radiology of a university hospital in Daejeon City. The research was conducted from Apr 5 to Apr 12, 2007. The number of microbes and the pollution level of bacteriology of IP Cassette and Table by laboratories were tested before and after using alcohol and tissue for disinfection including disinfectant. In order to collect specimens exactly, they were collected with the nurse who specialized in infection management of the hospital, and statistical processing was done with SPSS V13.0. To compare the results before and after using alcohol and tissue, T-test was implemented, and post-hoc test was conducted. Bacteria were detected in 19 cases of 24 subjects(79.2%), however, they were not detected in 5 cases(20.8%). 7 kinds of bacteria were detected as isolated bacteria, of which Methicillin Resistant coagulase-negative Staphylococci(MRCNS) were detected in 15 cases(62.5%), which was most, Methicillin Resistant Staphylococcus Aureus(MRSA) in 6 cases(16.7%), Enterococcus Faecium(EFM) in 5 cases(20.8%), Acinetobacter baumannii(ABA) in 2 cases(8.3%), and Bacillus sp, Coagulase-negative Staphylococci(CNS), and Enterococcus sp(ENT) in 1 case, respectively(4.2%). In all bacteria except ABA, Gram positive bacillia were detected in 30 cases(97%), and Gram negative bacilli were detected only in 1 case(3%). As for the kinds of bacteria and the number of groups before and after using 70% Alcohol by Groups, when the bacteria were identified after disinfecting IP Cassette and Table with 70% Alcohol, all the bacteria became

  20. Plasmodium falciparum Expressing Domain Cassette 5 Type PfEMP1 (DC5-PfEMP1) Bind PECAM1

    DEFF Research Database (Denmark)

    Berger, Sanne S; Turner, Louise; Wang, Christian W;

    2013-01-01

    Members of the Plasmodium falciparum Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been...... associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more...... likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5...

  1. WBC27, an Adenosine Tri-phosphate-binding Cassette Protein, Controls Pollen Wall Formation and Patterning in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ying Dou; Ke-Zhen Yang; Yi Zhang; Wei Wang; Xiao-Lei Liu; Li-Qun Chen; Xue-Qin Zhang; De Ye

    2011-01-01

    In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores.

  2. A Study on the Measurement of the Pollution Level of Bacteria and Disinfection of Table and IP Cassette

    International Nuclear Information System (INIS)

    For the number of microbes and the pollution level of bacteriology of IP Cassette and Table by laboratories, after identifying the bacteria before and after using alcohol and tissue including disinfectant and statistically testing, this research was intended to provide the basic data for the prevention and the right disinfection guideline for infection management of hospitals in diagnostic radiology. The subject of this research was the general room of diagnostic radiology of a university hospital in Daejeon City. The research was conducted from Apr 5 to Apr 12, 2007. The number of microbes and the pollution level of bacteriology of IP Cassette and Table by laboratories were tested before and after using alcohol and tissue for disinfection including disinfectant. In order to collect specimens exactly, they were collected with the nurse who specialized in infection management of the hospital, and statistical processing was done with SPSS V13.0. To compare the results before and after using alcohol and tissue, T-test was implemented, and post-hoc test was conducted. Bacteria were detected in 19 cases of 24 subjects(79.2%), however, they were not detected in 5 cases(20.8%). 7 kinds of bacteria were detected as isolated bacteria, of which Methicillin Resistant coagulase-negative Staphylococci(MRCNS) were detected in 15 cases(62.5%), which was most, Methicillin Resistant Staphylococcus Aureus(MRSA) in 6 cases(16.7%), Enterococcus Faecium(EFM) in 5 cases(20.8%), Acinetobacter baumannii(ABA) in 2 cases(8.3%), and Bacillus sp, Coagulase-negative Staphylococci(CNS), and Enterococcus sp(ENT) in 1 case, respectively(4.2%). In all bacteria except ABA, Gram positive bacillia were detected in 30 cases(97%), and Gram negative bacilli were detected only in 1 case(3%). As for the kinds of bacteria and the number of groups before and after using 70% Alcohol by Groups, when the bacteria were identified after disinfecting IP Cassette and Table with 70% Alcohol, all the bacteria became

  3. Functions of the conserved thrombospondin carboxy-terminal cassette in cell-extracellular matrix interactions and signaling.

    Science.gov (United States)

    Adams, Josephine C

    2004-06-01

    Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that function at cell surfaces, in extracellular matrix (ECM) and as bridging molecules in cell-cell interactions. TSPs are multifunctional and modulate cell behavior during development, wound-healing, immune response, tumor growth and in the homeostasis of adult tissues. TSPs are assembled as oligomers that are composed of homologous polypeptides. In all the TSP polypeptides, the most highly-conserved region is the carboxyl-region, which contains a characteristic set of domains comprising EGF domains, TSP type 3 repeats and a globular carboxy-terminal domain. This large region is termed here the thrombospondin carboxy-terminal cassette (TSP-CTC). The strong conservation of the TSP-CTC suggests that it may mediate ancestral functions that are shared by all TSPs. This review summarizes the current knowledge of the TSP-CTC and areas of future interest. PMID:15094125

  4. Virtual screening of ABCC1 transporter nucleotidebinding domains as a therapeutic target in multidrug resistant cancer

    OpenAIRE

    Rungsardthong, Kanin; Mares- Sámano, Sergio; Penny, Jeffrey

    2012-01-01

    ABCC1 is a member of the ATP-binding Cassette super family of transporters, actively effluxes xenobiotics from cells. Clinically, ABCC1 expression is linked to cancer multidrug resistance. Substrate efflux is energised by ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) and inhibition of these events may help combat drug resistance. The aim of this study is to identify potential inhibitors of ABCC1 through virtual screening of National Cancer Institute (NCI) compounds. A th...

  5. Interfering with bacterial gossip

    DEFF Research Database (Denmark)

    Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Givskov, Michael

    2011-01-01

    defense. Antibiotics exhibit a rather limited effect on biofilms. Furthermore, antibiotics have an ‘inherent obsolescence’ because they select for development of resistance. Bacterial infections with origin in bacterial biofilms have become a serious threat in developed countries. Pseudomonas aeruginosa...... that appropriately target bacteria in their relevant habitat with the aim of mitigating their destructive impact on patients. In this review we describe molecular mechanisms involved in “bacterial gossip” (more scientifically referred to as quorum sensing (QS) and c-di-GMP signaling), virulence, biofilm formation...

  6. In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice.

    Science.gov (United States)

    Amps, K J; Jones, M; Baker, D; Moore, H D

    2010-06-01

    The development of efficient and robust methods for the cryopreservation of human embryonic stem cells (hESCs) is important for the production of master and working cell banks for future clinical applications. Such methods must meet requirements of good manufacturing practice (GMP) and maintain genetic stability of the cell line. We investigated the culture of four Shef hESC lines in gas permeable 'culture cassettes' which met GMP compliance. hESCs adhered rapidly to the membrane and colonies displayed good proliferation and expansion. After 5-7 days of culture, hESCs were cryopreserved in situ using 10% dimethyl sulphoxide in foetal calf serum at approximately 1 degrees C/min. This method was compared with a control of standard flask culture and cryopreservation in vials. Post-thaw cassette culture displayed relative proliferation ratios (fold increase above flask/cryovial culture) of 114 (Shef 4), 8.2 (Shef 5), 195 (shef 6) and 17.5 (Shef 7). The proportion of cells expressing pluripotency markers after cryopreservation was consistently greater in cassette culture than for the control with the markers SSEA3 and SSEA4 exhibiting a significant increase (P> or =0.05). The efficiency of cell line culture in cassette was associated with the overall passage number of the cell line. The procedure enables cryopreservation of relatively large quantities of hESCs in situ, whilst returning high yields of viable, undifferentiated stem cells, thereby increasing capacity to scale up with greater efficacy.

  7. Application of Cassette Ultracentrifugation Using Non-labeled Compounds and Liquid Chromatography-Tandem Mass Spectrometry Analysis for High-Throughput Protein Binding Determination.

    Science.gov (United States)

    Kieltyka, Kasia; McAuliffe, Brian; Cianci, Christopher; Drexler, Dieter M; Shou, Wilson; Zhang, Jun

    2016-03-01

    Membrane-based devices typically used for serum protein binding determination are not fully applicable to highly lipophilic compounds because of nonspecific binding to the device membrane. Ultracentrifugation, however, completely eliminates the issue by using a membrane-free approach, although its wide application has been limited. This lack of utilization is mainly attributed to 2 factors: the high cost in acquiring and handling of radiolabeled compounds and low assay throughput owing to the difficulties in process automation. To overcome these challenges, we report a high-throughput workflow by cassette ultracentrifugation of nonradiolabeled compounds followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Twenty compounds with diverse physicochemical and protein binding properties were selected for the evaluation of the workflow. To streamline the working process, approaches of matrix balancing for all the samples for LC-MS/MS analysis and determining free fraction without analytical calibration curves were adopted. Both the discrete ultracentrifugation of individual compounds and cassette ultracentrifugation of all the test compounds followed by simultaneous LC-MS/MS analysis exhibited a linear correlation with literature values, demonstrating respectively the validity of the ultracentrifugation process and the cassette approach. The cassette ultracentrifugation using nonradiolabeled compounds followed by LC-MS/MS analysis has greatly facilitated its application for high-throughput protein binding screening in drug discovery. PMID:26886323

  8. Sterol transporter adenosine triphosphate-binding cassette transporter G8, gallstones, and biliary cancer in 62,000 individuals from the general population

    DEFF Research Database (Denmark)

    Stender, Stefan; Frikke-Schmidt, Ruth; Nordestgaard, Børge G;

    2011-01-01

    Gallstone disease, a risk factor for biliary cancer, has a strong heritable component, but the underlying genes are largely unknown. To test the hypothesis that ABCG8 (adenosine triphosphate-binding cassette transporter G8) Asp19His (D19H) genotype predicted risk of gallstones and biliary cancer ...

  9. Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1.

    Science.gov (United States)

    Isegawa, Y; Sheng, J; Sokawa, Y; Yamanishi, K; Nakagomi, O; Ueda, S

    1992-12-01

    A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Bacterial Wound Culture

    Science.gov (United States)

    ... Home Visit Global Sites Search Help? Bacterial Wound Culture Share this page: Was this page helpful? Also known as: Aerobic Wound Culture; Anaerobic Wound Culture Formal name: Culture, wound Related ...

  11. Bacterial surface adaptation

    Science.gov (United States)

    Utada, Andrew

    2014-03-01

    Biofilms are structured multi-cellular communities that are fundamental to the biology and ecology of bacteria. Parasitic bacterial biofilms can cause lethal infections and biofouling, but commensal bacterial biofilms, such as those found in the gut, can break down otherwise indigestible plant polysaccharides and allow us to enjoy vegetables. The first step in biofilm formation, adaptation to life on a surface, requires a working knowledge of low Reynolds number fluid physics, and the coordination of biochemical signaling, polysaccharide production, and molecular motility motors. These crucial early stages of biofilm formation are at present poorly understood. By adapting methods from soft matter physics, we dissect bacterial social behavior at the single cell level for several prototypical bacterial species, including Pseudomonas aeruginosa and Vibrio cholerae.

  12. Bacterial intermediate filaments

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Cabeen, M.; Jacobs-Wagner, C.

    2009-01-01

    Crescentin, which is the founding member of a rapidly growing family of bacterial cytoskeletal proteins, was previously proposed to resemble eukaryotic intermediate filament (IF) proteins based on structural prediction and in vitro polymerization properties. Here, we demonstrate that crescentin...

  13. Bacterial Meningitis in Infants

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-04-01

    Full Text Available A retrospective study of 80 infantile patients (ages 30-365 days; 47 male, 33 female with culture-proven bacterial meningitis seen over a 16 year period (1986-2001 is reported from Taiwan.

  14. Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

    Science.gov (United States)

    Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

    2010-03-01

    The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins. PMID:20208152

  15. Apo and ligand-bound structures of ModA from the archaeon Methanosarcina acetivorans.

    Science.gov (United States)

    Chan, Sum; Giuroiu, Iulia; Chernishof, Irina; Sawaya, Michael R; Chiang, Janet; Gunsalus, Robert P; Arbing, Mark A; Perry, L Jeanne

    2010-03-01

    The trace-element oxyanion molybdate, which is required for the growth of many bacterial and archaeal species, is transported into the cell by an ATP-binding cassette (ABC) transporter superfamily uptake system called ModABC. ModABC consists of the ModA periplasmic solute-binding protein, the integral membrane-transport protein ModB and the ATP-binding and hydrolysis cassette protein ModC. In this study, X-ray crystal structures of ModA from the archaeon Methanosarcina acetivorans (MaModA) have been determined in the apoprotein conformation at 1.95 and 1.69 A resolution and in the molybdate-bound conformation at 2.25 and 2.45 A resolution. The overall domain structure of MaModA is similar to other ModA proteins in that it has a bilobal structure in which two mixed alpha/beta domains are linked by a hinge region. The apo MaModA is the first unliganded archaeal ModA structure to be determined: it exhibits a deep cleft between the two domains and confirms that upon binding ligand one domain is rotated towards the other by a hinge-bending motion, which is consistent with the 'Venus flytrap' model seen for bacterial-type periplasmic binding proteins. In contrast to the bacterial ModA structures, which have tetrahedral coordination of their metal substrates, molybdate-bound MaModA employs octahedral coordination of its substrate like other archaeal ModA proteins.

  16. The bacterial lipocalins.

    Science.gov (United States)

    Bishop, R E

    2000-10-18

    The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with two of its closest homologues, apolipoprotein D and lazarillo. Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells.

  17. Diagnosis of bacterial vaginosis

    Directory of Open Access Journals (Sweden)

    Đukić Slobodanka

    2013-01-01

    Full Text Available Bacterial vaginosis is a common, complex clinical syndrome characterized by alterations in the normal vaginal flora. When symptomatic, it is associated with a malodorous vaginal discharge and on occasion vaginal burning or itching. Under normal conditions, lactobacilli constitute 95% of the bacteria in the vagina. Bacterial vaginosis is associated with severe reduction or absence of the normal H2O2­producing lactobacilli and overgrowth of anaerobic bacteria and Gardnerella vaginalis, Atopobium vaginae, Mycoplasma hominis and Mobiluncus species. Most types of infectious disease are diagnosed by culture, by isolating an antigen or RNA/DNA from the microbe, or by serodiagnosis to determine the presence of antibodies to the microbe. Therefore, demonstration of the presence of an infectious agent is often a necessary criterion for the diagnosis of the disease. This is not the case for bacterial vaginosis, since the ultimate cause of the disease is not yet known. There are a variety of methods for the diagnosis of bacterial vaginosis but no method can at present be regarded as the best. Diagnosing bacterial vaginosis has long been based on the clinical criteria of Amsel, whereby three of four defined criteria must be satisfied. Nugent’s scoring system has been further developed and includes validation of the categories of observable bacteria structures. Up­to­date molecular tests are introduced, and better understanding of vaginal microbiome, a clear definition for bacterial vaginosis, and short­term and long­term fluctuations in vaginal microflora will help to better define molecular tests within the broader clinical context.

  18. Bacterial glycosyltransferase toxins.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

  19. Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

    KAUST Repository

    Hill-Cawthorne, Grant A.

    2014-06-27

    Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

  20. Subtyping of Chilean Methicillin-Resistant Staphylococcus aureus strains carrying the staphylococcal cassette chromosome mec type I

    Directory of Open Access Journals (Sweden)

    Gustavo Medina

    2012-09-01

    Full Text Available The cassette chromosome mec (SCCmec present in methicillin-resistant Staphylococcus aureus (MRSA has two essential components, the ccr gene complex and the mec gene complex. Additionally, SCCmec has non-essential components called J regions which are used for MRSA subtyping. This study was performed to determine subtypes MRSA strains carrying SCCmec type I based on polymorphism of regions located downstream of the mecA gene. A total of 98 MRSA strains carrying SCCmec type I isolated from patients hospitalized at the County Hospital of Valdivia (Chile between May 2007 and May 2008, were analyzed by multiplex PCR designed to amplify the mecA gene and 7 DNA hypervariable regions located around the mecA gene. MRSA strains were classified into seventeen genotypes accordingly to amplification patterns of DNA hypervariable regions. Five genotypes showed amplification patterns previously described. The remaining twelve genotypes showed new amplification patterns. Genotypes 18 and Genotype 19 were the most frequently detected. Regions HVR, Ins117 and pI258 stand out as being present in more than 60% of tested isolates. The acquisition of hypervariable regions by MRSA is a continuous horizontal transfer process through which the SCCmec have been preserved intact, or even may give rise to new types and subtypes of SCCmec. Therefore it is possible to infer that most MRSA strains isolated at the County Hospital of Valdivia (Chile were originated from two local clones which correspond to Genotype 18 and Genotype 19.

  1. Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene Replacement with eg3 Gene Expression Cassette

    Institute of Scientific and Technical Information of China (English)

    Tian-Hong WANG; Ti LIU; Zhi-Hong WU; Shi-Li LIU; Yi LU; Yin-Bo QU

    2004-01-01

    To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.

  2. Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes

    Directory of Open Access Journals (Sweden)

    Mancini Cecilia

    2011-10-01

    Full Text Available Abstract In the preparation of transgenic murine ES cells it is important to verify the construct has a single insertion, because an ectopic neomycin phosphortransferase positive selection cassette (NEO may cause a position effect. During a recent work, where a knockin SCA28 mouse was prepared, we developed two assays based on Real-Time PCR using both SYBR Green and specific minor groove binder (MGB probes to evaluate the copies of NEO using the comparative delta-delta Ct method versus the Rpp30 reference gene. We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D., and were clearly distinguishable from clones with two or more NEO copies. This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.

  3. Methicillin-resistant Staphylococcus saprophyticus isolates carrying staphylococcal cassette chromosome mec have emerged in urogenital tract infections.

    Science.gov (United States)

    Higashide, Masato; Kuroda, Makoto; Omura, Carlos Takashi Neves; Kumano, Miyuki; Ohkawa, Saburo; Ichimura, Sadahiro; Ohta, Toshiko

    2008-06-01

    Staphylococcus saprophyticus is a uropathogenic bacterium that causes acute uncomplicated urinary tract infections, particularly in female outpatients. We investigated the dissemination and antimicrobial susceptibilities of 101 S. saprophyticus isolates from the genitourinary tracts of patients in Japan. Eight of these isolates were mecA positive and showed beta-lactam resistance. Pulsed-field gel electrophoresis showed that only some isolates were isogenic, indicating that the mecA gene was apparently acquired independently by mecA-positive isolates through staphylococcal cassette chromosome mec (SCCmec). Type determination of SCCmec by multiplex PCR showed a nontypeable element in the eight mecA-positive isolates. Sequence analysis of the entire SCCmec element from a prototype S. saprophyticus strain revealed that it was nontypeable with the current SCCmec classification due to the novel composition of the class A mec gene complex (IS431-mecA-mecR1-mecI genes) and the ccrA1/ccrB3 gene complex. Intriguingly, the attachment sites of SCCmec are similar to those of type I SCCmec in S. aureus NCTC 10442. Furthermore, the genes around the mec gene complex are similar to those of type II/III SCCmec in S. aureus, while those around the ccr gene complex are similar to those of SCC15305RM found in S. saprophyticus ATCC 15305. In comparison with known SCCmec elements, this S. saprophyticus SCCmec is a novel type.

  4. Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Grant A Hill-Cawthorne

    Full Text Available Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec. We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA, a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4% of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

  5. Diversity of staphylococcal cassette chromosome mec elements in predominant methicillin-resistant Staphylococcus aureus clones in a small geographic area.

    Science.gov (United States)

    Basset, Patrick; Senn, Laurence; Vogel, Valérie; Zanetti, Giorgio; Blanc, Dominique S

    2010-11-01

    Recent population genetic studies suggest that staphylococcal cassette chromosome mec (SCCmec) was acquired much more frequently than previously thought. In the present study, we aimed to investigate the diversity of SCCmec elements in a local methicillin-resistant Staphylococcus aureus (MRSA) population. Each MRSA isolate (one per patient) recovered in the Vaud canton of Switzerland from January 2005 to December 2008 was analyzed by the double-locus sequence typing (DLST) method and SCCmec typing. DLST analysis indicated that 1,884/2,036 isolates (92.5%) belong to four predominant clones. As expected from the local spread of a clone, most isolates within clones harbored an identical SCCmec type. However, three to seven SCCmec types have been recovered in every predominant DLST clone, suggesting that some of these elements might have been acquired locally. This pattern could also be explained by distinct importations of related isolates into the study region. The addition of a third highly variable locus to further increase the discriminatory power of typing as well as epidemiological data suggested that most ambiguous situations were explained by the second hypothesis. In conclusion, our study showed that even if the acquisition of new SCCmec elements at a local level likely occurs, it does not explain all the diversity observed in the study region. PMID:20713672

  6. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    Science.gov (United States)

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  7. Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

    Science.gov (United States)

    Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

    2008-04-01

    The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

  8. Construction of a Trp- commercial baker's yeast strain by using food-safe-grade dominant drug resistance cassettes.

    Science.gov (United States)

    Estruch, Francisco; Prieto, José Antonio

    2003-12-01

    We have designed a food-safe-grade module for gene disruptions in commercial baker's yeast strains, which contains the G418 resistance cassette, KanMX4, flanked by direct repeats from the MEL1 gene of Saccharomyces cerevisiae. This module was used to obtain a Trp(-) auxotrophic mutant of the polyploid HY strain by successive targeting to the TRP1 locus and later in vivo excision of the kan(r) marker. Southern blot analysis indicated that HY contains five copies of the TRP1 gene. However, after four disruption rounds, a strain named HYtrpM(4), unable to grow in the absence of tryptophan, was selected. Southern and Northern analysis of HYtrpM(4) cells showed that a remaining functional wild-type copy was still present, suggesting that the level of phosphoribosylanthranylate isomerase activity, resulting from a single copy of TRP1, is too low to sustain growth. Accordingly, a high reversion frequency of the Trp(-) phenotype, through gene conversion, was found in cells of the mutant strain. Nevertheless, this was not a drawback for its use as a recipient strain of heterologous genes. Indeed, YEpACT-X24 transformants were stable after 25 generations and expressed and secreted high levels of active recombinant xylanase. These data indicate that the new Trp(-) strain can be used to generate a stable recombinant yeast that fulfils all the requirements and market criteria for commercial utilisation.

  9. Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin.

    Directory of Open Access Journals (Sweden)

    Gwladys I Bertin

    Full Text Available BACKGROUND: Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1 is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains ("domain cassettes" (DC. The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection. METHODS: Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM. We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS. RESULTS: Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples. CONCLUSION: This is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis.

  10. 多重PCR检测MRSA的SCCmec基因分型%Staphylococcal chromosome cassette typing in MRSA by multiplex PCR

    Institute of Scientific and Technical Information of China (English)

    韩玉涛; 蒋燕群

    2008-01-01

    目的 了解我院MRSA的流行状况.方法 收集2005年1-6月65株社区感染MRSA及60株医院感染MRSA,应用多重PCR对MRSA染色体mec基因盒(Staphylococcal cassette chromosome SCCmec)分型及杀白细胞毒素(PVL)基因检测,应用K-B纸片法进行药敏分析.结果 125株MRSA的mecA基因阳性,其中SCCmecⅡ型1株,SCCmecⅢ型120株,SCCmecⅣ型3株,未分型1株;未发现携带PVL基因的MRSA.携带SCCmecⅡ型、SCCmecⅢ型的菌株均为多重耐药株,而携带SCCmecⅣ型的菌株除对β内酰胺类药物耐药外,对其他类别的抗菌药敏感.结论 本院分离的MRSA以SCCmecⅢ型为主,发现SCCmecⅣ型CA-MRSA,但不携带PVL基因;携带SCCmecⅡ、SCCmecⅢ的临床分离株耐药严重.

  11. Effect of genomic location on horizontal transfer of a recombinant gene cassette between Pseudomonas strains in the rhizosphere and spermosphere of barley seedlings

    DEFF Research Database (Denmark)

    Sengelov, G.; Kristensen, K. J.; Sørensen, Anders Morten Hay;

    2001-01-01

    , horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas strutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into...... a conjugative plasmid was 8.20 x 10(-3) transconjugants/(donors x recipients)(1/2) in the rhizosphere and 4.57 x 10(-2) transconjugants/(donors x recipients)(1/2) in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms...

  12. Seizures Complicating Bacterial Meningitis

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-09-01

    Full Text Available The clinical data of 116 patients, 1 month to <5 years of age, admitted for bacterial meningitis, and grouped according to those with and without seizures during hospitalization, were compared in a study at Buddhist Dalin Tzu Chi General Hospital, Chang Gung Memorial Hospital and other centers in Taiwan.

  13. Bacterial extracellular lignin peroxidase

    Science.gov (United States)

    Crawford, Donald L.; Ramachandra, Muralidhara

    1993-01-01

    A newly discovered lignin peroxidase enzyme is provided. The enzyme is obtained from a bacterial source and is capable of degrading the lignin portion of lignocellulose in the presence of hydrogen peroxide. The enzyme is extracellular, oxidative, inducible by lignin, larch wood xylan, or related substrates and capable of attacking certain lignin substructure chemical bonds that are not degradable by fungal lignin peroxidases.

  14. Bacterial Skin Infections

    Science.gov (United States)

    ... or scraped, the injury should be washed with soap and water and covered with a sterile bandage. Petrolatum may be applied to open areas to keep the tissue moist and to try to prevent bacterial invasion. Doctors recommend that people do not use ...

  15. Vimentin in Bacterial Infections

    DEFF Research Database (Denmark)

    Mak, Tim N; Brüggemann, Holger

    2016-01-01

    filaments (IFs). IFs have not only roles in maintaining the structural integrity of the cell, but they are also involved in many cellular processes including cell adhesion, immune signaling, and autophagy, processes that are important in the context of bacterial infections. Here, we summarize the knowledge...

  16. Bacterial microflora of nectarines

    Science.gov (United States)

    Microflora of fruit surfaces has been the best source of antagonists against fungi causing postharvest decays of fruit. However, there is little information on microflora colonizing surfaces of fruits other than grapes, apples, and citrus fruit. We characterized bacterial microflora on nectarine f...

  17. Modeling intraocular bacterial infections.

    Science.gov (United States)

    Astley, Roger A; Coburn, Phillip S; Parkunan, Salai Madhumathi; Callegan, Michelle C

    2016-09-01

    Bacterial endophthalmitis is an infection and inflammation of the posterior segment of the eye which can result in significant loss of visual acuity. Even with prompt antibiotic, anti-inflammatory and surgical intervention, vision and even the eye itself may be lost. For the past century, experimental animal models have been used to examine various aspects of the pathogenesis and pathophysiology of bacterial endophthalmitis, to further the development of anti-inflammatory treatment strategies, and to evaluate the pharmacokinetics and efficacies of antibiotics. Experimental models allow independent control of many parameters of infection and facilitate systematic examination of infection outcomes. While no single animal model perfectly reproduces the human pathology of bacterial endophthalmitis, investigators have successfully used these models to understand the infectious process and the host response, and have provided new information regarding therapeutic options for the treatment of bacterial endophthalmitis. This review highlights experimental animal models of endophthalmitis and correlates this information with the clinical setting. The goal is to identify knowledge gaps that may be addressed in future experimental and clinical studies focused on improvements in the therapeutic preservation of vision during and after this disease. PMID:27154427

  18. Synthesis of 5-oxyquinoline derivatives for reversal of multidrug resistance

    Directory of Open Access Journals (Sweden)

    Torsten Dittrich

    2012-10-01

    Full Text Available The inhibition of ABC (ATP binding cassette transporters is considered a powerful tool to reverse multidrug resistance. Zosuquidar featuring a difluorocyclopropyl-annulated dibenzosuberyl moiety has been found to be an inhibitor of the P-glycoprotein, one of the best-studied multidrug efflux pumps. Twelve 5-oxyisoquinoline derivatives, which are analogues of zosuquidar wherein the dibenzosuberyl-piperazine moiety is replaced by either a diarylaminopiperidine or a piperidone-derived acetal or thioacetal group, have been synthesized as pure enantiomers. Their inhibitory power has been evaluated for the bacterial multidrug-resistance ABC transporter LmrCD and fungal Pdr5. Four of the newly synthesized compounds reduced the transport activity to a higher degree than zosuquidar, being up to fourfold more efficient than the lead compound in the case of LmrCD and about two times better for Pdr5.

  19. Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

    International Nuclear Information System (INIS)

    Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr324 in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated

  20. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    OpenAIRE

    Tetsushi Sakuma; Mitsumasa Takenaga; Yoshinori Kawabe; Takahiro Nakamura; Masamichi Kamihira; Takashi Yamamoto

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, includi...

  1. Staphylococcal Cassette Chromosome mec (SCCmec) Typing of Methicillin-Resistant Staphylococcus aureus Strains Isolated in 11 Asian Countries: a Proposal for a New Nomenclature for SCCmec Elements

    OpenAIRE

    Chongtrakool, Piriyaporn; Ito, Teruyo; Ma, Xiao Xue; Kondo, Yoko; Trakulsomboon, Suwanna; Tiensasitorn, Chuntima; Jamklang, Mantana; Chavalit, Tavinun; Song, Jae-Hoon; Hiramatsu, Keiichi

    2006-01-01

    A description of staphylococcal cassette chromosome mec (SCCmec) elements carried by 615 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 11 Asian countries is reported, and a novel nomenclatural system based on their structures is proposed. The 615 strains were classified as type 3A (370 strains), type 2A (207 strains), type 2B (32 strains), type 1B (1 strain), and nontypeable (5 strains). The previously reported type III SCCmec (DDBJ/EMBL/GenBank accession no. AB037671...

  2. Novel Multiplex PCR Assay for Characterization and Concomitant Subtyping of Staphylococcal Cassette Chromosome mec Types I to V in Methicillin-Resistant Staphylococcus aureus

    OpenAIRE

    Zhang, Kunyan; McClure, Jo-Ann; Elsayed, Sameer; Louie, Thomas; Conly, John M

    2005-01-01

    Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). SCCmec elements are currently classified into types I to V based on the nature of the mec and ccr gene complexes, and are further classified into subtypes according to their junkyard region DNA segments. Previously described traditional SCCmec PCR typing schemes require multiple primer sets and PCR experiments, while a previous...

  3. Diversity of Staphylococcal Cassette Chromosome mec Elements in Predominant Methicillin-Resistant Staphylococcus aureus Clones in a Small Geographic Area ▿

    OpenAIRE

    Basset, Patrick; Senn, Laurence; Vogel, Valérie; Zanetti, Giorgio; Blanc, Dominique S.

    2010-01-01

    Recent population genetic studies suggest that staphylococcal cassette chromosome mec (SCCmec) was acquired much more frequently than previously thought. In the present study, we aimed to investigate the diversity of SCCmec elements in a local methicillin-resistant Staphylococcus aureus (MRSA) population. Each MRSA isolate (one per patient) recovered in the Vaud canton of Switzerland from January 2005 to December 2008 was analyzed by the double-locus sequence typing (DLST) method and SCCmec t...

  4. The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.

    Directory of Open Access Journals (Sweden)

    Peter M Jones

    Full Text Available ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs, which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.

  5. Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis.

    Science.gov (United States)

    Goldman, B S; Beckman, D L; Bali, A; Monika, E M; Gabbert, K K; Kranz, R G

    1997-05-16

    The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters. PMID:9175857

  6. Heme uptake in bacterial pathogens

    OpenAIRE

    Contreras, Heidi; Chim, Nicholas; Credali, Alfredo; Goulding, Celia W.

    2014-01-01

    Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within...

  7. Dicty_cDB: Contig-U15721-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 4 DQ223859_1( DQ223859 |pid:none) Danio rerio ATP-binding cassette t... 182 9e-44 ( P45843 ) RecName: Full=Protein scarlet...RecName: Full=ATP-binding cassette sub-family G member ... 172 6e-41 U39739_1( U39739 |pid:none) Drosophila melanogaster scarlet...omplex homolog prot... 169 8e-40 AY172185_1( AY172185 |pid:none) Bactrocera tryoni scarlet gene, co... 168 1

  8. Hedgehog Pathway Inhibitor HhAntag691 Is a Potent Inhibitor of ABCG2/BCRP and ABCB1/Pgp1

    OpenAIRE

    Zhang, Yimao; Laterra, John; Pomper, Martin G.

    2009-01-01

    HhAntag691 (GDC-0449), a low-molecular weight inhibitor of the tumor-promoting hedgehog (Hh) signaling pathway, has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors. Here, we show that HhAntag691 inhibits multiple ATP-binding cassette (ABC) transporters. ATP-binding cassette transporters are within a family of membrane proteins, the overexpression of which is associated with multidrug resistance, a major impediment to s...

  9. Bacterial chemoreceptors and chemoeffectors.

    Science.gov (United States)

    Bi, Shuangyu; Lai, Luhua

    2015-02-01

    Bacteria use chemotaxis signaling pathways to sense environmental changes. Escherichia coli chemotaxis system represents an ideal model that illustrates fundamental principles of biological signaling processes. Chemoreceptors are crucial signaling proteins that mediate taxis toward a wide range of chemoeffectors. Recently, in deep study of the biochemical and structural features of chemoreceptors, the organization of higher-order clusters in native cells, and the signal transduction mechanisms related to the on-off signal output provides us with general insights to understand how chemotaxis performs high sensitivity, precise adaptation, signal amplification, and wide dynamic range. Along with the increasing knowledge, bacterial chemoreceptors can be engineered to sense novel chemoeffectors, which has extensive applications in therapeutics and industry. Here we mainly review recent advances in the E. coli chemotaxis system involving structure and organization of chemoreceptors, discovery, design, and characterization of chemoeffectors, and signal recognition and transduction mechanisms. Possible strategies for changing the specificity of bacterial chemoreceptors to sense novel chemoeffectors are also discussed.

  10. Bacterial Colony Optimization

    Directory of Open Access Journals (Sweden)

    Ben Niu

    2012-01-01

    Full Text Available This paper investigates the behaviors at different developmental stages in Escherichia coli (E. coli lifecycle and developing a new biologically inspired optimization algorithm named bacterial colony optimization (BCO. BCO is based on a lifecycle model that simulates some typical behaviors of E. coli bacteria during their whole lifecycle, including chemotaxis, communication, elimination, reproduction, and migration. A newly created chemotaxis strategy combined with communication mechanism is developed to simplify the bacterial optimization, which is spread over the whole optimization process. However, the other behaviors such as elimination, reproduction, and migration are implemented only when the given conditions are satisfied. Two types of interactive communication schemas: individuals exchange schema and group exchange schema are designed to improve the optimization efficiency. In the simulation studies, a set of 12 benchmark functions belonging to three classes (unimodal, multimodal, and rotated problems are performed, and the performances of the proposed algorithms are compared with five recent evolutionary algorithms to demonstrate the superiority of BCO.

  11. [Bacterial diseases of rape].

    Science.gov (United States)

    Zakharova, O M; Mel'nychuk, M D; Dankevych, L A; Patyka, V P

    2012-01-01

    Bacterial destruction of the culture was described and its agents identified in the spring and winter rape crops. Typical symptoms are the following: browning of stem tissue and its mucilagization, chlorosis of leaves, yellowing and beginning of soft rot in the place of leaf stalks affixion to stems, loss of pigmentation (violet). Pathogenic properties of the collection strains and morphological, cultural, physiological, and biochemical properties of the agents of rape's bacterial diseases isolated by the authors have been investigated. It was found that all the isolates selected by the authors are highly or moderately aggressive towards different varieties of rape. According to the complex of phenotypic properties 44% of the total number of isolates selected by the authors are related to representatives of the genus Pseudomonas, 37% - to Xanthomonas and 19% - to Pectobacterium. PMID:23293826

  12. Bacterial transformation of terpenoids

    International Nuclear Information System (INIS)

    Data on the bacterial transformation of terpenoids published in the literature in the past decade are analyzed. Possible pathways for chemo-, regio- and stereoselective modifications of terpenoids are discussed. Considerable attention is given to new technological approaches to the synthesis of terpenoid derivatives suitable for the use in the perfume and food industry and promising as drugs and chiral intermediates for fine organic synthesis. The bibliography includes 246 references

  13. Supramolecular bacterial systems

    OpenAIRE

    Sankaran, Shrikrishnan

    2015-01-01

    For nearly over a decade, a wide variety of dynamic and responsive supramolecular architectures have been investigated and developed to address biological systems. Since the non-covalent interactions between individual molecular components in such architectures are similar to the interactions found in living systems, it was possible to integrate chemically-synthesized and naturally-occurring components to create platforms with interesting bioactive properties. Bacterial cells and recombinant ...

  14. Bacterial Colony Optimization

    OpenAIRE

    Ben Niu; Hong Wang

    2012-01-01

    This paper investigates the behaviors at different developmental stages in Escherichia coli (E. coli) lifecycle and developing a new biologically inspired optimization algorithm named bacterial colony optimization (BCO). BCO is based on a lifecycle model that simulates some typical behaviors of E. coli bacteria during their whole lifecycle, including chemotaxis, communication, elimination, reproduction, and migration. A newly created chemotaxis strategy combined with communication mechanism i...

  15. Conjugative DNA transfer induces the bacterial SOS response and promotes antibiotic resistance development through integron activation.

    Directory of Open Access Journals (Sweden)

    Zeynep Baharoglu

    2010-10-01

    Full Text Available Conjugation is one mechanism for intra- and inter-species horizontal gene transfer among bacteria. Conjugative elements have been instrumental in many bacterial species to face the threat of antibiotics, by allowing them to evolve and adapt to these hostile conditions. Conjugative plasmids are transferred to plasmidless recipient cells as single-stranded DNA. We used lacZ and gfp fusions to address whether conjugation induces the SOS response and the integron integrase. The SOS response controls a series of genes responsible for DNA damage repair, which can lead to recombination and mutagenesis. In this manuscript, we show that conjugative transfer of ssDNA induces the bacterial SOS stress response, unless an anti-SOS factor is present to alleviate this response. We also show that integron integrases are up-regulated during this process, resulting in increased cassette rearrangements. Moreover, the data we obtained using broad and narrow host range plasmids strongly suggests that plasmid transfer, even abortive, can trigger chromosomal gene rearrangements and transcriptional switches in the recipient cell. Our results highlight the importance of environments concentrating disparate bacterial communities as reactors for extensive genetic adaptation of bacteria.

  16. INHIBITION OF ACTIVATED K-RAS GENE BY SIRNA EXPRESSION CASSETTES IN HUMAN PANCREATIC CARCINOMA CELL LINE MIAPACA-2

    Institute of Scientific and Technical Information of China (English)

    WANG Wei; WANG Chun-you; DONG Ji-hua; CHEN Xiong; ZHANG Min; ZHAO Gang

    2005-01-01

    Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

  17. In70 of plasmid pAX22, a bla(VIM-1)-containing integron carrying a new aminoglycoside phosphotransferase gene cassette.

    Science.gov (United States)

    Riccio, M L; Pallecchi, L; Fontana, R; Rossolini, G M

    2001-04-01

    An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides. PMID:11257042

  18. The Agrobacterium-mediated transformation of common wheat (Triticum aestivum L.) and triticale (x Triticosecale Wittmack): role of the binary vector system and selection cassettes.

    Science.gov (United States)

    Bińka, Agnieszka; Orczyk, Wacław; Nadolska-Orczyk, Anna

    2012-02-01

    The influence of two binary vector systems, pGreen and pCAMBIA, on the Agrobacterium-mediated transformation ability of wheat and triticale was studied. Both vectors carried selection cassettes with bar or nptII driven by different promoters. Two cultivars of wheat, Kontesa and Torka, and one cultivar of triticale, Wanad, were tested. The transformation rates for the wheat cultivars ranged from 0.00 to 3.58% and from 0.00 to 6.79% for triticale. The best values for wheat were 3.58% for Kontesa and 3.14% for Torka, and these were obtained after transformation with the pGreen vector carrying the nptII selection gene under the control of 35S promoter. In the case of the bar selection system, the best transformation rates were, respectively, 1.46 and 1.79%. Such rates were obtained when the 35S::bar cassette was carried by the pCAMBIA vector; they were significantly lower with the pGreen vector. The triticale cultivar Wanad had its highest transformation rate after transformation with nptII driven by 35S in pCAMBIA. The bar selection system for the same triticale cultivar was better when the gene was driven by nos and the selection cassette was carried by pGreen. The integration of the transgenes was confirmed with at least three pairs of specific starters amplifying the fragments of nptII, bar, or gus. The expression of selection genes, measured by reverse transcriptase polymerase chain reaction (RT-PCR) in relation to the actin gene, was low, ranging from 0.00 to 0.63 for nptII and from 0.00 to 0.33 for bar. The highest relative transcript accumulation was observed for nptII driven by 35S and expressed in Kontesa that had been transformed with pGreen.

  19. Spontaneous bacterial peritonitis

    Institute of Scientific and Technical Information of China (English)

    Anastasios Koulaouzidis; Shivaram Bhat; Athar A Saeed

    2009-01-01

    Since its initial description in 1964, research has transformed spontaneous bacterial peritonitis (SBP) from a feared disease (with reported mortality of 90%) to a treatable complication of decompensated cirrhosis,albeit with steady prevalence and a high recurrence rate. Bacterial translocation, the key mechanism in the pathogenesis of SBP, is only possible because of the concurrent failure of defensive mechanisms in cirrhosis.Variants of SBP should be treated. Leucocyte esterase reagent strips have managed to shorten the 'tap-toshot' time, while future studies should look into their combined use with ascitic fluid pH. Third generation cephalosporins are the antibiotic of choice because they have a number of advantages. Renal dysfunction has been shown to be an independent predictor of mortality in patients with SBP. Albumin is felt to reduce the risk of renal impairment by improving effective intravascular volume, and by helping to bind proinflammatory molecules. Following a single episode of SBP, patients should have long-term antibiotic prophylaxis and be considered for liver transplantation.

  20. Antimicrobials for bacterial bioterrorism agents.

    Science.gov (United States)

    Sarkar-Tyson, Mitali; Atkins, Helen S

    2011-06-01

    The limitations of current antimicrobials for highly virulent pathogens considered as potential bioterrorism agents drives the requirement for new antimicrobials that are suitable for use in populations in the event of a deliberate release. Strategies targeting bacterial virulence offer the potential for new countermeasures to combat bacterial bioterrorism agents, including those active against a broad spectrum of pathogens. Although early in the development of antivirulence approaches, inhibitors of bacterial type III secretion systems and cell division mechanisms show promise for the future.

  1. Expression and clinical significance of thymidylate synthase and adenosine triphosphate-binding cassette superfamily G member protein in advanced gastric carcinoma%进展期胃癌胸苷酸合成酶和三磷酸腺苷结合转运蛋白G超家族成员2的表达及意义

    Institute of Scientific and Technical Information of China (English)

    程浩; 贾喜花; 郑淑君; 张晓伟; 张金库

    2013-01-01

    目的 检测胸苷酸合成酶(TS)和三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)在进展期胃癌中的表达,探讨其与临床病理特征的关系.方法 收集80例进展期胃癌患者的手术标本,采用免疫组织化学方法,在胃癌组织和相应癌旁正常组织中检测TS和ABCG2的表达,在胃癌组织中检测P-糖蛋白的表达.分析TS、ABCG2表达与临床病理特征、P-糖蛋白表达的相关性.两组间比较行x2检验,多组间比较行Kruskal-Wallis H检验.结果 胃癌组织中TS和ABCG2的总阳性率[85.0%(68/80)和90.0% (72/80)]均高于癌旁正常组织[62.5%(50/80)和78.7% (63/80)],差异均有统计学意义(x2=11.466和16.463,P=0.009和0.001).TS和ABCG2在胃癌组织中的表达水平均与肿瘤TNM分期、分化程度、浸润深度密切相关(TS的x2=30.686、49.823、40.545,ABCG2的x2=48.192、64.722、47.512;P均<0.01),肿瘤分期越晚、分化程度越差、浸润越深,二者的表达水平越高.胃癌组织中TS和ABCG2的表达水平均与P-糖蛋白表达水平相关(x2=43.977和29.509,P均<0.01).结论 TS和ABCG2有可能成为判断胃癌恶性程度、进展、耐药及预后的指标.%Objective To investigate the expressions of thymidylate synthase (TS) and adenosine triphosphate (ATP)-binding cassette superfamily G member 2 (ABCG2) in advanced gastric cancer (GC) and to explore their correlation with clinical pathological features.Methods A total of 80surgical specimens of advanced gastric cancer patients were collected.The expressions of TS and ABCG2 in gastric cancer tissues and adjacent normal gastric tissues were detected by immunohistochemical method.The expression of P-glycoprotein in gastric cancer tissues was also examined.The correlations between TS,ABCG2 and clinical pathological features and P-glycoprotein were analyzed.Chi-square test was performed for two groups comparison and Kruskal-Wallis H test were used for multi-groups comparison.Results The positive rates

  2. [Small intestine bacterial overgrowth].

    Science.gov (United States)

    Leung Ki, E L; Roduit, J; Delarive, J; Guyot, J; Michetti, P; Dorta, G

    2010-01-27

    Small intestine bacterial overgrowth (SIBO) is a condition characterised by nutrient malabsorption and excessive bacteria in the small intestine. It typically presents with diarrhea, flatulence and a syndrome of malabsorption (steatorrhea, macrocytic anemia). However, it may be asymptomatic in the eldery. A high index of suspicion is necessary in order to differentiate SIBO from other similar presenting disorders such as coeliac disease, lactose intolerance or the irritable bowel syndrome. A search for predisposing factor is thus necessary. These factors may be anatomical (stenosis, blind loop), or functional (intestinal hypomotility, achlorydria). The hydrogen breath test is the most frequently used diagnostic test although it lacks standardisation. The treatment of SIBO consists of eliminating predisposing factors and broad-spectrum antibiotic therapy. PMID:20214190

  3. Studying bacterial multispecies biofilms

    DEFF Research Database (Denmark)

    Røder, Henriette Lyng; Sørensen, Søren Johannes; Burmølle, Mette

    2016-01-01

    The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating...... the approaches used to study these complex communities. This review focuses on the establishment of multispecies biofilms in vitro, interspecies interactions in microhabitats, and how to select communities for evaluation. Studies have used different experimental approaches; here we evaluate the benefits...... and drawbacks of varying the degree of complexity. This review aims to facilitate multispecies biofilm research in order to expand the current limited knowledge on interspecies interactions. Recent technological advances have enabled total diversity analysis of highly complex and diverse microbial communities...

  4. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...

  5. Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells

    International Nuclear Information System (INIS)

    Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event

  6. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  7. Molecular approaches for bacterial azoreductases

    Directory of Open Access Journals (Sweden)

    Montira Leelakriangsak

    2013-12-01

    Full Text Available Azo dyes are the dominant types of synthetic dyes, widely used in textiles, foods, leather, printing, tattooing, cosmetics, and pharmaceutical industries. Many microorganisms are able to decolorize azo dyes, and there is increasing interest in biological waste treatment methods. Bacterial azoreductases can cleave azo linkages (-N=N- in azo dyes, forming aromatic amines. This review mainly focuses on employing molecular approaches, including gene manipulation and recombinant strains, to study bacterial azoreductases. The construction of the recombinant protein by cloning and the overexpression of azoreductase is described. The mechanisms and function of bacterial azoreductases can be studied by other molecular techniques discussed in this review, such as RT-PCR, southern blot analysis, western blot analysis, zymography, and muta-genesis in order to understand bacterial azoreductase properties, function and application. In addition, understanding the regulation of azoreductase gene expression will lead to the systematic use of gene manipulation in bacterial strains for new strategies in future waste remediation technologies.

  8. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    Science.gov (United States)

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  9. Universal stress protein Rv2624c alters abundance of arginine and enhances intracellular survival by ATP binding in mycobacteria

    Science.gov (United States)

    Jia, Qiong; Hu, Xinling; Shi, Dawei; Zhang, Yan; Sun, Meihao; Wang, Jianwei; Mi, Kaixia; Zhu, Guofeng

    2016-01-01

    The universal stress protein family is a family of stress-induced proteins. Universal stress proteins affect latency and antibiotic resistance in mycobacteria. Here, we showed that Mycobacterium smegmatis overexpressing M. tuberculosis universal stress protein Rv2624c exhibits increased survival in human monocyte THP-1 cells. Transcriptome analysis suggested that Rv2624c affects histidine metabolism, and arginine and proline metabolism. LC-MS/MS analysis showed that Rv2624c affects the abundance of arginine, a modulator of both mycobacteria and infected THP-1 cells. Biochemical analysis showed that Rv2624c is a nucleotide-binding universal stress protein, and an Rv2624c mutant incapable of binding ATP abrogated the growth advantage in THP-1 cells. Rv2624c may therefore modulate metabolic pathways in an ATP-dependent manner, changing the abundance of arginine and thus increasing survival in THP-1 cells. PMID:27762279

  10. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

    Directory of Open Access Journals (Sweden)

    Pene-Dumitrescu Teodora

    2012-03-01

    Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

  11. Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase.

    Science.gov (United States)

    Kubala, M; Plásek, J; Amler, E

    2004-01-01

    An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.

  12. An Allosteric Cross-Talk Between the Activation Loop and the ATP Binding Site Regulates the Activation of Src Kinase

    Science.gov (United States)

    Pucheta-Martínez, Encarna; Saladino, Giorgio; Morando, Maria Agnese; Martinez-Torrecuadrada, Jorge; Lelli, Moreno; Sutto, Ludovico; D'Amelio, Nicola; Gervasio, Francesco Luigi

    2016-04-01

    Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe.

  13. Deactivation of the Arabidopsis BRI1 receptor kinase by autophosphorylation within the glycine-rich loop involved in ATP binding

    Science.gov (United States)

    The activity of the dual-specificity brassinosteroid receptor kinase, BRI1, reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we identify regions of the juxtamembrane domain that are essential f...

  14. ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

    Directory of Open Access Journals (Sweden)

    Wei Sheng Chia

    Full Text Available p97/Valosin-containing protein (VCP is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD. It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

  15. Immune responses and protection induced by Brucella suis S2 bacterial ghosts in mice.

    Science.gov (United States)

    Liu, Jun; Li, Yi; Sun, Yang; Ji, Xue; Zhu, Lingwei; Guo, Xuejun; Zhou, Wei; Zhou, Bo; Liu, Shuang; Zhang, Ruian; Feng, Shuzhang

    2015-08-15

    With the purpose of generating Brucella suis bacterial ghosts and investigating the immunogenicity of bacterial ghosts as a vaccine candidate, the lysis gene E and temperature-sensitive regulator cassette were cloned into a shuttle plasmid, pBBR1MCS-2, for construction of a recombinant temperature-sensitive shuttle lysis plasmid, pBBR1MCS-E. pBBR1MCS-E was then introduced into attenuated B. suis live vaccine S2 bacteria, and the resultant transformants were used for production of B. suis ghosts (BSGs) by inducing lysis gene E expression. The BSGs were characterized by observing their morphology by transmission electron microscopy. The safety and immunogenicity of BSGs were further evaluated using a murine model, the result suggested that BSG was as safe as formalin-killed B. suis. In mice, BSG demonstrated a similar capacity of inducing pathogen-specific serum IgG antibody response, spleen CD3(+) and CD4(+) T cell responses, induce secretion of gamma interferon and interleukin-4, and protection levels against Brucella melitensis 16M challenge, as the attenuated B. suis live vaccine. These data suggesting that BSG could confer protection against Brucella infection in a mouse model of disease and may be developed as a new vaccine candidate against Brucella infection. PMID:26022514

  16. Positioning of bacterial chemoreceptors.

    Science.gov (United States)

    Jones, Christopher W; Armitage, Judith P

    2015-05-01

    For optimum growth, bacteria must adapt to their environment, and one way that many species do this is by moving towards favourable conditions. To do so requires mechanisms to both physically drive movement and provide directionality to this movement. The pathways that control this directionality comprise chemoreceptors, which, along with an adaptor protein (CheW) and kinase (CheA), form large hexagonal arrays. These arrays can be formed around transmembrane receptors, resulting in arrays embedded in the inner membrane, or they can comprise soluble receptors, forming arrays in the cytoplasm. Across bacterial species, chemoreceptor arrays (both transmembrane and soluble) are localised to a variety of positions within the cell; some species with multiple arrays demonstrate this variety within individual cells. In many cases, the positioning pattern of the arrays is linked to the need for segregation of arrays between daughter cells on division, ensuring the production of chemotactically competent progeny. Multiple mechanisms have evolved to drive this segregation, including stochastic self-assembly, cellular landmarks, and the utilisation of ParA homologues. The variety of mechanisms highlights the importance of chemotaxis to motile species.

  17. Evolution of Bacterial Suicide

    Science.gov (United States)

    Tchernookov, Martin; Nemenman, Ilya

    2013-03-01

    While active, controlled cellular suicide (autolysis) in bacteria is commonly observed, it has been hard to argue that autolysis can be beneficial to an individual who commits it. We propose a theoretical model that predicts that bacterial autolysis is evolutionarily advantageous to an individualand would fixate in physically structured environments for stationary phase colonies. We perform spatially resolved agent-based simulations of the model, which predict that lower mixing in the environment results in fixation of a higher autolysis rate from a single mutated cell, regardless of the colony's genetic diversity. We argue that quorum sensing will fixate as well, even if initially rare, if it is coupled to controlling the autolysis rate. The model does not predict a strong additional competitive advantage for cells where autolysis is controlled by quorum sensing systems that distinguish self from nonself. These predictions are broadly supported by recent experimental results in B. subtilisand S. pneumoniae. Research partially supported by the James S McDonnell Foundation grant No. 220020321 and by HFSP grant No. RGY0084/2011.

  18. Electromagnetism of Bacterial Growth

    Science.gov (United States)

    Ainiwaer, Ailiyasi

    2011-10-01

    There has been increasing concern from the public about personal health due to the significant rise in the daily use of electrical devices such as cell phones, radios, computers, GPS, video games and television. All of these devices create electromagnetic (EM) fields, which are simply magnetic and electric fields surrounding the appliances that simultaneously affect the human bio-system. Although these can affect the human system, obstacles can easily shield or weaken the electrical fields; however, magnetic fields cannot be weakened and can pass through walls, human bodies and most other objects. The present study was conducted to examine the possible effects of bacteria when exposed to magnetic fields. The results indicate that a strong causal relationship is not clear, since different magnetic fields affect the bacteria differently, with some causing an increase in bacterial cells, and others causing a decrease in the same cells. This phenomenon has yet to be explained, but the current study attempts to offer a mathematical explanation for this occurrence. The researchers added cultures to the magnetic fields to examine any effects to ion transportation. Researchers discovered ions such as potassium and sodium are affected by the magnetic field. A formula is presented in the analysis section to explain this effect.

  19. Pharmacological functions of multidrug transporters: studies employing combination transporter knockout mice

    NARCIS (Netherlands)

    Lagas, J.S.

    2009-01-01

    ATP-binding cassette (ABC) multidrug transporters are drug efflux pumps located in the plasma membrane that utilize the energy of ATP hydrolysis to extrude a wide spectrum of endogenous and exogenous compounds from cells, including numerous (anticancer) drugs and/or their metabolites. The studies de

  20. Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

    NARCIS (Netherlands)

    Hollenstein, K.; Comellas-Bigler, M.; Bevers, L.E.; Feiters, M.C.; Meyer-Klaucke, W.; Hagedoorn, P.-L.; Locher, K.P.

    2009-01-01

    Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO4 2−) and tungstate (WO4 2−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across th

  1. Functional analysis of ABC transporter genes from Botrytis cinerea identifies BcatrB as a transporter of eugenol

    NARCIS (Netherlands)

    Schoonbeek, H.; Nistelrooy, van J.G.M.; Waard, de M.A.

    2003-01-01

    The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expressio

  2. ABC transporters from Botrytis cinerea in biotic and abiotic interactions

    NARCIS (Netherlands)

    Schoonbeek, H.

    2004-01-01

    Botrytis cinereais the causal agent of grey mould disease on a wide variety of crop plants. It is relatively insensitive to natural and synthetic fungitoxic compounds. This thesis describes how ABC (ATP-binding cassette) transporters contribute to protection by actively secre

  3. Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

    Science.gov (United States)

    Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

  4. Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens

    Science.gov (United States)

    The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devastating fungal pathogens leaf rust, stripe rust, and powdery mildew. In previous studies, this broad-spectrum resistance was shown to be controlled by a single gene which encodes a putative ATP-binding cassette transport...

  5. Multidrug resistance in oncology and beyond : from imaging of drug efflux pumps to cellular drug targets

    NARCIS (Netherlands)

    Nagengast, Wouter B; Oude Munnink, Thijs H; Dijkers, Eli; Hospers, Geesiena; Brouwers, Adrienne H; Schröder, Carolien P; Lub-de Hooge, Marjolijn; de Vries, Elisabeth G E

    2010-01-01

    Resistance of tumor cells to several structurally unrelated classes of natural products, including anthracyclines, taxanes, and epipodophyllotoxines, is often referred as multidrug resistance (MDR). This is associated with ATP-binding cassette transporters, which function as drug efflux pumps such a

  6. Transport of topoisomerase I inhibitors by the breast cancer resistance protein - Potential clinical implications

    NARCIS (Netherlands)

    Schellens, JHM; Maliepaard, M; Scheper, RJ; Scheffer, GL; Jonker, JW; Smit, JW; Beijnen, JH; Schinkel, AH; Liehr, JG; Giovanella, BC; Verschaegen, CF

    2000-01-01

    The multidrug resistance protein BCRP (breast cancer resistance protein) is a member of the ATP-binding cassette family of drug transporters. Overexpression of BCRP caused by exposure of cells to mitoxantrone (MX) or doxorubicin/verapamil resulted in a resistance pattern that is different from what

  7. Sphingolipids in neuroblastoma : Their role in drug resistance mechanisms

    NARCIS (Netherlands)

    Sietsma, H; Dijkhuis, AJ; Kamps, W; Kok, JW

    2002-01-01

    Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g.,

  8. Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

    NARCIS (Netherlands)

    Meszaros, Peter; Klappe, Karin; Hummel, Ina; Hoekstra, Dick; Kok, Jan Willem

    2011-01-01

    MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly comple

  9. Are lipid rafts involved in ABC transporter-mediated drug resistance of tumor cells?

    NARCIS (Netherlands)

    Kok, Jan Willem; Klappe, Karin; Hummel, Ina; Kroesen, Bart-Jan; Sietsma, Hannie; Meszaros, Peter

    2008-01-01

    Since their discovery, lipid rafts have been implicated in several cellular functions, including protein transport in polarized cells and signal transduction. Also in multidrug resistance lipid rafts may be important with regard to the localization of ATP-binding cassette (ABC) transporters in these

  10. Lipid dependence of ABC transporter localization and function

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Hoekstra, Dick; Kok, Jan Willem

    2009-01-01

    Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and dis

  11. the role of the actin cytoskeleton and lipid rafts in the localization and function of the ABCC1 transporter

    NARCIS (Netherlands)

    Kok, Jan; Klappe, Katharina; Hummel, Ina

    2014-01-01

    ATP-binding cassette (ABC) transporters are known to be important factors in multidrug resistance of tumor cells. Lipid rafts have been implicated in their localization in the plasma membrane, where they function as drug efflux pumps. This specific localization in rafts may support the activity of A

  12. Dicty_cDB: Contig-U15342-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available _2( FJ710212 |pid:none) Targeting vector pP{white-STAR}, c... 50 2e-05 AE014298_482( AE014298 |pid:none) Dro...type bezz_... 50 2e-05 (Q99PE7) RecName: Full=ATP-binding cassette sub-family G member ... 50 2e-05 FJ710212

  13. Genetic and bibliographic information: Abcc9 [GenLibi

    Lifescience Database Archive (English)

    Full Text Available Abcc9 ATP-binding cassette, sub-family C (CFTR/MRP), member 9 rat Hypertension (MeS...H) Cardiovascular Diseases (C14) > Vascular Diseases (C14.907) > Hypertension (C14.907.489) 04A0394477; 05A0803372 ...

  14. Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

    NARCIS (Netherlands)

    Gajic, O; Buist, G; Kojic, M; Topisirovic, L; Kuipers, OP; Kok, J

    2003-01-01

    A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a

  15. Functional Diversity of Tandem Substrate-Binding Domains in ABC Transporters from Pathogenic Bacteria

    NARCIS (Netherlands)

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Vujicic - Zagar, Andreja; Guskov, Albert; Slotboom, Dirk-Jan; Poolman, Bert

    2013-01-01

    The ATP-binding cassette (ABC) transporter GInPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional transp

  16. The Liver X Receptor (LXR) and its Target Gene ABCA1 are Regulated Upon Low Oxygen in Human Trophoblast Cells : A Reason for Alterations in Preeclampsia?

    NARCIS (Netherlands)

    Plosch, T.; Gellhaus, A.; van Straten, E. M. E.; Wolf, N.; Huijkman, N. C. A.; Schmidt, M.; Dunk, C. E.; Kuipers, F.; Winterhager, E.

    2010-01-01

    Objectives: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in th

  17. Functional expression and characterization of plant ABC transporters in Xenopus laevis oocytes for transport engineering purposes

    DEFF Research Database (Denmark)

    Xu, Deyang; Veres, Dorottya; Belew, Zeinu Mussa;

    2016-01-01

    Transport engineering in bioengineering is aimed at efficient export of the final product to reduce toxicity and feedback inhibition and to increase yield. The ATP-binding cassette (ABC) transporters with their highly diverse substrate specificity and role in cellular efflux are potentially suita...

  18. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert; Rydström, Jan

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  19. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    NARCIS (Netherlands)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was

  20. Genetic Variation in ABCG1 and Risk of Myocardial Infarction and Ischemic Heart Disease

    DEFF Research Database (Denmark)

    Schou, Jesper; Frikke-Schmidt, Ruth; Kardassis, Dimitris;

    2012-01-01

    OBJECTIVE: ATP binding cassette transporter G1 (ABCG1) facilitates cholesterol efflux from macrophages to mature high-density lipoprotein particles. Whether genetic variation in ABCG1 affects risk of atherosclerosis in humans remains to be determined. METHODS AND RESULTS: We resequenced the core ...

  1. The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

    DEFF Research Database (Denmark)

    Christensen, P U; Davey, William John; Nielsen, O;

    1997-01-01

    In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is onl...

  2. Sequence Classification: 893776 [

    Lifescience Database Archive (English)

    Full Text Available ate and benzoate, regulated by War1p, mutants exhibit sorbate hypersensitivity; Pdr12p || http://www.ncbi.nlm.nih.gov/protein/6325199 ... ...brane weak-acid-inducible ATP-binding cassette (ABC) transporter, required for weak organic acid resistance, strongly induced by sorb

  3. Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines

    NARCIS (Netherlands)

    Scheffer, GL; Maliepaard, M; Pijnenborg, ACLM; van Gastelen, MA; Schroeijers, AB; Allen, JD; Ross, DD; van der Valk, P; Dalton, WS; Schellens, JHM; Scheper, RJ; de Jong, MC

    2000-01-01

    Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (,MDR1) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported sole

  4. Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium.

    Science.gov (United States)

    Half molecule ATP-binding cassette transporters of the HMT1(heavy metal tolerance factor 1)subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans and Chlamydomonas reinhardtii, and have homologs in other species, including plants and humans. Based on studies i...

  5. ABCC1 polymorphisms contribute to level and decline of lung function in two population-based cohorts

    NARCIS (Netherlands)

    Siedlinski, Mateusz; Boezen, H M; Boer, Jolanda M A; Smit, Henriette A; Postma, Dirkje S

    2009-01-01

    OBJECTIVE: The ATP-binding cassette transporter ABCC1 [i.e. multidrug resistance-associated protein 1 (MRP1)] is a membrane-bound pump excreting a variety of xenobiotics from the cell, and thus ABCC1 may play an important role in smoking-related lung function loss and development of chronic obstruct

  6. Effect of (mixtures of) flavonoids on the in vitro and in vivo bioavailability of 2-mino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) : a biologically based modelling approach

    NARCIS (Netherlands)

    Schutte, M.E.

    2007-01-01

    The transport of food ingredients across the intestinal epithelium is an important factor determining the absorption upon oral intake. Uptake of compounds in the intestine may be influenced by transport proteins such as the ATP binding cassette transporters (ABC transporters). ABC transporters have

  7. Human breast cancer resistance protein : Interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine

    NARCIS (Netherlands)

    Pavek, P; Merino, G; Wagenaar, E; Bolscher, E; Novotna, M; Jonker, JW; Schinkel, AH

    2005-01-01

    The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells, mediating drug resistance and affecting the pharmacological behavior of many compounds. To study the interaction of human wild-type BCRP with steroid drugs, hormo

  8. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    NARCIS (Netherlands)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was

  9. ABCA1 impacts athero-thrombotic risk and 10-year survival in a contemporary secondary prevention setting

    NARCIS (Netherlands)

    J.J. Regieli; P.A. Doevendans; D.E. Grobbee; A.H. Zwinderman; Y. van der Graaf; J.J.P. Kastelein; J.W. Jukema

    2011-01-01

    Objectives: We prospectively investigated the effects of ATP-binding cassette protein-1 (ABCA1) variants on long-term clinical outcome in patients with coronary artery disease (CAD). Background: ABCA1 is implicated in the etiology of atherothrombosis and may offer a target to reduce cardiovascular r

  10. sphingosine-1-phosphate transport and its role in immunology

    NARCIS (Netherlands)

    Reitsema, V.; Bouma, Hjalmar; Kok, Jan

    2014-01-01

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite with many important functions in cellular and systemic physiology, including the immune system. As it cannot traverse the membrane, it is exported from cells by transporters. Several members of the ATP-binding cassette (ABC) transporter fami

  11. Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells - Implications for milk cholesterol secretion

    DEFF Research Database (Denmark)

    Mani, O; Körner, M; Ontsouka, C E;

    2011-01-01

    The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used...

  12. Conformational changes and ligand recognition of Escherichia coli D-xylose binding protein revealed

    DEFF Research Database (Denmark)

    Sooriyaarachchi, Sanjeewani; Ubhayasekera, Wimal; Park, Chankyu;

    2010-01-01

    ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-r...

  13. Dietary plant sterols accumulate in the brain

    NARCIS (Netherlands)

    Jansen, PJ; Lutjohann, D; Abildayeva, K; Vanmierlo, T; Plosch, T; Plat, J; von Bergmann, K; Groen, AK; Ramaekers, FCS; Kuipers, F; Mulder, M

    2006-01-01

    Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood-brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8

  14. Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules

    NARCIS (Netherlands)

    Kruit, Janine K; Wijesekara, Nadeeja; Fox, Jocelyn E Manning; Dai, Xiao-Qing; Brunham, Liam R; Searle, Gavin J; Morgan, Garry P; Costin, Adam J; Tang, Renmei; Bhattacharjee, Alpana; Johnson, James D; Light, Peter E; Marsh, Brad J; Macdonald, Patrick E; Verchere, C Bruce; Hayden, Michael R

    2011-01-01

    OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from β-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking β-cell ABCA1. RESEARCH DESIGN AND METHODS: Calcium imaging, patch clamp

  15. Gclust Server: 76160 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 76160 SCE_YLR188W=MDL1 Cluster Sequences Related Sequences(340) 695 Half-type ATP-b...es(340) Sequence length 695 Representative annotation Half-type ATP-binding cassette (ABC) transporter of th

  16. Metabolism and transport of tamoxifen in relation to its effectiveness

    DEFF Research Database (Denmark)

    Cronin-Fenton, Deirdre P; Damkier, Per; Lash, Timothy L

    2014-01-01

    Tamoxifen reduces the rate of breast cancer recurrence by approximately a half. Tamoxifen is metabolized to more active metabolites by enzymes encoded by polymorphic genes, including cytochrome P450 2D6 (CYP2D6). Tamoxifen is a substrate for ATP-binding cassette transporter proteins. We review ta...

  17. PfMDR2 and PfMDR5 are dispensable for Plasmodium falciparum asexual parasite multiplication but change in vitro susceptibility to anti-malarial drugs

    NARCIS (Netherlands)

    Velden, M. van der; Rijpma, S.R.; Russel, F.G.M.; Sauerwein, R.W.; Koenderink, J.B.

    2015-01-01

    BACKGROUND: Membrane-associated ATP binding cassette (ABC) transport proteins hydrolyze ATP in order to translocate a broad spectrum of substrates, from single ions to macromolecules across membranes. In humans, members from this transport family have been linked to drug resistance phenotypes, e.g.,

  18. Bacterial Communities: Interactions to Scale

    Science.gov (United States)

    Stubbendieck, Reed M.; Vargas-Bautista, Carol; Straight, Paul D.

    2016-01-01

    In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities. PMID:27551280

  19. Bacterial Communities: Interactions to Scale

    Directory of Open Access Journals (Sweden)

    Reed M. Stubbendieck

    2016-08-01

    Full Text Available In the environment, bacteria live in complex multispecies communities. These communities span in scale from small, multicellular aggregates to billions or trillions of cells within the gastrointestinal tract of animals. The dynamics of bacterial communities are determined by pairwise interactions that occur between different species in the community. Though interactions occur between a few cells at a time, the outcomes of these interchanges have ramifications that ripple through many orders of magnitude, and ultimately affect the macroscopic world including the health of host organisms. In this review we cover how bacterial competition influences the structures of bacterial communities. We also emphasize methods and insights garnered from culture-dependent pairwise interaction studies, metagenomic analyses, and modeling experiments. Finally, we argue that the integration of multiple approaches will be instrumental to future understanding of the underlying dynamics of bacterial communities.

  20. Meningitis bacteriana Bacterial meningitis

    Directory of Open Access Journals (Sweden)

    Ana Teresa Alvarado Guevara

    2006-03-01

    causales son virales lo cual conlleva a las diferentes sub-clasificaciones. También en ciertos casos puede ser ocasionada por hongos, bacterias atípicas, micobacterias y parásitos.In Costa Rica the bacterial meningitis had turn into a high-priority subject in which to monitoring epidemiologist. It had been talked about in the last months, to dice an increase in the attention is published of this subject, due to this phenomenon it becomes necessary to make a revision of topic. Meningitis is an inflammation of leptomeninges and colonization of the subarachnoid cerebrospinal fluid (LCR due to different agents, which produces meningeal symptoms (ex. migraine, neck rigidity, and photophobia and pleocytosis in LCR. De pending on the variables to take into account is possible to group it in different classifications, taking into account the time of evolution are possible to be divided in acute or chronic, to first with few hours or days of beginning of the symptoms, whereas the chronicle also presents a silence course but of the disease of approximately 4 weeks of instauration. There is a difference according to its etiologic agent; they can be infectious and non-infectious. Examples of common non-infectious causes include medications (ex, nonsteroidal anti-inflammatory drugs, and antibiotics and carcinomatosis. A classification exists as well according to the causal agent. The acute bacterial meningitis remarks a bacterial origin of the syndrome, which characterizes by the by an acute onset of meningeal symptoms and neutrophilic pleocytosis. Each one of the bacteriological agents, parasitic or fungus finishes by characterizing the different presentations of the clinical features (ex, meningocóccica meningitis, Cryptococcus meningitis. Finally, there is also the aseptic meningitis, denominated in this form because it’s nonpyogenic cellular response caused by many types of agents. The patients show an acute beginning of symptoms, fever and lymphocytic pleocytosis. After

  1. Bacterial Culture of Neonatal Sepsis

    OpenAIRE

    AH Movahedian; R Moniri; Z Mosayebi

    2006-01-01

    Neonatal bacterial sepsis is one of the major cause of morbidity and mortality in neonates. This retrospective study was performed to determine the incidence of bacterial sepsis with focus on Gram negative organisms in neonates admitted at Beheshti Hospital in Kashan, during a 3-yr period, from September 2002 to September 2005. Blood culture was performed on all neonates with risk factors or signs of suggestive sepsis. Blood samples were cultured using brain heart infusion (BHI) broth accordi...

  2. Mast cells in bacterial infections

    OpenAIRE

    Rönnberg, Elin

    2014-01-01

    Mast cells are implicated in immunity towards bacterial infection, but the molecular mechanisms by which mast cells contribute to the host response are only partially understood. Previous studies have examined how mast cells react to purified bacterial cell wall components, such as peptidoglycan and lipopolysaccharide. To investigate how mast cells react to live bacteria we co-cultured mast cells and the gram-positive bacteria Streptococcus equi (S. equi) and Staphylococcus aureus (S. aureus)...

  3. Bacterial Alkaloids Prevent Amoebal Predation.

    Science.gov (United States)

    Klapper, Martin; Götze, Sebastian; Barnett, Robert; Willing, Karsten; Stallforth, Pierre

    2016-07-25

    Bacterial defense mechanisms have evolved to protect bacteria against predation by nematodes, predatory bacteria, or amoebae. We identified novel bacterial alkaloids (pyreudiones A-D) that protect the producer, Pseudomonas fluorescens HKI0770, against amoebal predation. Isolation, structure elucidation, total synthesis, and a proposed biosynthetic pathway for these structures are presented. The generation of P. fluorescens gene-deletion mutants unable to produce pyreudiones rendered the bacterium edible to a variety of soil-dwelling amoebae. PMID:27294402

  4. Bacterial cellulose/boehmite composites

    International Nuclear Information System (INIS)

    Composites based on bacterial cellulose membranes and boehmite were obtained. SEM results indicate that the bacterial cellulose (BC) membranes are totally covered by boehmite and obtained XRD patterns suggest structural changes due to this boehmite addition. Thermal stability is accessed through TG curves and is dependent on boehmite content. Transparency is high comparing to pure BC as can be seen through UV-vis absorption spectroscopy. (author)

  5. A Human t-PA Mutant cDNA Cassette Knocked in the Murine fgfr-4 Locus Targeting for Mammary Gland Expression

    Institute of Scientific and Technical Information of China (English)

    Fu-Yu LIN; Hong-Xing CHEN; Xuan CHENG; Xiao YANG; Ji-Xian DENG; Pei-Tang HUANG

    2004-01-01

    The expression of foreign gene in transgenic animals produced by pronuclear microinjection is often confounded by the position effects caused by not only the nature of chromosomal integration site but also the number and arrangement of multiple transgene copies. Gene targeting provides a new way to overcome these inhibitions by introducing single-copy transgene into a chosen site. The choice of a good chromosomal site will favor transgene expression in a predictable fashion. In this study, we tested a new site (fgfr-4) for foreign gene integration and expression. A t-PA mutant (t-PAm) expression cassette under bovine αs1-casein regulatory sequences was efficiently knocked-infgfr-4 site through homologous recombination. The t-PAm was expressed in the milk of all targeted mice. Our experiment indicates that the fgfr-4 may be a candidate site for knocking foreign gene to make transgenic animals.

  6. Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae.

    Science.gov (United States)

    Schröder, Gunnar; Schuelein, Ralf; Quebatte, Maxime; Dehio, Christoph

    2011-08-30

    Bacterial type IV secretion systems (T4SS) mediate interbacterial conjugative DNA transfer and transkingdom protein transfer into eukaryotic host cells in bacterial pathogenesis. The sole bacterium known to naturally transfer DNA into eukaryotic host cells via a T4SS is the plant pathogen Agrobacterium tumefaciens. Here we demonstrate T4SS-mediated DNA transfer from a human bacterial pathogen into human cells. We show that the zoonotic pathogen Bartonella henselae can transfer a cryptic plasmid occurring in the bartonellae into the human endothelial cell line EA.hy926 via its T4SS VirB/VirD4. DNA transfer into EA.hy926 cells was demonstrated by using a reporter derivative of this Bartonella-specific mobilizable plasmid generated by insertion of a eukaryotic egfp-expression cassette. Fusion of the C-terminal secretion signal of the endogenous VirB/VirD4 protein substrate BepD with the plasmid-encoded DNA-transport protein Mob resulted in a 100-fold increased DNA transfer rate. Expression of the delivered egfp gene in EA.hy926 cells required cell division, suggesting that nuclear envelope breakdown may facilitate passive entry of the transferred ssDNA into the nucleus as prerequisite for complementary strand synthesis and transcription of the egfp gene. Addition of an eukaryotic neomycin phosphotransferase expression cassette to the reporter plasmid facilitated selection of stable transgenic EA.hy926 cell lines that display chromosomal integration of the transferred plasmid DNA. Our data suggest that T4SS-dependent DNA transfer into host cells may occur naturally during human infection with Bartonella and that these chronically infecting pathogens have potential for the engineering of in vivo gene-delivery vectors with applications in DNA vaccination and therapeutic gene therapy. PMID:21844337

  7. Characterization of an Enterococcus gallinarum Isolate Carrying a Dual vanA and vanB Cassette.

    Science.gov (United States)

    Eshaghi, Alireza; Shahinas, Dea; Li, Aimin; Kariyawasam, Ruwandi; Banh, Philip; Desjardins, Marc; Melano, Roberto G; Patel, Samir N

    2015-07-01

    The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate of Enterococcus gallinarum exhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence of vanA and vanB genes in addition to the naturally carried vanC gene. vanA was identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. The vanB operon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn1549 and Tn5382. To the best of our knowledge, this is the first report of E. gallinarum carrying both vanA and vanB operons, indicating the importance of identifying the vancomycin resistance mechanism in non-E. faecium and non-E. faecalis enterococcal species.

  8. The Human Vaginal Bacterial Biota and Bacterial Vaginosis

    Directory of Open Access Journals (Sweden)

    Sujatha Srinivasan

    2008-01-01

    Full Text Available The bacterial biota of the human vagina can have a profound impact on the health of women and their neonates. Changes in the vaginal microbiota have been associated with several adverse health outcomes including premature birth, pelvic inflammatory disease, and acquisition of HIV infection. Cultivation-independent molecular methods have provided new insights regarding bacterial diversity in this important niche, particularly in women with the common condition bacterial vaginosis (BV. PCR methods have shown that women with BV have complex communities of vaginal bacteria that include many fastidious species, particularly from the phyla Bacteroidetes and Actinobacteria. Healthy women are mostly colonized with lactobacilli such as Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus iners, though a variety of other bacteria may be present. The microbiology of BV is heterogeneous. The presence of Gardnerella vaginalis and Atopobium vaginae coating the vaginal epithelium in some subjects with BV suggests that biofilms may contribute to this condition.

  9. Bacterial tactic responses.

    Science.gov (United States)

    Armitage, J P

    1999-01-01

    Many, if not most, bacterial species swim. The synthesis and operation of the flagellum, the most complex organelle of a bacterium, takes a significant percentage of cellular energy, particularly in the nutrient limited environments in which many motile species are found. It is obvious that motility accords cells a survival advantage over non-motile mutants under normal, poorly mixed conditions and is an important determinant in the development of many associations between bacteria and other organisms, whether as pathogens or symbionts and in colonization of niches and the development of biofilms. This survival advantage is the result of sensory control of swimming behaviour. Although too small to sense a gradient along the length of the cell, and unable to swim great distances because of buffetting by Brownian motion and the curvature resulting from a rotating flagellum, bacteria can bias their random swimming direction towards a more favourable environment. The favourable environment will vary from species to species and there is now evidence that in many species this can change depending on the current physiological growth state of the cell. In general, bacteria sense changes in a range of nutrients and toxins, compounds altering electron transport, acceptors or donors into the electron transport chain, pH, temperature and even the magnetic field of the Earth. The sensory signals are balanced, and may be balanced with other sensory pathways such as quorum sensing, to identify the optimum current environment. The central sensory pathway in this process is common to most bacteria and most effectors. The environmental change is sensed by a sensory protein. In most species examined this is a transmembrane protein, sensing the external environment, but there is increasing evidence for additional cytoplasmic receptors in many species. All receptors, whether sensing sugars, amino acids or oxygen, share a cytoplasmic signalling domain that controls the activity of a

  10. New Treatments for Bacterial Keratitis

    Directory of Open Access Journals (Sweden)

    Raymond L. M. Wong

    2012-01-01

    Full Text Available Purpose. To review the newer treatments for bacterial keratitis. Data Sources. PubMed literature search up to April 2012. Study Selection. Key words used for literature search: “infectious keratitis”, “microbial keratitis”, “infective keratitis”, “new treatments for infectious keratitis”, “fourth generation fluoroquinolones”, “moxifloxacin”, “gatifloxacin”, “collagen cross-linking”, and “photodynamic therapy”. Data Extraction. Over 2400 articles were retrieved. Large scale studies or publications at more recent dates were selected. Data Synthesis. Broad spectrum antibiotics have been the main stay of treatment for bacterial keratitis but with the emergence of bacterial resistance; there is a need for newer antimicrobial agents and treatment methods. Fourth-generation fluoroquinolones and corneal collagen cross-linking are amongst the new treatments. In vitro studies and prospective clinical trials have shown that fourth-generation fluoroquinolones are better than the older generation fluoroquinolones and are as potent as combined fortified antibiotics against common pathogens that cause bacterial keratitis. Collagen cross-linking was shown to improve healing of infectious corneal ulcer in treatment-resistant cases or as an adjunct to antibiotics treatment. Conclusion. Fourth-generation fluoroquinolones are good alternatives to standard treatment of bacterial keratitis using combined fortified topical antibiotics. Collagen cross-linking may be considered in treatment-resistant infectious keratitis or as an adjunct to antibiotics therapy.

  11. Antibiotic resistance of bacterial biofilms

    DEFF Research Database (Denmark)

    Hoiby, N.; Bjarnsholt, T.; Givskov, M.;

    2010-01-01

    A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis...... and other components of the body's defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains....... Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance...

  12. Phylogenetic organization of bacterial activity.

    Science.gov (United States)

    Morrissey, Ember M; Mau, Rebecca L; Schwartz, Egbert; Caporaso, J Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J; Liu, Cindy M; Hayer, Michaela; McHugh, Theresa A; Marks, Jane C; Price, Lance B; Hungate, Bruce A

    2016-09-01

    Phylogeny is an ecologically meaningful way to classify plants and animals, as closely related taxa frequently have similar ecological characteristics, functional traits and effects on ecosystem processes. For bacteria, however, phylogeny has been argued to be an unreliable indicator of an organism's ecology owing to evolutionary processes more common to microbes such as gene loss and lateral gene transfer, as well as convergent evolution. Here we use advanced stable isotope probing with (13)C and (18)O to show that evolutionary history has ecological significance for in situ bacterial activity. Phylogenetic organization in the activity of bacteria sets the stage for characterizing the functional attributes of bacterial taxonomic groups. Connecting identity with function in this way will allow scientists to begin building a mechanistic understanding of how bacterial community composition regulates critical ecosystem functions. PMID:26943624

  13. Bacterial Degradation of Aromatic Compounds

    Directory of Open Access Journals (Sweden)

    Qing X. Li

    2009-01-01

    Full Text Available Aromatic compounds are among the most prevalent and persistent pollutants in the environment. Petroleum-contaminated soil and sediment commonly contain a mixture of polycyclic aromatic hydrocarbons (PAHs and heterocyclic aromatics. Aromatics derived from industrial activities often have functional groups such as alkyls, halogens and nitro groups. Biodegradation is a major mechanism of removal of organic pollutants from a contaminated site. This review focuses on bacterial degradation pathways of selected aromatic compounds. Catabolic pathways of naphthalene, fluorene, phenanthrene, fluoranthene, pyrene, and benzo[a]pyrene are described in detail. Bacterial catabolism of the heterocycles dibenzofuran, carbazole, dibenzothiophene, and dibenzodioxin is discussed. Bacterial catabolism of alkylated PAHs is summarized, followed by a brief discussion of proteomics and metabolomics as powerful tools for elucidation of biodegradation mechanisms.

  14. Clinical applications of bacterial glycoproteins.

    Science.gov (United States)

    Fulton, Kelly M; Smith, Jeffrey C; Twine, Susan M

    2016-01-01

    There is an ongoing race between bacterial evolution and medical advances. Pathogens have the advantages of short generation times and horizontal gene transfer that enable rapid adaptation to new host environments and therapeutics that currently outpaces clinical research. Antibiotic resistance, the growing impact of nosocomial infections, cancer-causing bacteria, the risk of zoonosis, and the possibility of biowarfare all emphasize the increasingly urgent need for medical research focussed on bacterial pathogens. Bacterial glycoproteins are promising targets for alternative therapeutic intervention since they are often surface exposed, involved in host-pathogen interactions, required for virulence, and contain distinctive glycan structures. The potential exists to exploit these unique structures to improve clinical prevention, diagnosis, and treatment strategies. Translation of the potential in this field to actual clinical impact is an exciting prospect for fighting infectious diseases. PMID:26971465

  15. Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

    International Nuclear Information System (INIS)

    Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZα-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.

  16. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  17. Disease notes - Bacterial root rot

    Science.gov (United States)

    Bacterial root rot initiated by lactic acid bacteria, particularly Leuconostoc, occurs every year in Idaho sugarbeet fields. Hot fall weather seems to make the problem worse. Although Leuconostoc initiates the rot, other bacteria and yeast frequently invade the tissue as well. The acetic acid bac...

  18. Biotechnological applications of bacterial cellulases

    Directory of Open Access Journals (Sweden)

    Esther Menendez

    2015-08-01

    Full Text Available Cellulases have numerous applications in several industries, including biofuel production, food and feed industry, brewing, pulp and paper, textile, laundry, and agriculture.Cellulose-degrading bacteria are widely spread in nature, being isolated from quite different environments. Cellulose degradation is the result of a synergic process between an endoglucanase, an exoglucanase and a,β-glucosidase. Bacterial endoglucanases degrade ß-1,4-glucan linkages of cellulose amorphous zones, meanwhile exoglucanases cleave the remaining oligosaccharide chains, originating cellobiose, which is hydrolyzed by ß-glucanases. Bacterial cellulases (EC 3.2.1.4 are comprised in fourteen Glycosil Hydrolase families. Several advantages, such as higher growth rates and genetic versatility, emphasize the suitability and advantages of bacterial cellulases over other sources for this group of enzymes. This review summarizes the main known cellulolytic bacteria and the best strategies to optimize their cellulase production, focusing on endoglucanases, as well as it reviews the main biotechnological applications of bacterial cellulases in several industries, medicine and agriculture.

  19. A Program Against Bacterial Bioterrorism

    DEFF Research Database (Denmark)

    Kemp, Michael; Dargis, Rimtas; Andresen, Keld;

    2012-01-01

    In 2002 it was decided to establish laboratory facilities in Denmark for diagnosing agents associated with bioterrorism in order to make an immediate appropriate response to the release of such agents possible. Molecular assays for detection of specific agents and molecular and proteomic techniques...... for bacterial infections not associated with bioterrorism that are difficult to culture or identify....

  20. Molecular Mechanisms Underlying Bacterial Persisters

    DEFF Research Database (Denmark)

    Maisonneuve, Etienne; Gerdes, Kenn

    2014-01-01

    technological advances in microfluidics and reporter genes have improved this scenario. Here, we summarize recent progress in the field, revealing the ubiquitous bacterial stress alarmone ppGpp as an emerging central regulator of multidrug tolerance and persistence, both in stochastically and environmentally...

  1. Cognitive outcome in adults after bacterial meningitis.

    NARCIS (Netherlands)

    Hoogman, M.; Beek, D. van de; Weisfelt, M.; Gans, J. de; Schmand, B.

    2007-01-01

    OBJECTIVE: To evaluate cognitive outcome in adult survivors of bacterial meningitis. METHODS: Data from three prospective multicentre studies were pooled and reanalysed, involving 155 adults surviving bacterial meningitis (79 after pneumococcal and 76 after meningococcal meningitis) and 72 healthy c

  2. Filtration properties of bacterial cellulose membranes

    OpenAIRE

    Lehtonen, Janika

    2015-01-01

    Bacterial cellulose has the same molecular formula as cellulose from plant origin, but it is characterized by several unique properties including high purity, crystallinity and mechanical strength. These properties are dependent on parameters such as the bacterial strain used, the cultivation conditions and post-growth processing. The possibility to achieve bacterial cellulose membranes with different properties by varying these parameters could make bacterial cellulose an interesting materi...

  3. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

    Science.gov (United States)

    Sakuma, Tetsushi; Takenaga, Mitsumasa; Kawabe, Yoshinori; Nakamura, Takahiro; Kamihira, Masamichi; Yamamoto, Takashi

    2015-01-01

    Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins. PMID:26473830

  4. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    Science.gov (United States)

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.

  5. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos.

    Science.gov (United States)

    Wang, Liren; Shao, Yanjiao; Guan, Yuting; Li, Liang; Wu, Lijuan; Chen, Fangrui; Liu, Meizhen; Chen, Huaqing; Ma, Yanlin; Ma, Xueyun; Liu, Mingyao; Li, Dali

    2015-01-01

    The CRISPR-Cas RNA-guided system has versatile uses in many organisms and allows modification of multiple target sites simultaneously. Generating novel genetically modified mouse and rat models is one valuable application of this system. Through the injection of Cas9 protein instead of mRNA into embryos, we observed fewer off-target effects of Cas9 and increased point mutation knock-in efficiency. Large genomic DNA fragment (up to 95 kb) deletion mice were generated for in vivo study of lncRNAs and gene clusters. Site-specific insertion of a 2.7 kb CreERT2 cassette into the mouse Nfatc1 locus allowed labeling and tracing of hair follicle stem cells. In addition, we combined the Cre-Loxp system with a gene-trap strategy to insert a GFP reporter in the reverse orientation into the rat Lgr5 locus, which was later inverted by Cre-mediated recombination, yielding a conditional knockout/reporter strategy suitable for mosaic mutation analysis. PMID:26620761

  6. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    Directory of Open Access Journals (Sweden)

    Laura Ordovás

    2015-11-01

    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  7. On the Use of Molecular Weight Cutoff Cassettes to Measure Dynamic Relaxivity of Novel Gadolinium Contrast Agents: Example Using Hyaluronic Acid Polymer Complexes in Phosphate-Buffered Saline

    Directory of Open Access Journals (Sweden)

    Nima Kasraie

    2011-01-01

    Full Text Available The aims of this study were to determine whether standard extracellular contrast agents of Gd(III ions in combination with a polymeric entity susceptible to hydrolytic degradation over a finite period of time, such as Hyaluronic Acid (HA, have sufficient vascular residence time to obtain comparable vascular imaging to current conventional compounds and to obtain sufficient data to show proof of concept that HA with Gd-DTPA ligands could be useful as vascular imaging agents. We assessed the dynamic relaxivity of the HA bound DTPA compounds using a custom-made phantom, as well as relaxation rates at 10.72 MHz with concentrations ranging between 0.09 and 7.96 mM in phosphate-buffered saline. Linear dependences of static longitudinal relaxation rate (R1 on concentration were found for most measured samples, and the HA samples continued to produce high signal strength after 24 hours after injection into a dialysis cassette at 3T, showing superior dynamic relaxivity values compared to conventional contrast media such as Gd-DTPA-BMA.

  8. On the Use of Molecular Weight Cutoff Cassettes to Measure Dynamic Relaxivity of Novel Gadolinium Contrast Agents: Example Using Hyaluronic Acid Polymer Complexes in Phosphate-Buffered Saline

    International Nuclear Information System (INIS)

    The aims of this study were to determine whether standard extracellular contrast agents of Gd(III) ions in combination with a polymeric entity susceptible to hydrolytic degradation over a finite period of time, such as Hyaluronic Acid (HA), have sufficient vascular residence time to obtain comparable vascular imaging to current conventional compounds and to obtain sufficient data to show proof of concept that HA with Gd-DTPA ligands could be useful as vascular imaging agents. We assessed the dynamic relaxivity of the HA bound DTPA compounds using a custom-made phantom, as well as relaxation rates at 10.72 MHz with concentrations ranging between 0.09 and 7.96 mM in phosphate-buffered saline. Linear dependences of static longitudinal relaxation rate (R1) on concentration were found for most measured samples, and the HA samples continued to produce high signal strength after 24 hours after injection into a dialysis cassette at 3T, showing superior dynamic relaxivity values compared to conventional contrast media such as Gd-DTPA-BMA

  9. Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids

    Directory of Open Access Journals (Sweden)

    Tetsushi Sakuma

    2015-10-01

    Full Text Available Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc gene, in Chinese hamster ovary (CHO cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

  10. Design and application of locking hydraulic cylinder for roll cassette in straightener for heavy plate%中厚板矫直机辊盒锁紧液压缸的设计应用

    Institute of Scientific and Technical Information of China (English)

    曹一兵

    2013-01-01

    A security and reliable auto-locking device for the roll cassette of heavy plate straightener was designed to overcome the disadvantage that the locking device for roll cassette needs manual manipulation.The locking device works well in the straightener.It improved the operating reliability and security of straighteners.%针对中厚板矫直机上辊盒锁紧装置存在手动操作的问题,设计了一种安全可高的自动锁紧液压缸装置,该设装置在中厚板矫直机上的应用效果较好,提高了矫直机操作的可靠性及安全性.

  11. Distribution of Triplet Separators in Bacterial Genomes

    Institute of Scientific and Technical Information of China (English)

    HU Rui; ZHENG Wei-Mou

    2001-01-01

    Distributions of triplet separator lengths for two bacterial complete genomes are analyzed. The theoretical distributions for the independent random sequence and the first-order Markov chain are derived and compared with the distributions of the bacterial genomes. A prominent double band structure, which does not exist in the theoretical distributions, is observed in the bacterial distributions for most triplets.``

  12. Bacterial chromosome organization and segregation.

    Science.gov (United States)

    Badrinarayanan, Anjana; Le, Tung B K; Laub, Michael T

    2015-01-01

    If fully stretched out, a typical bacterial chromosome would be nearly 1 mm long, approximately 1,000 times the length of a cell. Not only must cells massively compact their genetic material, but they must also organize their DNA in a manner that is compatible with a range of cellular processes, including DNA replication, DNA repair, homologous recombination, and horizontal gene transfer. Recent work, driven in part by technological advances, has begun to reveal the general principles of chromosome organization in bacteria. Here, drawing on studies of many different organisms, we review the emerging picture of how bacterial chromosomes are structured at multiple length scales, highlighting the functions of various DNA-binding proteins and the impact of physical forces. Additionally, we discuss the spatial dynamics of chromosomes, particularly during their segregation to daughter cells. Although there has been tremendous progress, we also highlight gaps that remain in understanding chromosome organization and segregation. PMID:26566111

  13. Bacterial streamers in curved microchannels

    Science.gov (United States)

    Rusconi, Roberto; Lecuyer, Sigolene; Guglielmini, Laura; Stone, Howard

    2009-11-01

    Biofilms, generally identified as microbial communities embedded in a self-produced matrix of extracellular polymeric substances, are involved in a wide variety of health-related problems ranging from implant-associated infections to disease transmissions and dental plaque. The usual picture of these bacterial films is that they grow and develop on surfaces. However, suspended biofilm structures, or streamers, have been found in natural environments (e.g., rivers, acid mines, hydrothermal hot springs) and are always suggested to stem from a turbulent flow. We report the formation of bacterial streamers in curved microfluidic channels. By using confocal laser microscopy we are able to directly image and characterize the spatial and temporal evolution of these filamentous structures. Such streamers, which always connect the inner corners of opposite sides of the channel, are always located in the middle plane. Numerical simulations of the flow provide evidences for an underlying hydrodynamic mechanism behind the formation of the streamers.

  14. Bacterial survival in Martian conditions

    CERN Document Server

    D'Alessandro, Giuseppe Galletta; Giulio Bertoloni; Maurizio

    2010-01-01

    We shortly discuss the observable consequences of the two hypotheses about the origin of life on Earth and Mars: the Lithopanspermia (Mars to Earth or viceversa) and the origin from a unique progenitor, that for Earth is called LUCA (the LUCA hypothesis). To test the possibility that some lifeforms similar to the terrestrial ones may survive on Mars, we designed and built two simulators of Martian environments where to perform experiments with different bacterial strains: LISA and mini-LISA. Our LISA environmental chambers can reproduce the conditions of many Martian locations near the surface trough changes of temperature, pressure, UV fluence and atmospheric composition. Both simulators are open to collaboration with other laboratories interested in performing experiments on many kind of samples (biological, minerals, electronic) in situations similar to that of the red planet. Inside LISA we have studied the survival of several bacterial strains and endospores. We verified that the UV light is the major re...

  15. Collective Functionality through Bacterial Individuality

    Science.gov (United States)

    Ackermann, Martin

    According to the conventional view, the properties of an organism are a product of nature and nurture - of its genes and the environment it lives in. Recent experiments with unicellular organisms have challenged this view: several molecular mechanisms generate phenotypic variation independently of environmental signals, leading to variation in clonal groups. My presentation will focus on the causes and consequences of this microbial individuality. Using examples from bacterial genetic model systems, I will first discuss different molecular and cellular mechanisms that give rise to bacterial individuality. Then, I will discuss the consequences of individuality, and focus on how phenotypic variation in clonal populations of bacteria can promote interactions between individuals, lead to the division of labor, and allow clonal groups of bacteria to cope with environmental uncertainty. Variation between individuals thus provides clonal groups with collective functionality.

  16. Advances in the relationship between integron-gene cassette system and drug-resistance of bacteria%整合子-基因盒体系与细菌耐药关系及研究进展

    Institute of Scientific and Technical Information of China (English)

    张弘; 高伟利; 张双宅; 刘维华

    2013-01-01

    The bacteria resistance has posed a serious threat to the public health problem at present. Integron -gene cassette system played an important role in bacteria capturing and disseminating antibiotic resistance gene. Integron identified and captured free resistance gene box under the action of integrase. The spread of transposons and zygosity plasmid caused diffusion of drug - resistant genes and multiple resistance effects in different bacteria. This article reviewed the construction, class, distribution, source of integron and gene cassettes and spread mechanism of integron - gene cassette system.%目前,细菌耐药性已经对公共卫生问题构成严重威胁,整合子-基因盒体系是介导细菌获得耐药性与耐药基因播散的重要分子机制.整合子在整合酶作用下识别并俘获游离耐药基因盒,随着转座子或接合性质粒播散,造成细菌间耐药基因的扩散和多重耐药性的产生.本文就整合子-基因盒体系的结构、分类、分布、起源及播散机制等几方面研究近况开展讨论.

  17. A rev1-vpu polymorphism unique to HIV-1 subtype A and C strains impairs envelope glycoprotein expression from rev-vpu-env cassettes and reduces virion infectivity in pseudotyping assays

    International Nuclear Information System (INIS)

    Functional studies of HIV-1 envelope glycoproteins (Envs) commonly include the generation of pseudoviruses, which are produced by co-transfection of rev-vpu-env cassettes with an env-deficient provirus. Here, we describe six Env constructs from transmitted/founder HIV-1 that were defective in the pseudotyping assay, although two produced infectious virions when expressed from their cognate proviruses. All of these constructs exhibited an unusual gene arrangement in which the first exon of rev (rev1) and vpu were in the same reading frame without an intervening stop codon. Disruption of the rev1-vpu fusion gene by frameshift mutation, stop codon, or abrogation of the rev initiation codon restored pseudovirion infectivity. Introduction of the fusion gene into wildtype Env cassettes severely compromised their function. The defect was not due to altered env and rev transcription or a dominant negative effect of the expressed fusion protein, but seemed to be caused by inefficient translation at the env initiation codon. Although the rev1-vpu polymorphism affects Env expression only in vitro, it can cause problems in studies requiring Env complementation, such as analyses of co-receptor usage and neutralization properties, since 3% of subtype A, 20% of subtype C and 5% of CRF01A/E viruses encode the fusion gene. A solution is to eliminate the rev initiation codon when amplifying rev-vpu-env cassettes since this increases Env expression irrespective of the presence of the polymorphism.

  18. Bacterial communication and group behavior

    OpenAIRE

    Greenberg, E. Peter

    2003-01-01

    The existence of species-specific and interspecies bacterial cell-cell communication and group organization was only recently accepted. Researchers are now realizing that the ability of these microbial teams to communicate and form structures, known as biofilms, at key times during the establishment of infection significantly increases their ability to evade both host defenses and antibiotics. This Perspective series discusses the known signaling mechanisms, the roles they play in both chroni...

  19. The problem of bacterial diarrhoea.

    Science.gov (United States)

    Harries, J T

    1976-01-01

    The reported incidence of "pathogenic" bacteria, as judged by serotype, in the stools of children with acute diarrhoea has varied from 4 to 33% over the last twenty years. Techniques such as tissue culture provide a means for detecting enterotoxin-producing strains of bacteria, strains which often do not possess "pathogenic" serotypes. "Pathogenicity" requires redefinition, and the aetiological importance of bacteria in diarrhoea is probably considerably greater than previous reports have indicated. Colonization of the bowel by a pathogen will result in structural and/or mucosal abnormalities, and will depend on a series of complex interactions between the external environment, the pathogen, and the host and its resident bacterial flora. Enteropathogenic bacteria may be broadly classified as (i) invasive (e.g. Shigella, Salmonella and some Escherichia coli) which predominantly affect the distal bowel, or (ii) non-invasive (e.g. Vibrio cholerae and E. coli) which affect the proximal bowel. V. cholerae and E. coli elaborate heat-labile enterotoxins which activate adenylate cyclase and induce small intestinal secretion; the secretory effects of heat-stable E. coli and heat-labile Shigella dysenteriae enterotoxins are not accompanied by cyclase activation. The two major complications of acute diarrhoea are (i) hypernatraemic dehydration with its attendant neurological, renal and vascular lesions, and (ii) protracted diarrhoea which may lead to severe malnutrition. Deconjugation of bile salts and colonization of the small bowel with toxigenic strains of E. coli may be important in the pathophysiology of the protracted diarrhoea syndrome. The control of bacterial diarrhoea requires a corrdinated political, educational, social, public health and scientific attack. Bacterial diarrhoea is a major health problem throughout the world, and carries an appreciable morbidity and mortality. This is particularly the case during infancy, and in those developing parts of the world

  20. Bacterial survival in Martian conditions

    OpenAIRE

    Galletta, Giuseppe; Bertoloni, Giulio; D'Alessandro, Maurizio

    2010-01-01

    We shortly discuss the observable consequences of the two hypotheses about the origin of life on Earth and Mars: the Lithopanspermia (Mars to Earth or viceversa) and the origin from a unique progenitor, that for Earth is called LUCA (the LUCA hypothesis). To test the possibility that some lifeforms similar to the terrestrial ones may survive on Mars, we designed and built two simulators of Martian environments where to perform experiments with different bacterial strains: LISA and mini-LISA. ...

  1. Small intestinal bacterial overgrowth syndrome

    Institute of Scientific and Technical Information of China (English)

    Jan; Bures; Jiri; Cyrany; Darina; Kohoutova; Miroslav; Frstl; Stanislav; Rejchrt; Jaroslav; Kvetina; Viktor; Vorisek; Marcela; Kopacova

    2010-01-01

    Human intestinal microbiota create a complex polymi-crobial ecology. This is characterised by its high population density, wide diversity and complexity of interaction. Any dysbalance of this complex intestinal microbiome, both qualitative and quantitative, might have serious health consequence for a macro-organism, including small intestinal bacterial overgrowth syndrome (SIBO).SIBO is defined as an increase in the number and/or alteration in the type of bacteria in the upper gastro-intestinal tract. There...

  2. Population dynamics of bacterial persistence

    OpenAIRE

    Patra, Pintu

    2014-01-01

    The life of microorganisms is characterized by two main tasks, rapid growth under conditions permitting growth and survival under stressful conditions. The environments, in which microorganisms dwell, vary in space and time. The microorganisms innovate diverse strategies to readily adapt to the regularly fluctuating environments. Phenotypic heterogeneity is one such strategy, where an isogenic population splits into subpopulations that respond differently under identical environments. Bacteri...

  3. Immunization by a bacterial aerosol

    OpenAIRE

    Garcia-Contreras, Lucila; Wong, Yun-Ling; Muttil, Pavan; Padilla, Danielle; Sadoff, Jerry; DeRousse, Jessica; Germishuizen, Willem Andreas; Goonesekera, Sunali; Elbert, Katharina; Bloom, Barry R.; Miller, Rich; Fourie, P. Bernard; Hickey, Anthony; Edwards, David

    2008-01-01

    By manufacturing a single-particle system in two particulate forms (i.e., micrometer size and nanometer size), we have designed a bacterial vaccine form that exhibits improved efficacy of immunization. Microstructural properties are adapted to alter dispersive and aerosol properties independently. Dried “nanomicroparticle” vaccines possess two axes of nanoscale dimensions and a third axis of micrometer dimension; the last one permits effective micrometer-like physical dispersion, and the form...

  4. Rheumatoid arthritis and bacterial infections

    OpenAIRE

    N L Prokopjeva; N N Vesikova; I M Marusenko; V A Ryabkov

    2008-01-01

    To study features of bacterial infections course in pts with rheumatoid arthritis (RA) and changes of laboratory measures after focus of infection sanation. Material and methods. 46 pts with definite rheumatoid arthritis were examined at the time of comorbid infection (Cl) detection and after infection focus sanation. Bacteriological test with evaluation of flora sensitivity to antibiotics by disco-diffusion method was performed at baseline and after the course of antibacterial therapy to ass...

  5. Molecular approaches for bacterial azoreductases

    OpenAIRE

    Montira Leelakriangsak

    2013-01-01

    Azo dyes are the dominant types of synthetic dyes, widely used in textiles, foods, leather, printing, tattooing, cosmetics, and pharmaceutical industries. Many microorganisms are able to decolorize azo dyes, and there is increasing interest in biological waste treatment methods. Bacterial azoreductases can cleave azo linkages (-N=N-) in azo dyes, forming aromatic amines. This review mainly focuses on employing molecular approaches, including gene manipulation and recombinant strains, to study...

  6. Bacterial meningitis by streptococcus agalactiae

    OpenAIRE

    Villarreal-Velásquez Tatiana Paola; Cortés-Daza César Camilo

    2012-01-01

    Introduction: bacterial meningitis is an infectious disease considered a medicalemergency. The timely management has an important impact on the evolution of thedisease. Streptococcus agalactiae, a major causative agent of severe infections innewborns can colonize different tissues, including the central nervous system.Case report: Male patient 47 years old from rural areas, with work activity as amilker of cattle, referred to tertiary care, with disorientation, neck stiffness, and grandmal se...

  7. Bacterial sex in dental plaque

    Directory of Open Access Journals (Sweden)

    Ingar Olsen

    2013-06-01

    Full Text Available Genes are transferred between bacteria in dental plaque by transduction, conjugation, and transformation. Membrane vesicles can also provide a mechanism for horizontal gene transfer. DNA transfer is considered bacterial sex, but the transfer is not parallel to processes that we associate with sex in higher organisms. Several examples of bacterial gene transfer in the oral cavity are given in this review. How frequently this occurs in dental plaque is not clear, but evidence suggests that it affects a number of the major genera present. It has been estimated that new sequences in genomes established through horizontal gene transfer can constitute up to 30% of bacterial genomes. Gene transfer can be both inter- and intrageneric, and it can also affect transient organisms. The transferred DNA can be integrated or recombined in the recipient's chromosome or remain as an extrachromosomal inheritable element. This can make dental plaque a reservoir for antimicrobial resistance genes. The ability to transfer DNA is important for bacteria, making them better adapted to the harsh environment of the human mouth, and promoting their survival, virulence, and pathogenicity.

  8. Cytochemical Differences in Bacterial Glycocalyx

    Science.gov (United States)

    Krautgartner, Wolf Dietrich; Vitkov, Ljubomir; Hannig, Matthias; Pelz, Klaus; Stoiber, Walter

    2005-02-01

    To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid-metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances-probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

  9. Bacterial adhesion and biofilms on surfaces

    Institute of Scientific and Technical Information of China (English)

    Trevor Roger Garrett; Manmohan Bhakoo; Zhibing Zhang

    2008-01-01

    Bacterial adhesion has become a significant problem in industry and in the domicile,and much research has been done for deeper understanding of the processes involved.A generic biological model of bacterial adhesion and population growth called the bacterial biofilm growth cycle,has been described and modified many times.The biofilm growth cycle encompasses bacterial adhesion at all levels,starting with the initial physical attraction of bacteria to a substrate,and ending with the eventual liberation of cell dusters from the biofilm matrix.When describing bacterial adhesion one is simply describing one or more stages of biofilm development,neglecting the fact that the population may not reach maturity.This article provides an overview of bacterial adhesion.cites examples of how bac-terial adhesion affects industry and summarises methods and instrumentation used to improve our understanding of the adhesive prop-erties of bacteria.

  10. Molecular models of human P-glycoprotein in two different catalytic states

    Directory of Open Access Journals (Sweden)

    Tulkens Paul M

    2009-01-01

    Full Text Available Abstract Background P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies. Results We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants. Conclusion Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs, within the transmembrane domains (TMDs or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission. Our models may characterize different steps

  11. Emergence of sequence type 779 methicillin-resistant Staphylococcus aureus harboring a novel pseudo staphylococcal cassette chromosome mec (SCCmec)-SCC-SCCCRISPR composite element in Irish hospitals.

    LENUS (Irish Health Repository)

    Kinnevey, Peter M

    2013-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878\\/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods.

  12. Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

    Institute of Scientific and Technical Information of China (English)

    SHI Nong-nong; HE Guang-yuan; LI Ke-xiu; WANG Hui-zhong; CHEN Guan-ping; XU Ying

    2007-01-01

    1Ax1 high molecular weight glutenin subunit (HMW-GS) gene expression cassette (GEC) lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of AmpR gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest (GOI) and the selectable marker (MG), bombardment pressure and genotypes are vital for the expression of a transformed GEC.

  13. Automatic synthesis of 16{alpha}-[{sup 18}F]fluoro-17{beta}-estradiol using a cassette-type [{sup 18}F]fluorodeoxyglucose synthesizer

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Tetsuya [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Fukui 910-1193 (Japan)]. E-mail: morit@fmsrsa.fukui-med.ac.jp; Kasamatsu, Shingo [JFE P and S Fukui Branch, University of Fukui, Matsuoka, Fukui 910-1193 (Japan); Mosdzianowski, Christoph [GE Healthcare Technologies, Parc Scientifique du Sart Tilman Av. Pre-Aliy (c/o Socran) B-4031 Liege (Belgium); Welch, Michael J. [Mallinckrodt Institute of Radiology, Washington University Medical Center, St. Louis, MO 63110 (United States); Yonekura, Yoshiharu [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Fukui 910-1193 (Japan); Fujibayashi, Yasuhisa [Biomedical Imaging Research Center, University of Fukui, Matsuoka, Fukui 910-1193 (Japan)

    2006-02-15

    16{alpha}-[{sup 18}F]fluoro-17{beta}-estradiol ([{sup 18}F]FES) is a radiotracer for imaging estrogen receptors by positron emission tomography. We developed a clinically applicable automatic preparation system for [{sup 18}F]FES by modifying a cassette-type [{sup 18}F]fluorodeoxyglucose synthesizer. Two milligrams of 3-O-methoxymethyl-16,17-O-sulfuryl-16-epiestriol in acetonitrile was heated at 105{sup o}C for 10 min with dried [{sup 18}F]fluoride. The resultant solution was evaporated and hydrolyzed with 0.2 N HCl in 90% acetonitrile/water at 95{sup o}C for 10 min under pressurized condition. The neutralization was carried out with 2.8% NaHCO{sub 3}, and then the high-performance liquid chromatography (HPLC) purification was performed. The desired radioactive fraction was collected and the solvent was replaced by 10 ml of saline, and then passed through a 0.22-{mu}m filter into a pyrogen-free vial as the final product. The HPLC purification data demonstrated that [{sup 18}F]FES was synthesized with a yield of 76.4{+-}1.9% (n=5). The yield as the final product for clinical use was 42.4{+-}3.2% (n=5, decay corrected). The total preparation time was 88.2{+-}6.4 min, including the HPLC purification and the solvent replacement process. The radiochemical purity of the final product was >99%, and the specific activity was more than 111 GBq/{mu}mol. The final product was stable for more than 6 h in saline containing sodium ascorbate. This new preparation system enables us to produce [{sup 18}F]FES safe for clinical use with high and reproducible yield.

  14. Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

    Science.gov (United States)

    Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

    2016-08-01

    The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. PMID:27238459

  15. Structural characterization of two metastable ATP-bound states of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Megan L O'Mara

    Full Text Available ATP Binding Cassette (ABC transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs, the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+ with each NBD indicates that the coordination of ATP and Mg(2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation

  16. Periodontal diseases as bacterial infection

    Directory of Open Access Journals (Sweden)

    A. Bascones Martínez

    2005-12-01

    Full Text Available The periodontal disease is conformed by a group of illnesses affecting the gums and dental support structures. They are caused by certain bacteria found in the bacterial plaque. These bacteria are essential to the onset of illness; however, there are predisposing factors in both the host and the microorganisms that will have an effect on the pathogenesis of the illness. Periodontopathogenic bacterial microbiota is needed, but by itself, it is not enough to cause the illness, requiring the presence of a susceptible host. These diseases have been classified as gingivitis, when limited to the gums, and periodontitis, when they spread to deeper tissues. Classification of periodontal disease has varied over the years.The one used in this work was approved at the International Workshop for a Classification of Periodontal Diseases and Conditions, held in 1999. This study is an overview of the different periodontal disease syndromes. Later, the systematic use of antibiotic treatment consisting of amoxicillin, amoxicillinclavulanic acid, and metronidazole as first line coadjuvant treatment of these illnesses will be reviewed.

  17. Bacterial mutagenicity assays: test methods.

    Science.gov (United States)

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  18. BACTERIAL DESEASES IN SEA FISH

    Directory of Open Access Journals (Sweden)

    Ivančica Strunjak-Perović

    1997-10-01

    Full Text Available With development of the fish culturing in the sea, the interest in their health also increased. The reason for this are diseases or rather mortality that occur in such controlled cultures and cause great economic losses. By growing large quantities of fish in rather small species, natural conditions are changed, so fish is more sensitive and prone to infection agents (viruses, bacteria, parasites. Besides, a large fish density in the cultural process accelerates spreading if the diseases, but also enables a better perception of them. In wild populations sick specimen very quickly become predator’s prey, witch makes it difficult to note any pathological changes in such fish. There are lots of articles on viral, bacterial and parasitic diseases nowdays, but this work deals exclusively with bacterial deseases that occur in the controlled sea cultures (vibriosis, furunculosis, pastherelosis, nocardiosis, mycobaceriosis, edwardsielosis, yersiniosis, deseases caused by bacteria of genera Flexibacter, Pseudomonas, Aeromonas, Streptococus and bacteria nephryithis. Yet, the knowledge of these deseases vary, depending on wether a fish species is being cultured for a longer period of time or is only being introduced in the controlled culture.

  19. Bioinformatic Comparison of Bacterial Secretomes

    Institute of Scientific and Technical Information of China (English)

    Catharine Song; Aseem Kumar; Mazen Saleh

    2009-01-01

    The rapid increasing number of completed bacterial genomes provides a good op-portunity to compare their proteomes. This study was undertaken to specifically compare and contrast their secretomes-the fraction of the proteome with pre-dicted N-terminal signal sequences, both type Ⅰ and type Ⅱ. A total of 176 theoreti-cal bacterial proteomes were examined using the ExProt program. Compared with the Gram-positives, the Gram-negative bacteria were found, on average, to con-tain a larger number of potential Sec-dependent sequences. In the Gram-negative bacteria but not in the others, there was a positive correlation between proteome size and secretome size, while there was no correlation between secretome size and pathogenicity. Within the Gram-negative bacteria, intracellular pathogens were found to have the smallest secretomes. However, the secretomes of certain bacte-ria did not fit into the observed pattern. Specifically, the secretome of Borrelia burgdoferi has an unusually large number of putative lipoproteins, and the signal peptides of mycoplasmas show closer sequence similarity to those of the Gram-negative bacteria. Our analysis also suggests that even for a theoretical minimal genome of 300 open reading frames, a fraction of this gene pool (up to a maximum of 20%) may code for proteins with Sec-dependent signal sequences.

  20. Bacterial Culture of Neonatal Sepsis

    Directory of Open Access Journals (Sweden)

    AH Movahedian

    2006-08-01

    Full Text Available Neonatal bacterial sepsis is one of the major cause of morbidity and mortality in neonates. This retrospective study was performed to determine the incidence of bacterial sepsis with focus on Gram negative organisms in neonates admitted at Beheshti Hospital in Kashan, during a 3-yr period, from September 2002 to September 2005. Blood culture was performed on all neonates with risk factors or signs of suggestive sepsis. Blood samples were cultured using brain heart infusion (BHI broth according to standard method. From the 1680 neonates 36% had positive blood culture for Pseudomans aeruginosa, 20.7% for Coagulase negative Staphylococci, and 17% for Klebsiella spp. Gram-negative organisms accounted for 72.1% of all positive cultures. The overall mortality rate was 19.8% (22 /111 of whom 63.6% (14 /22 were preterm. Pseudomona aeruginosa and Klebsiella spp. showed a high degree of resistance to commonly used antibiotics (ampicillin, gentamicin as well as third generation cephalosporins. Continued local surveillance studies are urged to monitor emerging antimicrobial resistance and to guide interventions to minimize its occurrence.