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Sample records for atp-binding cassette subfamily

  1. 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...

  2. 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.

  3. Modulating the function of ATP-binding cassette subfamily G member 2 (ABCG2) with inhibitor cabozantinib.

    Science.gov (United States)

    Zhang, Guan-Nan; Zhang, Yun-Kai; Wang, Yi-Jun; Barbuti, Anna Maria; Zhu, Xi-Jun; Yu, Xin-Yue; Wen, Ai-Wen; Wurpel, John N D; Chen, Zhe-Sheng

    2017-01-25

    Cabozantinib (XL184) is a small molecule tyrosine kinase receptor inhibitor, which targets c-Met and VEGFR2. Cabozantinib has been approved by the Food and Drug Administration to treat advanced medullary thyroid cancer and renal cell carcinoma. In the present study, we evaluated the ability of cabozantinib to modulate the function of the ATP-binding cassette subfamily G member 2 (ABCG2) by sensitizing cells that are resistant to ABCG2 substrate antineoplastic drugs. We used a drug-selected resistant cell line H460/MX20 and three ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2, and ABCG2-482-T7, which overexpress ABCG2. Cabozantinib, at non-toxic concentrations (3 or 5μM), sensitized the ABCG2-overexpressing cells to mitoxantrone, SN-38, and topotecan. Our results indicate that cabozantinib reverses ABCG2-mediated multidrug resistance by antagonizing the drug efflux function of the ABCG2 transporter instead of downregulating its expression. The molecular docking analysis indicates that cabozantinib binds to the drug-binding site of the ABCG2 transporter. Overall, our findings demonstrate that cabozantinib inhibits the ABCG2 transporter function and consequently enhances the effect of the antineoplastic agents that are substrates of ABCG2. Cabozantinib may be a useful agent in anticancer treatment regimens for patients who are resistant to ABCG2 substrate drugs.

  4. Afatinib reverses multidrug resistance in ovarian cancer via dually inhibiting ATP binding cassette subfamily B member 1.

    Science.gov (United States)

    Wang, Sheng-qi; Liu, Shi-ting; Zhao, Bo-xin; Yang, Fu-heng; Wang, Ya-tian; Liang, Qian-Ying; Sun, Ya-bin; Liu, Yuan; Song, Zhi-hua; Cai, Yun; Li, Guo-feng

    2015-09-22

    ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro (p afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo. More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR.

  5. Inherited surfactant deficiency due to uniparental disomy of rare mutations in the surfactant protein-B and ATP binding cassette, subfamily A, member 3 genes

    Science.gov (United States)

    Hamvas, Aaron; Nogee, Lawrence M.; Wegner, Daniel J.; DePass, Kelcey; Christodoulou, John; Bennetts, Bruce; McQuade, Leon R.; Gray, Peter H.; Deterding, Robin R.; Carroll, Travis R.; Kammesheidt, Anja; Kasch, Laura M.; Kulkarni, Shashikant; Cole, F. Sessions

    2009-01-01

    Objective To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in the surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. Study design We resequenced parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only one heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). Results We identified one infant homozygous for the c.1549C>GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the three ABCA3 deficient infants. Two ABCA3 deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and two infants died. None exhibited any non-pulmonary phenotype. Conclusions Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles. PMID:19647838

  6. 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.

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

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

    NARCIS (Netherlands)

    Rijpma, S.R.; Heuvel, J.J.; Velden, M. van der; 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 involve

  9. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires elabor

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

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    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.

  11. 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

  12. 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 i...

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

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    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.

  14. Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo*

    OpenAIRE

    Raichaudhuri, Ayan; Peng, Mingsheng; Naponelli, Valeria; Chen, Sixue; Sánchez-Fernández, Rocío; Gu, Honglan; Gregory, Jesse F.; Hanson, Andrew D; Rea, Philip A.

    2009-01-01

    The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuol...

  15. Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera.

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    Çakır, Birsen; Kılıçkaya, Ozan

    2013-01-01

    The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 "full-size," 41 "half-size," and 15 "soluble" putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.

  16. Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera.

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    Birsen Çakır

    Full Text Available The ATP-binding cassette (ABC protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 "full-size," 41 "half-size," and 15 "soluble" putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog and ABCC (multidrug resistance-associated protein. We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.

  17. ATP-binding cassette transporters in tumor endothelial cells and resistance to metronomic chemotherapy.

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    Hida, Kyoko; Kikuchi, Hiroshi; Maishi, Nako; Hida, Yasuhiro

    2017-02-16

    Drug resistance is a major problem in anticancer therapy. ATP-binding cassette (ABC) transporters have a role in the multidrug resistance. A new regimen of chemotherapy has been proposed, called "metronomic chemotherapy". Metronomic chemotherapy is the frequent, regular administration of drug doses designed to maintain low, but active, concentrations of chemotherapeutic drugs over prolonged periods of time, without causing serious toxicities. Metronomic chemotherapy regimens were developed to optimize the antitumor efficacy of agents that target the tumor vasculature instead of tumor cells, and to reduce toxicity of antineoplastic drugs [1]. Nevertheless, recent studies revealed that ABC transporters are expressed at a higher level in the endothelium in the tumor. To avoid resistance to metronomic anti-angiogenic chemotherapy, ABC transporter inhibition of tumor endothelial cells may be a promising strategy. In this mini-review, we discuss the possible mechanism of resistance to metronomic chemotherapy from the viewpoint of tumor endothelial cell biology, focusing on ABC transporters.

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

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    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.

  19. Transport in technicolor: mapping ATP-binding cassette transporters in sea urchin embryos.

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    Gökirmak, Tufan; Shipp, Lauren E; Campanale, Joseph P; Nicklisch, Sascha C T; Hamdoun, Amro

    2014-09-01

    One quarter of eukaryotic genes encode membrane proteins. These include nearly 1,000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multidrug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease.

  20. 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.

  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. 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

  3. The role of ATP-binding cassette (ABC) transporters in pathogenesis and multidrug resistance of the wheat pathogen Mycosphaerella graminicola

    NARCIS (Netherlands)

    Stergiopoulos, I.

    2003-01-01

    ATP-binding cassette (ABC) transporters are membrane proteins that utilise the energy derived from the hydrolysis of ATP to drive the transport of compounds over biological membranes. They are members of one of the largest protein families to date, present in both pro- and eukaryotic

  4. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

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    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

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

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    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.

  6. A Survey of the ATP-Binding Cassette (ABC Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis.

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    Greta Carmona-Antoñanzas

    Full Text Available Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837, are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences, C (11 and G (2. The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

  7. The role of ATP-binding cassette transporters in neuro-inflammation: relevance for bioactive lipids

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    Gijs eKooij

    2012-04-01

    Full Text Available ATP-binding cassette (ABC transporters are highly expressed by brain endothelial cells that form the blood-brain barrier (BBB. These efflux pumps play an important role in maintaining brain homeostasis as they actively hinder the entry of unwanted blood-derived compounds into the central nervous system (CNS. Consequently, their high activity at the BBB has been a major hurdle for the treatment of several brain diseases, as they prevent numerous drugs to reach their site of action within the brain. Importantly, recent data indicate that endogenous substrates for ABC transporters may include inflammatory mediators, such as prostaglandins, leukotrienes, cytokines, chemokines and bioactive lipids, suggesting a potential role for ABC transporters in immunological responses, and more specifically in inflammatory brain disorders, such as multiple sclerosis (MS. In this review, we will give a comprehensive overview of recent findings that illustrate this novel role for ABC transporters in neuro-inflammatory processes. Moreover, we will provide first insights into underlying mechanisms and focus on the importance for bioactive lipids, in particular platelet-activating factor (PAF, herein. A thorough understanding of these events may form the basis for the development for selective treatment modalities to dampen the neuro-inflammatory attack in MS and thereby reducing tissue damage.

  8. A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly.

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    Schaedler, Theresia A; Thornton, Jeremy D; Kruse, Inga; Schwarzländer, Markus; Meyer, Andreas J; van Veen, Hendrik W; Balk, Janneke

    2014-08-22

    An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe(2+) alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.

  9. Caveolin-1 and ATP binding cassette transporter A1 and G1-mediated cholesterol efflux.

    Science.gov (United States)

    Wang, Faqi; Gu, Hong-mei; Zhang, Da-wei

    2014-01-01

    Atherosclerosis is one major cause of cardiovascular diseases, the leading cause of death in industrialized countries. Reverse cholesterol transport (RCT) is thought to be one primary pathway to protect against atherosclerosis. The first and rate-limiting step of RCT is ATP-binding cassette transport A1 (ABCA1) and ABCG1-mediated cholesterol efflux from the cells. Recently, caveolin-1 (CAV1), a scaffolding protein that organizes and concentrates certain caveolin-interacting signaling molecules and receptors within caveolae membranes, has been shown to regulate ABCA1 and ABCG1-mediated cholesterol efflux probably via interacting with them. In the present review, we summarize the current knowledge and views on the regulatory role of CAV1 on the cholesterol homeostasis with emphasis on the association of CAV1 with ABCA1 and ABCG1. We conclude that the dominance of the positive regulation by CAV1 on the ABCA1 and ABCG1-mediated cholesterol efflux is depending on the species, cell types, as well as the levels of CAV1 expression.

  10. 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.

  11. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance

    OpenAIRE

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting fr...

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

    Directory of Open Access Journals (Sweden)

    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

  13. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance

    Science.gov (United States)

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols. PMID:25254091

  14. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance.

    Science.gov (United States)

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols.

  15. 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.

  16. Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

    Science.gov (United States)

    Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

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

    Directory of Open Access Journals (Sweden)

    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

  18. ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

    Directory of Open Access Journals (Sweden)

    Ruimin Huang

    2011-05-01

    Full Text Available 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. To investigate the effect of ATP-binding cassette (ABC transporters on BLI readout, we generated click beetle (cLuc, firefly (fLuc, Renilla (rLuc, and Gaussia (gLuc luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2. In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type–independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

  19. 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...

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

    Science.gov (United States)

    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-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

  1. ABCC4与人类肿瘤%ATP-binding cassette transporter family class C4 and human cancer

    Institute of Scientific and Technical Information of China (English)

    石妮; 赵晓航

    2011-01-01

    ATP-binding cassette transporter family class C4 (ABCC4) is known as a member of the ATP-binding cassette transporter super-family, involved in the active transport of endogenous anions and xenobiotic, which is not normally produced or expected to be present in human, such as antibiotics. Recently it has been found that the copy number variations of Abcc4 gene and overexpression of ABCC4 protein in many kinds of human cancers, which might involved in tumorigenesis, progress and chemotherapeutic response. This review will focus on the ectopic expression of Abcc4 in human cancer and the potential role of ABCC4 in tumorigenesis and progress.%ABCC4(ATP-binding cassette transporter family class C4,ABCC4)是ABC蛋白家族成员,主要参与转运机体物质代谢中产生的有机阴离子和一些异型生物质等生物学功能.近年研究发现某些人类肿瘤存在Abcc4基因的拷贝数变异,主要表现为Abcc4基因拷贝数增加和ABCC4蛋白过表达,这些改变与肿瘤发生发展、耐药,以及治疗疗效具有相关性.该文综述了Abcc4基因的拷贝数变异和异常表达与肿瘤生物学特性的关系,探讨ABCC4在肿瘤发生发展中的作用机制.

  2. 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.

  3. Structure, function, and evolution of bacterial ATP-binding cassette systems

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, A.L.; Dassa, E.; Orelle, C.; Chen, J. (Purdue)

    2010-07-27

    The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed in Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420). ABC systems couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena, comprising not only transmembrane (TM) transport, for which they are best known, but also several non-transport-related processes, such as translation elongation (62) and DNA repair (174). Although ABC systems deserve much attention because they are involved in severe human inherited diseases (107), they were first discovered and characterized in detail in prokaryotes, as early as the 1970s (13, 148, 238, 468). The most extensively analyzed systems were the high-affinity histidine and maltose uptake systems of Salmonella enterica serovar Typhimurium and Escherichia coli. Over 2 decades ago, after the completion of the nucleotide sequences encoding these transporters in the respective laboratories of Giovanna Ames and Maurice Hofnung, Hiroshi Nikaido and colleagues noticed that the two systems displayed a global similarity in the nature of their components and, moreover, that the primary sequences of MalK and

  4. 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.

  5. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei

    2017-02-23

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  6. 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

  7. 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

    DEFF Research Database (Denmark)

    Hansen, Morten Ejby; Fredslund, Folmer; Andersen, Joakim Mark

    2016-01-01

    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 -(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide...

  8. Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters

    NARCIS (Netherlands)

    Roohparvar, R.; Huser, A.; Zwiers, L.H.; Waard, de M.A.

    2007-01-01

    Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due

  9. 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 (...

  10. 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

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

    Science.gov (United States)

    Zhiyi, Nie; Guijuan, Kang; Yu, Li; Longjun, Dai; Rizhong, Zeng

    2015-01-01

    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.

  12. Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

    Science.gov (United States)

    Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

    2017-02-01

    The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. ATP-Binding Cassette Genes Genotype and Expression: A Potential Association with Pancreatic Cancer Development and Chemoresistance?

    Directory of Open Access Journals (Sweden)

    Li Pang

    2014-01-01

    Full Text Available Genetic polymorphisms in ABC (ATP-binding cassette transporter genes are associated with differential responses to chemotherapy in various cancers including pancreatic cancer. In this study, four SNPs in the ABCB1, ABCC1, and ABCG2 genes were investigated in normal and pancreatic cancerous specimens. The expression of the three transporters was also analyzed. The TT genotypes of G2677T and C3435T in ABCB1 gene were associated with lower risk of developing pancreatic cancer (P=0.013, OR = 0.35 and P=0.015, OR = 0.29, resp.. To our knowledge, this is the first report of the common polymorphisms in the ABCB1 gene affecting the genetic risk of developing pancreatic cancer. Moreover, the expression of ABCB1 in 2677TT and 3435TT carriers was lower compared to the wild-type homozygotes and heterozygotes. A cell viability assay, using standard pancreatic cancer cell lines, revealed that the ABCB1 2677TT-3455TT haplotype was more sensitive than the other haplotypes to gemcitabine. Conclusion. Polymorphisms in ABCB1 G2677T and G3435T were associated with differential susceptibility to pancreatic cancer and may predict responses to chemotherapy.

  19. 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.

  20. ATP结合盒蛋白E1在真核生物中的作用%Role of ATP-binding cassette protein E1 in eukaryote

    Institute of Scientific and Technical Information of China (English)

    尤锋; 李燕

    2012-01-01

    ATP结合盒(ATP-binding cassette,ABC)蛋白超家族是真核生物进化过程中最为保守的一类蛋白.ABCE1是ABC超家族中E亚家族的唯一成员,除可抑制哺乳动物核糖核酸酶L外,还参与真核生物蛋白合成的翻译起始、终止及核糖体循环,并有可能成为新的抗肿瘤靶点.本文对ABCE1的基本生物学特性及其生物学作用作一介绍.%ATP-binding cassette ( ABC) proteins are one of the most conserved protein families in eukaryote evolution. ABCE1 is the only member in ABC E subfamily. It was found that ABCE1, a ribonuclease L inhibitor in mammals, participates in the protein translation's initiation, termination and ribosome recycling of eukaryote, and may become a new antitumor target. This article reviews the basic biological characteristics and functions of ABCE1.

  1. 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...

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

    Directory of Open Access Journals (Sweden)

    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. Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.

    Science.gov (United States)

    Hebbeln, Peter; Rodionov, Dmitry A; Alfandega, Anja; Eitinger, Thomas

    2007-02-20

    BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the alpha-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

  4. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    Science.gov (United States)

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  5. 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

    2016-11-05

    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.

  6. 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

    The ATP-binding cassette (ABC) transporter family transports various molecules across the enterocytes in the gut protecting the intestine against potentially harmful substances. Moreover, ABC transporters are involved in mucosal immune defence through interaction with cytokines. The study aimed...

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

    Directory of Open Access Journals (Sweden)

    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

  8. 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

  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. 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...

  11. Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

    Science.gov (United States)

    Atsumi, Shogo; Miyamoto, Kazuhisa; Yamamoto, Kimiko; Narukawa, Junko; Kawai, Sawako; Sezutsu, Hideki; Kobayashi, Isao; Uchino, Keiro; Tamura, Toshiki; Mita, Kazuei; Kadono-Okuda, Keiko; Wada, Sanae; Kanda, Kohzo; Goldsmith, Marian R; Noda, Hiroaki

    2012-06-19

    Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

  12. 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......We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines...... resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated...

  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. 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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Luty Adrian JF

    2009-08-01

    Full Text Available Abstract 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 cassette (ABC protein, as an important determinant of resistance. In the Plasmodium falciparum genome, there are several ABC transporters some of which could be putative drug transporting proteins. In order to understand the molecular mechanisms underlying drug resistance, characterization of these transporters is essential. The aim of this study was to characterize and localize putative ABC transporters. Methods In the plasmoDB database, 16 members of the P. falciparum ABC family can be identified, 11 of which are putative transport proteins. A phylogenetic analysis of the aligned NBDs of the PfABC genes was performed. Antibodies against PfMRP1 (PfABCC1, PfMRP2 (PfABCC2, and PfMDR5 (PfABCB5 were generated, affinity purified and used in immunocytochemistry to localize the proteins in the asexual stages of the parasite. Results The ABC family members of P. falciparum were categorized into subfamilies. The ABC B subfamily was the largest and contained seven members. Other family members that could be involved in drug transport are PfABCC1, PfABCC2, PfABCG1, and PfABCI3. The expression and localization of three ABC transport proteins was determined. PfMRP1, PfMRP2, and PfMDR5 are localized to the plasma membrane in all asexual stages of the parasite. Conclusion In conclusion, 11 of the 16 ABC proteins in the P. falciparum genome are putative transport proteins, some of which might be involved in drug resistance. Moreover, it was demonstrated that three of these proteins are expressed on the parasite's plasma membrane.

  17. 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.

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

    Science.gov (United States)

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    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.

  19. Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

    Science.gov (United States)

    Li, Xiao-Rui; Yang, Liu-Zhong; Huo, Xiao-Qing; Wang, Ying; Yang, Qing-Hui; Zhang, Qing-Qin

    2015-07-01

    In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (Pmigration capacity of the cells in the experimental group was significantly decreased (Pmigration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

  20. Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

    Science.gov (United States)

    Schäfer-Somi, S; Palme, N

    2016-04-01

    The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as 40% morphologically abnormal sperm and/or Bad freezers were older than good freezers (5.3 vs 3.4 years, p bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p good freezers (p bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings.

  1. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.

    Science.gov (United States)

    Iqbal, Jahangir; Parks, John S; Hussain, M Mahmood

    2013-10-18

    We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92-95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations.

  2. Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica

    Science.gov (United States)

    Shitan, Nobukazu; Bazin, Ingrid; Dan, Kazuyuki; Obata, Kazuaki; Kigawa, Koji; Ueda, Kazumitsu; Sato, Fumihiko; Forestier, Cyrille; Yazaki, Kazufumi

    2003-01-01

    Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome. PMID:12524452

  3. Lobular Distribution and Variability in Hepatic ATP Binding Cassette Protein B1 (ABCB1, P-gp): Ontogenetic Differences and Potential for Toxicity

    Science.gov (United States)

    Abanda, Ngu Njei; Riches, Zoe; Collier, Abby C.

    2017-01-01

    The ATP Binding Cassette B1 (ABCB1) transporter has critical roles in endo- and xenobiotic efficacy and toxicity. To understand population variability in hepatic transport we determined ABCB1 mRNA and protein levels in total liver lysates sampled from 8 pre-defined sites (n = 24, 18–69 years), and in S9 from randomly acquired samples (n = 87, 7 days–87 years). ABCB1 levels did not differ significantly throughout individual livers and showed 4.4-fold protein variation between subjects. Neither mRNA nor protein levels varied with sex, ethnicity, obesity or triglycerides in lysates or S9 (that showed the same relationships), but protein levels were lower in pediatric S9 (p < 0.0001), with 76% of adult ABCB1 present at birth and predicted to mature in 5 years. Pediatric total liver lysates were not available. In summary, opportunistic collection for studying human hepatic ABCB1 is acceptable. Additionally, ABCB1 may be lower in children, indicating differential potential for toxicity and response to therapy in this special population. PMID:28218636

  4. Effect of Apolipoprotein A-I on ATP Binding Cassette Transporter A1 Degradation and Cholesterol Efflux in THP-1 Macrophage-derived Foam Cells

    Institute of Scientific and Technical Information of China (English)

    Chao-Ke TANG; Chang-Geng RUAN; Yong-Zong YANG; Guo-Hua TANG; Guang-Hui IY; Zuo WANG; Lu-Shan LIU; Shuang WAN; Zhong-Hua YUAN; Xiu-Sheng HE; Jun-Hao YANG

    2004-01-01

    Cholesterol-loaded macrophage foam cells are a central componentof atherosclerotic lesions ATP binding cassette transporter A1(ABCA1),the defective molecule in Tangier disease,mediates the efflux ofphospholipid and cholesterol from cells to apolipoprotein A-I(apoA-I),reversing foam cell formation.This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophagederived foam cells.After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time,cholesterol efflux,ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator,RT-PCR and Western blot,respectively.The mean ABCA1 fluorescence intensity on THP-1macrophage-derived foam cells was detected by flow cytometry.Results showed that apoA-I markedly increased ABCAl-mediated cholesterol efflux from THP-1 macrophage-derived foam cells.This was accompanied by an increase in the content of ABCA1.ApoA-I did not alter ABCA 1 mRNA abundance.Significantly,thiol protease inhibitors increased the level ofABCA1 protein and slowed its decay in THP-1macrophage-derived foam cells,whereas none of the proteosome-specific inhibitor lactacystin,other protease inhibitors,or the lysosomal inhibitor NH4Cl showed such effects.The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors.Our results suggested that thiol protease inhibitors mightprovide an alternative way to upregulate ABCA1 protein.This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

  5. 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.

  6. 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.

  7. 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.

  8. Polycyclic aromatic hydrocarbons (PAHs) mediate transcriptional activation of the ATP binding cassette transporter ABCB6 gene via the aryl hydrocarbon receptor (AhR).

    Science.gov (United States)

    Chavan, Hemantkumar; Krishnamurthy, Partha

    2012-09-14

    Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

  9. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  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. 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.

  13. 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.

  14. 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

  15. The bovine ATP-binding cassette transporter ABCG2 Tyr581Ser single-nucleotide polymorphism increases milk secretion of the fluoroquinolone danofloxacin.

    Science.gov (United States)

    Otero, Jon A; Real, Rebeca; de la Fuente, Álvaro; Prieto, Julio G; Marqués, Margarita; Álvarez, Ana I; Merino, Gracia

    2013-03-01

    The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues.

  16. Binding of PDZ-RhoGEF to ATP-binding cassette transporter A1 (ABCA1) induces cholesterol efflux through RhoA activation and prevention of transporter degradation.

    Science.gov (United States)

    Okuhira, Keiichiro; Fitzgerald, Michael L; Tamehiro, Norimasa; Ohoka, Nobumichi; Suzuki, Kazuhiro; Sawada, Jun-ichi; Naito, Mikihiko; Nishimaki-Mogami, Tomoko

    2010-05-21

    ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

  17. Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

    Science.gov (United States)

    Merino, Gracia; Real, Rebeca; Baro, Marta F; Gonzalez-Lobato, Lucia; Prieto, Julio G; Alvarez, Ana I; Marques, Margarita M

    2009-01-01

    ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

  18. 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.

  19. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

  20. 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.

  1. Kaempferol suppresses lipid accumulation in macrophages through the downregulation of cluster of differentiation 36 and the upregulation of scavenger receptor class B type I and ATP-binding cassette transporters A1 and G1.

    Science.gov (United States)

    Li, Xiu-Ying; Kong, Ling-Xi; Li, Juan; He, Hai-Xia; Zhou, Yuan-Da

    2013-02-01

    The accumulation of foam cells in atherosclerotic lesions is a hallmark of early-stage atherosclerosis. Kaempferol has been shown to inhibit oxidized low-density lipoprotein (oxLDL) uptake by macrophages; however, the underlying molecular mechanisms are not yet fully investigated. In this study, we shown that treatment with kaempferol markedly suppresses oxLDL-induced macrophage foam cell formation, which occurs due to a decrease in lipid accumulation and an increase in cholesterol efflux from THP-1-derived macrophages. Additionally, the kaempferol treatment of macrophages led to the downregulation of cluster of differentiation 36 (CD36) protein levels, the upregulation of ATP-binding cassette (ABC) transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI) and ABCG1 protein levels, while no effects on scavenger receptor A (SR-A) expression were observed. Kaempferol had similar effects on the mRNA and protein expression of ABCA1, SR-BI, SR-A, CD36 and ABCG1. The reduced CD36 expression following kaempferol treatment involved the inhibition of c-Jun-activator protein-1 (AP-1) nuclear translocation. The inhibition of AP-1 using the inhibitor, SP600125, confirmed this involvement, as the AP-1 inhibition significantly augmented the kaempferol-induced reduction in CD36 expression. Accordingly, the kaempferol-mediated suppression of lipid accumulation in macrophages was also augmented by SP600125. The increased expression of ABCA1, SR-BI and ABCG1 following kaempferol treatment was accompanied by the enhanced protein expression of heme oxygenase-1 (HO-1). This increase was reversed following the knockdown of the HO-1 gene using small hairpin RNA (shRNA). Moreover, the kaempferol-mediated attenuation of lipid accumulation and the promotion of cholesterol efflux was also inhibited by HO-1 shRNA. In conclusion, the c-Jun-AP‑1-dependent downregulation of CD36 and the HO-1-dependent upregulation of ABCG1, SR-BI and ABCA1 may mediate the beneficial effects of

  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. 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.

    Science.gov (United States)

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

    2014-07-07

    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 minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure-activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [(125)I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

  4. 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

  5. Moving out: from sterol transport to drug resistance - the ABCG subfamily of efflux pumps.

    Science.gov (United States)

    Moitra, Karobi; Silverton, Latoya; Limpert, Katy; Im, Kate; Dean, Michael

    2011-01-01

    The ATP binding cassette (ABC) proteins are typically ATP-driven transmembrane pumps that have been evolutionarily conserved from bacteria to humans. In humans these transporters are subdivided into seven subfamilies, ranging from A to G. The ABCG subfamily of transporters is the primary focus of this review. This subfamily of proteins has been conserved throughout evolution and plays a central role in several cellular processes, such as sterol homeostasis and multidrug resistance. Functional polymorphisms/mutations in some of these G-subfamily transporters have clinical consequences in humans.

  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. 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.

  8. 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.

  9. Effect of 5-fluorouracil on the expression of ATP-binding cassette superfamily G member 2 in human colon cancer cell SW480%氟尿嘧啶对人结肠癌SW480细胞ABCG2表达的影响

    Institute of Scientific and Technical Information of China (English)

    瞿金妙; 尤捷; 刘海光; 黄奇迪; 郭贵龙

    2013-01-01

    目的 观察氟尿嘧啶(5-FU)对人结肠癌SW480细胞ABCG2表达的影响.方法 用不同药物浓度的5-FU处理SW480细胞,用CCK8法检测5-FU在SW480中的IC50,流式细胞仪检测SW480细胞ABCG2的阳性表达率,RT-PCR检测ABCG2的mRNA在SW480细胞中的表达差异.结果 5-FU对SW480细胞的IC50随着药物浓度的增加而升高(P<0.05).流式细胞仪检测发现,正常SW480细胞(A组)中ABCG2阳性表达率为(6.26±0.86)%;在药物处理48 h后即刻检测时(B组)的阳性表达率下降至(3.43±1.18)%(P<0.05);在药物处理48 h后的第2代细胞检测时(C组)则升高至(12.91±3.42)%(P<0.05).3组ABCG2 mRNA表达趋势与流式细胞仪检测结果的趋势一致.结论 不同浓度的5-FU可以影响人结肠癌SW480细胞ABCG2的表达.%Objective To investigate the effect of 5-fluorouracil (5-FU) on the expression of ATP-binding cassette superfamily G member 2(ABCG2) in human colon cancer cell SW480.Methods SW480 cells were treated with various concentrations of 5-FU.CCK8 assay was utilized to detect the 5-FU IC50 to SW480 cells.Positive expression of ABCG2 was detected by flow cytometry,and mRNA expression of ABCG2 was detected by real time polymerase chain reaction (RT-PCR).Results The 5-FU IC50 to SW480 cells increased as the drug concentration increased(P<0.05).Flow cytometry revealed that positive expression rate of ABCG2 in normal SW480 cells (group A) was (6.26±0.86)%.Immediately after treatment with 5-FU for 48 hours,the positive expression rate of ABCG2 (group B) was (3.43±1.18)%(P<0.05).In the second passage of cells after treatment with 5-FU for 48 hours,the positive expression rate of ABCG2 (group C) was (12.91±3.42)%(P<0.05).The mRNA expression of ABCG2 detected by RT-PCR was in accordance with the results from flow cytometry.Conclusion Expression of ABCG2 in SW480 cells can be affected by various concentrations of 5-FU.

  10. 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.

  11. 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.

  12. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.

    Science.gov (United States)

    Bompadre, Silvia G; Hwang, Tzyh-Chang

    2007-08-25

    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 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

  13. Recent advances regarding the role of ABC subfamily C member 10 (ABCC10) in the efflux of antitumor drugs

    Institute of Scientific and Technical Information of China (English)

    Rishil J. Kathawala; Yi-Jun Wang; Charles R. Ashby Jr; Zhe-Sheng Chen

    2014-01-01

    ABCC10, also known as multidrug-resistant protein 7 (MRP7), is the tenth member of the C subfamily of the ATP-binding cassette (ABC) superfamily. ABCC10 mediates multidrug resistance (MDR) in cancer cells by preventing the intracellular accumulation of certain antitumor drugs. The ABCC10 transporter is a 171-kDa protein that is localized on the basolateral cell membrane. ABCC10 is a broad-specificity transporter of xenobiotics, including antitumor drugs, such as taxanes, epothilone B, vinca alkaloids, and cytarabine, as wel as modulators of the estrogen pathway, such as tamoxifen. In recent years, ABCC10 inhibitors, including cepharanthine, lapatinib, erlotinib, nilotinib, imatinib, sildenafil, and vardenafil, have been reported to overcome ABCC10-mediated MDR. This review discusses some recent and clinically relevant aspects of the ABCC10 drug efflux transporter from the perspective of current chemotherapy, particularly its inhibition by tyrosine kinase inhibitors and phosphodiesterase type 5 inhibitors.

  14. ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena.

    Science.gov (United States)

    Ning, YingZhi; Dang, Huai; Liu, GuangLong; Xiong, Jie; Yuan, DongXia; Feng, LiFang; Miao, Wei

    2015-03-01

    The reuse of dichlorodiphenyltrichloroethane (DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down (abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L(-1) DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

    Science.gov (United States)

    Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

    2008-01-01

    Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6422, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å3 Da−1, which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan. PMID:18540059

  20. Equilibrated Atomic Models of Outward-Facing P-glycoprotein and Effect of ATP Binding on Structural Dynamics

    Science.gov (United States)

    Pan, Lurong; Aller, Stephen G.

    2015-01-01

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms. PMID:25600711

  1. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    Science.gov (United States)

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-06-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH. radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD.-, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396-. Its negative charge could trigger conformational changes necessary for signal transduction.

  2. 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

  3. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L; Kirk, Kevin L

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

  4. 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.

  5. 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.

  6. Enhancement of avermectin and ivermectin production by overexpression of the maltose ATP-binding cassette transporter in Streptomyces avermitilis.

    Science.gov (United States)

    Li, Meng; Chen, Zhi; Zhang, Xuan; Song, Yuan; Wen, Ying; Li, Jilun

    2010-12-01

    We investigated the function of maltose ABC transporter system encoded by malEFG-a and the effect of its overexpression on antibiotic production in Streptomyces avermitilis. A malEFG-a deletion mutant was unable to grow in a minimal medium with maltose as sole carbon source and produce avermectin. Maltose utilization and avermectin production were restored by introduction of a single copy of malEFG-a. RT-PCR analysis showed that the expression of malE-a was induced by maltose, and was strongly repressed by glucose. When multi-copy, integrative malEFG-a gene expression vectors were introduced into wild-type strain ATCC31267 and ivermectin-producer OI-31, antibiotic production increased by 2.6- to 3.3-fold and the time required for fermentation decreased by about 10%. The overexpression of malEFG-a improved the utilization rate of starch, and thereby enhanced avermectin production. Such an approach would be useful for the improvement of commercial antibiotic production using starch as the main carbon source in the fermentation process.

  7. 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...

  8. Function and regulation of ATP-binding cassette transport proteins involved in hepatobiliary transport (vol 12, pg 13, 2000)

    NARCIS (Netherlands)

    Hooiveld, GJEJ; van Montfoort, JE; Meijer, DKF; Muller, M

    2001-01-01

    Hepatobiliary transport of endogenous and exogenous compounds is mediated by the coordinated action of multiple transport systems present at the sinusoidal (basolateral) and canalicular (apical) membrane domains of hepatocytes. During the last few years many of these transporters have been cloned an

  9. ATP binding and aspartate protonation enhance photoinduced electron transfer in plant cryptochrome.

    Science.gov (United States)

    Cailliez, Fabien; Müller, Pavel; Gallois, Michaël; de la Lande, Aurélien

    2014-09-17

    Cryptochromes are flavoproteins encountered in most vegetal and animal species. They play a role of blue-light receptors in plants and in invertebrates. The putative resting state of the FAD cofactor in these proteins is its fully oxidized form, FADox. Upon blue-light excitation, the isoalloxazine ring (ISO) may undergo an ultrafast reduction by a nearby tryptophan residue W400. This primary reduction triggers a cascade of electron and proton transfers, ultimately leading to the formation of the FADH° radical. A recent experimental study has shown that the yield of FADH° formation in Arabidopsis cryptochrome can be strongly modulated by ATP binding and by pH, affecting the protonation state of D396 (proton donor to FAD°(-)). Here we provide a detailed molecular analysis of these effects by means of combined classical molecular dynamics simulations and time-dependent density functional theory calculations. When ATP is present and D396 protonated, FAD remains in close contact with W400, thereby enhancing electron transfer (ET) from W400 to ISO*. In contrast, deprotonation of D396 and absence of ATP introduce flexibility to the photoactive site prior to FAD excitation, with the consequence of increased ISO-W400 distance and diminished tunneling rate by almost two orders of magnitude. We show that under these conditions, ET from the adenine moiety of FAD becomes a competitive relaxation pathway. Overall, our data suggest that the observed effects of ATP and pH on the FAD photoreduction find their roots in the earliest stage of the photoreduction process; i.e., ATP binding and the protonation state of D396 determine the preferred pathway of ISO* relaxation.

  10. 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.

  11. 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.

  12. Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis.

    Science.gov (United States)

    Mao, Lisong; Wang, Yanli; Liu, Yuemin; Hu, Xiche

    2004-02-20

    intermolecular interactions of large biomolecular systems becomes computationally feasible. The establishment of the molecular basis for recognition of the adenine moiety of ATP in proteins will directly impact molecular design of ATP-binding site targeted enzyme inhibitors such as kinase inhibitors.

  13. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    Directory of Open Access Journals (Sweden)

    Wee Tek Tay

    2015-11-01

    Full Text Available The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the

  14. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    Science.gov (United States)

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

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

    Science.gov (United States)

    Kim, Jae-Young; Stewart, Paul A; Borne, Adam L; Fang, Bin; Welsh, Eric A; Chen, Yian Ann; Eschrich, Steven A; Koomen, John M; Haura, Eric B

    2016-06-01

    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.

  16. 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.

  17. ATP binding and hydrolysis-driven rate-determining events in the RFC-catalyzed PCNA clamp loading reaction.

    Science.gov (United States)

    Sakato, Miho; Zhou, Yayan; Hingorani, Manju M

    2012-02-17

    The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp·DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at ~2 s(-1)) and bind primer-template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC·ATP·PCNA(open)·ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s(-1)). Dissociation of PCNA·ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.

  18. 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

  19. AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene

    OpenAIRE

    Lu, Yu-Ping; Li, Ze-Sheng; Rea, Philip A.

    1997-01-01

    Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione S-transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of g...

  20. ABC转运蛋白提升四膜虫对DDT的耐受能力%ATP-binding cassette transporter enhances Tetrahymena tolerance to DDT

    Institute of Scientific and Technical Information of China (English)

    宁应之; 党怀; 刘光龙; 熊杰; 袁冬霞; 冯立芳; 缪炜

    2014-01-01

    世界卫生组织允许DDT在部分国家和地区使用.大规模频繁使用杀虫剂造成了蚊子的抗药性,大大降低了DDT的防治效果.目前对于DDT的耐受主要集中在蚊子,但对于喷洒的DDT进入水生态系统后,造成食物链底层水生生物体对DDT耐受还未见报道.本研究对DDT胁迫下的四膜虫全基因组中筛选出一个ATP结合盒转运蛋白基因(abcb15),它能够识别DDT作为其转运的底物.绿色荧光蛋白标记显示ABCB 15蛋白定位于细胞膜上,是一个细胞膜泵蛋白.基因敲降技术造成的abcb15基因敲降株在DDT胁迫下表现出更低的生长速率,这表明ABCB15膜泵蛋白能够将进入细胞内的DDT转运至细胞膜外,藉以提高四膜虫对DDT的耐受能力,以减轻DDT对细胞的损伤.

  1. 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.

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    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.

  2. 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.

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

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    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.

  4. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells

    NARCIS (Netherlands)

    Hinrichs, JWJ; Klappe, K; Hummel, [No Value; Kok, JW

    2004-01-01

    In this study we show that P-glycoprotein in multi-drug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multi-drug-resistant HT29(col) human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched

  5. ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

    Science.gov (United States)

    Nowyhed, Heba N.; Chandra, Shilpi; Kiosses, William; Marcovecchio, Paola; Andary, Farah; Zhao, Meng; Fitzgerald, Michael L.; Kronenberg, Mitchell; Hedrick, Catherine C.

    2017-01-01

    ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function. PMID:28091533

  6. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

    NARCIS (Netherlands)

    Sakamoto, K; Margolles, A; van Veen, HW; Konings, WN

    2001-01-01

    Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino

  7. 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

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

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    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.

  10. A man-made ATP-binding protein evolved independent of nature causes abnormal growth in bacterial cells.

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    Joshua M Stomel

    Full Text Available Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology.

  11. 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.

  12. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

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    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  13. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    Energy Technology Data Exchange (ETDEWEB)

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    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. 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

  15. 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.

  16. Simulated microgravity alters the expression of cytoskeleton- and ATP-binding-related genes in MLO-Y4 osteocytes

    Science.gov (United States)

    Chen, Zhihao; Zhao, Fan; Qi, Yiduo; Hu, Lifang; Li, Dijie; Yin, Chong; Su, Peihong; Zhang, Yan; Ma, Jianhua; Qian, Jing; Zhou, Hongpo; Zou, Yiwei; Qian, Airong

    2016-12-01

    Bone undergoes dynamic modelling and remodelling processes, and it requires gravity-mediated mechanical stimulation for the maintenance of mineral content and structure. Osteocytes are the most commonly found cells in the mature bone, and they are sensitive to mechanical changes. The purpose of this study was to investigate the effects of microgravity simulated with a random position machine (RPM) on the gene expression profile of osteocytes. Genes sensitive to RPM treatment were sorted on the basis of biological processes, interactions and signalling pathways. Overall, 504 differentially expressed genes (DEGs) in osteocytes cultured under RPM conditions were found. The DEGs were further analysed using bioinformatics tools such as DAVID and iReport. A total of 15 ATP-binding and cytoskeleton-related genes were further confirmed by quantitative real-time PCR (qRT-PCR). Our findings demonstrate that the RPM affected the expression of genes involved in cytoskeleton remodelling and the energy-transfer process in osteocytes. The identification of mechanosensitive genes may enhance our understanding of the roles of osteocytes in mechanosensation and may provide some potential targets for preventing and treating bone-related diseases.

  17. 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

  18. 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.

  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.

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    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. The rem Mutations in the ATP-Binding Groove of the Rad3/XPD Helicase Lead to Xeroderma pigmentosum-Cockayne Syndrome-Like Phenotypes

    Science.gov (United States)

    Montelone, Beth A.; Aguilera, Andrés

    2014-01-01

    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. PMID:25500814

  1. Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

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    Zhongshan Wang

    2011-01-01

    Full Text Available The cloning, expression and purification of the glutathione (sulfur import system ATP-binding protein (gsiA was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3 was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3 provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

  2. 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.

  3. Automated protein subfamily identification and classification.

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    Duncan P Brown

    2007-08-01

    Full Text Available Function prediction by homology is widely used to provide preliminary functional annotations for genes for which experimental evidence of function is unavailable or limited. This approach has been shown to be prone to systematic error, including percolation of annotation errors through sequence databases. Phylogenomic analysis avoids these errors in function prediction but has been difficult to automate for high-throughput application. To address this limitation, we present a computationally efficient pipeline for phylogenomic classification of proteins. This pipeline uses the SCI-PHY (Subfamily Classification in Phylogenomics algorithm for automatic subfamily identification, followed by subfamily hidden Markov model (HMM construction. A simple and computationally efficient scoring scheme using family and subfamily HMMs enables classification of novel sequences to protein families and subfamilies. Sequences representing entirely novel subfamilies are differentiated from those that can be classified to subfamilies in the input training set using logistic regression. Subfamily HMM parameters are estimated using an information-sharing protocol, enabling subfamilies containing even a single sequence to benefit from conservation patterns defining the family as a whole or in related subfamilies. SCI-PHY subfamilies correspond closely to functional subtypes defined by experts and to conserved clades found by phylogenetic analysis. Extensive comparisons of subfamily and family HMM performances show that subfamily HMMs dramatically improve the separation between homologous and non-homologous proteins in sequence database searches. Subfamily HMMs also provide extremely high specificity of classification and can be used to predict entirely novel subtypes. The SCI-PHY Web server at http://phylogenomics.berkeley.edu/SCI-PHY/ allows users to upload a multiple sequence alignment for subfamily identification and subfamily HMM construction. Biologists wishing to

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

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    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

  5. 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.

  6. 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.

  7. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

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    Gracian Tejral

    2017-03-01

    Full Text Available Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

  8. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

    Science.gov (United States)

    Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm

    2017-01-01

    Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis. PMID:28316890

  9. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

    Science.gov (United States)

    Betzenhauser, Matthew J; Wagner, Larry E; Park, Hyung Seo; Yule, David I

    2009-06-12

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

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

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    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.

  11. Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins.

    Science.gov (United States)

    Hollenstein, Kaspar; Comellas-Bigler, Mireia; Bevers, Loes E; Feiters, Martin C; Meyer-Klaucke, Wolfram; Hagedoorn, Peter-Leon; Locher, Kaspar P

    2009-06-01

    Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO(4) (2-)) and tungstate (WO(4) (2-)). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria.

  12. 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.

  13. 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.

  14. Whatever happened to cassette-dosing pharmacokinetics?

    Science.gov (United States)

    Manitpisitkul, Prasarn; White, Ronald E

    2004-08-01

    Cassette dosing is a procedure that is used for rapidly assessing the pharmacokinetics of a series of discovery drug candidates by dosing a mixture of compounds rather than a single compound. Cassette dosing has advantages and disadvantages associated with its use, which leads to controversy about how and if it should be used. To assess the current practices of the pharmaceutical industry regarding cassette dosing, a survey of several pharmaceutical companies was conducted. Analysis of the survey revealed that opinion on this subject is divided within the pharmaceutical industry. In addition, it was determined that approximately only a half of those companies that perform in vivo pharmacokinetic screening use cassette dosing for this purpose.

  15. 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

  16. 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 ...

  17. 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

  18. AcEST: DK962825 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ly A member 1... 34 0.43 sp|Q8WWZ4|ABCAA_HUMAN ATP-binding cassette sub-family A ...ctase chain 5 OS=C... 31 3.6 sp|Q54R52|ABCAA_DICDI ABC transporter A family member 10 OS=Dict... 31 3.7 sp|P

  19. Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

    Science.gov (United States)

    While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (ATP-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baselin...

  20. Concept design of the cassette toroidal mover

    Energy Technology Data Exchange (ETDEWEB)

    Maekinen, H., E-mail: harri.makinen@vtt.fi [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Jaervenpaeae, J. [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Valkama, P.; Vaeyrynen, J.; Amjad, F. [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); Siuko, M. [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Mattila, J. [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); Semeraro, L.; Esque, S. [Fusion for Energy, Torres Diagonal Litoral B3, Josep Pla 2, 08019 Barcelona (Spain)

    2011-10-15

    A full scale physical development and test facility, Divertor Test Platform 2 (DTP2), has been established in Finland for the purpose of demonstrating and developing the remote handling (RH) equipment designs for ITER using prototypes and virtual models. The major objective of the DTP2 environment is to verify and develop ITER divertor RH devices and operations. In practice this means various test trials and measurements of performance characteristics. This paper describes the design process of the Cassette Toroidal Mover (CTM). The main purpose of this design task was the development of the CTM concept. The goal of the design process was to achieve compatibility between CTM and the latest ITER divertor design. The design process was based on using a variety of tools, i.e. Catia V5, Delmia, Ansys, Mathcad and project management tools. Applicable European Standards were applied to the concept design. CTM is the cassette transporter, which carries divertor cassettes on the toroidal rails inside the ITER Vacuum Vessel (VV) during the divertor maintenance. The operation environment differs from a common industrial environment. Radiation level is 100 Gy/h. The temperature during RH operations can be 50 {sup o}C. Clearances are less than 20 mm and the loads carried weigh 9000 kg. These conditions require special solutions during the product development process. The design process consisted of defining and developing of the CTM operational sequence. This sequence includes the procedure of how the CTM - with it is onboard manipulator - prepares for and handles the divertor cassettes during RH operations. RH operations are essential part when defining CTM functions. High reliability is required in order to carry out RH tasks successfully. The recoverability of CTM is also an important design criteria. This paper describes the design process and the structure of the CTM concept.

  1. 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.

  2. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

    Science.gov (United States)

    Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G

    2013-02-07

    CaMKII (Ca²⁺/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.

  3. Tetrahymena metallothioneins fall into two discrete subfamilies.

    Directory of Open Access Journals (Sweden)

    Silvia Díaz

    Full Text Available BACKGROUND: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals. METHODOLOGY/PRINCIPAL FINDINGS: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5. Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported. CONCLUSION/SIGNIFICANCE: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

  4. 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.

  5. Estimating diversence times among subfamilies in Nymphalidae

    Institute of Scientific and Technical Information of China (English)

    ZHANG Min; CAO TianWen; JIN Ke; REN ZhuMei; GUO YaPing; SHI Jing; ZHONG Yang; MA EnBo

    2008-01-01

    The mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear elongation factor 1α (EF-1α) gene were sequenced from 13 species of Nymphalidae. Phylogenetic trees of Nymphalidae, which is the largest family in butterflies, were constructed based on the sequences determined from 13 species sequenced in our laboratory and an additional 43 species obtained from GenBank using the maximum likelihood (ML) and Bayesian methods. Relative-rate tests between lineages in these phylogenetic trees were performed. On the basis of the results of the relative-rate tests and fossil information of Satyrinae, Nymphalinae and Biblidinae, the average divergence times among the subfamilies are estimated as 44.2-87.1 million years ago (Ma). These results will be helpful for better understanding of the origin and evolution of this family, as well as the divergence time of butterflies and other complex taxa.

  6. ATP-Binding Cassettee Transporter Signals Salt-Induced Stomatal Closure in Arabidopsis thaliana L. by H2S Pathway%ABC转运体位于H2S上游参与盐胁迫诱导的拟南芥气孔关闭

    Institute of Scientific and Technical Information of China (English)

    吴延朋; 李洪旺; 侯丽霞; 张丹丹; 刘新

    2014-01-01

    本文以拟南芥野生型、ABC转运体缺失突变体(Atmrp4、Atmrp5和Atmrp4/5)为材料研究了硫化氢(hydrogen sulfide, H2S)和ABC转运体在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片AtMRP4及AtMRP5表达量显著升高,诱导野生型拟南芥叶片气孔关闭,但对Atmrp4、Atmrp5及Atmrp4/5气孔开度无显著影响;而ABC转运体抑制剂格列本脲(glibenclamide, Gli)可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明ABC转运体参与盐胁迫诱导的拟南芥气孔关闭过程。盐胁迫能够引起野生型拟南芥H2S合成相关酶L-/D-半胱氨酸脱巯基酶(L-/D-CDes)活性及H2S含量显著升高,而ABC转运体抑制剂格列本脲处理后则没有这种变化,同时盐胁迫也不能引起Atmrp4、Atmrp5及Atmrp4/5的L-/D-CDes活性及H2S含量显著升高,表明ABC转运体位于H2S上游参与盐胁迫诱导气孔关闭过程。%Using Arabidopsis thaliana wild type, ATP-binding cassette (ABC) transporter deficient mutants Atmrp4, Atmrp5, Atmrp4/5 as materials, the participation of hydrogen sulifde (H2S) and ABC transporter signal pathway in salt-induced stomatal closure were studied. These results showed that AtMRP4 and AtMRP5 transcription rate in Arabidopsis leaves increased dramaticly under salt stress, and salt stress induced stomata closure in wild type, but had no effect on Atmrp4, Atmrp5 and Atmrp4/5. The ABC transporter inhibitor glibenclamide (Gli) inhibited the inducing effects of salt stress on stomata closure in wild type, it could be deduced that ABC transporter participate in salt-induced stomatal closure in Arabidopsis. Salt-induced increase in L-/D-cysteine desulfhydrase (L-/D-CDes, enzymes participate in H2S production) activities and H2S content could be seen in wild type, but had no effect on wild type treated by Gli as well as Atmrp4, Atmrp5, Atmrp4/5. All these results indicated that ABC transporter functions upstream

  7. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B;

    1999-01-01

    -AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change...... in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...

  8. 4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIα targeting the ATP binding site.

    Science.gov (United States)

    Pogorelčnik, Barbara; Janežič, Matej; Sosič, Izidor; Gobec, Stanislav; Solmajer, Tom; Perdih, Andrej

    2015-08-01

    Human DNA topoisomerase IIα (htIIα) is a validated target for the development of novel anticancer agents. Starting from our discovered 4-amino-1,3,5-triazine inhibitors of htIIα, we investigated a library of 2,4,6-trisubstituted-1,3,5-triazines for novel inhibitors that bind to the htIIα ATP binding site using a combination of structure-based and ligand-based pharmacophore models and molecular docking. 4,6-substituted-1,3,5-triazin-2(1H)-ones 8, 9 and 14 were identified as novel inhibitors with activity comparable to the established drug etoposide (1). Compound 8 inhibits the htIIα decatenation in a superior fashion to etoposide. Cleavage assays demonstrated that selected compounds 8 and 14 do not act as poisons and antagonize the poison effect of etoposide. Microscale thermophoresis (MST) confirmed binding of compound 8 to the htIIα ATPase domain and compound 14 effectively inhibits the htIIα mediated ATP hydrolysis. The molecular dynamics simulation study provides further insight into the molecular recognition. The 4,6-disubstituted-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the to the htIIα ATPase domain.

  9. The structural biology of the FGF19 subfamily.

    Science.gov (United States)

    Beenken, Andrew; Mohammadi, Moosa

    2012-01-01

    The ability of the Fibroblast Growth Factor (FGF) 19 subfamily to signal in an endocrine fashion sets this subfamily apart from the remaining five FGF subfamilies known for their paracrine functions during embryonic development. Compared to the members of paracrine FGF subfamiles, the three members of the FGF19 subfamily, namely FGF19, FGF21 and FGF23, have poor affinity for heparan sulfate (HS) and therefore can diffuse freely in the HS-rich extracellular matrix to enter into the bloodstream. In further contrast to paracrine FGFs, FGF19 subfamily members have unusually poor affinity for their cognate FGF receptors (FGFRs) and therefore cannot bind and activate them in a solely HS-dependent fashion. As a result, the FGF19 subfamily requires α/βklotho coreceptor proteins in order to bind, dimerize and activate their cognate FGFRs. This klotho-dependency also determines the tissue specificity of endocrine FGFs. Recent structural and biochemical studies have begun to shed light onto the molecular basis for the klotho-dependent endocrine mode of action of the FGF19 subfamily. Crystal structures of FGF19 and FGF23 show that the topology of the HS binding site (HBS) of FGF19 subfamily members deviates drastically from the common topology adopted by the paracrine FGFs. The distinct topologies of the HBS of FGF19 and FGF23 prevent HS from direct hydrogen bonding with the backbone atoms of the HBS of these ligands and accordingly decrease the HS binding affinity of this subfamily. Recent biochemical data reveal that the ?klotho ectodomain binds avidly to the ectodomain of FGFR1c, the main cognate FGFR of FGF23, creating a de novo high affinity binding site for the C-terminal tail of FGF23. The isolated FGF23 C-terminus can be used to effectively inhibit the formation of the FGF23-FGFR1c-αklotho complex and alleviate hypophosphatemia in renal phosphate disorders due to elevated levels of FGF23.

  10. Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export

    OpenAIRE

    Evers, R.; Haas, M; Sparidans, R; Beijnen, J.; Wielinga, P R; Lankelma, J.; Borst, P

    2000-01-01

    The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters expo...

  11. Sertraline and Its Metabolite Desmethylsertraline, but not Bupropion or Its Three Major Metabolites, Have High Affinity for P-Glycoprotein

    OpenAIRE

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-01-01

    The ATP-binding cassette (ABC) transporter protein subfamily Bl line (ABCBl) transporter P-glycoprotein (P-gp) plays an important role in the blood–brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertralin, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alchhol (EB), and hydroxy metabolite (HB) was studied using an ATPase ass...

  12. 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...

  13. 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

  14. A Suggested New Bacteriophage Genus, “Kp34likevirus”, within the Autographivirinae Subfamily of Podoviridae

    Directory of Open Access Journals (Sweden)

    Harald Eriksson

    2015-04-01

    Full Text Available Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503 and vB_KpnP_SU552A (SU552A are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649, F19 (NC_023567 and NTUH-K2044-K1-1 (NC_025418. Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1, morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.

  15. 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.

  16. 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.

  17. 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.

  18. 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。

  19. Minimal subfamilies and the probabilistic interpretation for modulus on graphs

    CERN Document Server

    Albin, Nathan

    2016-01-01

    The notion of $p$-modulus of a family of objects on a graph is a measure of the richness of such families. We develop the notion of minimal subfamilies using the method of Lagrangian duality for $p$-modulus. We show that minimal subfamilies have at most $|E|$ elements and that these elements carry a weight related to their "importance" in relation to the corresponding $p$-modulus problem. When $p=2$, this measure of importance is in fact a probability measure and modulus can be thought as trying to minimize the expected overlap in the family.

  20. 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.

  1. Identification and Characterization of the Populus AREB/ABF Subfamily

    Institute of Scientific and Technical Information of China (English)

    Lexiang Ji; Jia Wang; Meixia Ye; Ying Li; Bin Guo; Zhong Chen; Hao Li; Xinmin An

    2013-01-01

    Abscisic acid (ABA) is a major plant hormone that plays an important role in responses to abiotic stresses.The ABA-responsive element binding proteinlABRE-binding factor (AREB/ABF) gene subfamily contains crucial transcription factors in the ABA-mediated signaling pathway.In this study,a total of 14 putative AREB/ABF members were identified in the Populus trichocarpa Torr.& Gray.genome using five AREB/ABF amino acid sequences from Arabidopsis thaliana L.as probes.The 14 putative Populus subfamily members showed high protein similarities,especially in the basic leucine zipper (bZlP) domain region.A neighbor-joining analysis combined with gene structure data revealed homology among the 14 genes.The expression patterns of the Populus AREB/ABF subfamily suggested that the most abundant transcripts of 11 genes occurred in leaf tissues,while two genes were most transcribed in root tissues.Significantly,eight Populus AREB/ABF gene members were upregulated after treatment with 100 μM exogenous ABA,while the other six members were downregulated.We identified the expression profiles of the subfamily members in Populus tissues and elucidated different response patterns of Populus AREB/ABF members to ABA stress.This study provided insight into the roles of Populus AREB/ABF homologues in plant response to abiotic stresses.

  2. Vaillantellinae, A New Subfamily Of Cobitidae (Pisces, Cypriniformes)

    NARCIS (Netherlands)

    Nalbant, T.T.; Banarescu, P.M.

    1977-01-01

    INTRODUCTION It is generally accepted that the Eurasian freshwater fish family Cobitidae includes three main divisions, usually considered as subfamilies: Cobitinae, Botiinae and Noemacheilinae (Berg, 1940; Ramaswami, 1953; Nalbant, 1963). The two first named, in which the lateral ethmoids are movab

  3. First molecular phylogeny of the subfamily Polycerinae (Mollusca, Nudibranchia, Polyceridae)

    Science.gov (United States)

    Palomar, Gemma; Pola, Marta; Garcia-Vazquez, Eva

    2014-03-01

    The subfamily Polycerinae includes four genera with around 46 species described to date. This subfamily is characterized by a limaciform body, which may have simple tentacular processes on the margin of the oral veil. Phylogenetic relationships between the genera of the subfamily Polycerinae (Polyceridae) have not yet been studied, and therefore, the only available information is based on morphological descriptions. The present study reports the first phylogenetic analysis of Polycerinae based on the mitochondrial genes cytochrome oxidase subunit I and the large ribosomal subunit (16S rRNA) using maximum likelihood and Bayesian methods. Our results showed that Polycerinae is monophyletic, but the relationships within the subfamily as well as within Polycera remain unresolved. A key finding of this study is that there are clearly two sympatric species of Polycera present in South Africa: Polycera capensis Quoy and Gaimard, 1824 also found in Australia and an undescribed Polycera sp. On the other hand, the studied specimens of the genus Gymnodoris were clustered within Polycerinae, reopening the problem of the systematic position of this genus. Additional genes and species of Polycerinae and Gymnodoris would provide more information and probably fully resolve this situation.

  4. 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.

  5. 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...

  6. Expression of antibiotic resistance genes in the integrated cassettes of integrons.

    Science.gov (United States)

    Collis, C M; Hall, R M

    1995-01-01

    Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter P(ant), and alterations in the sequence of P(ant) affected the level of resistance expressed by cassette genes. When both P(ant) and the secondary promoter P2 were present, transcription from both promoters was detected. When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene. In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P(ant), and was reduced to different extents by the presence of individual upstream cassettes. In Northern (RNA) blots, multiple discrete transcripts originating at P(ant) were detected, and only the longer transcripts contained the distal genes. Together, these data suggest that premature transcription termination occurs within the cassettes. The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3' to the coding region) not only function as recombination sites but may also function as transcription terminators. PMID:7695299

  7. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M;

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

  8. A simplified erythromycin resistance cassette for Treponema denticola mutagenesis

    Science.gov (United States)

    Goetting-Minesky, M. Paula; Fenno, J. Christopher

    2010-01-01

    The primary selectable marker for genetic studies of Treponema denticola is a hybrid gene cassette containing both ermF and ermAM (ermB) genes. ErmB functions in Escherichia coli, while ErmF has been assumed to confer resistance in T. denticola. We demonstrate here that ErmB is sufficient for erythromycin selection in T. denticola and that the native ermB promoter drives ErmB expression. PMID:20691222

  9. Cassette less SOFC stack and method of assembly

    Science.gov (United States)

    Meinhardt, Kerry D

    2014-11-18

    A cassette less SOFC assembly and a method for creating such an assembly. The SOFC stack is characterized by an electrically isolated stack current path which allows welded interconnection between frame portions of the stack. In one embodiment electrically isolating a current path comprises the step of sealing a interconnect plate to a interconnect plate frame with an insulating seal. This enables the current path portion to be isolated from the structural frame an enables the cell frame to be welded together.

  10. Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae).

    Science.gov (United States)

    Comer, Jason R; Zomlefer, Wendy B; Barrett, Craig F; Stevenson, Dennis Wm; Heyduk, Karolina; Leebens-Mack, James H

    2016-04-01

    Palms (Arecaceae) include economically important species such as coconut, date palm, and oil palm. Resolution of the palm phylogeny has been problematic due to rapid diversification and slow rates of molecular evolution. The focus of this study is on relationships of the 14 tribes of subfamily Arecoideae and their inferred ancestral areas. A targeted sequencing approach was used to generate a data set of 168 single/low copy nuclear genes for 34 species representing the Arecoideae tribes and the other palm subfamilies. Species trees from the concatenated and coalescent based analyses recovered largely congruent topologies. Three major tribal clades were recovered: the POS clade (Podococceae, Oranieae, Sclerospermeae), the RRC clade (Roystoneeae, Reinhardtieae, Cocoseae), and the core arecoid clade (Areceae, Euterpeae, Geonomateae, Leopoldinieae, Manicarieae, Pelagodoxeae). Leopoldinieae was sister to the rest of the core arecoids (Geonomateae, Manicarieae+Pelagodoxeae, and Areceae+Euterpeae). The nuclear phylogeny supported a North American origin for subfamily Arecoideae, with most tribal progenitors diversifying within the Americas. The POS clade may have dispersed from the Americas into Africa, with tribe Oranieae subsequently spreading into the Indo-Pacific. Two independent dispersals into the Indo-Pacific were inferred for two tribes within the core arecoids (tribes Areceae and Pelagodoxeae).

  11. Cytogenetics and genome evolution in the subfamily Triatominae (Hemiptera, Reduviidae).

    Science.gov (United States)

    Panzera, F; Pérez, R; Panzera, Y; Ferrandis, I; Ferreiro, M J; Calleros, L

    2010-01-01

    The subfamily Triatominae (Hemiptera, Reduviidae), vectors of Chagas disease, includes over 140 species. Karyotypic information is currently available for 80 of these species. This paper summarizes the chromosomal variability of the subfamily and how it may reveal aspects of genome evolution in this group. The Triatominae present a highly conserved chromosome number. All species, except 3, present 20 autosomes. The differences in chromosome number are mainly caused by variation in the number of sex chromosomes, due to the existence of 3 sex systems in males (XY, X(1)X(2)Y and X(1)X(2)X(3)Y). However, inter- and intraspecific differences in the position, quantity and meiotic behavior of constitutive heterochromatin, in the total genome size, and in the location of ribosomal 45S rRNA clusters, have revealed considerable cytogenetic variability within the subfamily. This cytogenetic diversity offers the opportunity to perform cytotaxonomic and phylogenetic studies, as well as structural, evolutionary, and functional analyses of the genome. The imminent availability of the complete genome of Rhodnius prolixus also opens new perspectives for understanding the evolution and genome expression of triatomines. The application of fluorescence in situ hybridization for the mapping of genes and sequences, as well as comparative analyses of genome homology by comparative genomic hybridization will be useful tools for understanding the genomic changes in relation to evolutionary processes such as speciation and adaptation to different environments.

  12. Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Érica L. Fonseca

    2015-11-01

    Full Text Available The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF, a short 5’ untranslated region (UTR and a 3’ recombination site (attC. Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW, had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription

  13. Cassettes for solid-oxide fuel cell stacks and methods of making the same

    Science.gov (United States)

    Weil, K. Scott; Meinhardt, Kerry D; Sprenkle, Vincent L

    2012-10-23

    Solid-oxide fuel cell (SOFC) stack assembly designs are consistently investigated to develop an assembly that provides optimal performance, and durability, within desired cost parameters. A new design includes a repeat unit having a SOFC cassette and being characterized by a three-component construct. The three components include an oxidation-resistant, metal window frame hermetically joined to an electrolyte layer of a multi-layer, anode-supported ceramic cell and a pre-cassette including a separator plate having a plurality of vias that provide electrical contact between an anode-side collector within the pre-cassette and a cathode-side current collector of an adjacent cell. The third component is a cathode-side seal, which includes a standoff that supports a cathode channel spacing between each of the cassettes in a stack. Cassettes are formed by joining the pre-cassette and the window frame.

  14. 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...... 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...

  15. The HRASLS (PLA/AT) subfamily of enzymes.

    Science.gov (United States)

    Mardian, Emily B; Bradley, Ryan M; Duncan, Robin E

    2015-10-26

    The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1-5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A1/2 (PLA1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.

  16. Revision of the monogenean subfamily Neothoracocotylinae Lebedev, 1969 (Polyopisthocotylea: Thoracocotylidae).

    Science.gov (United States)

    Hayward, C J; Rohde, K

    1999-11-01

    Members of the subfamily Neothoracocotylinae are gastrocotylinean monogeneans on the gills of scombrid fishes of the genera Scomberomorus and Acanthocybium, and reportedly of a coryphaenid fish belonging to the genus Coryphaena. We revise the diagnosis of the subfamily and its two genera and accept only two species as valid. Neothoracocotyle acanthocybii (Meserve, 1938) Hargis, 1956 is known from Acanthocybium solandri throughout the Pacific Ocean and in the western Atlantic. N. coryphaenae (Yamaguti, 1938) Hargis, 1956, known only from a single specimen and described from Coryphaena hippurus in Japan, is synonymised with N. acanthocybii. The sole member of Scomberocotyle, S. scomberomori (Koratha, 1955) Hargis, 1956, infects five species of Scomberomorus in the eastern Pacific Ocean and the western and castern Atlantic. We record this worm from several new hosts and/or localities, including S. sierra and S. concolor in the eastern Pacific (Mexico to Colombia), S. maculatus and S. cavalla in the western Atlantic (USA to Brazil), and S. tritor in the eastern Atlantic (Sierra Leone to Nigeria).

  17. 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.

  18. Subfamily logos: visualization of sequence deviations at alignment positions with high information content

    Directory of Open Access Journals (Sweden)

    Beitz Eric

    2006-06-01

    Full Text Available Abstract Background Recognition of relevant sequence deviations can be valuable for elucidating functional differences between protein subfamilies. Interesting residues at highly conserved positions can then be mutated and experimentally analyzed. However, identification of such sites is tedious because automated approaches are scarce. Results Subfamily logos visualize subfamily-specific sequence deviations. The display is similar to classical sequence logos but extends into the negative range. Positive, upright characters correspond to residues which are characteristic for the subfamily, negative, upside-down characters to residues typical for the remaining sequences. The symbol height is adjusted to the information content of the alignment position. Residues which are conserved throughout do not appear. Conclusion Subfamily logos provide an intuitive display of relevant sequence deviations. The method has proven to be valid using a set of 135 aligned aquaporin sequences in which established subfamily-specific positions were readily identified by the algorithm.

  19. The Formation of the Polyploid Hybrids From Different Subfamily Fish Crossings and Its Evolutionary Significance

    OpenAIRE

    Liu, Shaojun; Qin, Qinbo; Xiao, Jun; Lu, Wenting; Shen, Jiamin; Li, Wei; Liu, Jifang; Duan, Wei; Zhang, Chun; Tao, De Min; Zhao, Rurong; Yan, Jinpeng; Liu, Yun

    2007-01-01

    This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp × blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (♀) × blunt snout bream (BSB) (♂) as w...

  20. The Caenorhabditis elegans parvulin gene subfamily and their expression under cold or heat stress along with the fkb subfamily.

    Science.gov (United States)

    Fasseas, Michael K; Dimou, Maria; Katinakis, Panagiotis

    2012-07-06

    Parvulins and FKBPs are members of the peptidyl-prolyl cis/trans isomerases (PPIase) enzyme family whose role is to catalyze the interconversion between the cis trans forms of a peptide bond preceding internal proline residues in a polypeptide substrate. Members of the parvulin subfamily have been found to be involved in a variety of diseases, including Alzheimer's disease and cancer and are also considered possible antiparasitic targets. Genes Y110A2AL.13 (pin-1) and Y48C3A.16 (pin-4) were found in the worm's genome, possibly encoding parvulins. One is homologous to human and fly PIN1 whereas the other is homologous to human and fly PIN4. Both were expressed in Escherichia coli, purified and found to have in vitro PPIase activity. Expression levels of both genes, as well as the fkb genes (that encode FK506-binding proteins) were measured during development and under cold or heat stress conditions. The results revealed a potential role for these genes under temperature-related stress. RNAi silencing was performed for wild type and mutant strain worms under normal and cold or heat stress conditions. A reduced lifespan was observed when pin-4 dsRNA was fed to the fkb-5 deficient worms. Our work presents a first attempt to characterize the Caenorhabditis elegans parvulins and may present an interesting starting point for further experimentation concerning their role, along with the FKBP subfamily, in nematode physiology and their possible use as antiparasitic targets.

  1. Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

    Directory of Open Access Journals (Sweden)

    Gillings Michael R

    2006-01-01

    Full Text Available Abstract Background Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be. Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains. Results The complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes. It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons. Conclusion Our systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene

  2. 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

  3. Actions and mode of actions of FGF19 subfamily members.

    Science.gov (United States)

    Fukumoto, Seiji

    2008-03-01

    Fibroblast growth factors (FGFs) are humoral factors with diverse biological functions. While most FGFs were shown to work as local factors regulating cell growth and differentiation, recent investigations indicated that FGF19 subfamily members, FGF15/19, FGF21 and FGF23 work as systemic factors. FGF15/19 produced by intestine inhibits bile acid synthesis and FGF21from liver is involved in carbohydrate and lipid metabolism. In addition, FGF23 was shown to be produced by bone and regulate phosphate and vitamin D metabolism. Furthermore, these FGFs require klotho or betaklotho for their actions in addition to canonical FGF receptors. It is possible that these FGFs together with their receptor systems might be targets for novel therapeutic measures in the future.

  4. Two new nematode species of the subfamily Brittonematinae (Dorylaimida: Actinolaimidae

    Directory of Open Access Journals (Sweden)

    Andrássy, I.

    2010-10-01

    Full Text Available Abstract. Two new species of actinolaimoid nematodes of the subfamily Brittonematinae are described and illustrated.Actinca marisae sp. n. from Brazil is characterized by the long (on average 2.92 mm and slender body, 30–32 distinct longitudinalridges on cuticle, narrow head, slender odontostyle, onchial tips facing each other, cylindrus occupying somewhat lessthan one-half of pharynx, broad vulval lips, and by medum long tail. Afractinca eburnea sp. n. from Côte d’Ivoire can bedistinguished by a relatively long body (on average 1.88 mm, thin cuticle provided with 14 longitudinal ridges, cap-like offsetlabial ring, very slender odontostyle, long prerectum, vulva sunk in body contour, and by the elongate-conoid female tail. Mainmorphological structures of Actinca and Afractinca species are summarized. Some comments on further brittonematine speciesare added.

  5. Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology.

    Science.gov (United States)

    Jiao, Guan-Sheng; Thoresen, Lars H; Kim, Taeg Gyum; Haaland, Wade C; Gao, Feng; Topp, Michael R; Hochstrasser, Robin M; Metzker, Michael L; Burgess, Kevin

    2006-10-16

    We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.

  6. Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology.

    Science.gov (United States)

    Gestal, Alicia M; Liew, Elissa F; Coleman, Nicholas V

    2011-12-01

    Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm(R) or Lac(+)) were obtained at frequencies ranging from 10(1) to 10(6) c.f.u. (µg DNA)(-1). Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P(C) were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

  7. On the correct name for some subfamilies of Mustelidae (Mammalia, Carnivora

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    Fabio Oliveira do Nascimento

    2014-01-01

    Full Text Available Mustelids (Mustelidae exhibit a wide morphological and ecological diversity, ranging from aquatic to semi arboreal and fossorial forms. It is the most diversity family in Carnivora, and this has promoted a great number of taxonomic arrangements for subfamilies, which can range from two to 15 depending on the author. The relatively recent use of molecular data has helped to elucidate the classification of mustelids, and eight subfamilies are currently recognized: Mustelinae, Galictinae, Helictidinae, Martinae, Melinae, Mellivorinae, Taxidiinae and Lutrinae. However, some of these subfamilies have nomenclatural problems, not receiving the oldest available name. The subfamily that includes martens (Martes, Charronia and Pekania, tayra (Eira and wolverine (Gulo has received the name of Martinae Wagner, 1841, but the oldest available name is Guloninae Gray, 1825. This problem also occurs for the subfamily that includes the grisons (Galictis, Patagonian weasel (Lyncodon, marbled polecat (Vormela and striped weasels (Ictonyx and Poecilogale, which are known as Grisoninae Pocock, 1921, but the correct name for this group is Ictonychinae, Pocock, 1921. The subfamily that includes ferret badgers (Melogale retains the name Helictidinae Gray, 1865, because its validity is not affected when the type-genus of the subfamily becomes a junior synonym of another genus. Furthermore, a list of the extant subfamilies of Mustelidae and their respective synonyms and included genera is provided.

  8. Historical legacies in the geographical diversity patterns of New World palm (Arecaceae) subfamilies

    DEFF Research Database (Denmark)

    Bjorholm, Stine; Svenning, Jens-Christian; Baker, William

    2006-01-01

    The extent to which species richness patterns of the major palm subfamilies in the Americas are controlled by lineage history was studied. Based on the fossil record, we suggest that the subfamily Coryphoideae has followed a boreotropical dispersal route into Central and South America, whereas ca...

  9. Phantoms of Gondwana?-phylogeny of the spider subfamily Mynogleninae (Araneae: Linyphiidae)

    DEFF Research Database (Denmark)

    Frick, Holger; Scharff, Nikolaj

    2014-01-01

    This is the first genus-level phylogeny of the subfamily Mynogleninae. It is based on 190 morphological characters scored for 44 taxa: 37 mynoglenine taxa (ingroup) representing 15 of the 17 known genera and seven outgroup taxa representing the subfamilies Stemonyphantinae, Linyphiinae (Linyphiin...

  10. Phylogenetic relationships of subfamilies in the family Hesperiidae (Lepidoptera: Hesperioidea) from China.

    Science.gov (United States)

    Yuan, Xiangqun; Gao, Ke; Yuan, Feng; Wang, Ping; Zhang, Yalin

    2015-06-10

    Hesperiidae is one of the largest families of butterflies. Our knowledge of the higher systematics on hesperiids from China is still very limited. We infer the phylogenetic relationships of the subfamilies of Chinese skippers based on three mitochondrial genes (cytochrome b (Cytb), the NADH dehydrogenase subunit 1 (ND1) and cytochrome oxidase I (COI)). In this study, 30 species in 23 genera were included in the Bayesian and maximum likelihood analyses. The subfamily Coeliadinae, Eudaminae, Pyrginae and Heteropterinae were recovered as a monophyletic clade with strong support. The subfamily Hesperiinae formed a clade, but support for monophyly was weak. Our results imply that the five subfamilies of Chinese Hesperiidae should be divided into: Coeliadinae, Eudaminae, Pyrginae, Heteropterinae and Hesperiinae. The relationships of the five subfamilies should be as follows: Coeliadinae + (Eudaminae + (Pyrginae + (Heteropterinae + Hesperiinae))).

  11. The superintegron integrase and the cassette promoters are co-regulated in Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Evelyne Krin

    Full Text Available Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

  12. Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression.

    Science.gov (United States)

    Fernandes, Fabiana; Vidigal, João; Dias, Mafalda M; Prather, Kristala L J; Coroadinha, Ana S; Teixeira, Ana P; Alves, Paula M

    2012-11-01

    Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

  13. VAMP subfamilies identified by specific R-SNARE motifs.

    Science.gov (United States)

    Rossi, Valeria; Picco, Raffaella; Vacca, Marcella; D'Esposito, Maurizio; D'Urso, Michele; Galli, Thierry; Filippini, Francesco

    2004-05-01

    In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.

  14. Bacterial lipoxygenases, a new subfamily of enzymes? A phylogenetic approach.

    Science.gov (United States)

    Hansen, Jhoanne; Garreta, Albert; Benincasa, Maria; Fusté, M Carmen; Busquets, Montserrat; Manresa, Angeles

    2013-06-01

    Lipoxygenases (EC. 1.13.11.12) are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. They catalyze the dioxygenation of 1Z,4Z-pentadiene moieties of polyunsaturated fatty acids obtaining hydroperoxy fatty acids. For years, the presence of lipoxygenases was considered a eukaryotic feature, present in mammals, plants, small marine invertebrates, and fungi, but now, some lipoxygenase sequences have been detected on prokaryotic organisms, changing the idea that lipoxygenases are exclusively a eukaryotic affair. Lipoxygenases are involved in different types of reactions on eukaryote organisms where the biological role and the structural characteristics of these enzymes are well studied. However, these aspects of the bacterial lipoxygenases have not yet been elucidated and are unknown. This revision discusses biochemical aspects, biological applications, and some characteristics of these enzymes and tries to determine the existence of a subfamily of bacterial lipoxygenases in the context of the phylogeny of prokaryotic lipoxygenases, supporting the results of phylogenetic analyzes with the comparison and discussion of structural information of the first prokaryotic lipoxygenase crystallized and other eukaryotic lipoxygenases structures.

  15. Radiation exposure reduction by use of Kevlar cassettes in the neonatal nursery.

    Science.gov (United States)

    Herman, M W; Mak, H K; Lachman, R S

    1987-05-01

    A study was performed to determine whether the use of Kevlar cassettes in the neonatal intensive care nursery would reduce radiation exposure to patients. The radiation dose to the neonates was measured by using thermoluminescent dosimeters. In addition, the attenuation of the Kevlar cassettes and the sensitivity of the film-screen combination were compared with the previously used system. The greatest radiation reduction using a mobile X-ray unit was 27%; based on sensitivity measurements, the theoretical reduction averaged 38%. The reduction in radiation exposure resulted from reduced attenuation by the Kevlar cassette.

  16. Mid-tertiary dispersal, not Gondwanan vicariance explains distribution patterns in the wax palm subfamily (Ceroxyloideae: Arecaceae)

    DEFF Research Database (Denmark)

    Trénel, Philipp; Gustafsson, Mats; Baker, W.J.

    2007-01-01

    The Ceroxyloideae is a small but heterogeneous subfamily of palms (Arecaceae, Palmae). It includes a Caribbean lineage (tribe Cyclospathae), a southern hemisphere disjunction (tribe Ceroxyleae), and an amphi-Andean element (tribe Phytelepheae), until recently considered a distinct subfamily...

  17. Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Masatsugu; Hiraki; Yoshihiko; Kitajima; Seiji; Sato; Jun; Nakamura; Kazuyoshi; Hashiguchi; Hirokazu; Noshiro; Kohji; Miyazaki

    2010-01-01

    AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain r...

  18. Association of allelic variation in genes mediating aspects of energy homeostasis with weight gain during administration of antipsychotic drugs (CATIE Study

    Directory of Open Access Journals (Sweden)

    Hemant K Tiwari

    2011-09-01

    Full Text Available Antipsychotic drugs are widely used in treating schizophrenia, bipolar disorder, and other psychiatric disorders. Many of these drugs, despite their therapeutic advantages, substantially increase body weight. We assessed the association of alleles of 31 genes implicated in body weight regulation with weight gain among patients being treated with specific antipsychotic medications in the CATIE trial, we found that rs2237988 in ATP-binding cassette subfamily C member 8 (ABCC8 , and rs11643744 and rs9922047 in Fat Mass and Obesity Associated (FTO were associated with such weight gain.

  19. The function of integron-associated genes cassettes in Vibrio species: the tip of the iceberg

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    Rita A Rapa

    2013-12-01

    Full Text Available The integron is a genetic element that incorporates mobile genes termed gene cassettes into a reserved genetic site via site-specific recombination. It is best known for its role in antibiotic resistance with one type of integron, the class 1 integron, a major player in the dissemination of antibiotic resistance genes across Gram negative pathogens and commensals. However, integrons are ancient structures with over 100 classes (including class 1 present in bacteria from the broader environment. While, the class 1 integron is only one example of an integron being mobilised into the clinical environment, it is by far the most successful. Unlike clinical class 1 integrons which are largely found on plasmids, other integron classes are found on the chromosomes of bacteria and carry diverse gene cassettes indicating a non-antibiotic resistance role(s. However, there is very limited knowledge on what these alternative roles are. This is particularly relevant to Vibrio species where gene cassettes make up approximately 1-3% of their entire genome. In this review, we discuss how emphasis on class 1 integron research has resulted in a limited understanding by the wider research community on the role of integrons in the broader environment. This has the capacity to be counterproductive in solving or improving the antibiotic resistance problem into the future. Furthermore, there is still a significant lack of knowledge on how gene cassettes in Vibrio species drive adaptation and evolution. From research in V. rotiferianus DAT722, new insight on how gene cassettes affect cellular physiology offers new alternative roles for the gene cassette resource. At least a subset of gene cassettes are involved in host surface polysaccharide modification suggesting that gene cassette may be important in processes such as bacteriophage resistance, adhesion/biofilm formation, protection from grazers and bacterial aggregation.

  20. 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.

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

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    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.

  2. Cassette loaded with a resilient packing to connect socket ends on the sea bed

    Energy Technology Data Exchange (ETDEWEB)

    Tuson, S.P.R.

    1983-06-08

    A cassette loaded with a resilient packing is used to connect a pipe on the sea bed to one end of a hollow shaft forming part of the crosspiece of a cardan joint at the base of an articulated columns mounted on the sea bed. The resilient packing comprises a stack of rubber rings and metallic washers disposed between end rings and capable of being derformed in torsion, the packing in use being compressed between an abutment on the end of the hollow shaft in the cardan joint and an abutment on the adjacent end of the pipe. The cassette comprises a tubular body which can be fitted in or removed from a housing for a bearing of the cardan joint, the end rings of the packing projecting through the opposite ends of the tubular body of the cassette. The cassette is fitted with jacks for compressing the packing, and wedges for retaining the packing in a compressed state until the cassette is fitted in the bearing housing, whereupon the jacks are again operated to compress further the packing and enable the wedges to be removed. Release of the jacks then allows the packing to expand within the cassette and engage the ends of the packing against the ends of the hollow shaft and pipe so as to form a connection therebetween. 2 drawings.

  3. Analysis of the features and source gene composition of the AluYg6 subfamily of human retrotransposons

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    Brookfield John FY

    2007-07-01

    Full Text Available Abstract Background Alu elements are a family of SINE retrotransposons in primates. They are classified into subfamilies according to specific diagnostic mutations from the general Alu consensus. It is now believed that there may be several retrotranspositionally-competent source genes within an Alu subfamily. To investigate the evolution of young Alu elements it is critical to have access to complete subfamilies, which, following the release of the final human genome assembly, can now be obtained using in silico methods. Results 380 elements belonging to the young AluYg6 subfamily were identified in the human genome, a number significantly exceeding prior expectations. An AluYg6 element was also identified in the chimpanzee genome, indicating that the subfamily is older than previously estimated, and appears to have undergone a period of dormancy before its expansion. The relative contributions of back mutation and gene conversion to variation at the six diagnostic positions are examined, and cases of complete forward gene conversion events are reported. Two small subfamilies derived from AluYg6 have been identified, named AluYg6a2 and AluYg5b3, which contain 40 and 27 members, respectively. These small subfamilies are used to illustrate the ambiguity regarding Alu subfamily definition, and to assess the contribution of secondary source genes to the AluYg6 subfamily. Conclusion The number of elements in the AluYg6 subfamily greatly exceeds prior expectations, indicating that the current knowledge of young Alu subfamilies is incomplete, and that prior analyses that have been carried out using these data may have generated inaccurate results. A definition of primary and secondary source genes has been provided, and it has been shown that several source genes have contributed to the proliferation of the AluYg6 subfamily. Access to the sequence data for the complete AluYg6 subfamily will be invaluable in future computational analyses investigating

  4. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Tardigrade, Hypsibius dujardini

    Science.gov (United States)

    Reinsch, Sigrid; Myers, Zachary Alan; DeSimone, Julia Carol; Freeman, John L.; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. We performed ground testing to determine whether ARC EMCS seed cassettes could be adapted for use with tardigrades for future spaceflight experiments. Tardigrades (water bears) are small invertebrates that enter the tun state in response to desiccation or other environmental stresses. Tardigrade tuns have suspended metabolism and have been shown to be survive exposure to space vacuum, high pressure, temperature and other stresses. For spaceflight experiments using the EMCS, the organisms ideally must be able to survive desiccation and storage in the cassette at ambient temperature for several weeks prior to the initiation of the experiment by the infusion of water to the cassette during spaceflight. The ability of tardigrades to survive extremes by entering the tun state make them ideal candidates for growth experiments in the EMCS cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membrane contains dried growth medium. The goals of our study were to (1) determine whether tardigrades survive and reproduce on PES membranes, (2) develop a consistent method for dehydration of the tardigrades with high recovery rates upon rehydration, (3) to determine an appropriate food source for the tardigrades that can also be dehydrated/rehydrated and (4) successful mock rehydration experiment in cassettes with appropriate food source. We present results that show successful multigenerational growth of tardigrades on PES membranes with a variety of wet food sources. We have successfully performed a mock rehydration with tardigrades and at least one candidate food, protonema of the moss Polytrichum, that supports multigenerational growth and whose spores germinate quickly enough to match tardigrade feeding patterns post rehydration. Our results indicate that experiments on the ISS using the tardigrade, Hypsibius dujardini

  5. 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

  6. The effects of ABCG5/G8 polymorphisms on HDL-cholesterol concentrations depend on ABCA1 genetic variants in the Boston Puerto Rican health study

    Science.gov (United States)

    Background and aims: ATP-binding cassette transporters G5/G8 (ABCG5/G8) are associated with HDL-C concentrations. To assess whether the effect of ABCG5/G8 genetic variants on HDL-C concentrations is dependent on ATP-binding cassette transporters A1 (ABCA1), we studied potential interactions between ...

  7. 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% ...

  8. 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% ...

  9. Phylogenetic relationships and protein modelling revealed two distinct subfamilies of group II HKT genes between crop and model grasses.

    Science.gov (United States)

    Ariyarathna, H A Chandima K; Francki, Michael G

    2016-07-01

    Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies.

  10. Datziinae as a new subfamily name for the unavailable name Protopsychodinae Stebner et al., 2015, (Diptera: Psychodidae

    Directory of Open Access Journals (Sweden)

    Frauke Stebner

    2015-11-01

    Full Text Available In a recent paper a new subfamily of Psychodidae was inadequately named Protopsychodinae. This nomenclatural act cannot be considered as a valid name under ICZN regulations because the subfamily name is not based on the type genus Datzia Stebner et al., 2015, and furthermore the fossil genus Protopsychoda Azar et al., 1999 was originally described under the subfamily Psychodinae. Therefore, the new family-group name Datziinae is herein proposed.

  11. Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.

    Science.gov (United States)

    Real, R; González-Lobato, L; Baro, M F; Valbuena, S; de la Fuente, A; Prieto, J G; Alvarez, A I; Marques, M M; Merino, G

    2011-12-01

    In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0

  12. Control model for compressible cake filtration of green liquor in cassette filter

    OpenAIRE

    Bornefelt, Kajsa

    2006-01-01

    In the closed chemical recovery cycle in the sulphate pulp mill it is important to remove non-process elements. This is done by clarification of the green liquor, either in clarifiers or in filters. This project focuses on a cassette filter developed by Kvaerner Pulping AB. The cassette filter is semi-continuous and the aim of the project was to model the filter in order to be able to control cycle time and feed towards optimization of the capacity. The green liquor sludge forms a compressibl...

  13. Construction of New Campylobacter Cloning Vectors and a New Mutational Cat Cassette

    Science.gov (United States)

    1993-01-01

    mutational cat cassette PE - 61102A PR - 3M161102 6. AUTHOR(S) TA - BS13AK Yao R, Aim RA, Trust TJ, Guerry P WU- 1291 7. PERFORMING ORGANIZATION NAME(S) AND...mutational cat cassette %~ccesion For (Site-specific mutagenesis; recombinant DNA; multiple cloning site; PCR; shuttle vectors) NTIS CRA&I OTIC TAB E...incompatibility plasmids can mobilize shuttle vectors containing E. coi and C. coi plasmid Systems of experimental genetics are in the early stages replicons

  14. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures

    Science.gov (United States)

    Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J.; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K.; Geer, Renata C.; Gonzales, Noreen R.; Gwadz, Marc; Hurwitz, David I.; Lu, Fu; Marchler, Gabriele H.; Song, James S.; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A.; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y.; Bryant, Stephen H.

    2017-01-01

    NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. PMID:27899674

  15. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.

    Science.gov (United States)

    Sharma, Deepak; Jankowsky, Eckhard

    2014-01-01

    In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.

  16. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Chauvet, Sylvain; Guo, Jingyu; Hartman, John L; Kirk, Kevin L

    2014-07-18

    ATP-binding cassette (ABC) transporters are an ancient family of transmembrane proteins that utilize ATPase activity to move substrates across cell membranes. The ABCC subfamily of the ABC transporters includes active drug exporters (the multidrug resistance proteins (MRPs)) and a unique ATP-gated ion channel (cystic fibrosis transmembrane conductance regulator (CFTR)). The CFTR channel shares gating principles with conventional ligand-gated ion channels, but the allosteric network that couples ATP binding at its nucleotide binding domains (NBDs) with conformational changes in its transmembrane helices (TMs) is poorly defined. It is also unclear whether the mechanisms that govern CFTR gating are conserved with the thermodynamically distinct MRPs. Here we report a new class of gain of function (GOF) mutation of a conserved proline at the base of the pore-lining TM6. Multiple substitutions of this proline promoted ATP-free CFTR activity and activation by the weak agonist, 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). TM6 proline mutations exhibited additive GOF effects when combined with a previously reported GOF mutation located in an outer collar of TMs that surrounds the pore-lining TMs. Each TM substitution allosterically rescued the ATP sensitivity of CFTR gating when introduced into an NBD mutant with defective ATP binding. Both classes of GOF mutations also rescued defective drug export by a yeast MRP (Yor1p) with ATP binding defects in its NBDs. We conclude that the conserved TM6 proline helps set the energy barrier to both CFTR channel opening and MRP-mediated drug efflux and that CFTR channels and MRP pumps utilize similar allosteric mechanisms for coupling conformational changes in their translocation pathways to ATP binding at their NBDs.

  17. A new subfamily of the grasshopper (Orthoptera: Acridoidea:Gomphoceridae) from the Tibetan Plateau of China

    Institute of Scientific and Technical Information of China (English)

    Dao-Chuan Zhang; Yu-Long Zhang; Xiang-Chu Yin

    2012-01-01

    This paper reports a new subfamily,a new genus and a new species,that is,Pacrinae subfam.nov.,Pacris gen.nov and Pacris xizangensis sp.nov in Gomphoceridae.The new subfamily is allied to Orinhippinae of Gomphoceridae and it differs from the latter by wings and tympanum absent.The new genus is similar to Orinhippus Uvarov,1921 but differs from the latter in: (i) foveolae absent; (ii) tegmina absent; (iii) tympanum absent;(iv) hind margin ofpronotum with incised in the middle.Type specimens are deposited in the Museum of Hebei University,Baoding,China.

  18. Nine novel microsatellite markers for the army ant Simopelta pergandei (subfamily Ponerinae)

    DEFF Research Database (Denmark)

    Kronauer, D.J.C.; Boomsma, J.J.; Pierce, N.E.

    2011-01-01

    Simopelta (subfamily Ponerinae) army ants are specialized predators of other ants in New World tropical forests. Although they show a striking convergence in overall life-history with the well known army ants of the subfamilies Aenictinae, Dorylinae, and Ecitoninae, the genus has been little.......0) and expected heterozygosities between 0.32 and 0.85 (mean: 0.65). These genetic markers will be useful in studying the sociobiology and molecular ecology of Simopelta army ants and in elucidating convergent evolutionary trajectories that have culminated in the army ant lifestyle...

  19. 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.

  20. amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Solis-Escalante, D.; Kuijpers, N.G.A.; Bongaerts, N.; Bolat, I.; Bosman, L.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.A.S.

    2012-01-01

    Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, ar

  1. 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...

  2. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes

    NARCIS (Netherlands)

    Romano, A.; Raemakers, C.J.J.M.; Bernardi, J.; Visser, R.G.F.; Mooibroek, A.

    2003-01-01

    Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transf

  3. Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

    Directory of Open Access Journals (Sweden)

    McMahon James B

    2007-10-01

    Full Text Available Abstract Background Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. Methods In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. Results We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. Conclusion The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

  4. 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

  5. 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.

  6. 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

  7. New taxa of the subfamily Doryctinae Foerster (Hymenoptera: Braconidae) from French Guiana and Brazil

    NARCIS (Netherlands)

    Braet, Y.; Achterberg, van C.

    2001-01-01

    Three genera of the subfamily Doryctinae Foerster, 1862 (Hymenoptera: Braconidae) are treated and keyed: Ptesimogaster Marsh, 1965, Caingangia Marsh, 1993, and Leptodoryctes Barbalho & Penteado- Dias, 1999. The latter genus is characterised by the presence of an apical setal comb on the hind tibia.

  8. A new genus and subgenus of the subfamily Euphorinae (Hymenoptera: Braconidae) from East Asia

    NARCIS (Netherlands)

    Belokobylskij, S.A.

    1999-01-01

    Three new taxa belonging to the subfamily Euphorinae Foerster (Hymenoptera; Braconidae) are described and illustrated. Mama mariae gen. nov. & spec. nov. from southern Far East Russia and two species of the subgenus Chaetocentistes nov. of the genus Centistes Haliday. A key to species (i.e. Centiste

  9. Classification of leaf beetles from Korea. Part V. Subfamily Megalopodinae (Coleoptera: Chrysomelidae)

    OpenAIRE

    Seung Lak An

    2015-01-01

    Korean members of the subfamily Megalopodinae are redescribed; three species are recognized, among which Temnaspis bonneuili Pic is reported for the first time in Korea. Keys, diagnoses, collection locations (domestic), distributions, host plants, and biological information about all known species are provided.

  10. Classification of leaf beetles from Korea. Part V. Subfamily Megalopodinae (Coleoptera: Chrysomelidae

    Directory of Open Access Journals (Sweden)

    Seung Lak An

    2015-12-01

    Full Text Available Korean members of the subfamily Megalopodinae are redescribed; three species are recognized, among which Temnaspis bonneuili Pic is reported for the first time in Korea. Keys, diagnoses, collection locations (domestic, distributions, host plants, and biological information about all known species are provided.

  11. Generic revision of the subfamily Betylobraconinae (Hymenoptera: Braconidae) and other groups with modified fore tarsus

    NARCIS (Netherlands)

    Achterberg, van C.

    1995-01-01

    The genera of the subfamilies Betylobraconinae, Hormiinae, Lysiterminae, Pambolinae-Chremylini, and Doryctinae-Ypsistocerini are revised. A key is given to the genera of groups belonging to the cyclostome grade including species with shortened and/or (partly) widened fore tarsus. Sixteen new genera

  12. Seed morphology and anatomy and its utility in recognizing subfamilies and tribes of Zingiberaceae

    Energy Technology Data Exchange (ETDEWEB)

    Benedict, John C.; Smith, Selena Y.; Collinson, Margaret E.; Leong-Skornickova, Jana; Specht, Chelsea D.; Marone, Federica; Xiao, Xianghui; Parkinson, Dilworth Y.

    2015-11-01

    PREMISE OF THE STUDY: Recent phylogenetic analyses based on molecular data suggested that the monocot family Zingiberaceae be separated into four subfamilies and four tribes. Robust morphological characters to support these clades are lacking. Seeds were analyzed in a phylogenetic context to test independently the circumscription of clades and to better understand evolution of seed characters within Zingiberaceae. METHODS: Seventy-five species from three of the four subfamilies were analyzed using synchrotron based x-ray tomographic microscopy (SRXTM) and scored for 39 morphoanatomical characters. KEY RESULTS: Zingiberaceae seeds are some of the most structurally complex seeds in angiosperms. No single seed character was found to distinguish each subfamily, but combinations of characters were found to differentiate between the subfamilies. Recognition of the tribes based on seeds was possible for Globbeae, but not for Alpinieae, Riedelieae, or Zingibereae, due to considerable variation. CONCLUSIONS: SRXTM is an excellent, nondestructive tool to capture morphoanatomical variation of seeds and allows for the study of taxa with limited material available. Alpinioideae, Siphonochiloideae, Tamijioideae, and Zingiberoideae are well supported based on both molecular and morphological data, including multiple seed characters. Globbeae are well supported as a distinctive tribe within the Zingiberoideae, but no other tribe could be differentiated using seeds due to considerable homoplasy when compared with currently accepted relationships based on molecular data. Novel seed characters suggest tribal affinities for two currently unplaced Zingiberaceae taxa: Siliquamomum may be related to Riedelieae and Monolophus to Zingibereae, but further work is needed before formal revision of the family.

  13. Leaf and Stem CO2 Uptake in the Three Subfamilies of the Cactaceae 1

    Science.gov (United States)

    Nobel, Park S.; Hartsock, Terry L.

    1986-01-01

    Net CO2 uptake over 24-hour periods was examined for the leaves and for the stems of 11 species of cacti representing all three subfamilies. For Pereskia aculeata, Pereskia grandifolia, and Maihuenia poeppigii (subfamily Pereskioideae), all the net shoot CO2 uptake was by the leaves and during the daytime. In contrast, for the leafless species Carnegiea gigantea, Ferocactus acanthodes, Coryphantha vivipara, and Mammillaria dioica (subfamily Cactoideae), all the shoot net CO2 uptake was by the stems and at night. Similarly, for leafless Opuntia ficus-indica (subfamily Opuntioideae), all net CO2 uptake occurred at night. For leafy members of the Opuntioideae (Pereskiopsis porteri, Quiabentia chacoensis, Austrocylindropuntia subulata), at least 88% of the shoot CO2 uptake over 24 hours was by the leaves and some CO2 uptake occurred at night. Leaves responded to the instantaneous level of photosynthetically active radiation (PAR) during the daytime, as occurs for C3 plants, whereas nocturnal CO2 uptake by stems of O. ficus-indica and F. acanthodes responded to the total daily PAR, as occurs for Crassulacean acid metabolism (CAM) plants. Thus, under the well-watered conditions employed, the Pereskioideae behaved as C3 plants, the Cactoideae behaved as CAM plants, and the Opuntioideae exhibited characteristics of both pathways. PMID:16664741

  14. New Drosophila P-like elements and reclassification of Drosophila P-elements subfamilies.

    Science.gov (United States)

    Loreto, Elgion L S; Zambra, Francis M B; Ortiz, Mauro F; Robe, Lizandra J

    2012-07-01

    Genomic searches for P-like transposable elements were performed (1) in silico in the 12 available Drosophila genomes and (2) by PCR using degenerate primers in 21 Neotropical Drosophila species. In silico searches revealed P-like sequences only in Drosophila persimilis and Drosophila willistoni. Sixteen new P-like elements were obtained by PCR. These sequences were added to sequences of previously described P-like elements, and a phylogenetic analysis was performed. The subfamilies of P-elements described in the literature (Canonical, M, O, T, and K) were included in the reconstructed tree, and all were monophyletic. However, we suggest that some subfamilies can be enlarged, other subdivided, and some new subfamilies may be proposed, totalizing eleven subfamilies, most of which contain new P-like sequences. Our analyses support the monophyly of P-like elements in Drosophilidae. We suggest that, once these elements need host-specific factors to be mobilizable, the horizontal transfer (HT) of P-like elements may be inhibited among more distant taxa. Nevertheless, HT among Drosophilidae species appears to be a common phenomenon.

  15. Four new species of the subfamily Psilodercinae (Araneae: Ochyroceratidae) from Southwest China.

    Science.gov (United States)

    Wang, Chunxia; Li, Shuqiang

    2013-01-01

    Four new species of the family Ochyroceratidae are described from Southwest China: Althepus christae sp. nov., Lecler- cera undulatus sp. nov., Psiloderces incomptus sp. nov. from Yunnan; and P. exilis sp. nov. from Guangxi. The four spe- cies belong to the subfamily Psilodercinae.

  16. Wood anatomy of the Euphorbiaceae, in particular of the subfamily Phyllanthoideae

    NARCIS (Netherlands)

    Mennega, Alberta M.W.

    1985-01-01

    The great variety in wood structure of the large family Euphorbiaceae makes it impossible to describe briefly a general wood pattern. Nevertheless, a more or less clear division into four anatomical groups can be made. A short overview is given of the wood structure of the uni-ovulate subfamilies Ac

  17. Leaf and Stem CO(2) Uptake in the Three Subfamilies of the Cactaceae.

    Science.gov (United States)

    Nobel, P S; Hartsock, T L

    1986-04-01

    Net CO(2) uptake over 24-hour periods was examined for the leaves and for the stems of 11 species of cacti representing all three subfamilies. For Pereskia aculeata, Pereskia grandifolia, and Maihuenia poeppigii (subfamily Pereskioideae), all the net shoot CO(2) uptake was by the leaves and during the daytime. In contrast, for the leafless species Carnegiea gigantea, Ferocactus acanthodes, Coryphantha vivipara, and Mammillaria dioica (subfamily Cactoideae), all the shoot net CO(2) uptake was by the stems and at night. Similarly, for leafless Opuntia ficus-indica (subfamily Opuntioideae), all net CO(2) uptake occurred at night. For leafy members of the Opuntioideae (Pereskiopsis porteri, Quiabentia chacoensis, Austrocylindropuntia subulata), at least 88% of the shoot CO(2) uptake over 24 hours was by the leaves and some CO(2) uptake occurred at night. Leaves responded to the instantaneous level of photosynthetically active radiation (PAR) during the daytime, as occurs for C(3) plants, whereas nocturnal CO(2) uptake by stems of O. ficus-indica and F. acanthodes responded to the total daily PAR, as occurs for Crassulacean acid metabolism (CAM) plants. Thus, under the well-watered conditions employed, the Pereskioideae behaved as C(3) plants, the Cactoideae behaved as CAM plants, and the Opuntioideae exhibited characteristics of both pathways.

  18. 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

  19. Nanoparticle-delivered VEGF-silencing cassette and suicide gene expression cassettes inhibit colon carcinoma growth in vitro and in vivo.

    Science.gov (United States)

    Leng, Aimin; Yang, Jing; Liu, Ting; Cui, Jianfang; Li, Xiu-Hua; Zhu, Yanan; Xiong, Ting; Chen, Yuxiang

    2011-12-01

    The strategies for tumor-specific expression of suicide genes and target tumor angiogenesis have been tested in tumors. However, the anti-tumor efficacy of the combination of these two strategies, particularly, delivering suicide gene and anti-angiogenesis agent by nanoparticles, has not yet been evaluated in colon carcinoma. We constructed a cassette to silence VEGF-A expression and express a fused yCDglyTK gene driven by tumor-specific promoter (shVEGF-CDTK). The DNA carrying shVEGF-CDTK was delivered into colon carcinoma cells by calcium phosphate nanoparticles (CPNPs). Cell proliferation was measured by MTT assay, and apoptosis was detected by flow cytometry. The anti-tumor effect of the combined cassette was tested in xenograft animal model. With 5-fluorocytosine (5-FC), CPNP-delivered shVEGF-CDTK DNA (CPNP-shVEGF-CDTK) showed high expression of fused yCDglyTK gene and effectively silenced VEGF-A expression in vitro and in vivo, which significantly inhibited colon carcinoma cell proliferation and induced apoptosis in vitro. With 5-FC, the systemic delivery of CPNP-shVEGF-CDTK significantly inhibited tumor growth in the colon carcinoma xenograft animal model. The combined cassette is obviously effective in inhibiting tumor cell proliferation and inducing apoptosis in vitro and tumor growth in vivo than the CPNP-shVEGF or CPNP-CDTK alone. The combination of VEGF-A-silencing and tumor-specific expression of suicide gene is an effective strategy for colon carcinoma treatment.

  20. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

    Science.gov (United States)

    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  1. 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.

  2. 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...

  3. Odontomariinae, a new middle paleozoic subfamily of slit-bearing euophaloidean gastropods (Euophalomorpha, Gastropoda)

    Science.gov (United States)

    Fryda, J.; Heidelberger, D.; Blodgett, R.B.

    2006-01-01

    A new subfamily, the Odontomariinae subfam. nov., is established herein for a distinctive group of uncoiled, slit-bearing Middle Devonian euomphalid gastropods. Its taxonomic position is based on the recent discovery of open coiled protoconchs and it is placed within the Euomphalomorpha. The genera Odontomaria Odontomaria C. F. Roemer and Tubiconcha n. gen. belonging to this new subfamily are enlarged based on studies on new material of the following species: Odontomaria semiplicata (Sandberger & Sandberger), Odontomaria gracilis n. sp., Odontomaria jankei n. sp., Odontomaria cheeneetnukensis n. sp., Odontomaria cindiprellerae n. sp. and Tubiconcha leunissi (Heidelberger, 2001). Members of the Odontomariinae were mainly sedentary organisms in high-energy, moderately shallow water. ?? 2006 E. Schweizerbart'sche Verlagsbuchhandlung.

  4. 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/.

  5. 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.

  6. 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.

  7. Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

    Science.gov (United States)

    Das, Sabyasachi; Li, Jianxu; Holland, Stephen J; Iyer, Lakshminarayan M; Hirano, Masayuki; Schorpp, Michael; Aravind, L; Cooper, Max D; Boehm, Thomas

    2014-10-14

    Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

  8. Circulating cathodic antigen cassette test versus haematuria strip test in diagnosis of urinary schistosomiasis.

    Science.gov (United States)

    El-Ghareeb, Azza S; Abd El Motaleb, Ghada S; Waked, Nevien Maher; Osman Hany Kamel, Nancy; Aly, Nagwa Shaban

    2016-12-01

    Urinary schistosomiasis caused by Schistosoma haematobium constitutes a major public health problem in many tropical and sub-tropical countries. This study was conducted to evaluate circulating cathodic antigen cassette test and haematuria strip test for detection of S. haematobium in urine samples and to evaluate their screening performance among the study population. Microscopy was used as a gold standard. A total of 600 urine samples were examined by microscopy for detection of S. haematobium eggs, screened for microhaematuria using Self-Stik reagent strips and screened for circulating cathodic antigen (CCA) using the urine-CCA cassette test. The specificity of CCA, microhaematuria and macrohaematuria was 96.4, 40.6 and 31.2 % respectively while the sensitivity was 88.2, 99.3 and 100 % respectively which was statistically significant (P < 0.001). These findings suggest that using of urine-CCA cassette test in diagnosis of urinary schistosomiasis is highly specific (96.4 %) compared with the highly sensitive haematuria strip test (100 %). The degree of agreement between microscopic examination and CCA detection was 99.3 % with highly statistically significant difference (P < 0.001). The combination of two techniques could potentially use for screening and mapping of S. haematobium infection.

  9. Data supporting the nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae).

    Science.gov (United States)

    Comer, Jason R; Zomlefer, Wendy B; Barrett, Craig F; Stevenson, Dennis Wm; Heyduk, Karolina; Leebens-Mack, James H

    2016-06-01

    This data article provides data and supplemental materials referenced in "Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae)" (Comer et al., 2016) [1]. Raw sequence reads generated for this study are available through the Sequence Read Archive (SRA Study Accession: SRP061467). An aligned supermatrix of 168 nuclear genes for 35 taxa (34 palms and one outgroup taxon) is provided. Also provided are individual maximum likelihood gene trees used for the coalescent based analyses, output from the maximum parsimony analyses, and two figures.

  10. The gymnosperm Pinus pinea contains both AOX gene subfamilies, AOX1 and AOX2.

    Science.gov (United States)

    Frederico, António Miguel; Zavattieri, Maria Amely; Campos, Maria Doroteia; Cardoso, Hélia Guerra; McDonald, Allison E; Arnholdt-Schmitt, Birgit

    2009-12-01

    The gymnosperm Pinus pinea L. (stone pine) is a typical Mediterranean pine used for nuts and timber production, and as an ornamental around the world. Pine genomes are large in comparison to other species. The hypothesis that retrotransposons, such as gymny, made a large contribution to this alteration in genome size was recently confirmed. However, P. pinea is unique in other various aspects. P. pinea demonstrates a different pattern of gymny organization than other Pinus subgenera. Additionally, P. pinea has a highly recalcitrant behaviour in relation to standard conifer protocols for the induction of somatic embryogenesis or rooting. Because such types of cell reprogramming can be explained as a reaction of plant cells to external stress, it is of special interest to study sequence peculiarities in stress-inducible genes, such as the alternative oxidase (AOX). This is the first report containing molecular evidence for the existence of AOX in gymnosperms at the genetic level. P. pinea AOXs were isolated by a polymerase chain reaction (PCR) approach and three genes were identified. Two of the genes belong to the AOX1 subfamily and one belongs to the AOX2 subfamily. The existence of both AOX subfamilies in gymnosperms is reported here for the first time. This discovery supports the hypothesis that AOX1 and AOX2 subfamilies arose prior to the separation of gymnosperms and angiosperms, and indicates that the AOX2 is absent in monocots because of subsequent gene loss events. Polymorphic P. pinea AOX1 sequences from a selected genetic clone are presented indicating non-allelic, non-synonymous and synonymous translation products.

  11. 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.

  12. Clonal Expansion and Cytotoxicity of TCRVβ Subfamily T Cells Induced by CML and K562 Cells

    Institute of Scientific and Technical Information of China (English)

    YupingZHang; YangqiuLi; ShaohuaChen; LijianYang; GengxinLuo; XueliZhang

    2004-01-01

    OBJECTIVE To investigate the anti-leukemia effect, the distribution and clonal expansion of TCRVβ subfamily T cells in T cells from cord blood and adult peripheral blood induced by CML cells and K562 cells in vitro. METHODS Peripheral blood T cells from one adult donor and 3 cases of cord blood were stimulated with CML cells and K562 cells and further amplified by a suspended T cell-bulk culture,in order to induce CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the specific cytotoxicity in CML by LDH assay, the phenotype identification by indirect immunofiuorescence technique and the distribution and clonal expansion of TCRVβ subfamily by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan analysis, respectively. RESULTS Oligoclonal and oligoclonal tendency T cells with higher specific cytotoxicity from cord blood and adult peripheral blood could be induced by stimulation with CML cells and K562 cells. CONCLUSIONS Specific cytotoxic T cells for an anti-CML effect could be induced by CML cells and K562 cells .The induced T cells which have the characteristic of specific cytotoxicity against CML cells may come from the clonal expansion of TCRVβ subfamily T cells.

  13. Computational identification and evolutional analysis of Piwi subfamily in 11 Drosophila species

    Institute of Scientific and Technical Information of China (English)

    Xue Zhou; Ya-Long Xu; Luo-Gen Cheng; Fei Li

    2008-01-01

    The complete genome sequences of 11 Drosophila species provide an opportu-nity to investigate the gene family evolution in closely related species.Drosophila Piwi subfamily,including three mcmbers,piwi,Aub and Ago3,has attracted increasing attention as it participates in the biogenesis of piRNA.Here,we identified 33 Piwi homologs from the genome of 11 Drosophila species.The full-length cDNA sequences of piwi and Aub genes were obtained by using New GENSCAN Web Server.The Ago3 homologs were diffcult regarding full-length information because they had long introns.The genomic structure of Piwf subfamily genes are highly conserved among diverse Drosophila species.Insect piwi and Aub genes have long first introns.The average length of the first intmn is 1 284 bp for piwf and 840 bp for Aub,Which is much larger than those of other introns(93 bp for Piwf and 54 bp for Aub).However,this phenomenon is not observed in mammalian piwi genes.We alSO found that there were abundant repeat sequences in both exons and introns of insect Ago3 genes.DHe to recent insertions of long terminal repeat elements in four Drosophila species.part of the third introns exhibit higher conservation thall adjacent exons and other introns.An evolutional tree created by Minimum Evolution method indicates that mamma-lian piwf genes are more closely related to the insect Ag03 Piwi subfamily.

  14. Designing exons for human olfactory receptor gene subfamilies using a mathematical paradigm

    Indian Academy of Sciences (India)

    Sk Sarif Hassan; Pabitra Pal Choudhury; Amita Pal; R L Brahmachary; Arunava Goswami

    2010-09-01

    Ligands for only two human olfactory receptors are known. One of them, OR1D2, binds to Bourgeonal, a volatile chemical constituent of the fragrance of the mythical flower, Lily of the valley or Our Lady’s tears, Convallaria majalis (also the national flower of Finland). OR1D2, OR1D4 and OR1D5 are three full-length olfactory receptors present in an olfactory locus in the human genome. These receptors are more than 80% identical in DNA sequences and have 108 base pair mismatches among them. Apparently, these mismatch positions show no striking pattern using computer pattern recognition tools. In an attempt to find a mathematical rule in those mismatches, we find that an L-system generated sequence can be inserted into the OR1D2 subfamily-specific star model and novel full-length olfactory receptors can be generated. This remarkable mathematical principle could be utilized for making new subfamily olfactory receptor members from any olfactory receptor subfamily. The aroma and electronic nose industry might utilize this rule in future.

  15. Gene Structures, Classification, and Expression Models of the DREB Transcription Factor Subfamily in Populus trichocarpa

    Directory of Open Access Journals (Sweden)

    Yunlin Chen

    2013-01-01

    Full Text Available We identified 75 dehydration-responsive element-binding (DREB protein genes in Populus trichocarpa. We analyzed gene structures, phylogenies, domain duplications, genome localizations, and expression profiles. The phylogenic construction suggests that the PtrDREB gene subfamily can be classified broadly into six subtypes (DREB A-1 to A-6 in Populus. The chromosomal localizations of the PtrDREB genes indicated 18 segmental duplication events involving 36 genes and six redundant PtrDREB genes were involved in tandem duplication events. There were fewer introns in the PtrDREB subfamily. The motif composition of PtrDREB was highly conserved in the same subtype. We investigated expression profiles of this gene subfamily from different tissues and/or developmental stages. Sixteen genes present in the digital expression analysis had high levels of transcript accumulation. The microarray results suggest that 18 genes were upregulated. We further examined the stress responsiveness of 15 genes by qRT-PCR. A digital northern analysis showed that the PtrDREB17, 18, and 32 genes were highly induced in leaves under cold stress, and the same expression trends were shown by qRT-PCR. Taken together, these observations may lay the foundation for future functional analyses to unravel the biological roles of Populus’ DREB genes.

  16. Enhanced performance of methamphetamine lateral flow cassettes using an electronic lateral flow reader.

    Science.gov (United States)

    Smith, Jerome P; Sammons, Deborah L; Robertson, Shirley A; Snawder, John E

    2015-01-01

    Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton-tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0-10 ng/ml for calibration solutions and 0-25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point.

  17. Organization and evolution of two SIDER retroposon subfamilies and their impact on the Leishmania genome

    Directory of Open Access Journals (Sweden)

    Bringaud Frédéric

    2009-05-01

    Full Text Available Abstract Background We have recently identified two large families of extinct transposable elements termed Short Interspersed DEgenerated Retroposons (SIDERs in the parasitic protozoan Leishmania major. The characterization of SIDER elements was limited to the SIDER2 subfamily, although members of both subfamilies have been shown to play a role in the regulation of gene expression at the post-transcriptional level. Apparent functional domestication of SIDERs prompted further investigation of their characterization, dissemination and evolution throughout the Leishmania genus, with particular attention to the disregarded SIDER1 subfamily. Results Using optimized statistical profiles of both SIDER1 and SIDER2 subgroups, we report the first automated and highly sensitive annotation of SIDERs in the genomes of L. infantum, L. braziliensis and L. major. SIDER annotations were combined to in-silico mRNA extremity predictions to generate a detailed distribution map of the repeat family, hence uncovering an enrichment of antisense-oriented SIDER repeats between the polyadenylation and trans-splicing sites of intergenic regions, in contrast to the exclusive sense orientation of SIDER elements within 3'UTRs. Our data indicate that SIDER elements are quite uniformly dispersed throughout all three genomes and that their distribution is generally syntenic. However, only 47.4% of orthologous genes harbor a SIDER element in all three species. There is evidence for species-specific enrichment of SIDERs and for their preferential association, especially for SIDER2s, with different metabolic functions. Investigation of the sequence attributes and evolutionary relationship of SIDERs to other trypanosomatid retroposons reveals that SIDER1 is a truncated version of extinct autonomous ingi-like retroposons (DIREs, which were functional in the ancestral Leishmania genome. Conclusion A detailed characterization of the sequence traits for both SIDER subfamilies unveils

  18. Genesis of the vertebrate FoxP subfamily member genes occurred during two ancestral whole genome duplication events.

    Science.gov (United States)

    Song, Xiaowei; Tang, Yezhong; Wang, Yajun

    2016-08-22

    The vertebrate FoxP subfamily genes play important roles in the construction of essential functional modules involved in physiological and developmental processes. To explore the adaptive evolution of functional modules associated with the FoxP subfamily member genes, it is necessary to study the gene duplication process. We detected four member genes of the FoxP subfamily in sea lampreys (a representative species of jawless vertebrates) through genome screenings and phylogenetic analyses. Reliable paralogons (i.e. paralogous chromosome segments) have rarely been detected in scaffolds of FoxP subfamily member genes in sea lampreys due to the considerable existence of HTH_Tnp_Tc3_2 transposases. However, these transposases did not alter gene numbers of the FoxP subfamily in sea lampreys. The coincidence between the "1-4" gene duplication pattern of FoxP subfamily genes from invertebrates to vertebrates and two rounds of ancestral whole genome duplication (1R- and 2R-WGD) events reveal that the FoxP subfamily of vertebrates was quadruplicated in the 1R- and 2R-WGD events. Furthermore, we deduced that a synchronous gene duplication process occurred for the FoxP subfamily and for three linked gene families/subfamilies (i.e. MIT family, mGluR group III and PLXNA subfamily) in the 1R- and 2R-WGD events using phylogenetic analyses and mirror-dendrogram methods (i.e. algorithms to test protein-protein interactions). Specifically, the ancestor of FoxP1 and FoxP3 and the ancestor of FoxP2 and FoxP4 were generated in 1R-WGD event. In the subsequent 2R-WGD event, these two ancestral genes were changed into FoxP1, FoxP2, FoxP3 and FoxP4. The elucidation of these gene duplication processes shed light on the phylogenetic relationships between functional modules of the FoxP subfamily member genes.

  19. Frames of exponentials:lower frame bounds for finite subfamilies, and approximation of the inverse frame operator

    DEFF Research Database (Denmark)

    Christensen, Ole; Lindner, Alexander M

    2001-01-01

    We give lower frame bounds for finite subfamilies of a frame of exponentials {e(i lambdak(.))}k is an element ofZ in L-2(-pi,pi). We also present a method for approximation of the inverse frame operator corresponding to {e(i lambdak(.))}k is an element ofZ, where knowledge of the frame bounds for...... for finite subfamilies is crucial. (C) 2001 Elsevier Science Inc. All rights reserved....

  20. 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

  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. The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance.

    Science.gov (United States)

    Liu, Shaojun; Qin, Qinbo; Xiao, Jun; Lu, Wenting; Shen, Jiamin; Li, Wei; Liu, Jifang; Duan, Wei; Zhang, Chun; Tao, Min; Zhao, Rurong; Yan, Jinpeng; Liu, Yun

    2007-06-01

    This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp x blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (female symbol) x blunt snout bream (BSB) (male symbol) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.

  3. Phylogeny and evolutionary patterns in the Dwarf crayfish subfamily (Decapoda: Cambarellinae.

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    Carlos Pedraza-Lara

    Full Text Available The Dwarf crayfish or Cambarellinae, is a morphologically singular subfamily of decapod crustaceans that contains only one genus, Cambarellus. Its intriguing distribution, along the river basins of the Gulf Coast of United States (Gulf Group and into Central México (Mexican Group, has until now lacked of satisfactory explanation. This study provides a comprehensive sampling of most of the extant species of Cambarellus and sheds light on its evolutionary history, systematics and biogeography. We tested the impact of Gulf Group versus Mexican Group geography on rates of cladogenesis using a maximum likelihood framework, testing different models of birth/extinction of lineages. We propose a comprehensive phylogenetic hypothesis for the subfamily based on mitochondrial and nuclear loci (3,833 bp using Bayesian and Maximum Likelihood methods. The phylogenetic structure found two phylogenetic groups associated to the two main geographic components (Gulf Group and Mexican Group and is partially consistent with the historical structure of river basins. The previous hypothesis, which divided the genus into three subgenera based on genitalia morphology was only partially supported (P = 0.047, resulting in a paraphyletic subgenus Pandicambarus. We found at least two cases in which phylogenetic structure failed to recover monophyly of recognized species while detecting several cases of cryptic diversity, corresponding to lineages not assigned to any described species. Cladogenetic patterns in the entire subfamily are better explained by an allopatric model of speciation. Diversification analyses showed similar cladogenesis patterns between both groups and did not significantly differ from the constant rate models. While cladogenesis in the Gulf Group is coincident in time with changes in the sea levels, in the Mexican Group, cladogenesis is congruent with the formation of the Trans-Mexican Volcanic Belt. Our results show how similar allopatric

  4. Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

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    Wang Ya-Ping

    2008-03-01

    Full Text Available Abstract Background Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp in length, with a 342 bp open reading frame (ORF encoding a putative protein of 113 amino acids (aa. Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

  5. Reassessment of Species Diversity of the Subfamily Denticollinae (Coleoptera: Elateridae through DNA Barcoding.

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    Taeman Han

    Full Text Available The subfamily Denticollinae is a taxonomically diverse group in the family Elateridae. Denticollinae includes many morphologically similar species and crop pests, as well as many undescribed species at each local fauna. To construct a rapid and reliable identification system for this subfamily, the effectiveness of molecular species identification was assessed based on 421 cytochrome c oxidase subunit I (COI sequences of 84 morphologically identified species. Among the 84 morphospecies, molecular species identification of 60 species (71.4% was consistent with their morphological identifications. Six cryptic and/or pseudocryptic species with large genetic divergence (>5% were confirmed by their sympatric or allopatric distributions. However, 18 species, including a subspecies, had ambiguous genetic distances and shared overlapping intra- and interspecific genetic distances (range: 2.12%-3.67% suggesting incomplete lineage sorting, introgression of mitochondrial genome, or affection by endosymbionts, such as Wolbachia infection, between species and simple genetic variation within species. In this study, we propose a conservative threshold of 3.6% for convenient molecular operational taxonomic unit (MOTU identification in the subfamily Denticollinae based on the results of pairwise genetic distances analyses using neighbor-joining, mothur, Automatic Barcode Gap Discovery analysis, and tree-based species delimitation by Poisson Tree Processes analysis. Using the 3.6% threshold, we identified 87 MOTUs and found 8 MOTUs in the interval between 2.5% to 3.5%. Evaluation of MOTUs identified in this range requires integrative species delimitation, including review of morphological and ecological differences as well as sensitive genetic markers. From this study, we confirmed that COI sequence is useful for reassessing species diversity for polymorphic and polytypic species occurring in sympatric and allopatric distributions, and for a single species having

  6. 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.

  7. Data supporting the nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae

    Directory of Open Access Journals (Sweden)

    Jason R. Comer

    2016-06-01

    Full Text Available This data article provides data and supplemental materials referenced in “Nuclear phylogenomics of the palm subfamily Arecoideae (Arecaceae” (Comer et al., 2016 [1]. Raw sequence reads generated for this study are available through the Sequence Read Archive (SRA Study Accession: SRP061467. An aligned supermatrix of 168 nuclear genes for 35 taxa (34 palms and one outgroup taxon is provided. Also provided are individual maximum likelihood gene trees used for the coalescent based analyses, output from the maximum parsimony analyses, and two figures.

  8. Phthiria sharafi sp. nov., a new record of the subfamily Phthiriinae (Bombyliidae, Diptera) from Saudi Arabia.

    Science.gov (United States)

    El-Hawagry, Magdi S; Al Dhafer, Hathal M

    2014-10-10

    This new species (Phthiria sharafi sp. nov.) represents the first record of the subfamily Phthiriinae (Bombyliidae, Diptera) from Saudi Arabia. The species was collected from Garf Raydah Protected Area, Abha, Asir Province, south-western part of Saudi Arabia, using a Malaise trap erected in a site rich in olive, cactus and Juniper trees. The type locality has an Afrotropical influence, with the Afrotropical elements predominant, and a closer affiliation to the Afrotropical region than to the Palearctic region or the Eremic zone. 

  9. Lactobacillus plantarum CUL66 can impact cholesterol homeostasis in Caco-2 enterocytes.

    Science.gov (United States)

    Michael, D R; Moss, J W E; Calvente, D Lama; Garaiova, I; Plummer, S F; Ramji, D P

    2016-06-01

    Hypercholesterolemia drives the development of cardiovascular disease, the leading cause of mortality in western society. Supplementation with probiotics that interfere with cholesterol metabolism may provide a contribution to disease prevention. Lactobacillus plantarum CUL66 (NCIMB 30280) has been assessed in vitro for its ability to impact cholesterol absorption. L. plantarum CUL66 tested positive for bile salt hydrolase activity and the ability to assimilate cholesterol from culture media. RT-qPCR analysis showed that the bacterium significantly decreased the expression of Niemann-Pick C1-like 1 and ATP-binding cassette transporter-1 in polarised Caco-2 cells after 6 h exposure. Conversely, the expression of ATP-binding cassette sub-family G member (ABCG)-5 and ABCG-8, and 3-hydroxy-3-methylglutaryl-CoA reductase were significantly increased. Using a radiolabelled assay, we also observed significant reductions in the uptake and basolateral efflux of cholesterol by Caco-2 cells exposed to L. plantarum CUL66. This in vitro study identified L. plantarum CUL66 as a cholesterol lowering bacteria by highlighting its ability to beneficially regulate multiple in vitro events associated with intestinal cholesterol metabolism and provides evidence of efficacy for its inclusion in future in vivo studies.

  10. Characteristics of the LrhA subfamily of transcriptional regulators from Sinorhizobium meliloti

    Institute of Scientific and Technical Information of China (English)

    Mingsheng Qi; Li Luo; Haiping Cheng; Jiabi Zhu; Guanqiao Yu

    2008-01-01

    In our previous work, we identified 94 putative genes encoding LysR-type transcriptional regulators from Sinorhizobium meliloti. All of these putative lysR genes were mutagenized using plasmid insertions to determine their phenotypes. Six LysR-type regulators, encoded by mutants SMa1979, SMb20715, SMc00820, SMc04163, SMc03975,and SMc04315, showed similar amino acid sequences (30%)and shared the conserved DNA-binding domain with LrhA,HexA, or DgdR. Phenotype analysis of these gene mutants indicated that the regulators control the swimming behaviors of the bacteria, production of quorum-sensing signals, and secretion of extracellular proteins. These characteristics are very similar to those of LrhA, HexA, and DgdR.Thus, we refer to this group as the LrhA subfamily. Sequence analysis showed that a great number of homologous genes of the LrhA subfamily were distributed in the α,β, and γsubdivisions of proteobacteria, and a few in actinobacteria. These findings could provide new clues to the roles of the LysR gene family.

  11. Nonlinear interaction of intense hypergeometric Gaussian subfamily laser beams in plasma

    Science.gov (United States)

    Sobhani, H.; Vaziri (Khamedi), M.; Rooholamininejad, H.; Bahrampour, A. R.

    2016-07-01

    Propagation of Hypergeometric-Gaussian laser beam in a nonlinear plasma medium is investigated by considering the Source Dependent Expansion method. A subfamily of Hypergeometric-Gaussian beams with a non-negative, even and integer radial index, can be expressed as the linear superposition of finite number of Laguerre-Gaussian functions. Propagation of Hypergeometric-Gaussian beams in a nonlinear plasma medium depends on the value of radial index. The bright rings' number of these beams is changed during the propagation in plasma medium. The effect of beam vortex charge number l and initial (input) beam intensity on the self-focusing of Hypergeometric-Gaussian beams is explored. Also, by choosing the suitable initial conditions, Hypergeometric-Gaussian subfamily beams can be converted to one or more mode components that a typical of mode conversion may be occurred. The self-focusing of these winding beams can be used to control the focusing force and improve the electron bunch quality in laser plasma accelerators.

  12. Flight patterns and sex ratio of beetles of the subfamily Dynastinae (Coleoptera, Melolonthidae

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    Larissa Simões Corrêa de Albuquerque

    2016-09-01

    Full Text Available ABSTRACT Dynastinae is one of the most representative subfamilies of Melolonthidae (Scarabaeoidea and has considerable ecological importance due mainly to interactions with plants of the families Araceae and Annonaceae. This relationship has led to the evolution of nocturnal activity patterns, which are influenced by environmental conditions. In the present study, abiotic factors were investigated to comprehend the influence on the flight patterns and identify the sex ratio of beetles from this subfamily. A study was conducted at Campo de Instrução Marechal Newton Cavalcanti in northeastern Brazil between December 2010 and November 2011. Thirteen species of Dynastinae were identified, most of which were from the genus Cyclocephala. Abundance and richness were greater in the dry season. Six species exhibited peak flight activity at specific periods of the night. More females than males were recorded for Cyclocephala distincta and C. paraguayensis. The present findings suggest that rainfall reduces the flight activity of these beetles and different time schedules may be related to mating behavior, foraging behavior and the avoidance of interspecific resource competition.

  13. A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae.

    Science.gov (United States)

    Amiche, Mohamed; Ladram, Ali; Nicolas, Pierre

    2008-11-01

    A growing number of cationic antimicrobial peptides have been isolated from the skin of hylid frogs belonging to the Phyllomedusinae subfamily. The amino acid sequences of these peptides are currently located in several databases under identifiers with no consistent system of nomenclature to describe them. In order to provide a workable terminology for antimicrobial peptides from Phyllomedusid frogs, we have made a systematic effort to collect, analyze, and classify all the Phyllomedusid peptide sequences available in databases. We propose that frogs belonging to the Phyllomedusinae subfamily should be described by the species names set out in Amphibian Species of the World: http://research.amnh.org/herpetology/amphibia/index.php, American Museum of Natural History, New York, USA. Multiple alignments analysis of at least 80 antimicrobial peptides isolated from 12 Phyllomedusinae species were distributed in seven distinct peptide families including dermaseptin, phylloseptin, plasticin, dermatoxin, phylloxin, hyposin and orphan peptides, and will be considered as the name of the headgroup of each family. The parent peptide's name should be followed by the first upper letter of the species for orthologous peptides and publication date determines priority. For example, the abbreviation B for bicolor and H for hypochondrialis. When two species begin with the same letter, two letters in upper case should be used (the first letter followed by the second or the third letter and so on). For example, the abbreviation DI for distincta, DU for duellmani, VA for vaillanti and VN for vanzolinii. Paralogous peptides should bear letter(s) in upper case followed by numbers.

  14. Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

    Science.gov (United States)

    Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

    2008-11-01

    Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

  15. Myosin Binding Protein-C Slow: An Intricate Subfamily of Proteins

    Directory of Open Access Journals (Sweden)

    Maegen A. Ackermann

    2010-01-01

    Full Text Available Myosin binding protein C (MyBP-C consists of a family of thick filament associated proteins. Three isoforms of MyBP-C exist in striated muscles: cardiac, slow skeletal, and fast skeletal. To date, most studies have focused on the cardiac form, due to its direct involvement in the development of hypertrophic cardiomyopathy. Here we focus on the slow skeletal form, discuss past and current literature, and present evidence to support that: (i MyBP-C slow comprises a subfamily of four proteins, resulting from complex alternative shuffling of the single MyBP-C slow gene, (ii the four MyBP-C slow isoforms are expressed in variable amounts in different skeletal muscles, (iii at least one MyBP-C slow isoform is preferentially found at the periphery of M-bands and (iv the MyBP-C slow subfamily may play important roles in the assembly and stabilization of sarcomeric M- and A-bands and regulate the contractile properties of the actomyosin filaments.

  16. Phylogenetic relationships between flies of the Tephritinae subfamily (Diptera, Tephritidae) and their symbiotic bacteria.

    Science.gov (United States)

    Mazzon, Luca; Martinez-Sañudo, Isabel; Simonato, Mauro; Squartini, Andrea; Savio, Claudia; Girolami, Vincenzo

    2010-07-01

    The Tephritinae is considered the most specialized subfamily of fruit flies, predominantly infesting flowerheads of Asteraceae. Some species are known to host specific non-culturable symbiont bacteria ("Candidatus Stammerula spp.") in the midgut. In this work we (i) examined the phylogenetic relationships among the insect hosts, (ii) investigated the presence of bacteria in other hitherto unexamined species, and (iii) evaluated the phylogenetic congruence between insects and symbionts. A total of 33 Tephritinae species in 17 different genera were analyzed. Two regions of the mitochondrial DNA (16S rDNA and COI-tRNALeu-COII) were examined in the insect host, while the 16S was analyzed in the bacteria. From the phylogenetic trees, four of the five tribes considered were statistically supported by each of the clustering methods used. Species belonging to the tribe Noeetini never clustered at significant levels. The phylogenetic COI-tRNALeu-COII tree showed internal nodes more highly supported than the 16S phylogeny. The analysis of the distribution of symbiosis across the subfamily has highlighted the presence of bacteria only in the tribe Tephritini and in the genus Noeeta from the tribe Noeetini. A cophylogenetic analysis revealed a substantial congruence between hosts and symbionts. The interesting exceptions can be justified by events like losses, duplications and hosts switching opportunities, which are likely to arise during the biological cycle of the fly in consideration of the extracellular status of these symbionts.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Site-specific integration and tailoring of cassette design for sustainable gene transfer.

    Science.gov (United States)

    Lombardo, Angelo; Cesana, Daniela; Genovese, Pietro; Di Stefano, Bruno; Provasi, Elena; Colombo, Daniele F; Neri, Margherita; Magnani, Zulma; Cantore, Alessio; Lo Riso, Pietro; Damo, Martina; Pello, Oscar M; Holmes, Michael C; Gregory, Philip D; Gritti, Angela; Broccoli, Vania; Bonini, Chiara; Naldini, Luigi

    2011-08-21

    Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

  2. 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.

  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. A Self-deleting Cre-lox-ermAM Cassette, CHESHIRE, for marker-less gene deletion in Streptococcus pneumoniae

    Science.gov (United States)

    Weng, Liming; Biswas, Indranil; Morrison, Donald A.

    2009-01-01

    Although targeted mutagenesis of Streptococcus pneumoniae is readily accomplished with the aid of natural genetic transformation and chimeric donor DNA constructs assembled in vitro, the drug resistance markers often employed for selection of recombinant products can themselves be undesirable by-products of the genetic manipulation. A new cassette carrying the erythromycin-resistance marker ermAM is described that can be used as a temporary marker for selection of desired recombinants. The cassette may subsequently be removed at will by virtue of an embedded fucose-regulated Cre recombinase gene and terminal lox66 and lox71 Cre recognition sites, with retention of 34 bp from the cassette as an inert residual double-mutant lox72 site. PMID:19850089

  5. The Evolutionary History of R2R3-MYB Proteins Across 50 Eukaryotes: New Insights Into Subfamily Classification and Expansion.

    Science.gov (United States)

    Du, Hai; Liang, Zhe; Zhao, Sen; Nan, Ming-Ge; Tran, Lam-Son Phan; Lu, Kun; Huang, Yu-Bi; Li, Jia-Na

    2015-06-05

    R2R3-MYB proteins (2R-MYBs) are one of the main transcription factor families in higher plants. Since the evolutionary history of this gene family across the eukaryotic kingdom remains unknown, we performed a comparative analysis of 2R-MYBs from 50 major eukaryotic lineages, with particular emphasis on land plants. A total of 1548 candidates were identified among diverse taxonomic groups, which allowed for an updated classification of 73 highly conserved subfamilies, including many newly identified subfamilies. Our results revealed that the protein architectures, intron patterns, and sequence characteristics were remarkably conserved in each subfamily. At least four subfamilies were derived from early land plants, 10 evolved from spermatophytes, and 19 from angiosperms, demonstrating the diversity and preferential expansion of this gene family in land plants. Moreover, we determined that their remarkable expansion was mainly attributed to whole genome and segmental duplication, where duplicates were preferentially retained within certain subfamilies that shared three homologous intron patterns (a, b, and c) even though up to 12 types of patterns existed. Through our integrated distributions, sequence characteristics, and phylogenetic tree analyses, we confirm that 2R-MYBs are old and postulate that 3R-MYBs may be evolutionarily derived from 2R-MYBs via intragenic domain duplication.

  6. Evaluation of D-1 tape and cassette characteristics: Moisture content of Sony and Ampex D-1 tapes when delivered

    Science.gov (United States)

    Ashton, Gary

    Commercial D-1 cassette tapes and their associated recorders were designed to operate in broadcast studios and record in accordance with the International Radio Consultative Committee (CCIR) 607 digital video standards. The D-1 recorder resulted in the Society of Motion Picture and Television Engineers (SMPTE) standards 224 to 228 and is the first digital video recorder to be standardized for the broadcast industry. The D-1 cassette and associated media are currently marketed for broadcast use. The recorder was redesigned for data applications and is in the early stages of being evaluated. The digital data formats used are specified in MIL-STD-2179 and the American National Standards Institute (ANSI) X3.175-190 standard. In early 1990, the National Media Laboratory (NML) was asked to study the effects of time, temperature, and relative humidity on commercial D-1 cassettes. The environmental range to be studied was the one selected for the Advanced Tactical Air Reconnaissance System (ATARS) program. Several discussions between NML personnel, ATARS representatives, recorder contractors, and other interested parties were held to decide upon the experimental plan to be implemented. Review meetings were held periodically during the course of the experiment. The experiments were designed to determine the dimensional stability of the media and cassette since this is one of the major limiting factors of helical recorders when the media or recorders are subjected to non-broadcasting environments. Measurements were also made to characterize each sample of cassettes to give preliminary information on which purchase specifications could be developed. The actual tests performed on the cassettes and media before and after aging fall into the general categories listed.

  7. The role of morpho-anatomical traits of the leaves in the taxonomy of Kalanchoideae Berg. subfamily (Crassulaceae DC.

    Directory of Open Access Journals (Sweden)

    Myhajlo A. Chernetskyy

    2012-03-01

    Full Text Available The paper presents the results of the analysis of the morphological and anatomical structure of leaves of 35 species of the genus Kalanchoë Adans. (Crassulaceae DC. in the taxonomic aspect of the subfamily Kalanchoideae Berg. Based on own studies and literature analyses of the flower morphology, embryology, karyology, vascular anatomy of stem and molecular genetics, the author has found that the most appropriate taxonomic system of the subfamily Kalanchoideae assumes existence of one genus Kalanchoë divided into three sections:Bryophyllum (Salisb. Boit. & Mann.,Eukalanchoë Boit. & Mann. and Kitchingia (Bak. Boit. & Mann. Distinguishing three separate genera Bryophyllum Salisb., Kalanchoё Adans. and Kitchingia Bak., as has been the case throughout the history of the subfamily Kalanchoideae, is hardly possible due to existence of “intermediate” species.

  8. Nasal mites of the subfamily Speleognathinae (Ereynetidae) from birds in Texas.

    Science.gov (United States)

    Pence, D B; Casto, S D

    1976-06-01

    Nasal mites of the subfamily Speleognathinae were recovered from several species of birds in Texas. New host records include Ophthalmophagus striatus (Crossley) 1952 from Columbigallina passerina, Boydaia clarki Fain 1963 from Callipepla squamata, Boydaia falconis Fain 1956 from Falco sparverius, and Boydaia tyrannus Ford 1959 from Myiarchus cinerascens. Also recovered was Astrida coccyzae Pence 1972 from Coccyzus americanus. Boydaia pheucticola sp. n. from Pheucticus melanocephalus is described. It differs from similar species in the adult female by having the coxal setae formula 2-1-2-0, sensillae clavate but not globose or subglobose, and interior seta on coxa I reduced in size but not vestigial. The larva is differentiated by the modified legs II with one long recurved hooklike claw and a shorter curved claw.

  9. Quill mites of the subfamily Picobiinae (Acari: Syringophilidae) associated with woodpeckers (Aves: Piciformes: Picidae).

    Science.gov (United States)

    Skoracki, Maciej; Unsoeld, Markus; Kavetska, Katarzyna; Kaszewska, Katarzyna

    2014-03-01

    The paper contains a review of quill mites of the subfamily Picobiinae (Acari: Prostigmata: Syringophilidae) associated with woodpeckers (Aves: Piciformes: Picidae). Three new species are described: Picobia mentalis Skoracki et Unsoeld sp. nov. from Picus mentalis Temminck, Neopicobia ea Skoracki et Unsoeld sp. nov. from Celeus flavus (St. Mueller) (type host), C. elegans (St. Mueller), C. torquatus (Boddaert), and Neopicobia freya Skoracki et Unsoeld sp. nov. from Dryocopus galeatus (Temminck) (type host) and Piculus rubiginosus (Swainson). Additionally, six new host species for Picobia heeri Haller, 1878 and 12 new host species for Picobia dryobatis (Fritsch, 1956) are reported. A complete list of the picobiines parasitising birds of the family Picidae is presented in the tabular form.

  10. Relevant use of Klotho in FGF19 subfamily signaling system in vivo.

    Science.gov (United States)

    Tomiyama, Ken-ichi; Maeda, Ryota; Urakawa, Itaru; Yamazaki, Yuji; Tanaka, Tomohiro; Ito, Shinji; Nabeshima, Yoko; Tomita, Tsutomu; Odori, Shinji; Hosoda, Kiminori; Nakao, Kazuwa; Imura, Akihiro; Nabeshima, Yo-ichi

    2010-01-26

    Alpha-Klotho (alpha-Kl) and its homolog, beta-Klotho (beta-Kl) are key regulators of mineral homeostasis and bile acid/cholesterol metabolism, respectively. FGF15/ humanFGF19, FGF21, and FGF23, members of the FGF19 subfamily, are believed to act as circulating metabolic regulators. Analyses of functional interactions between alpha- and beta-Kl and FGF19 factors in wild-type, alpha-kl(-/-), and beta-kl(-/-) mice revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of alpha-Kl, FGF23, and 1,25(OH)(2)D and an analogous regulatory network composed of beta-Kl, FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Contrary to in vitro data, beta-Kl is not essential for FGF21 signaling in adipose tissues in vivo, because (i) FGF21 signals are transduced in the absence of beta-Kl, (ii) FGF21 could not be precipitated by beta-Kl, and (iii) essential phenotypes in Fgf21(-/-) mice (decreased expressions of Hsl and Atgl in WAT) were not replicated in beta-kl(-/-) mice. These findings suggest the existence of Klotho-independent FGF21 signaling pathway(s) where undefined cofactors are involved. One-to-one functional interactions such as alpha-Klotho/FGF23, beta-Klotho/FGF15 (humanFGF19), and undefined cofactor/FGF21 would result in tissue-specific signal transduction of the FGF19 subfamily.

  11. Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

    Science.gov (United States)

    Levitz, Joshua; Royal, Perrine; Comoglio, Yannick; Wdziekonski, Brigitte; Schaub, Sébastien; Clemens, Daniel M; Isacoff, Ehud Y; Sandoz, Guillaume

    2016-04-12

    Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

  12. That awkward age for butterflies: insights from the age of the butterfly subfamily Nymphalinae (Lepidoptera: Nymphalidae).

    Science.gov (United States)

    Wahlberg, Niklas

    2006-10-01

    The study of the historical biogeography of butterflies has been hampered by a lack of well-resolved phylogenies and a good estimate of the temporal span over which butterflies have evolved. Recently there has been surge of phylogenetic hypotheses for various butterfly groups, but estimating ages of divergence is still in its infancy for this group of insects. The main problem has been the sparse fossil record for butterflies. In this study I have used a surprisingly good fossil record for the subfamily Nymphalinae (Lepidoptera: Nymphalidae) to estimate the ages of diversification of major lineages using Bayesian relaxed clock methods. I have investigated the effects of varying priors on posterior estimates in the analyses. For this data set, it is clear that the prior of the rate of molecular evolution at the ingroup node had the largest effect on the results. Taking this into account, I have been able to arrive at a plausible history of lineage splits, which appears to be correlated with known paleogeological events. The subfamily appears to have diversified soon after the K/T event about 65 million years ago. Several splits are coincident with major paleogeological events, such as the connection of the African and Asian continents about 21 million years ago and the presence of a peninsula of land connecting the current Greater Antilles to the South American continent 35 to 33 million years ago. My results suggest that the age of Nymphalidae is older than the 70 million years speculated to be the age of butterflies as a whole.

  13. Comparative Mitogenomic Analysis of Species Representing Six Subfamilies in the Family Tenebrionidae

    Directory of Open Access Journals (Sweden)

    Hong-Li Zhang

    2016-05-01

    Full Text Available To better understand the architecture and evolution of the mitochondrial genome (mitogenome, mitogenomes of ten specimens representing six subfamilies in Tenebrionidae were selected, and comparative analysis of these mitogenomes was carried out in this study. Ten mitogenomes in this family share a similar gene composition, gene order, nucleotide composition, and codon usage. In addition, our results show that nucleotide bias was strongly influenced by the preference of codon usage for A/T rich codons which significantly correlated with the G + C content of protein coding genes (PCGs. Evolutionary rate analyses reveal that all PCGs have been subjected to a purifying selection, whereas 13 PCGs displayed different evolution rates, among which ATPase subunit 8 (ATP8 showed the highest evolutionary rate. We inferred the secondary structure for all RNA genes of Tenebrio molitor (Te2 and used this as the basis for comparison with the same genes from other Tenebrionidae mitogenomes. Some conserved helices (stems and loops of RNA structures were found in different domains of ribosomal RNAs (rRNAs and the cloverleaf structure of transfer RNAs (tRNAs. With regard to the AT-rich region, we analyzed tandem repeat sequences located in this region and identified some essential elements including T stretches, the consensus motif at the flanking regions of T stretch, and the secondary structure formed by the motif at the 3′ end of T stretch in major strand, which are highly conserved in these species. Furthermore, phylogenetic analyses using mitogenomic data strongly support the relationships among six subfamilies: ((Tenebrionidae incertae sedis + (Diaperinae + Tenebrioninae + (Pimeliinae + Lagriinae, which is consistent with phylogenetic results based on morphological traits.

  14. 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.

  15. A precisely regulated gene expression cassette potently modulates metastasis and survival in multiple solid cancers.

    Directory of Open Access Journals (Sweden)

    Kun Yu

    Full Text Available Successful tumor development and progression involves the complex interplay of both pro- and anti-oncogenic signaling pathways. Genetic components balancing these opposing activities are likely to require tight regulation, because even subtle alterations in their expression may disrupt this balance with major consequences for various cancer-associated phenotypes. Here, we describe a cassette of cancer-specific genes exhibiting precise transcriptional control in solid tumors. Mining a database of tumor gene expression profiles from six different tissues, we identified 48 genes exhibiting highly restricted levels of gene expression variation in tumors (n = 270 compared to nonmalignant tissues (n = 71. Comprising genes linked to multiple cancer-related pathways, the restricted expression of this "Poised Gene Cassette" (PGC was robustly validated across 11 independent cohorts of approximately 1,300 samples from multiple cancer types. In three separate experimental models, subtle alterations in PGC expression were consistently associated with significant differences in metastatic and invasive potential. We functionally confirmed this association in siRNA knockdown experiments of five PGC genes (p53CSV, MAP3K11, MTCH2, CPSF6, and SKIP, which either directly enhanced the invasive capacities or inhibited the proliferation of AGS cancer cells. In primary tumors, similar subtle alterations in PGC expression were also repeatedly associated with clinical outcome in multiple cohorts. Taken collectively, these findings support the existence of a common set of precisely controlled genes in solid tumors. Since inducing small activity changes in these genes may prove sufficient to potently influence various tumor phenotypes such as metastasis, targeting such precisely regulated genes may represent a promising avenue for novel anti-cancer therapies.

  16. 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...

  17. 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.

  18. 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.

  19. Emissies uit een ligboxenstal voor melkvee met roostervloer voorzien van cassettes in de roosterspleten : meetprogramma Integraal Duurzame Stallen

    NARCIS (Netherlands)

    Mosquera, J.; Hol, J.M.G.; Huis in 't Veld, J.W.H.; Ploegaert, J.P.M.; Ogink, N.W.M.

    2012-01-01

    In dit onderzoek zijn de emissies bepaald van ammoniak, geur, fijn stof (PM10, PM2,5), methaan en lachgas uit een ligboxenstal voor melkvee met roostervloer voorzien van cassettes in de roosterspleten.This study reports the emissions of ammonia, odour, fine dust (PM10 and PM2.5), methane and nitrous

  20. Identification of antibiotic resistance cassettes in class 1 integrons in Aeromonas spp. strains isolated from fresh fish (Cyprinus carpio L.).

    Science.gov (United States)

    Sarria-Guzmán, Yohanna; López-Ramírez, María Patricia; Chávez-Romero, Yosef; Ruiz-Romero, Erick; Dendooven, Luc; Bello-López, Juan Manuel

    2014-05-01

    Forty-six Aeromonas spp. strains were isolated from fresh fish and investigated for their antimicrobial susceptibility, detection of Class 1 integrons by PCR, and arrangement of gene cassettes. Selected isolates were further characterized by enterobacterial repetitive intergenic consensus-PCR. Twenty isolates were found to carry Class 1 integrons. Amplification of the variable regions of the integrons revealed diverse bands ranging in size from 150 to 1,958 pb. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as adenylyl transferases (aadA2 and aadA5), dihydrofolate reductases (dfrA17 and dfrA1), chloramphenicol acetyl transferase (catB3), β-lactamase (oxa2), lincosamide nucleotidil transferase (linF), aminoglycoside-modifying enzyme (apha15), and oxacillinase (bla OXA-10). Two open reading frames with an unknown function were identified as orfC and orfD. The aadA2 cassette was the most common integron found in this study. Interestingly, five integrons were detected in the plasmids that might be involved in the transfer of resistance genes to other bacteria. This is a first report of cassette encoding for lincosamides (linF) resistance in Aeromonas spp. Implications on the incidence of integrons in isolates of Aeromonas spp. from fresh fish for human consumption, and its possible consequences to human health are discussed.

  1. Endotoxin deposits on the inner surfaces of closed-face cassettes during bioaerosol sampling: a field investigation at composting facilities.

    Science.gov (United States)

    Duquenne, Philippe; Simon, Xavier; Demange, Valérie; Harper, Martin; Wild, Pascal

    2015-05-01

    A set of 270 bioaerosol samples was taken from 15 composting facilities using polystyrene closed-face filter cassettes (CFCs). The objective was to measure the quantity of endotoxin deposits on the inner surfaces of the cassettes (sometimes referred to as 'wall deposits'). The results show that endotoxins are deposited on the inner surfaces of the CFCs through sampling and/or handling of samples. The quantity of endotoxins measured on inner surfaces range between 0.05 (the limit of detection of the method) and 3100 endotoxin units per cassette. The deposits can represent a large and variable percentage of the endotoxins sampled. More than a third of the samples presented a percentage of inner surface deposits >40% of the total quantity of endotoxins collected (filter + inner surfaces). Omitting these inner surface deposits in the analytical process lead to measurement errors relative to sampling all particles entering the CFC sampler, corresponding to a developing consensus on matching the inhalable particulate sampling convention. The result would be underestimated exposures and could affect the decision as to whether or not a result is acceptable in comparison to airborne concentration limits defined in terms of the inhalability convention. The results of this study suggest including the endotoxins deposited on the inner surfaces of CFCs during analysis. Further researches are necessary to investigate endotoxin deposits on the inner cassette surfaces in other working sectors.

  2. Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.

    Science.gov (United States)

    Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L

    2014-05-01

    Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein.

  3. 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....

  4. 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

  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.

  6. Australian water mites of the subfamily Notoaturinae Besch (Acari: Hydrachnidia: Aturidae), with the description of 24 new species

    NARCIS (Netherlands)

    H. Smit

    2010-01-01

    New data are presented on the subfamily Notoaturinae from Australia. Twenty-four new species are described: Austraturus aculeatus n. sp., A. canaliculatus n. sp., A. lamingtonensis n. sp., A. longigenitalis n. sp., A. montanus n. sp., A. otwayensis n. sp., A. tasmanicus n. sp., Azugaturus gibberipal

  7. Mid-Tertiary dispersal, not Gondwanan vicariance explains distribution patterns in the wax palm subfamily (Ceroxyloideae: Arecaceae).

    Science.gov (United States)

    Trénel, Philipp; Gustafsson, Mats H G; Baker, William J; Asmussen-Lange, Conny B; Dransfield, John; Borchsenius, Finn

    2007-10-01

    The Ceroxyloideae is a small but heterogeneous subfamily of palms (Arecaceae, Palmae). It includes a Caribbean lineage (tribe Cyclospathae), a southern hemisphere disjunction (tribe Ceroxyleae), and an amphi-Andean element (tribe Phytelepheae), until recently considered a distinct subfamily (Phytelephantoideae) due to its highly derived morphology. A variety of hypotheses have been proposed to account for the biogeography of the subfamily, involving Gondwanan vicariance, austral interplate dispersal from South America to Australia via Antarctica, Andean orogeny, and Pleistocene refuges. We assessed the systematic classification and biogeography of the group based on a densely sampled phylogeny using >5.5kb of DNA sequences from three plastid and two nuclear genomic regions. The subfamily and each of its three tribes were resolved as monophyletic with high support. Divergence time estimates based on penalized likelihood and Bayesian dating methods indicate that Gondwanan vicariance is highly unlikely as an explanation for basic disjunctions in tribe Ceroxyleae. Alternative explanations include a mid-Tertiary trans-Atlantic/trans-African dispersal track and the "lemurian stepping stones" hypothesis. Austral interplate dispersal of Oraniopsis to Australia could have occurred, but apparently only in the mid-Eocene/early Oligocene interval after global cooling had begun. Our data do not support Pleistocene climatic changes as drivers for speciation in the Andean-centered Phytelepheae as previously proposed. Radiation in this tribe coincides largely with the major uplift of the Andes, favoring Andean orogeny over Pleistocene climatic changes as a possible speciation-promoting factor in this tribe.

  8. Cloning of MASK, a novel member of the mammalian germinal center kinase III subfamily, with apoptosis-inducing properties

    DEFF Research Database (Denmark)

    Dan, Ippeita; Ong, Shao-En; Watanabe, Norinobu M

    2002-01-01

    We have cloned a novel human GCK family kinase that has been designated as MASK (Mst3 and SOK1-related kinase). MASK is widely expressed and encodes a protein of 416 amino acid residues, with an N-terminal kinase domain and a unique C-terminal region. Like other GCK-III subfamily kinases, MASK do...

  9. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    Science.gov (United States)

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies.

  10. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    Science.gov (United States)

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  11. Taxonomic study on the family Mitridae (Mollusca: Gastropoda: Neogastropoda) from the China's seas. Subfamilies: Imbricariinae and Cylindromitrinae

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Ninteen species of subfamilies Imbricariinae and Cylindromitrinae, family Mitridae, are recorded from the China's seas. Of which, one genus and six species are recorded for the first time from China' s seas, i.e., genus Ziba Adams H and Adams A, Cancilla (Cancilla)carnicolor, Ziba duplilirata, Z. insculpta, Neocancilla circula, Scabricola (Scabricola) desetangsii, Scabricola (Swainsonia) ocellata ocellata.

  12. Highly conserved salt bridge stabilizes a proteinase K subfamily enzyme, Aqualysin I, from Thermus aquaticus YT-1.

    Science.gov (United States)

    Sakaguchi, Masayoshi; Osaku, Kanae; Maejima, Susumu; Ohno, Nao; Sugahara, Yasusato; Oyama, Fumitaka; Kawakita, Masao

    2014-01-01

    The proteinase K subfamily enzymes, thermophilic Aqualysin I (AQN) from Thermus aquaticus YT-1 and psychrophilic serine protease (VPR) from Vibrio sp. PA-44, have six and seven salt bridges, respectively. To understand the possible significance of salt bridges in the thermal stability of AQN, we prepared mutant proteins in which amino acid residues participating in salt bridges common to proteinase K subfamily members and intrinsic to AQN were replaced to disrupt the bridges one at a time. Disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant resulted in a significant reduction in thermal stability, and a massive change in the content of the secondary structure was observed, even at 70°C, in the circular dichroism (CD) analysis. These results indicate that the common salt bridge Asp183-Arg12 is important in maintaining the conformation of proteinase K subfamily enzymes and suggest the importance of proximity between the regions around Asp183 and the N-terminal region around Arg12. Of the three mutants that lack an AQN intrinsic salt bridge, D212N was more prone to unfolding at 80°C than the wild-type enzyme. Similarly, D17N and E237Q were less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. The AQN intrinsic salt bridges appear to confer additional thermal stability to this enzyme. These findings will further our understanding of the factors involved in stabilizing protein structure.

  13. Whole genome identification, phylogeny and evolution of the cytochrome P450 family 2 (CYP2) sub-families in birds

    DEFF Research Database (Denmark)

    Almeida, Daniela; Maldonado, Emanuel; Khan, Imran

    2016-01-01

    The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 novel avian whole...

  14. Three new species in the subfamily Eriopeltinae Sulc from Italy (Hemiptera, Coccoidea, Coccidae) with comments on the genus Lecanopsis.

    Science.gov (United States)

    Pellizzari, Giuseppina

    2013-01-01

    Three new coccid species, namely Hadzibejliaspis ferenci Pellizzari n. sp., Lecanopsis sicula Pellizzari n. sp. and L. salvatorei Pellizzari n. sp. are described and illustrated. Identification keys for the genera in the subfamily Eriopeltinae Sulc and to species in the genera Hadzibejliaspis Koteja and Lecanopsis Targioni Tozzetti are provided.

  15. Sea snakes in Australian waters (Serpentes: subfamilies Hydrophiinae and Laticaudinae)—a review with an updated identification key

    DEFF Research Database (Denmark)

    Redsted Rasmussen, Arne; Sanders, Kate Laura; Guinea, Michael L

    2014-01-01

    Sea snakes (Elapidae, subfamilies Hydrophiinae and Laticaudinae) reach high species richness in the South China Sea and in the Australian region; however, most countries in the two regions still lack up-to-date checklists and identification tools for these snakes. We present an updated reviewed...

  16. Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals

    DEFF Research Database (Denmark)

    Kase, E T; Thoresen, G H; Westerlund, S;

    2007-01-01

    with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). METHODS: Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied...... with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers......-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). CONCLUSIONS/INTERPRETATION: T0901317-induced...

  17. Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation and epidermal organelles

    Science.gov (United States)

    Borkowski, Andrew W.; Park, Kyungho; Uchida, Yoshikazu; Gallo, Richard L.

    2013-01-01

    Injury to the skin, and the subsequent release of non-coding double-stranded RNA from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). Since changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. Double-stranded RNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with double-stranded RNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by oil-red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that double-stranded RNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance. PMID:23353987

  18. ABCG2 Inhibition as a Therapeutic Approach for Overcoming Multidrug Resistance in Cancer

    Indian Academy of Sciences (India)

    Maryam Hosseini Hasanabady; Fatemeh Kalalinia

    2016-06-01

    Breast cancer resistance protein (BCRP, ABCP or MXR) / ATP-binding cassette subfamily G member 2 (ABCG2) was characterized as a multidrug resistance efflux transporter in 1998. ABCG2 physiologically acts as a part of a self-defense mechanism for the organism; it enhances eliminating of toxic xenobiotic substances and harmful agents in the intestine, as well as through the blood-brain barrier and placental. ABCG2 recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and new targeted small therapeutic molecules in clinical usage. Development of ABCG2 inhibitors for clinical usage may allow increased penetration of therapeutic agents into sanctuary sites and increased their intestinal absorption. In this report, we review the mechanisms that modulate MDR mediated by the ABC transporter ABCG2 in normal and cancer cells by different levels including, epigenetic modifications, transcriptional, posttranscriptional, translation and posttranslational regulation. Some clinical applications of ABCG2 inhibitors, also is explained.

  19. AcEST: BP914455 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ed protein OS=Trich... 111 3e-23 tr|B0XAF4|B0XAF4_CULQU Peroxisomal membrane protein 70 abcd3 OS=... 110 4e-...7GS0_AEDAE Peroxisomal membrane protein 70 abcd3 OS=... 108 2e-22 >tr|A9T1K4|A9T1K4_PHYPA ATP-binding casset...a patens subsp. patens GN=ppabcd5 PE=3 SV=1 Length = 1271 Score = 300 bits (769), Expect = 3e-80 Identities ...nding cassette transporter, subfamily D, member 6, group PMP protein PpABCD6 OS=Physcomitrella patens subsp. patens GN=ppabcd

  20. Biodiversity of Food Species of the Solanaceae Family: A Preliminary Taxonomic Inventory of Subfamily Solanoideae

    Directory of Open Access Journals (Sweden)

    John Samuels

    2015-05-01

    Full Text Available Over the last fifty years there has been a continual reduction in horticultural and agricultural biodiversity of nutritionally important plants, including those of the Solanaceae family. To add to this, the broad range of traditional crops, previously grown on a sustainable scale in some parts of the world, has been replaced by a narrow range of major crops grown as large-scale monocultures. In order to counteract this trend, and to help maintain a broad wealth of genetic resources, conservation is essential. This, in turn, helps to safeguard food security. A taxonomic inventory, covering the diversity of species in a plant group, is an important first step in conservation. The Solanaceae is one of the major plant families providing food species. A survey of the biodiversity, ethnobotany and taxonomy of subfamily Solanoideae was undertaken and is presented here as an inventory of food species. Fifteen genera provide species that are utilised for food across the world. Of these, only four genera contain economically significant cultivated food cropspecies. The majority of these are in the genus Solanum, whilst Capsicum, Physalis and Lycium contribute the remainder of cultivated crop species. These genera and others also comprise species that are semi-cultivated, tolerated as useful weeds, or gathered from the wild.

  1. A review of the subfamily Picobiinae Johnston and Kethley, 1973 (Acariformes: Prostigmata: Syringophilidae).

    Science.gov (United States)

    Skoracki, Maciej; Sikora, Bozena; Spicer, Greg S

    2016-05-19

    The fauna of quill mites of the subfamily Picobiinae Johnston and Kethley, 1973 (Acariformes: Cheyletoidea: Syringophilidae) is comprehensively revised. All of 78 known species, which are grouped into 11 genera, are examined and diagnosed or redescribed. Data on picobiine hosts and distribution are summarized, including new host and locality records. The following new species are described: Charadriineopicobia apricaria sp. nov. ex Pluvialis apricaria (Linnaeus) (Charadriiformes: Charadriidae) from France, Neopicobia pari sp. nov. ex Periparus venustulus Swinhoe (type host) (Passeriformes: Paridae) from China, Parus major Linnaeus (Paridae) from Macedonia and Finland, and Poecile varius Temminck and Schlegel (Paridae) from Japan, Picobia magellani sp. nov. ex Scytalopus magellanicus (Gmelin) (Passeriformes: Rhinocryptidae) from Colombia, Picobia lonchura sp. nov. ex Lonchura leucogastra (Blyth) (Passeriformes: Estrildidae) from Indonesia, Picobia makoli sp. nov. ex Xiphocolaptes promeropirhynchus (Lesson) (Passeriformes: Furnariidae) from Colombia. The species Picobia polonica Skoracki, Magowski and Dabert, 2001 syn. nov. is a junior synonym of C. khulkhaskhani Kivganov and Sharafat, 1995. The following new combinations are proposed: Neopicobia ictericus (Skoracki and Glowska, 2010) comb. nov., Rafapicobia brotogeris (Fain, Bochkov and Mironov, 2000) comb. nov., and Rafapicobia ramphastos (Fain, Bochkov and Mironov, 2000) comb. nov. Keys to the all picobiine genera and species are presented, along with a check-list of picobiine species and their hosts.

  2. The exodus subfamily of CC chemokines inhibits the proliferation of chronic myelogenous leukemia progenitors.

    Science.gov (United States)

    Hromas, R; Cripe, L; Hangoc, G; Cooper, S; Broxmeyer, H E

    2000-02-15

    Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in disease processes. Among the biologic activities of chemokines is inhibition of proliferation of normal hematopoietic progenitors. However, chemokines that inhibit normal progenitors rarely inhibit proliferation of hematopoietic progenitors from patients with chronic myelogenous leukemia (CML). We and others recently cloned a subfamily of CC chemokines that share similar amino-terminal peptide sequences and a remarkable ability to chemoattract T cells. These chemokines, Exodus-1/LARC/MIP-3alpha, Exodus-2/SLC/6Ckine/TCA4, and Exodus-3/CKbeta11/MIP-3beta, were found to inhibit proliferation of normal human marrow progenitors. The study described here found that these chemokines also inhibited the proliferation of progenitors in every sample of marrow from patients with CML that was tested. This demonstration of consistent inhibition of CML progenitor proliferation makes the 3 Exodus chemokines unique among chemokines. (Blood. 2000;95:1506-1508)

  3. Identification of the Alternative Promoters of the KChIP4 Subfamily

    Institute of Scientific and Technical Information of China (English)

    Xiao-Yun DENG; Jia-Hui XIA; Fang CAI; Kun XIA; Qian PAN; Zhi-Gao LONG; Ling-Qian WU; De-Sheng LIANG; He-Ping DAI; Zhuo-Hua ZHANG

    2005-01-01

    The subfamily of voltage-dependent potassium (Kv) channel interacting protein 4 (KChIP4)is made up of the auxiliary interacting protein of voltage-dependent potassium channels. In this study, the structure of four splicing variants of the human KChIP4 gene was analyzed. Three of the four isoforms of the KChIP4 gene, KChIP4.1, KChIP4.2 and KChIP4.4, were amplified from mouse and human fetal brain tissues by reverse transcription-polymerase chain reaction and then identified. Based on the bioinformatics analysis of the genomic sequences of the gene, we cloned and characterized two promoter fragments from the KChIP4 gene. One was a 325 bp fragment upstream of the 5' end of the KChIP4.1 mRNA sequence and the other was an 818 bp fragment located immediately at the 5' end of the KChIP4.4 variant. Both of them can initiate the transcription of the reporter gene in HT1080 cells and Sprague-Dawley (SD) rat fetal brain neurons, and they contain C+G islands, except typical TATA boxes and CAAT boxes. This shows that the KChIP4 gene expression is regulated by an alternative promoter.

  4. Molecular phylogeny and biogeography of the weevil subfamily Platypodinae reveals evolutionarily conserved range patterns.

    Science.gov (United States)

    Jordal, Bjarte H

    2015-11-01

    Platypodinae is a peculiar weevil subfamily of species that cultivate fungi in tunnels excavated in dead wood. Their geographical distribution is generally restricted, with genera confined to a single continent or large island, which provides a useful system for biogeographical research. This study establishes the first detailed molecular phylogeny of the group, with the aim of testing hypotheses on classification, diversification, and biogeography. A phylogeny was reconstructed based on 3648 nucleotides from COI, EF-1α, CAD, ArgK, and 28S. Tree topology was well resolved and indicated a strong correlation with geography, more so than predicted by previous morphology-based classifications. Tesserocerini was paraphyletic, with Notoplatypus as the sister group to a clade consisting of three main lineages of Tesserocerini and the recently evolved Platypodini. Austroplatypus formed the sister group to all remaining Platypodini and hence confirmed its separate status from Platypus. The Indo-Australian genera of Platypodini were strikingly paraphyletic, suggesting that the taxonomy of this tribe needs careful revision. Ancestral-area reconstructions in Lagrange and S-DIVA were ambiguous for nodes roughly older than 80 Ma. More recent events were firmly assessed and involved post-Gondwanan long-distance dispersal. The Neotropics was colonized three times, all from the Afrotropical region, with the latest event less than 25 Ma that included the ancestor of all Neotropical Platypodini.

  5. Adaptive evolution after gene duplication in alpha-KT x 14 subfamily from Buthus martensii Karsch.

    Science.gov (United States)

    Cao, Zhijian; Mao, Xin; Xu, Xiuling; Sheng, Jiqun; Dai, Chao; Wu, Yingliang; Luo, Feng; Sha, Yonggang; Jiang, Dahe; Li, Wenxin

    2005-07-01

    A series of isoforms of alpha-KT x 14 (short chain potassium channel scorpion toxins) were isolated from the venom of Buthus martensii Karsch by RACE and screening cDNA library methods. These isoforms adding BmKK1--3 and BmSKTx1--2 together shared high homology (more than 97%) with each other. The result of genomic sequence analysis showed that a length 79 bp intron is inserted Ala codes between the first and the second base at the 17th amino acid of signal peptide. The introns of these isoforms also share high homology with those of BmKK2 and BmSKT x 1 reported previously. Sequence analysis of many clones of cDNA and genomic DNA showed that a species population or individual polymorphism of alpha-KT x 14 genes took place in scorpion Buthus martensii Karsch and accelerated evolution played an important role in the forming process of alpha-KT x 14 scorpion toxins subfamily. The result of southern hybridization indicated that alpha-KT x 14 toxin genes existed in scorpion chromosome with multicopies. All findings maybe provided an important evidence for an extensive evolutionary process of the scorpion "pharmacological factory": at the early course of evolution, the ancestor toxic gene duplicated into a series of multicopy genes integrated at the different chromosome; at the late course of evolution, subsequent functional divergence of duplicate genes was generated by mutations, deletions and insertion.

  6. Cytogenetic analysis of five species of the subfamily Corydoradinae (Teleostei: Siluriformes: Callichthyidae

    Directory of Open Access Journals (Sweden)

    Cristiane Kioko Shimabukuro-Dias

    2004-01-01

    Full Text Available In the present study, five callichthyid species belonging to the subfamily Corydoradinae were karyotyped: three species of Aspidoras and two of Corydoras. The three species of Aspidoras had the same diploid number, 2n = 46 chromosomes, similar karyotypic formulae, with most chromosomes metacentric or submetacentric, single interstitial Ag-NORs and C-band positive segments mainly found in the centromeric position. The comparative analysis of cytogenetic data available for the genus Aspidoras and other species of Corydoradinae suggest that several events of centric fusion occurred in the origin of the species of Aspidoras. The two analyzed species of Corydoras showed high diploid numbers, 2n = 74 in C. sodalis and 2n = 90 in C. britskii. While C. sodalis exhibited single Ag-NORs and terminal, interstitial and centromeric C-band positive segments in almost all chromosomes, C. britskii showed multiple Ag-NORs and a small number of C-band positive segments found in the terminal position in one acrocentric (A pair and in the interstitial position in one subtelocentric (ST pair. The occurrence of high diploid numbers and many ST and A chromosomes are uncommon among the Corydoradinae, suggesting the occurrence of a high number of chromosome rearrangements, mainly centric fissions, in the origin of the Corydoras species studied.

  7. Contribution of transient receptor potential vanilloid subfamily 1 to endothelin-1-induced thermal hyperalgesia.

    Science.gov (United States)

    Kawamata, T; Ji, W; Yamamoto, J; Niiyama, Y; Furuse, S; Namiki, A

    2008-06-26

    Endothelin-1 (ET-1) plays an important role in peripheral pain processing. However, the mechanisms of the nociceptive action of ET-1 have not been fully elucidated. In this study, we investigated the contribution of transient receptor potential vanilloid subfamily 1 (TRPV1) to ET-1-induced thermal hyperalgesia. Intraplantar ET-1-induced thermal hyperalgesia was examined by assessing the paw withdrawal latency to noxious heat stimuli. In electrophysiological study, whole-cell patch-clamp recordings were performed to investigate the interaction of ET-1 and TRPV1 using human embryonic kidney 293 (HEK293) cells expressing endothelin type A receptor (ET(A)) and TRPV1. Intraplantar ET-1 (3, 10 and 30 pmol) produced thermal hyperalgesia in a dose-dependent manner. Thermal hyperalgesia was attenuated by the inhibition of ET(A) and protein kinase C (PKC) but not that of ET(B). ET-1-induced thermal hyperalgesia was significantly attenuated in TRPV1-deficient mice compared with that in wild-type mice. In voltage-clamp experiments, 10 nM capsaicin evoked small inward currents in HEK293 cells expressing TRPV1 and ET(A). In the presence of ET-1, capsaicin produced much larger current responses (Pthermal hyperalgesia.

  8. Modulation of the Rat Hepatic Cytochrome P4501A Subfamily Using Biotin Supplementation

    Directory of Open Access Journals (Sweden)

    M. D. Ronquillo-Sánchez

    2013-01-01

    Full Text Available Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated daily with biotin (2 mg/kg, i.p., while the control groups were treated with saline. All of the rats were sacrificed by cervical dislocation after 1, 3, 5, or 7 days of treatment. CYP1A1 and CYP1A2 mRNAs were modified by biotin while enzyme activity and protein concentration were not affected. The lack of an effect of biotin on CYP1A activity was confirmed using other experimental strategies, including (i cotreatment of the animals with biotin and a known CYP1A inducer; (ii the addition of biotin to the reaction mixtures for the measurement of CYP1A1 and CYP1A2 activities; and (iii the use of an S9 mixture that was prepared from control and biotin-treated rats to analyze the activation of benzo[a]pyrene (BaP into mutagenic metabolites using the Ames test. The results suggest that biotin does not influence the CYP1A-mediated metabolism of xenobiotics.

  9. Out of Africa:Miocene Dispersal, Vicariance, and Extinction within Hyacinthaceae Subfamily Urgineoideae

    Institute of Scientific and Technical Information of China (English)

    Syed Shujait Ali; Martin Pfosser; Wolfgang Wetschnig; Mario MartnezAzorn; Manuel B. Crespo; Yan Yu

    2013-01-01

    Disjunct distribution patterns in plant lineages are usually explained according to three hypotheses:vicariance, geodispersal, and long-distance dispersal. The role of these hypotheses is tested in Urgineoideae (Hyacinthaceae), a subfamily disjunctly distributed in Africa, Madagascar, India, and the Mediterranean region. The potential ancestral range, dispersal routes, and factors responsible for the current distribution in Urgineoideae are investigated using divergence time estimations. Urgineoideae originated in Southern Africa approximately 48.9 Mya. Two independent dispersal events in the Western Mediterranean region possibly occurred during Early Oligocene and Miocene (29.9-8.5 Mya) via Eastern and Northwestern Africa. A dispersal from Northwestern Africa to India could have occurred between 16.3 and 7.6 Mya. Vicariance and extinction events occurred approximately 21.6 Mya. Colonization of Madagascar occurred between 30.6 and 16.6 Mya, after a single transoceanic dispersal event from Southern Africa. The current disjunct distributions of Urgineoideae are not satisfactorily explained by Gondwana fragmentation or dispersal via boreotropical forests, due to the younger divergence time estimates. The flattened winged seeds of Urgineoideae could have played an important role in long-distance dispersal by strong winds and big storms, whereas geodispersal could have also occurred from Southern Africa to Asia and the Mediterranean region via the so-called arid and high-altitude corridors.

  10. 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.

  11. Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

    Science.gov (United States)

    Close, Dan M.; Patterson, Stacey S.; Ripp, Steven; Baek, Seung J.; Sanseverino, John; Sayler, Gary S.

    2010-01-01

    Background 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. Methodology/Principal Findings 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 (FMNH2) 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. FMNH2 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. Conclusions/Significance 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. PMID:20805991

  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. Patients' preferences for video cassette recorded information: effect of age, sex and ethnic group.

    Science.gov (United States)

    Thomas, R; Deary, A; Kaminski, E; Stockton, D; De Zueew, N

    1999-06-01

    The emotional turmoil patients endure following a diagnosis of cancer can impair their ability to retain complex treatment-related information. Manoeuvres which increase the intensity of information have been shown to increase the amount retained. Providing details of treatment in a video format is one method of intensifying information provision, but the attitudes of patients to this format have not previously been evaluated. In this pilot study, the attitudes of 300 patients to video directed information were evaluated via questionnaires, of which 210 (70%) were returned. Eighty-nine per cent had easy access to a video cassette player. A highly significant number felt that the video would be very helpful or helpful (78%) compared to not helpful, worrying or equivocal 21% (P < 0.0001). This trend was particularly strong in patients < 60 years (83% versus 17%) (P < 0.0001) and those from ethnic groups (95% versus 5%) (P < 0.0001). As a result of this trial, a 20-min film (HEP) has been commissioned. It describes details of the two main treatments for cancer after surgery, namely chemotherapy and radiotherapy, shows patients actually having treatment, and explains the common side-effects and ways to alleviate them. Patients satisfaction with the film and its effect on anxiety and depression are currently being evaluated in an international prospective randomized trial. If it proves advantageous for patients--in view of the ethnic group bias in this study--it will be translated into the ethnic languages of the UK.

  14. 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).

  15. 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.

  16. Structural Comparison of Three Types of Staphylococcal Cassette Chromosome mec Integrated in the Chromosome in Methicillin-Resistant Staphylococcus aureus

    OpenAIRE

    Ito, Teruyo; Katayama, Yuki; Asada, Kazumi; Mori, Namiko; Tsutsumimoto, Kanae; Tiensasitorn, Chuntima; Hiramatsu, Keiichi

    2001-01-01

    The β-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the ele...

  17. Characteriz ation of integrons and associated gene cassettes in Acinetobacter baumannii strains isolated from intensive care unit in Tehran, Iran

    Directory of Open Access Journals (Sweden)

    Hossein Goudarzi

    2016-09-01

    Full Text Available Objective: To determine the antimicrobial susceptibility patterns, the frequency of integrons and associated gene cassettes in Acinetobacter baumannii (A. baumannii strains isolated from selected hospital intensive care units. Methods: During a ten-month period, 120 A. baumannii isolates were studied. The resistance rates to different classes of antimicrobial agents were determined. PCR was used to detect different types of integrons and associated gene cassettes. Results: The resistance rates to the majority of antibiotics tested were found to be between 39.3% and 99.1%. No isolate was observed to be resistant to colistin and polymyxin B. The rate of extensive drug-resistance among these clinical isolates was 62.5%. The prevalence of class 1 and 2 integrons was found to be 74.1% and 12.5%, respectively. Seven different gene cassettes (ampC, aacA4-catB8, ISAba1-blaOXA-23-GES-14, aadA2-cm1A6-GES-14-qacF, VIM-25-GES-24-qacF, dfrA5-ISAba1-blaOXA-51-blaOXA-40 and aadA2-GES-11-IMP-1 were observed in Class 1 integron-carrying strains. Three gene cassettes (IMP-4, VIM-2-VEB-aacA4 and dfrA2-sat-2-aadA4 were detected in class 2 integron-bearing A. baumannii strains. Conclusions: A high prevalence of integron was described among multidrug resistant A. baumannii in the hospital. The findings highlighted the need for continuous surveillance in order to prevent dissemination of multidrug resistance among A. baumannii strains in Iran.

  18. Characterization of integrons and associated gene cassettes in Acinetobacter baumannii strains isolated from intensive care unit in Tehran, Iran

    Institute of Scientific and Technical Information of China (English)

    Hossein Goudarzi; Mehdi Azad; Sima Sadat Seyedjavadi; Hadi Azimi; Alireza Salimi Chirani; Vahid Fallah Omrani; Mehdi Goudarzi

    2016-01-01

    Objective: To determine the antimicrobial susceptibility patterns, the frequency of integrons and associated gene cassettes in Acinetobacter baumannii (A. baumannii) strains isolated from selected hospital intensive care units. Methods: During a ten-month period, 120 A. baumannii isolates were studied. The resistance rates to different classes of antimicrobial agents were determined. PCR was used to detect different types of integrons and associated gene cassettes. Results: The resistance rates to the majority of antibiotics tested were found to be be-tween 39.3% and 99.1%. No isolate was observed to be resistant to colistin and poly-myxin B. The rate of extensive drug-resistance among these clinical isolates was 62.5%. The prevalence of class 1 and 2 integrons was found to be 74.1% and 12.5%, respec-tively. Seven different gene cassettes (ampC, aacA4-catB8, ISAba1-blaOXA-23-GES-14, aadA2-cm1A6-GES-14-qacF, VIM-25-GES-24-qacF, dfrA5-ISAba1-blaOXA-51-blaOXA-40 and aadA2-GES-11-IMP-1) were observed in Class 1 integron-carrying strains. Three gene cassettes (IMP-4, VIM-2-VEB-aacA4 and dfrA2-sat-2-aadA4) were detected in class 2 integron-bearing A. baumannii strains. Conclusions: A high prevalence of integron was described among multidrug resistant A. baumannii in the hospital. The findings highlighted the need for continuous surveillance in order to prevent dissemination of multidrug resistance among A. baumannii strains in Iran.

  19. Aepyosciurinae -- a new subfamily of Sciuridae (Rodentia, Mammalia) from basal loess deposits at the northeastern border of Tibetan Plateau

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Aepyosciurinae, a new subfamily of Sciuridae, were found at the base of the early Pleistocene loess deposits in Dongxiang County, Gansu Province. Its unilaterally hypsodont and lophodont cheek teeth are unique among the sciurids so far known all over the world. Certain degree of similarity can be observed between the cheek teeth of the new subfamily and the Anomalurinae living in tropical and subtropical forests in central and western Africa. Aepyosciuris orientalis gen. et sp. nov. might have lived in montaneous woodland or grassland and lived on harder leaves, barks, or even grass. This tends to show that the northeastern border area of the Tibetan Plateau had been lifted considerably high in early Pleistocene (ca. 2 Ma), with drier climate, becoming a suitable habitat for Aepyosciurus orientalis.

  20. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  1. Type 2 diabetes - Tuberculosis co-morbidity is associated with diminished circulating levels of IL-20 subfamily of cytokines.

    Science.gov (United States)

    Kumar, Nathella Pavan; Banurekha, Vaithilingam V; Nair, Dina; Kumaran, Paul; Dolla, Chandra Kumar; Babu, Subash

    2015-12-01

    IL-20 subfamily of cytokines play an important role in both host defense mechanisms and glucose metabolism. Since, the interaction between tuberculosis (TB) and diabetes (DM) involves both of the above processes, we examined the association of IL-20 subfamily of cytokines in TB-DM co-morbidity. We examined circulating plasma cytokine levels in individuals with active TB with (PTB-DM) or without (PTB) diabetes and also those with latent TB with (LTB-DM) or without (LTB) diabetes. PTB-DM is characterized by diminished circulating levels of IL-19, IL-20, IL-22 and IL-24 but increased levels of IL-10. Similarly, LTB-DM was also characterized by diminished circulating levels of IL-10, IL-19, IL-20 and IL-24 but increased levels of IL-22. Moreover, there was a significant negative correlation of IL-10, IL-19, IL-20, IL-22 and IL-24 levels with hemoglobin A1C (HbA1c) levels in both PTB and/or LTB individuals. Finally, PTB is characterized by diminished levels of IL-19, IL-20, IL-22 and IL-24 in comparison to LTB individuals. Our data reveal that coincident diabetes in either PTB or LTB is characterized by decreased production of the IL-20 subfamily of cytokines and suggest that these cytokines might play an important role in pathogenesis or protection.

  2. Evolutionary relationships among primary endosymbionts of the mealybug subfamily phenacoccinae (hemiptera: Coccoidea: Pseudococcidae).

    Science.gov (United States)

    Gruwell, Matthew E; Hardy, Nate B; Gullan, Penny J; Dittmar, Katharina

    2010-11-01

    Mealybugs (Coccoidea: Pseudococcidae) are sap-sucking plant parasites that harbor bacterial endosymbionts within specialized organs. Previous studies have identified two subfamilies, Pseudococcinae and Phenacoccinae, within mealybugs and determined the primary endosymbionts (P-endosymbionts) of the Pseudococcinae to be Betaproteobacteria ("Candidatus Tremblaya princeps") containing Gammaproteobacteria secondary symbionts. Here, the P-endosymbionts of phenacoccine mealybugs are characterized based on 16S rRNA from the bacteria of 20 species of phenacoccine mealybugs and four outgroup Puto species (Coccoidea: Putoidae) and aligned to more than 100 published 16S rRNA sequences from symbiotic and free-living bacteria. Phylogenetic analyses recovered three separate lineages of bacteria from the Phenacoccinae, and these are considered to be the P-endosymbionts of their respective mealybug hosts, with those from (i) the mealybug genus Rastrococcus belonging to the Bacteroidetes, (ii) the subterranean mealybugs, tribe Rhizoecini, also within Bacteroidetes, in a clade sister to cockroach endosymbionts (Blattabacterium), and (iii) the remaining Phenacoccinae within the Betaproteobacteria, forming a well-supported sister group to "Candidatus Tremblaya princeps." Names are proposed for two strongly supported lineages: "Candidatus Brownia rhizoecola" for P-endosymbionts of Rhizoecini and "Candidatus Tremblaya phenacola" for P-endosymbionts of Phenacoccinae excluding Rastrococcus and Rhizoecini. Rates of nucleotide substitution among lineages of Tremblaya were inferred to be significantly faster than those of free-living Betaproteobacteria. Analyses also recovered a clade of Gammaproteobacteria, sister to the P-endosymbiont lineage of aphids ("Candidatus Buchnera aphidicola"), containing the endosymbionts of Putoidae, the secondary endosymbionts of pseudococcine mealybugs, and the endosymbionts of several other insect groups.

  3. Comparative genome analysis between Agrostis stolonifera and members of the Pooideae subfamily, including Brachypodium distachyon.

    Directory of Open Access Journals (Sweden)

    Loreto Araneda

    Full Text Available Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28 is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum, oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs of creeping bentgrass relative to the Triticeae (3 LGs, oat (4 LGs, and rice (8 LGs. However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST. We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.

  4. The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

    Energy Technology Data Exchange (ETDEWEB)

    Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.; Parker, William B.; Piskur, Jure; Serra, I.; Terreni, M.; Ealick, Steven E. (Cornell); (Pavia); (Lund); (Southern Research)

    2011-09-20

    Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

  5. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  6. The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter.

    Science.gov (United States)

    Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

    2012-01-01

    The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Takao, E-mail: hayashi.takao@jaea.go.jp; Sakurai, Shinji; Shibanuma, Kiyoshi; Sakasai, Akira

    2014-10-15

    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.

  9. The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter

    Science.gov (United States)

    Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

    2012-01-01

    The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted. PMID:22368493

  10. The Evolution of the Bacterial Luciferase Gene Cassette (lux as a Real-Time Bioreporter

    Directory of Open Access Journals (Sweden)

    Gary Sayler

    2012-01-01

    Full Text Available The bacterial luciferase gene cassette (lux is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.

  11. Sampling efficiency of modified 37-mm sampling cassettes using computational fluid dynamics.

    Science.gov (United States)

    Anthony, T Renée; Sleeth, Darrah; Volckens, John

    2016-01-01

    In the U.S., most industrial hygiene practitioners continue to rely on the closed-face cassette (CFC) to assess worker exposures to hazardous dusts, primarily because ease of use, cost, and familiarity. However, mass concentrations measured with this classic sampler underestimate exposures to larger particles throughout the inhalable particulate mass (IPM) size range (up to aerodynamic diameters of 100 μm). To investigate whether the current 37-mm inlet cap can be redesigned to better meet the IPM sampling criterion, computational fluid dynamics (CFD) models were developed, and particle sampling efficiencies associated with various modifications to the CFC inlet cap were determined. Simulations of fluid flow (standard k-epsilon turbulent model) and particle transport (laminar trajectories, 1-116 μm) were conducted using sampling flow rates of 10 L min(-1) in slow moving air (0.2 m s(-1)) in the facing-the-wind orientation. Combinations of seven inlet shapes and three inlet diameters were evaluated as candidates to replace the current 37-mm inlet cap. For a given inlet geometry, differences in sampler efficiency between inlet diameters averaged less than 1% for particles through 100 μm, but the largest opening was found to increase the efficiency for the 116 μm particles by 14% for the flat inlet cap. A substantial reduction in sampler efficiency was identified for sampler inlets with side walls extending beyond the dimension of the external lip of the current 37-mm CFC. The inlet cap based on the 37-mm CFC dimensions with an expanded 15-mm entry provided the best agreement with facing-the-wind human aspiration efficiency. The sampler efficiency was increased with a flat entry or with a thin central lip adjacent to the new enlarged entry. This work provides a substantial body of sampling efficiency estimates as a function of particle size and inlet geometry for personal aerosol samplers.

  12. Cephalocteinae Mulsant et Rey, 1866 (Hemiptera, Heteroptera), a subfamily of Cydnidae new for the Italian fauna: first record of Cephalocteus scarabaeoides (Fabricius, 1807) from Sardinia.

    Science.gov (United States)

    Fancello, Luca; Cillo, Davide; Bazzato, Erika

    2016-01-25

    Cephalocteus scarabaeoides is recorded from the south-western coast of Sardinia, in sandy habitat (marine dunes near the beach), for the first time. The species and the subfamily are new for the Italian fauna.

  13. Comparison of lead and tin concentrations in air at a solder manufacturer from the closed-face 37-mm cassette with and without a custom cellulose-acetate cassette insert.

    Science.gov (United States)

    Lee, Eun Gyung; Chisholm, William P; Burns, Dru A; Nelson, John H; Kashon, Michael L; Harper, Martin

    2014-01-01

    A polyvinyl chloride (PVC) cassette insert with PVC filter (ACCU-CAP) in a 37-mm closed-face cassette (CFC) was designed for gravimetric analysis. A customized version of the ACCU-CAP, also to be used in the CFC, was manufactured from an acid-digestible cellulose-acetate cassette insert joined to a mixed cellulose ester (MCE) filter for wet chemical analysis. The aim of this study was to compare metal particle concentrations as sampled by the customized insert (CI) in a CFC sampler with the traditional sampling method using only a MCE filter in the CFC. Thirty-nine personal and 13 area samples were taken using paired filter-based CFC and the CI in CFC samplers at a solder manufacturing plant. The CI was removed from its CFC, and digested and analyzed as a whole. The MCE filter from the typical CFC was removed for analysis and then the interior of the cassette was wiped with Ghost Wipe for a separate analysis. The MCE filter only, Ghost Wipe, and CI were separately dissolved in heated nitric acid for ICP-MS analysis. Overall, the geometric mean concentration of the filter-only (FO) samples was considerably lower than that of the CI samples, by 53% for lead and 32% for tin. However, if the FO analysis was added to the corresponding Ghost Wipe analysis, i.e., filter+interior wipe (FW), the geometric mean concentrations of the FW results were similar to those of the CI results (by 113% for lead and 98% for tin). For both lead and tin the comparison of (log-transformed) metal concentrations between the FW and CI results showed no statistically significant difference (p-value = 0.3009 for lead and 0.800 for tin), while the comparison between the FO and CI results shows statistically significant differences (all p-values < 0.05). In conclusion, incorporating the sampler internal non-filter deposits by wiping or use of an internal filter capsule gave higher results than analyzing only the filter. Close agreement between the two methods of including non-filter deposits is

  14. 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.

  15. 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.

  16. 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.

  17. Members of rice plasma membrane intrinsic proteins subfamily are involved in arsenite permeability and tolerance in plants.

    Science.gov (United States)

    Mosa, Kareem A; Kumar, Kundan; Chhikara, Sudesh; Mcdermott, Joseph; Liu, Zijuan; Musante, Craig; White, Jason C; Dhankher, Om Parkash

    2012-12-01

    Rice accumulates high level of arsenic (As) in its edible parts and thus plays an important role in the transfer of As into the food chain. However, the mechanisms of As uptake and its detoxification in rice are not well understood. Recently, members of the Nodulin 26-like intrinsic protein (NIP) subfamily of plant aquaporins were shown to transport arsenite in rice and Arabidopsis. Here we report that members of the rice plasma membrane intrinsic protein (PIP) subfamily are also involved in As tolerance and transport. Based on the homology search with the mammalian AQP9 and yeast Fps1 arsenite transporters, we identified and cloned five rice PIP gene subfamily members. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to arsenite treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis oocytes significantly increased the uptake of arsenite. Overexpression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis yielded enhanced arsenite tolerance and higher biomass accumulation. Further, these transgenic plants showed no significant accumulation of As in shoot and root tissues in long term uptake assays. Whereas, short duration exposure to arsenite caused both active influx and efflux of As in the roots. The data suggests a bidirectional arsenite permeability of rice PIPs in plants. These rice PIPs genes will be highly useful for engineering important food and biofuel crops for enhanced crop productivity on contaminated soils without increasing the accumulation of toxic As in the biomass or edible tissues.

  18. Protein complex interactor analysis and differential activity of KDM3 subfamily members towards H3K9 methylation.

    Directory of Open Access Journals (Sweden)

    Michael Brauchle

    Full Text Available Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins.

  19. Whole genome identification, phylogeny and evolution of the cytochrome P450 family 2 (CYP2) sub-families in birds

    DEFF Research Database (Denmark)

    Almeida, Daniela; Maldonado, Emanuel; Khan, Imran;

    2016-01-01

    The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 novel avian whole......0, SRS2_SRS3 and SRS3.1) and heme binding areas that influence CYP2 structure and function of functional importance as under significant positive selection. Some of the positively selected sites in avian CYP2D are located within the same SRS1 region that was previously linked with the metabolism...

  20. Sea snakes in Australian waters (Serpentes: subfamilies Hydrophiinae and Laticaudinae)--a review with an updated identification key.

    Science.gov (United States)

    Rasmussen, Arne Redsted; Sanders, Kate Laura; Guinea, Michael L; Amey, Andrew P

    2014-10-02

    Sea snakes (Elapidae, subfamilies Hydrophiinae and Laticaudinae) reach high species richness in the South China Sea and in the Australian region; however, most countries in the two regions still lack up-to-date checklists and identification tools for these snakes. We present an updated reviewed checklist and a new complete identification key to sea snakes in Australian waters. The identification key includes 29 species documented and 4 possibly occurring taxa and is based mostly on easy-to-use external characters. We find no evidence for breeding populations of Laticauda in Australian waters, but include the genus on the list of possibly occurring taxa. 

  1. Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

    DEFF Research Database (Denmark)

    Dueholm, Bjørn; Krieger, Celia; Drew, Damian;

    2015-01-01

    Background: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants...... and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution...

  2. New species and records of the mite genus Prolistrophorus (Acariformes: Listrophoridae) from rodents of the subfamily Sigmodontinae (Rodentia: Cricetidae).

    Science.gov (United States)

    Bochkov, Andre V; Lareschi, Marcela; Barreto, Mauricio

    2014-10-01

    Six fur-mite species of the genus Prolistrophorus Fain, 1970 (Acariformes: Listrophoridae) were recorded from Central and South American rodents of the subfamily Sigmodontinae (Rodentia: Cricetidae). Among them, Prolistrophorus (Aprolistrophorus) parabidentatus sp. nov. from Akodon azarae from Argentina and Prolistrophorus (Aprolistrophorus) tylomys sp. nov. from Tylomys nudicaudus from Guatemala are described as new for science. New hosts are recorded for the following species: Prolistrophorus (Prolistrophorus) grassii (Radford, 1954) from Zygodontomys brevicauda from Colombia, P. (P.) frontalis (Hirst, 1921) from Oligoryzomys sp. from Argentina, P. (P.) argentinus (Hirst, 1921) from Melanomys caliginosus, Akodon affinis from Colombia and Scapteromys aquaticus from Argentina, Prolistrophorus (Beprolistrophorus) hirstianus Fain, 1973 from Scapteromys aquaticus from Argentina.

  3. A genomic view of the NOD-like receptor family in teleost fish: Identification of a novel NLR subfamily in zebrafish

    Science.gov (United States)

    Laing, K.J.; Purcell, M.K.; Winton, J.R.; Hansen, J.D.

    2008-01-01

    Background. A large multigene family of NOD-like receptor (NLR) molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression. Results. Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A) resembles mammalian NODs, the second (NLR-B) resembles mammalian NALPs, while the third (NLR-C) appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT)-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues. Conclusion. Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish. ?? 2008 Laing et al; licensee BioMed Central Ltd.

  4. A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish

    Directory of Open Access Journals (Sweden)

    Winton James R

    2008-02-01

    Full Text Available Abstract Background A large multigene family of NOD-like receptor (NLR molecules have been described in mammals and implicated in immunity and apoptosis. Little information, however, exists concerning this gene family in non-mammalian taxa. This current study, therefore, provides an in-depth investigation of this gene family in lower vertebrates including extensive phylogenetic comparison of zebrafish NLRs with orthologs in tetrapods, and analysis of their tissue-specific expression. Results Three distinct NLR subfamilies were identified by mining genome databases of various non-mammalian vertebrates; the first subfamily (NLR-A resembles mammalian NODs, the second (NLR-B resembles mammalian NALPs, while the third (NLR-C appears to be unique to teleost fish. In zebrafish, NLR-A and NLR-B subfamilies contain five and six genes respectively. The third subfamily is large, containing several hundred NLR-C genes, many of which are predicted to encode a C-terminal B30.2 domain. This subfamily most likely evolved from a NOD3-like molecule. Gene predictions for zebrafish NLRs were verified using sequence derived from ESTs or direct sequencing of cDNA. Reverse-transcriptase (RT-PCR analysis confirmed expression of representative genes from each subfamily in selected tissues. Conclusion Our findings confirm the presence of multiple NLR gene orthologs, which form a large multigene family in teleostei. Although the functional significance of the three major NLR subfamilies is unclear, we speculate that conservation and abundance of NLR molecules in all teleostei genomes, reflects an essential role in cellular control, apoptosis or immunity throughout bony fish.

  5. Probing the ATP-Binding Pocket of Protein Kinase DYRK1A with Benzothiazole Fragment Molecules.

    Science.gov (United States)

    Rothweiler, Ulli; Stensen, Wenche; Brandsdal, Bjørn Olav; Isaksson, Johan; Leeson, Frederick Alan; Engh, Richard Alan; Svendsen, John S Mjøen

    2016-11-10

    DYRK1A has emerged as a potential target for therapies of Alzheimer's disease using small molecules. On the basis of the observation of selective DYRK1A inhibition by firefly d-luciferin, we have explored static and dynamic structural properties of fragment sized variants of the benzothiazole scaffold with respect to DYRK1A using X-ray crystallography and NMR techniques. The compounds have excellent ligand efficiencies and show a remarkable diversity of binding modes in dynamic equilibrium. Binding geometries are determined in part by interactions often considered "weak", including "orthogonal multipolar" types represented by, for example, F-CO, sulfur-aromatic, and halogen-aromatic interactions, together with hydrogen bonds that are modulated by variation of electron withdrawing groups. These studies show how the benzothiazole scaffold is highly promising for the development of therapeutic DYRK1A inhibitors. In addition, the subtleties of the binding interactions, including dynamics, show how full structural studies are required to fully interpret the essential physical determinants of binding.

  6. Conserved inhibitory mechanism and competent ATP binding mode for adenylyltransferases with Fic fold.

    Directory of Open Access Journals (Sweden)

    Arnaud Goepfert

    Full Text Available The ubiquitous FIC domain is evolutionarily conserved from bacteria to human and has been shown to catalyze AMP transfer onto protein side-chain hydroxyl groups. Recently, it was predicted that most catalytically competent Fic proteins are inhibited by the presence of an inhibitory helix αinh that is provided by a cognate anti-toxin (class I, or is part of the N- or C-terminal part of the Fic protein itself (classes II and III. In vitro, inhibition is relieved by mutation of a conserved glutamate of αinh to glycine. For the class III bacterial Fic protein NmFic from Neisseria meningitidis, the inhibitory mechanism has been elucidated. Here, we extend above study by including bacterial class I and II Fic proteins VbhT from Bartonella schoenbuchensis and SoFic from Shewanella oneidensis, respectively, and the respective E->G mutants. Comparative enzymatic and crystallographic analyses show that, in all three classes, the ATP substrate binds to the wild-type FIC domains, but with the α-phosphate in disparate and non-competent orientations. In the E->G mutants, however, the tri-phosphate moiety is found reorganized to the same tightly bound structure through a unique set of hydrogen bonds with Fic signature motif residues. The γ-phosphate adopts the location that is taken by the inhibitory glutamate in wild-type resulting in an α-phosphate orientation that can be attacked in-line by a target side-chain hydroxyl group. The latter is properly registered to the Fic active center by main-chain β-interactions with the β-hairpin flap. These data indicate that the active site motif and the exposed edge of the flap are both required to form an adenylylation-competent Fic protein.

  7. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Ito, T; Katayama, Y; Asada, K; Mori, N; Tsutsumimoto, K; Tiensasitorn, C; Hiramatsu, K

    2001-05-01

    The beta-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA-carrying genetic elements found in the MRSA strains isolated in other countries of the world. There were substantial differences in the size and nucleotide sequences between the elements and the SCCmec. However, new elements shared the chromosomal integration site with the SCCmec. Structural analysis of the new elements revealed that they possessed all of the salient features of the SCCmec: conserved terminal inverted repeats and direct repeats at the integration junction points, conserved genetic organization around the mecA gene, and the presence of cassette chromosome recombinase (ccr) genes responsible for the movements of SCCmec. The elements, therefore, were considered to comprise the SCCmec family of staphylococcal mobile genetic elements together with the previously identified SCCmec. Among 38 epidemic MRSA strains isolated in 20 countries, 34 were shown to possess one of the three typical SCCmec elements on the chromosome. Our findings indicated that there are at least three distinct MRSA clones in the world with different types of SCCmec in their chromosome.

  8. 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.

  9. Class Ⅰ integron with a novel cassette array in an ESBL-producing multidrug-resistant Klebsiella pneumoniae isolate

    Institute of Scientific and Technical Information of China (English)

    Bing Gu; Mingqing Tong; Wangsheng Zhao; Shiyang Pan; Yuanhua Wei; Peijun Huang

    2006-01-01

    Objective: To analyze the molecular mechanism of integron mediated multi-resistance in an ESBL-producingK. Pneumoniae NJ 12 isolate. Methods: Susceptibility test was carried out by Kirby-Bauer method. Class Ⅰ, Ⅱ and Ⅲ integrons were detected by integrase gene PCR with primers that annealed to conserved regions of integron-encoded integrase genes intIl, intI2 and intI3.The variable region of integron was amplified by integron PCR with primers that targeted the conserved flanking regions, and the PCR product was sequenced. Six aminoglycoside modifying-enzyme genes, including ant ( 2")- Ⅰ , ant ( 3")- Ⅰ , aac (3)- Ⅰ , aac ( 3 )- Ⅱ , aac (6')- Ⅰ , and aac (6')- Ⅱ, were detected. Results: K. Pneumoniae NJ 12 was resistant to nine antibiotics, including piperacillin,ampicillin, cefuroxime, ceftazidime, cefotaxime, aztreonam, streptomycin, gentamicin and amikacin. This isolate was shown that there was positive with class Ⅰ integron, ant(2")- Ⅰ , ant(3")- Ⅰ , aac(3)-Ⅱ and aac(6')- Ⅰ modifying-enzyme genes. Neither class Ⅱ nor Ⅲ integron was detected; DNA sequencing of the fragment amplified by integron PCR revealed a novel cassette array aadB-cat-blaoxa-10/aadA1. Conclusion: Class Ⅰ integron with a novel cassette array in an ESBL-producing multidrug-resistant K. Pneumoniae NJ 12 isolate was reported from Nanjing area of China, with the GenBank accession number DQ141319.

  10. Structural and Functional Elucidation of Peptide Ts11 Shows Evidence of a Novel Subfamily of Scorpion Venom Toxins

    Science.gov (United States)

    Cremonez, Caroline M.; Maiti, Mohitosh; Peigneur, Steve; Cassoli, Juliana Silva; Dutra, Alexandre A. A.; Waelkens, Etienne; Lescrinier, Eveline; Herdewijn, Piet; de Lima, Maria Elena; Pimenta, Adriano M. C.; Arantes, Eliane C.; Tytgat, Jan

    2016-01-01

    To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, β, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or β-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx. PMID:27706049

  11. Crystal structure of Proteus mirabilis lipase, a novel lipase from the Proteus/psychrophilic subfamily of lipase family I.1.

    Directory of Open Access Journals (Sweden)

    Tyler P Korman

    Full Text Available Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca²⁺ coordination may explain how these lipases can fold without specific chaperones.

  12. Linked 5S and 45S rDNA sites are highly conserved through the subfamily Aurantioideae (Rutaceae).

    Science.gov (United States)

    Barros E Silva, A E; Dos Santos Soares Filho, W; Guerra, M

    2013-01-01

    Sites of 5S and 45S rDNA are more commonly located on different chromosomes of most angiosperms. Previous investigations have shown that in the subfamily Aurantioideae these sites may appear closely linked (adjacent sites), as in Poncirustrifoliata, or completely isolated, as in some species of Citrus. In the present work, the distribution of rDNA sites was investigated in representatives of 9 genera of Aurantioideae by FISH and CMA banding, aiming to understand the evolution of adjacent sites in the subfamily. A total of 57 rDNA sites were observed, 40 of them being adjacent to each other. All adjacent sites displayed the 45S rDNA array more terminally located. Assuming that the linked 5S-45S rDNA arrangement was the ancestral condition in Aurantioideae, the isolated rDNA sites observed in Clausena excavata,Bergera koenigii, and Fortunella obovata, as well as the complete linkage loss in Citrus maxima and C. medica indicates that unlinked sites arose independently several times in the evolution of the group. The linkage loss may be due to independent dispersion of one or both rDNA sequence families followed by deletion of the corresponding array in the adjacent site. The possible mechanisms behind these events and their occurrence in other groups are discussed.

  13. Analysis of Comparative Sequence and Genomic Data to Verify Phylogenetic Relationship and Explore a New Subfamily of Bacterial Lipases.

    Directory of Open Access Journals (Sweden)

    Malihe Masomian

    Full Text Available Thermostable and organic solvent-tolerant enzymes have significant potential in a wide range of synthetic reactions in industry due to their inherent stability at high temperatures and their ability to endure harsh organic solvents. In this study, a novel gene encoding a true lipase was isolated by construction of a genomic DNA library of thermophilic Aneurinibacillus thermoaerophilus strain HZ into Escherichia coli plasmid vector. Sequence analysis revealed that HZ lipase had 62% identity to putative lipase from Bacillus pseudomycoides. The closely characterized lipases to the HZ lipase gene are from thermostable Bacillus and Geobacillus lipases belonging to the subfamily I.5 with ≤ 57% identity. The amino acid sequence analysis of HZ lipase determined a conserved pentapeptide containing the active serine, GHSMG and a Ca(2+-binding motif, GCYGSD in the enzyme. Protein structure modeling showed that HZ lipase consisted of an α/β hydrolase fold and a lid domain. Protein sequence alignment, conserved regions analysis, clustal distance matrix and amino acid composition illustrated differences between HZ lipase and other thermostable lipases. Phylogenetic analysis revealed that this lipase represented a new subfamily of family I of bacterial true lipases, classified as family I.9. The HZ lipase was expressed under promoter Plac using IPTG and was characterized. The recombinant enzyme showed optimal activity at 65 °C and retained ≥ 97% activity after incubation at 50 °C for 1h. The HZ lipase was stable in various polar and non-polar organic solvents.

  14. Recognition of two major clades and early diverged groups within the subfamily Cyperoideae (Cyperaceae) including Korean sedges.

    Science.gov (United States)

    Jung, Jongduk; Choi, Hong-Keun

    2013-05-01

    We aim to present phylogenetic major groups within the subfamily Cyperoideae (Cyperaceae) on the basis of three molecular data sets; nuclear ribosomal internal transcribed spacer and 5.8S ribosomal RNA region, the ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit gene, and trnL intron and trnL-F intergenic spacer. Three molecular data and two combined data sets were used to obtain robust and detailed phylogenetic trees by using maximum parsimony and Bayesian inference, respectively. We analyzed 81 genera and 426 species of Cyperaceae, including Korean species. We suggest one early diverged group (EDGs), and two major clades (FAEC and SDC) within the subfamily Cyperoideae. And the clade EDGs comprises six tribes (Schoeneae, Bisboeckelereae, Sclerieae, Cryptangieae, Trilepideae, and Rhynchosporeae) at the basal nodes of Cyperoideae. The FAEC clade (posterior probability [PP]/bootstrap value [BS] = 1.00/85) comprises four tribes (Fuireneae, Abildgaardieae, Eleocharideae, Cypereae), and the SDC clade (PP/BS = 1.00/86) comprises three tribes (Scirpeae, Dulichieae, Cariceae). These three clades used for phylogenetic groups in our study will be useful for establishing the major lineage of the sedge family. The phylogeny of Korean sedges was also investigated within the whole phylogeny of Cyperaceae. The 20 genera of Korean sedges were placed in 10 tribes forming 14 clades.

  15. Resolution of phylogenetic relationships of the major subfamilies of the Delphacidae (Homoptera: Fulgoroidea) using the mitochondrial ribosomal DNA

    Institute of Scientific and Technical Information of China (English)

    EDDY DIJKSTRA; MICHEL A. SLOTMAN; RORY J. POST

    2006-01-01

    Delphacid relationships from the genus level to the subfamily have been completely resolved (among those taxa examined) using sequence data from the 3' end of the 12S gene. Monophyly of the non-asiracine subfamilies was strongly supported and the asiracine Ugyops was placed in the most basal position of the tree. Support levels for monophyly of the Delphacini increased after weighting transversions more heavily than transitions and after removing the cixiid outgroup from the dataset. Among the Delphacini,Conomelus and Megamelus were more closely related to each other than either was to Chloriona. These results are in agreement with the tree based on morphological characters. However, in contrast to morphological data our results strongly supported a sister group relationship between the Stenocraninae and the Kelisiinae. Although the 12S gene fragment gave some information about the species relationships within Chloriona, neither this fragment nor the 5' end of the 16S gene appear to be very useful for this level. Molecular evolutionary patterns provided evidence that there has been a shift in base composition from T to A during the early evolution of the non-Asiracinae. The non-Asiracinae also had comparatively fast substitution rates and these two observations are possibly correlated. In the 'modern' delphacid Chloriona, the AT content was comparatively low in regions free of constraints but this was not the case for 'non-modern' delphacids. The tRNA for valine has been translocated elsewhere, probably before the Delphacidae and Cixiidae diverged from each other.

  16. Testing the reliability of standard and complementary DNA barcodes for the monocot subfamily Alooideae from South Africa.

    Science.gov (United States)

    Daru, Barnabas H; van der Bank, Michelle; Bello, Abubakar; Yessoufou, Kowiyou

    2016-11-23

    Although a standard DNA barcode has been identified for plants, it does not always provide species-level specimen identifications for investigating important ecological questions. In this study, we assessed the species-level discriminatory power of standard (rbcLa + matK) and complementary barcodes (ITS1 and trnH-psbA) within the subfamily Alooideae (Asphodelaceae), a large and recent plant radiation, whose species are important in horticulture yet are threatened. Alooideae has its centre of endemism in southern Africa, with some outlier species occurring elsewhere in Africa and Madagascar. We sampled 360 specimens representing 235 species within all 11 genera of the subfamily. With three distance-based methods, all markers performed poorly for our combined data set, with the highest proportion of correct species-level specimen identifications (30%) found for ITS1. However, when performance was assessed across genera, the discriminatory power varied from 0% for all single markers and combinations in Gasteria to 63% in Haworthiopsis, again for ITS1, suggesting that DNA barcoding success may be related to the evolutionary history of the lineage considered. Although ITS1 could be a good barcode for Haworthiopsis, the generally poor performance of all markers suggests that Alooideae remains a challenge. As species boundaries within Alooideae remain controversial, we call for continued search for suitable markers or the use of genomics approaches to further explore species discrimination in the group.

  17. Relationships among the cyclostome braconid (Hymenoptera: Braconidae) subfamilies inferred from a mitochondrial tRNA gene rearrangement.

    Science.gov (United States)

    Dowton, M

    1999-03-01

    The arrangement of mitochondrial tRNA genes for lysine (K) and aspartate (D) from the junction of the cytochrome oxidase II and ATPase 8 genes was determined in a range of hymenopteran taxa. This indicated that the ancestral arrangement for the order is 'KD', as found in the Diptera (represented by Drosophila and Anopheles) and basal Orthoptera. Most Hymenoptera that evolved after the appearance of parasitism also have the 'KD' arrangement, including noncyclostome braconids. However, most cyclostome braconids have either a 'DK' or a 'DHK' arrangement (where 'H' refers to the tRNA gene for Histidine). In both cases, the aspartate tRNA gene is encoded on the mitochondrial N-strand, rather than the J-strand as is usually the case. This rearrangement identified a monophyletic group not previously recognized, consisting of Rogadinae + Braconinae + Gnamptodontinae + Histeromerinae + Rhyssalinae + Betylobraconinae + Opiinae + Alysiinae. Only one cyclostome subfamily (Doryctinae) retained the 'KD' arrangement, suggesting this to be the most basal of the cyclostome subfamilies, consistent with ectoparasitism being plesiomorphic for the cyclostomes. However, the Aphidiinae also retained the 'KD' arrangement, leaving unresolved the issue of whether they should be included within the cyclostomes.

  18. Xenobiotic, bile acid, and cholesterol transporters: function and regulation.

    Science.gov (United States)

    Klaassen, Curtis D; Aleksunes, Lauren M

    2010-03-01

    Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of

  19. Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3.

    Science.gov (United States)

    Floor, Stephen N; Condon, Kendall J; Sharma, Deepak; Jankowsky, Eckhard; Doudna, Jennifer A

    2016-01-29

    DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

  20. HCV Proteins and Immunoglobulin Variable Gene (IgV Subfamilies in HCV-Induced Type II Mixed Cryoglobulinemia: A Concurrent Pathogenetic Role

    Directory of Open Access Journals (Sweden)

    Giuseppe Sautto

    2012-01-01

    Full Text Available The association between hepatitis C virus (HCV infection and type II mixed cryoglobulinemia (MCII is well established, but the role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to be clearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selected immunoglobulin variable (IgV gene subfamilies frequently endowed with rheumatoid factor (RF activity. Moreover, the same IgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In this paper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, with particular attention reserved to the single IgV subfamilies most frequently involved.

  1. Impact of Genetic Polymorphisms of ABCB1 (MDR1, P-Glycoprotein) on Drug Disposition and Potential Clinical Implications: Update of the Literature.

    Science.gov (United States)

    Wolking, Stefan; Schaeffeler, Elke; Lerche, Holger; Schwab, Matthias; Nies, Anne T

    2015-07-01

    ATP-binding cassette transporter B1 (ABCB1; P-glycoprotein; multidrug resistance protein 1) is an adenosine triphosphate (ATP)-dependent efflux transporter located in the plasma membrane of many different cell types. Numerous structurally unrelated compounds, including drugs and environmental toxins, have been identified as substrates. ABCB1 limits the absorption of xenobiotics from the gut lumen, protects sensitive tissues (e.g. the brain, fetus and testes) from xenobiotics and is involved in biliary and renal secretion of its substrates. In recent years, a large number of polymorphisms of the ABCB1 [ATP-binding cassette, sub-family B (MDR/TAP), member 1] gene have been described. The variants 1236C>T (rs1128503, p.G412G), 2677G>T/A (rs2032582, p.A893S/T) and 3435C>T (rs1045642, p.I1145I) occur at high allele frequencies and create a common haplotype; therefore, they have been most widely studied. This review provides an overview of clinical studies published between 2002 and March 2015. In summary, the effect of ABCB1 variation on P-glycoprotein expression (messenger RNA and protein expression) and/or activity in various tissues (e.g. the liver, gut and heart) appears to be small. Although polymorphisms and haplotypes of ABCB1 have been associated with alterations in drug disposition and drug response, including adverse events with various ABCB1 substrates in different ethnic populations, the results have been majorly conflicting, with limited clinical relevance. Future research activities are warranted, considering a deep-sequencing approach, as well as well-designed clinical studies with appropriate sample sizes to elucidate the impact of rare ABCB1 variants and their potential consequences for effect sizes.

  2. 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

  3. Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette

    Science.gov (United States)

    Boonyaves, Kulaporn; Wu, Ting-Ying; Gruissem, Wilhelm; Bhullar, Navreet K.

    2017-01-01

    Micronutrient malnutrition is widespread, especially in poor populations across the globe, and iron deficiency anemia is one of the most prevalent forms of micronutrient deficiencies. Iron deficiency anemia has severe consequences for human health, working ability, and quality of life. Several interventions including iron supplementation and food fortification have been attempted and met with varied degrees of success. Rice, which is a staple food for over half of the world’s population, is an important target crop for iron biofortification. The genetic variability of iron content in the rice germplasm is very narrow, and thus, conventional breeding has not been successful in developing high iron rice varieties. Therefore, genetic engineering approaches have targeted at increasing iron uptake, translocation, and storage in the rice endosperm. We previously reported that AtIRT1, when expressed together with AtNAS1 and PvFERRITIN (PvFER) in high-iron (NFP) rice, has a synergistic effect of further increasing the iron concentration of polished rice grains. We have now engineered rice expressing AtIRT1, AtNAS1, and PvFER as a single locus gene cassette and compared the resulting lines with transgenic lines expressing AtIRT1 and PvFER gene cassettes. We also evaluated the efficacies of the MsENOD12B and native AtIRT1 promoters for the expression of AtIRT1 in rice in both types of gene cassettes, and found the native AtIRT1 promoter to be a better choice for driving the AtIRT1 expression in our biofortification strategy. All the single insertion transgenic lines have significant increases of iron concentration, both in polished and unpolished grains, but the concerted expression of AtIRT1, AtNAS1, and PvFER resulted to be a more effective strategy in achieving the highest iron increases of up to 10.46 μg/g dry weight. Furthermore, the transformed high iron lines grew better under iron deficiency growth conditions and also have significantly increased grain zinc

  4. NR-2L: a two-level predictor for identifying nuclear receptor subfamilies based on sequence-derived features.

    Directory of Open Access Journals (Sweden)

    Pu Wang

    Full Text Available Nuclear receptors (NRs are one of the most abundant classes of transcriptional regulators in animals. They regulate diverse functions, such as homeostasis, reproduction, development and metabolism. Therefore, NRs are a very important target for drug development. Nuclear receptors form a superfamily of phylogenetically related proteins and have been subdivided into different subfamilies due to their domain diversity. In this study, a two-level predictor, called NR-2L, was developed that can be used to identify a query protein as a nuclear receptor or not based on its sequence information alone; if it is, the prediction will be automatically continued to further identify it among the following seven subfamilies: (1 thyroid hormone like (NR1, (2 HNF4-like (NR2, (3 estrogen like, (4 nerve growth factor IB-like (NR4, (5 fushi tarazu-F1 like (NR5, (6 germ cell nuclear factor like (NR6, and (7 knirps like (NR0. The identification was made by the Fuzzy K nearest neighbor (FK-NN classifier based on the pseudo amino acid composition formed by incorporating various physicochemical and statistical features derived from the protein sequences, such as amino acid composition, dipeptide composition, complexity factor, and low-frequency Fourier spectrum components. As a demonstration, it was shown through some benchmark datasets derived from the NucleaRDB and UniProt with low redundancy that the overall success rates achieved by the jackknife test were about 93% and 89% in the first and second level, respectively. The high success rates indicate that the novel two-level predictor can be a useful vehicle for identifying NRs and their subfamilies. As a user-friendly web server, NR-2L is freely accessible at either http://icpr.jci.edu.cn/bioinfo/NR2L or http://www.jci-bioinfo.cn/NR2L. Each job submitted to NR-2L can contain up to 500 query protein sequences and be finished in less than 2 minutes. The less the number of query proteins is, the shorter the time will

  5. NR-2L: a two-level predictor for identifying nuclear receptor subfamilies based on sequence-derived features.

    Science.gov (United States)

    Wang, Pu; Xiao, Xuan; Chou, Kuo-Chen

    2011-01-01

    Nuclear receptors (NRs) are one of the most abundant classes of transcriptional regulators in animals. They regulate diverse functions, such as homeostasis, reproduction, development and metabolism. Therefore, NRs are a very important target for drug development. Nuclear receptors form a superfamily of phylogenetically related proteins and have been subdivided into different subfamilies due to their domain diversity. In this study, a two-level predictor, called NR-2L, was developed that can be used to identify a query protein as a nuclear receptor or not based on its sequence information alone; if it is, the prediction will be automatically continued to further identify it among the following seven subfamilies: (1) thyroid hormone like (NR1), (2) HNF4-like (NR2), (3) estrogen like, (4) nerve growth factor IB-like (NR4), (5) fushi tarazu-F1 like (NR5), (6) germ cell nuclear factor like (NR6), and (7) knirps like (NR0). The identification was made by the Fuzzy K nearest neighbor (FK-NN) classifier based on the pseudo amino acid composition formed by incorporating various physicochemical and statistical features derived from the protein sequences, such as amino acid composition, dipeptide composition, complexity factor, and low-frequency Fourier spectrum components. As a demonstration, it was shown through some benchmark datasets derived from the NucleaRDB and UniProt with low redundancy that the overall success rates achieved by the jackknife test were about 93% and 89% in the first and second level, respectively. The high success rates indicate that the novel two-level predictor can be a useful vehicle for identifying NRs and their subfamilies. As a user-friendly web server, NR-2L is freely accessible at either http://icpr.jci.edu.cn/bioinfo/NR2L or http://www.jci-bioinfo.cn/NR2L. Each job submitted to NR-2L can contain up to 500 query protein sequences and be finished in less than 2 minutes. The less the number of query proteins is, the shorter the time will

  6. Biogeography of the leafhopper subfamily Stegelytrinae (Hemiptera: Cicadellidae), based on a cluster analysis of geographical distribution in areas of endemism combined with phylogeny of the subfamily%世界秀头叶蝉亚科生物地理学

    Institute of Scientific and Technical Information of China (English)

    魏琮; 程若琳; 张雅林

    2012-01-01

    Biogeography of the leafhopper subfamily Stegelytrinae Baker is studied based on an analysis of geographical distribution of this subfamily worldwide using a cluster analysis of the zoological distribution of areas of endemism as well as the phylogeny of representatives of this subfamily.Results show that the Stegelytrinae mainly occur in the Oriental Region and in the Mediterranean area of the Palaearctic Region,and this extends to the east side of both Wallace's and Weber's lines.Eleven areas of endemism of this subfamily are recognized.The proportions of endemic taxa in different areas of endemism are generally very high in comparison with other leafhopper groups,but distinct differences could be found among the different areas of endemism of Stegelytrinae.This subfamily is most intensively diversified in the Indochina Peninsula (INCN).This is the stegelytrine distribution center,having the highest biodiversity at both generic and species levels.The dendrogram of endemic areas of Stegelytrinae constructed using cluster analysis of the zoological distribution of Stegelytrinae at generic level shows the endemic areas of Stegelytrinae can be divided into 4 large groups.Relationships among different endemic areas of Stegelytrinae correspond largely to the geologic history of related areas,which indicates that the evolution and vicariance of this subfamily have been closely related to the history of continental drift and climate changes.It is deduced that the presumed monophyletic Stegelytrinae originated in the Oriental Region after North America had separated from Eurasia; this is the case in the monophyletic genera group which is supported by the lateral frontal sutures extending dorsally well beyond the corresponding ocellus.In addition,two expanding traces of the Stegelytrinae are presumed,which remain plausible explanations for the dispersal of Stegelytrinae:(1) New Guinea (and probably (+ Australia)) -Kalimantan-Sumatra-Malay Peninsula-Indochina Peninsula

  7. The Subfamily-Specific Interaction between Kv2.1 and Kv6.4 Subunits Is Determined by Interactions between the N- and C-termini

    OpenAIRE

    Elke Bocksteins; Evy Mayeur; Abbi Van Tilborg; Glenn Regnier; Jean-Pierre Timmermans; Snyders, Dirk J

    2014-01-01

    The "silent" voltage-gated potassium (KvS) channel subunit Kv6.4 does not form electrically functional homotetramers at the plasma membrane but assembles with Kv2.1 subunits, generating functional Kv2.1/Kv6.4 heterotetramers. The N-terminal T1 domain determines the subfamily-specific assembly of Kv1-4 subunits by preventing interactions between subunits that belong to different subfamilies. For Kv6.4, yeast-two-hybrid experiments showed an interaction of the Kv6.4 N-terminus with the Kv2.1 N-...

  8. TCR gene segments from at least one third of V alpha subfamilies rearrange at the delta locus.

    Science.gov (United States)

    Genevée, C; Chung, V; Diu, A; Hercend, T; Triebel, F

    1994-02-01

    Using PCR and an experimentally validated V alpha subfamily-specific oligonucleotide panel (V alpha 1-w29), we have investigated whether the TCR delta chain may increase its combinatorial diversity by using V genes considered as alpha chain-specific. We show that at least 10 distinct human V alpha segments rearrange at the J delta locus, leading to scrambling of the two V gene repertoires. Fifty-five per cent of the V alpha/J delta transcripts characterized here were in frame. The 17 V alpha/C delta chains analysed included an extended CDR3 region with up to 18 aa encoded by the junctional region. In addition, a new J delta segment (J delta 4) has been characterized. Together, these findings demonstrate that combinatorial diversity in the human delta locus is larger than previously thought.

  9. The structure of SAV1646 from Staphylococcus aureus belonging to a new `ribosome-associated' subfamily of bacterial proteins.

    Science.gov (United States)

    Chirgadze, Yuri N; Clarke, Teresa E; Romanov, Vladimir; Kisselman, Gera; Wu-Brown, Jean; Soloveychik, Maria; Chan, Tiffany S Y; Gordon, Roni D; Battaile, Kevin P; Pai, Emil F; Chirgadze, Nickolay Y

    2015-02-01

    The crystal structure of the SAV1646 protein from the pathogenic microorganism Staphylococcus aureus has been determined at 1.7 Å resolution. The 106-amino-acid protein forms a two-layer sandwich with α/β topology. The protein molecules associate as dimers in the crystal and in solution, with the monomers related by a pseudo-twofold rotation axis. A sequence-homology search identified the protein as a member of a new subfamily of yet uncharacterized bacterial `ribosome-associated' proteins with at least 13 members to date. A detailed analysis of the crystal protein structure along with the genomic structure of the operon containing the sav1646 gene allowed a tentative functional model of this protein to be proposed. The SAV1646 dimer is assumed to form a complex with ribosomal proteins L21 and L27 which could help to complete the assembly of the large subunit of the ribosome.

  10. 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.

  11. Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)

    DEFF Research Database (Denmark)

    Jakobsen, Jannik Ejnar; Johansen, Marianne G; Schmidt, Mette;

    2013-01-01

    Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange...... (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using...... minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven...

  12. Review: Gerrit Herlyn & Thomas Overdick (Eds. (2003. Kassettengeschichten. Von Menschen und ihren Mixtapes [Cassette Stories. Men and Their Mix Tapes

    Directory of Open Access Journals (Sweden)

    Dirk Ducar

    2006-03-01

    Full Text Available This book deals with the phenomenon of mix tapes, actual audio cassettes containing music which are privately produced and distributed. The book attempts to explain how such mix tapes are used and how meaning is ascribed to them by their producers and recipients, and to contribute to the fields of cultural studies and media research. For two related reasons these aims are not achieved. First, the book is stuck in the obsolete research traditions of "Retten und Bewahren" [retain and rescue] and thus is afflicted with a typical obsession with the artifact and latently utopian views on its subject. Second, blatant deficiencies in the underlying code of research devalue the findings of the authors. URN: urn:nbn:de:0114-fqs0602146

  13. Adenosine triphosphate-binding cassette member A3 gene mutation in children from one family from Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Gawahir Mohamed Ahmed Mukhtar

    2016-01-01

    Full Text Available Mutation in ABCA3, which is adenosine triphosphate-binding cassette member A3, a member of protein transporter family for phospholipids into the lamellar bodies during synthesis of surfactant, can cause lung disease related to surfactant dysfunction with autosomal recessive pattern. We reported three cases from same family with ABCA3 mutation, their gene, clinical course, and outcomes mentioning that one patient had successful lung transplantation, one started the process of the lung transplantation while the third one died during infancy. We concluded that the patients with ABCA3 gene mutations are increasing in numbers may be due to the availability of the genetic testing and high index of suspicion among physicians. Lung transplantation is the definitive treatment, but availability is limited in our region.

  14. Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand.

    Science.gov (United States)

    Chanchaithong, Pattrarat; Prapasarakul, Nuvee; Perreten, Vincent; Schwendener, Sybille

    2016-02-01

    A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmecAI16-SCCczrAI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmecAI16, 19,400-bp SCCczrAI16, and 102-bp SCC-likeAI16 elements. SCCmecAI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczrAI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmecAI16-SCCczrAI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.

  15. 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

  16. Structural and Functional Analysis of a New Subfamily of Glycosyltransferases Required for Glycosylation of Serine-rich Streptococcal Adhesins

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Fan; Erlandsen, Heidi; Ding, Lei; Li, Jingzhi; Huang, Ying; Zhou, Meixian; Liang, Xiaobo; Ma, Jinbiao; Wu, Hui (UAB)

    2011-09-16

    Serine-rich repeat glycoproteins (SRRPs) are a growing family of bacterial adhesins found in many streptococci and staphylococci; they play important roles in bacterial biofilm formation and pathogenesis. Glycosylation of this family of adhesins is essential for their biogenesis. A glucosyltransferase (Gtf3) catalyzes the second step of glycosylation of a SRRP (Fap1) from an oral streptococcus, Streptococcus parasanguinis. Although Gtf3 homologs are highly conserved in SRRP-containing streptococci, they share minimal homology with functionally known glycosyltransferases. We report here the 2.3 {angstrom} crystal structure of Gtf3. The structural analysis indicates that Gtf3 forms a tetramer and shares significant structural homology with glycosyltransferases from GT4, GT5, and GT20 subfamilies. Combining crystal structural analysis with site-directed mutagenesis and in vitro glycosyltransferase assays, we identified residues that are required for UDP- or UDP-glucose binding and for oligomerization of Gtf3 and determined their contribution to the enzymatic activity of Gtf3. Further in vivo studies revealed that the critical amino acid residues identified by the structural analysis are crucial for Fap1 glycosylation in S. parasanguinis in vivo. Moreover, Gtf3 homologs from other streptococci were able to rescue the gtf3 knock-out mutant of S. parasanguinis in vivo and catalyze the sugar transfer to the modified SRRP substrate in vitro, demonstrating the importance and conservation of the Gtf3 homologs in glycosylation of SRRPs. As the Gtf3 homologs only exist in SRRP-containing streptococci, we conclude that the Gtf3 homologs represent a unique subfamily of glycosyltransferases.

  17. A Species-Level Phylogeny of Extant Snakes with Description of a New Colubrid Subfamily and Genus

    Science.gov (United States)

    McKelvy, Alexander D.; Grismer, L. Lee; Bell, Charles D.; Lailvaux, Simon P.

    2016-01-01

    Background With over 3,500 species encompassing a diverse range of morphologies and ecologies, snakes make up 36% of squamate diversity. Despite several attempts at estimating higher-level snake relationships and numerous assessments of generic- or species-level phylogenies, a large-scale species-level phylogeny solely focusing on snakes has not been completed. Here, we provide the largest-yet estimate of the snake tree of life using maximum likelihood on a supermatrix of 1745 taxa (1652 snake species + 7 outgroup taxa) and 9,523 base pairs from 10 loci (5 nuclear, 5 mitochondrial), including previously unsequenced genera (2) and species (61). Results Increased taxon sampling resulted in a phylogeny with a new higher-level topology and corroborate many lower-level relationships, strengthened by high nodal support values (> 85%) down to the species level (73.69% of nodes). Although the majority of families and subfamilies were strongly supported as monophyletic with > 88% support values, some families and numerous genera were paraphyletic, primarily due to limited taxon and loci sampling leading to a sparse supermatrix and minimal sequence overlap between some closely-related taxa. With all rogue taxa and incertae sedis species eliminated, higher-level relationships and support values remained relatively unchanged, except in five problematic clades. Conclusion Our analyses resulted in new topologies at higher- and lower-levels; resolved several previous topological issues; established novel paraphyletic affiliations; designated a new subfamily, Ahaetuliinae, for the genera Ahaetulla, Chrysopelea, Dendrelaphis, and Dryophiops; and appointed Hemerophis (Coluber) zebrinus to a new genus, Mopanveldophis. Although we provide insight into some distinguished problematic nodes, at the deeper phylogenetic scale, resolution of these nodes may require sampling of more slowly-evolving nuclear genes. PMID:27603205

  18. The search for therapeutic bacteriophages uncovers one new subfamily and two new genera of Pseudomonas-infecting Myoviridae.

    Directory of Open Access Journals (Sweden)

    Marine Henry

    Full Text Available In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article.

  19. Phylogenetic analysis and expression patterns of Pax genes in the onychophoran Euperipatoides rowelli reveal a novel bilaterian Pax subfamily.

    Science.gov (United States)

    Franke, Franziska Anni; Schumann, Isabell; Hering, Lars; Mayer, Georg

    2015-01-01

    Pax family genes encode a class of transcription factors that regulate various developmental processes. To shed light on the evolutionary history of these genes in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we analyzed the Pax repertoire in the embryonic and adult transcriptomes of the onychophoran Euperipatoides rowelli. Our data revealed homologs of all five major bilaterian Pax subfamilies in this species, including Pax2/5/8, Pax4/6, Pox-neuro, Pax1/9/Pox-meso, and Pax3/7. In addition, we identified a new Pax member, pax-α, which does not fall into any other known Pax subfamily but instead clusters in the heterogenic Pax-α/β clade containing deuterostome, ecdysozoan, and lophotrochozoan gene sequences. These findings suggest that the last common bilaterian ancestor possessed six rather than five Pax genes, which have been retained in the panarthropod lineage. The expression data of Pax orthologs in the onychophoran embryo revealed distinctive patterns, some of which might be related to their ancestral roles in the last common panarthropod ancestor, whereas others might be specific to the onychophoran lineage. The derived roles include, for example, an involvement of pax2/5/8, pox-neuro, and pax3/7 in onychophoran nephridiogenesis, and an additional function of pax2/5/8 in the formation of the ventral and preventral organs. Furthermore, our transcriptomic analyses suggest that at least some Pax genes, including pax6 and pax-α, are expressed in the adult onychophoran head, although the corresponding functions remain to be clarified. The remarkable diversity of the Pax expression patterns highlights the functional and evolutionary plasticity of these genes in panarthropods.

  20. In silico cloning and characterization of the TGA (TGACG MOTIF-BINDING FACTOR) transcription factors subfamily in Carica papaya.

    Science.gov (United States)

    Idrovo Espín, Fabio Marcelo; Peraza-Echeverria, Santy; Fuentes, Gabriela; Santamaría, Jorge M

    2012-05-01

    The TGA transcription factors belong to the subfamily of bZIP group D that play a major role in disease resistance and development. Most of the TGA identified in Arabidopsis interact with the master regulator of SAR, NPR1 that controls the expression of PR genes. As a first approach to determine the possible involvement of these transcription factors in papaya defense, we characterized Arabidopsis TGA orthologs from the genome of Carica papaya cv. SunUp. Six orthologs CpTGA1 to CpTGA6, were identified. The predicted CpTGA proteins were highly similar to AtTGA sequences and probably share the same DNA binding properties and transcriptional regulation features. The protein sequences alignment evidenced the presence of conserved domains, characteristic of this group of transcription factors. The phylogeny showed that CpTGA evolved into three different subclades associated with defense and floral development. This is the first report of basal expression patterns assessed by RT-PCR, from the whole subfamily of CpTGA members in different tissues from papaya cv. Maradol mature plants. Overall, CpTGA1, CpTGA3 CpTGA6 and CpTGA4 showed a basal expression in all tissues tested; CpTGA2 expressed strongly in all tissues except in petioles while CpTGA5 expressed only in petals and to a lower extent in petioles. Although more detailed studies in anthers and other floral structures are required, we suggest that CpTGA5 might be tissue-specific, and it might be involved in papaya floral development. On the other hand, we report here for the first time, the expression of the whole family of CpTGA in response to salicylic acid (SA). The expression of CpTGA3, CpTGA4 and CpTGA6 increased in response to SA, what would suggest its involvement in the SAR response in papaya.

  1. Sonorensin: an antimicrobial peptide, belonging to the heterocycloanthracin subfamily of bacteriocins, from a new marine isolate, Bacillus sonorensis MT93.

    Science.gov (United States)

    Chopra, Lipsy; Singh, Gurdeep; Choudhary, Vikas; Sahoo, Debendra K

    2014-05-01

    Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized.

  2. 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.

  3. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.

    Science.gov (United States)

    Robinson, Andrew; Wu, Peter S-C; Harrop, Stephen J; Schaeffer, Patrick M; Dosztányi, Zsuzsanna; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Otting, Gottfried; Dixon, Nicholas E; Curmi, Paul M G; Mabbutt, Bridget C

    2005-03-11

    The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.

  4. In vivo transfer of an incFIB plasmid harbouring a class 1 integron with gene cassettes dfrA1-aadA1

    NARCIS (Netherlands)

    Essen-Zandbergen, van A.; Smith, H.; Veldman, K.T.; Mevius, D.J.

    2009-01-01

    Transfer of resistance genes from bacteria from food producing animals to human pathogens is a potential risk to human health. The aim of this study was to determine in vivo transfer of a plasmid harbouring a class 1 integron containing gene cassettes dfrA1-aadA1 from Salmonella to Escherichia coli

  5. 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.

  6. 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 ...

  7. Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells.

    Science.gov (United States)

    Zhu, Haibao; Lau, Cia-Hin; Goh, Sal-Lee; Liang, Qingle; Chen, Can; Du, Shouhui; Phang, Rui-Zhe; Tay, Felix Chang; Tan, Wee-Kiat; Li, Zhendong; Tay, Johan Chin-Kang; Fan, Weimin; Wang, Shu

    2013-10-01

    Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

  8. 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)

  9. Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution.

    Science.gov (United States)

    Yu, Xianxian; Duan, Xiaoshan; Zhang, Rui; Fu, Xuehao; Ye, Lingling; Kong, Hongzhi; Xu, Guixia; Shan, Hongyan

    2016-01-01

    AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies.

  10. Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

    OpenAIRE

    Wang, Xiaohui; Bompadre, Silvia G.; Li, Min; Hwang, Tzyh-Chang

    2009-01-01

    The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G55...

  11. Design factors that influence the performance of flight intercept traps for the capture of longhorned beetles (Coleoptera: Cerambycidae) from the subfamilies Lamiinae and Cerambycinae.

    Science.gov (United States)

    Allison, Jeremy D; Bhandari, Basu D; McKenney, Jessica L; Millar, Jocelyn G

    2014-01-01

    In North America, cerambycid beetles can have significant ecological and economic effects on forest ecosystems, and the rate of introduction and/or detection of exotic species is increasing. Detection and survey programs rely on semiochemical-baited intercept traps which are often ineffective for large woodborers like cerambycid beetles. This study examined the effects of flight intercept trap design on the capture of cerambycid beetles in the subfamilies Lamiinae and Cerambycinae. These subfamilies are the two largest in the Cerambycidae and they include many of the most damaging cerambycid pests and species on regulatory watch lists in North America. This study demonstrates that intercept trap design, treatment of trap surfaces with a lubricant, and the type of collection cup all influence the capture of beetles from the subfamilies Lamiinae and Cerambycinae. It also demonstrates that the addition of a large lubricant-treated collar to the bottom funnel of a multiple-funnel trap significantly increases the capture of some Lamiinae. The best trap design for both subfamilies was a lubricant treated multiple-funnel [MF] trap equipped with a wet cup and lubricant treated large collar on the bottom funnel. This design captured between 4 and 14 times more Lamiinae and Cerambycinae than commercially-available MF and panel traps.

  12. New case of lateral asymmetry in fishes: A new subfamily, genus and species of deep water clingfishes from Papua New Guinea, western Pacific Ocean.

    Science.gov (United States)

    Fricke, Ronald; Chen, Jhen-Nien; Chen, Wei-Jen

    2017-01-01

    The unusual clingfish Protogobiesox asymmetricus n. gen, n. sp. is described on the basis of four specimens collected in deep water off the north coast of Papua New Guinea in 2012. The species is characterized by its 9-10 dorsal rays, 8 anal rays, 17-24 pectoral-fin rays, 15 principal caudal-fin rays, 3 gills, third arch with 3 gill rakers, 34-35 total vertebrae, with asymmetrical lateral bending starting behind the skull, bent at an angle of 85°-92°; skull asymmetrical in frontal view; skin naked, surface of head and body without striae; disc without adhesive papillae. A new subfamily Protogobiesocinae is described for this species and Lepadicyathus mendeleevi Prokofiev, 2005, which is redescribed. The new subfamily is compared within the family; keys to the subfamilies of Gobiesocidae and the species within the new subfamily are presented; its phylogenetic relationship to other gobiesocids is inferred based on a multi-locus DNA dataset.

  13. Discovery of a living fossil: a new xylastodorine species from New Caledonia (Heteroptera: Thaumastocoridae) and first record of the subfamily from the eastern Hemisphere

    NARCIS (Netherlands)

    Doesburg, van P.H.; Cassis, G.; Monteith, G.B.

    2010-01-01

    A new species belonging to the genus Proxylastodoris Heiss & Popov, 2002, P. kuscheli spec. nov., of the subfamily Xylastodorinae Barber, 1920 (Heteroptera: Thaumastocoridae) is described from New Caledonia. It is the first recent record outside the western Hemisphere of the Xyalstodorinae and is th

  14. Evolution of C2H2-zinc finger genes and subfamilies in mammals: Species-specific duplication and loss of clusters, genes and effector domains

    Directory of Open Access Journals (Sweden)

    Aubry Muriel

    2008-06-01

    Full Text Available Abstract Background C2H2 zinc finger genes (C2H2-ZNF constitute the largest class of transcription factors in humans and one of the largest gene families in mammals. Often arranged in clusters in the genome, these genes are thought to have undergone a massive expansion in vertebrates, primarily by tandem duplication. However, this view is based on limited datasets restricted to a single chromosome or a specific subset of genes belonging to the large KRAB domain-containing C2H2-ZNF subfamily. Results Here, we present the first comprehensive study of the evolution of the C2H2-ZNF family in mammals. We assembled the complete repertoire of human C2H2-ZNF genes (718 in total, about 70% of which are organized into 81 clusters across all chromosomes. Based on an analysis of their N-terminal effector domains, we identified two new C2H2-ZNF subfamilies encoding genes with a SET or a HOMEO domain. We searched for the syntenic counterparts of the human clusters in other mammals for which complete gene data are available: chimpanzee, mouse, rat and dog. Cross-species comparisons show a large variation in the numbers of C2H2-ZNF genes within homologous mammalian clusters, suggesting differential patterns of evolution. Phylogenetic analysis of selected clusters reveals that the disparity in C2H2-ZNF gene repertoires across mammals not only originates from differential gene duplication but also from gene loss. Further, we discovered variations among orthologs in the number of zinc finger motifs and association of the effector domains, the latter often undergoing sequence degeneration. Combined with phylogenetic studies, physical maps and an analysis of the exon-intron organization of genes from the SCAN and KRAB domains-containing subfamilies, this result suggests that the SCAN subfamily emerged first, followed by the SCAN-KRAB and finally by the KRAB subfamily. Conclusion Our results are in agreement with the "birth and death hypothesis" for the evolution of

  15. Phylogeny of Bromelioideae (Bromeliaceae) inferred from nuclear and plastid DNA loci reveals the evolution of the tank habit within the subfamily.

    Science.gov (United States)

    Schulte, Katharina; Barfuss, Michael H J; Zizka, Georg

    2009-05-01

    Phylogenetic relationships within subfamily Bromelioideae (Bromeliaceae, Poales) were inferred using DNA sequence data from the low-copy nuclear gene phosphoribulokinase (PRK) and five plastid loci (matK gene, 3'trnK intron, trnL intron, trnL-trnF spacer, atpB-rbcL spacer). The PRK dataset exhibited a considerably higher proportion of potentially informative characters than the plastid dataset (16.9% vs. 3.1%), leading to a higher resolution and improved nodal support of the resulting phylogenies. Bromelia is resolved as sister to the remainder of the subfamily, albeit this relationship receives only weak nodal support. The basal position of Bromelia, as well as Deinacanthon, Greigia, Ochagavia, Fascicularia and Fernseea within the subfamily is corroborated and the remainder of the subfamily forms a highly supported clade (the eu-bromelioids). By the inclusion of nuclear data the sister group position of Fernseea to the eu-bromelioids is now highly supported. Within the eu-bromelioids the resolution of the clade representing the more advanced core bromelioids has increased and further demonstrates the highly problematic generic concept of Aechmea as well as Quesnelia. Moreover, the data were used to examine the evolution of sepal symmetry and the tank habit. Tracing of character transitions onto the molecular phylogeny implies that both characters have undergone only few transitions within the subfamily and thus are not as homoplasious as previously assumed. The character state reconstruction reveals the great importance of the evolution of the tank habit for the diversification of the core bromelioids.

  16. Strong stimulation of recombinant protein production in Escherichia coli by combining stimulatory control elements in an expression cassette

    Directory of Open Access Journals (Sweden)

    Zwick Friederike

    2012-10-01

    Full Text Available Abstract Background The XylS/Pm expression system has been used to produce recombinant proteins at industrial levels in Escherichia coli. Activation of transcription from the Pm promoter takes place in the presence of benzoic acid or derivatives of it. Previous mutagenesis studies resulted in identification of several variants of the expression control elements xylS (X, Pm (P and the 5'-untranslated region (U that individually gave rise to strongly stimulated expression. The goal of this study was to test if combination of such stimulatory mutations in the same expression vectors would lead to further increase of expression levels. Results We combined X, P and U variants that were originally identified due to their ability to strongly stimulate expression of the reporter gene bla (resistance to penicillin. Combination of optimized elements stimulated bla expression up to 75-fold (X, P and U combined relative to the wild-type system, while accumulated transcript levels increased about 50-fold. This is much more than for the elements individually. We also tested combination of the variant elements on two other and unrelated genes, celB (encoding phosphoglucomutase and the human growth factor gene gm-csf. Protein production from these genes is much more efficient than from bla in the wild-type system, but expression was still significantly stimulated by the combination of X, P and U variants, although not to the same extent as for bla. We also integrated a single copy of the expression cassette with each gene into the E. coli chromosome and found that the expression level from this single copy was higher for bla than for the wild-type plasmid system, while it was lower for celB and gm-csf. Conclusion Our results show that combination of stimulatory expression control elements can be used to further increase production of different proteins in E. coli. For one reporter gene (bla this allowed for more protein production from a single gene copy

  17. Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

    Institute of Scientific and Technical Information of China (English)

    Fei Ren; Hong Zhang; Chao Qi; Mei-ling Gao; Hong Wang; Xia-qing Li

    2015-01-01

    The transient receptor potential cation channel subfamily V member 1 (TRPV1) provides the sensation of pain (nociception). However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517 (300 mg/kg), a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immuno-lfuorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clus-ters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

  18. TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum subsp. pallidum

    Science.gov (United States)

    Giacani, Lorenzo; Godornes, Charmie; Puray-Chavez, Maritza; Guerra-Giraldez, Cristina; Tompa, Martin; Lukehart, Sheila A.; Centurion-Lara, Arturo

    2009-01-01

    Transcriptional regulation in Treponema pallidum subsp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidum repeat) genes (tprE, tprG, and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames (ORFs) with similarity to known bacterial transcription factors (TFs), including four catabolite activator protein (CAP) homologs. In this work, sequences matching the E. coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG, and tprJ. Using elecrophoretic mobility shift assay (EMSA) and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homolog, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator which influences tpr promoter activity. PMID:19432808

  19. Differential expression of cytochrome P450 enzymes from the CYP2C subfamily in the human brain.

    Science.gov (United States)

    Booth Depaz, Iris M; Toselli, Francesca; Wilce, Peter A; Gillam, Elizabeth M J

    2015-03-01

    Cytochrome P450 enzymes from the CYP2C subfamily play a prominent role in the metabolic clearance of many drugs. CYP2C enzymes have also been implicated in the metabolism of arachidonic acid to vasoactive epoxyeicosatrienoic acids. CYP2C8, CYP2C9, and CYP2C19 are expressed in the adult liver at significant levels; however, the expression of CYP2C enzymes in extrahepatic tissues such as the brain is less well characterized. Form-specific antibodies to CYP2C9 and CYP2C19 were prepared by affinity purification of antibodies raised to unique peptides. CYP2C9 and CYP2C19 were located in microsomal fractions of all five human brain regions examined, namely the frontal cortex, hippocampus, basal ganglia, amygdala, and cerebellum. Both CYP2C9 and CYP2C19 were detected predominantly within the neuronal soma but with expression extending down axons and dendrites in certain regions. Finally, a comparison of cortex samples from alcoholics and age-matched controls suggested that CYP2C9 expression was increased in alcoholics.

  20. Phylogenetic relationships, character evolution, and biogeography of the subfamily Lygosominae (Reptilia: Scincidae) inferred from mitochondrial DNA sequences.

    Science.gov (United States)

    Honda, M; Ota, H; Kobayashi, M; Nabhitabhata, J; Yong, H S; Hikida, T

    2000-06-01

    Phylogenetic relationships among the lygosomine skinks were inferred from 1249 base positions of mitochondrial DNA sequences of 12S and 16S rRNA genes. The monophyly of this subfamily was confirmed and the presence of five distinct infrasubfamilial lineages detected. Of these, the Sphenomorphus group appears to have diverged first, followed by the Lygosoma and Egernia groups in order, leaving the Eugongylus and Mabuya groups as sister groups. Our results did not support monophyly of the Mabuya group sensu lato (i.e., an assemblage of the Lygosoma, Egernia, and Mabuya groups), for which a number of morphological and karyological studies demonstrated a considerable similarity. Our results also contradict the previous hypothesis, formulated on the basis of morphological and immunological data, which argued for the sister relationship between the Egernia and the Eugongylus groups. Morphological and karyological characters used to define the Mabuya group (sensu lato) may actually represent plesiomorphic states. The phylogenetic diversity of lygosomine skinks in the Australian region appears to have increased through multiple colonizations from Southeast Asia.

  1. Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

    Directory of Open Access Journals (Sweden)

    De Las Rivas Javier

    2007-03-01

    Full Text Available Abstract Background The pyridine nucleotide disulfide reductase (PNDR is a large and heterogeneous protein family divided into two classes (I and II, which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. Results A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE and the type II NADH dehydrogenases (NDH-2. In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II-reductase activity is detected in a specific subset of NDH-2. Conclusion The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2.

  2. TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum.

    Science.gov (United States)

    Giacani, Lorenzo; Godornes, Charmie; Puray-Chavez, Maritza; Guerra-Giraldez, Cristina; Tompa, Martin; Lukehart, Sheila A; Centurion-Lara, Arturo

    2009-06-01

    Transcriptional regulation in Treponema pallidum ssp. pallidum is poorly understood, primarily because this organism cannot be cultivated in vitro or genetically manipulated. We have recently shown a phase variation mechanism controlling transcription initiation of Subfamily II tpr (T. pallidumrepeat) genes (tprE, tprG and tprJ), a group of virulence factor candidates. Furthermore, the same study suggested that additional mechanisms might influence the level of transcription of these tprs. The T. pallidum genome sequence has revealed a few open reading frames with similarity to known bacterial transcription factors, including four catabolite activator protein homologues. In this work, sequences matching the Escherichia coli cAMP receptor protein (CRP) binding motif were identified in silico upstream of tprE, tprG and tprJ. Using elecrophoretic mobility shift assay and DNaseI footprinting assay, recombinant TP0262, a T. pallidum CRP homologue, was shown to bind specifically to amplicons obtained from the tpr promoters containing putative CRP binding motifs. Using a heterologous reporter system, binding of TP0262 to these promoters was shown to either increase (tprE and tprJ) or decrease (tprG) tpr promoter activity. This is the first characterization of a T. pallidum transcriptional modulator that influences tpr promoter activity.

  3. 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.

  4. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti.

    Science.gov (United States)

    Häcker, Irina; Harrell Ii, Robert A; Eichner, Gerrit; Pilitt, Kristina L; O'Brochta, David A; Handler, Alfred M; Schetelig, Marc F

    2017-03-07

    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Production of rotavirus core-like particles in Sf9 cells using recombinase-mediated cassette exchange.

    Science.gov (United States)

    Fernandes, Fabiana; Dias, Mafalda M; Vidigal, João; Sousa, Marcos F Q; Patrone, Marco; Teixeira, Ana P; Alves, Paula M

    2014-02-10

    A flexible Sf9 insect cell line was recently developed leveraging the recombinase-mediated cassette exchange (RMCE) technology, which competes with the popular baculovirus expression vector system (BEVS) in terms of speed to produce new proteins. Herein, the ability of this cell platform to produce complex proteins, such as rotavirus core-like particles, was evaluated. A gene construct coding for a VP2-GFP fusion protein was targeted to a pre-characterized high recombination efficiency locus flanked by flipase (Flp) recognition target sites and, after three weeks in selection, an isogenic cell population was obtained. Despite the lower cell specific productivities with respect to those obtained by baculovirus infection, the titers of VP2-GFP reached in shake flask batch cultures were comparable as a result of higher cell densities. To further improve the VP2-GFP levels from stable expression, analysis of exhausted medium was undertaken to design feeding strategies enabling higher cell densities as well as increased culture duration. The implementation of the best strategy allowed reaching 20 million cells per ml in bioreactor cultures; the integrity of the rotavirus core-like particles could be confirmed by electron microscopy. Overall, we show that this Sf9-Flp cell platform represents a valuable alternative to the BEVS for producing complex recombinant proteins, such as rotavirus core-like particles.

  10. Cre/lox-Recombinase-Mediated Cassette Exchange for Reversible Site-Specific Genomic Targeting of the Disease Vector, Aedes aegypti

    Science.gov (United States)

    Häcker, Irina; Harrell II, Robert A.; Eichner, Gerrit; Pilitt, Kristina L.; O’Brochta, David A.; Handler, Alfred M.; Schetelig, Marc F.

    2017-01-01

    Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting. PMID:28266580

  11. 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.

  12. 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.

  13. 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.

  14. ABCB1, ABCC2, SCN1A, SCN2A, GABRA1 gene polymorphisms and drug resistant epilepsy in the Chinese Han population.

    Science.gov (United States)

    Zhou, Luo; Cao, Yuze; Long, Hongyu; Long, Lili; Xu, Lin; Liu, Zhaoqian; Zhang, Ying; Xiao, Bo

    2015-06-01

    Drug resistance is common in epilepsy despite multiple available medications. Single nucleotide polymorphisms (SNP) may influence drug efficacy in epilepsy. We therefore aimed to clarify the association between polymorphisms of several controversial SNP loci and drug resistance in Chinese Han epilepsy patients from central China. Among all the 391 recruited subjects, 235 and 156 patients were classified into a drug responsive and resistant group, respectively, according to the definition of drug resistance proposed by the International League Against Epilepsy. The candidate SNP loci, including ATP-binding cassette (ABC) subfamily gene ABCB1 rs2032582 and rs1045642; ABC subfamily gene ABCC2 rs717620 and rs2273697; sodium channel subunit gene SCN1A rs3812718, SCN2A rs2304016; γ-amino butyric acid type A (GABAA) receptor subunit subtype gene GABRA1 rs2279020 were genotyped following the Illumina protocols. There were no significant differences in allelic or genotypic frequencies between the drug responsive and resistant patients. The polymorphisms of the above SNP loci may not be associated with drug resistance of epilepsy in the Chinese Han population.

  15. A hybrid dynamic Bayesian network approach for modelling temporal associations of gene expressions for hypertension diagnosis.

    Science.gov (United States)

    Akutekwe, Arinze; Seker, Huseyin

    2014-01-01

    Computational and machine learning techniques have been applied in identifying biomarkers and constructing predictive models for diagnosis of hypertension. Strategies such as improved classification rules based on decision trees have been proposed. Other techniques such as Fuzzy Expert Systems (FES) and Neuro-Fuzzy Systems (NFS) have recently been applied. However, these methods lack the ability to detect temporal relationships among biomarker genes that will aid better understanding of the mechanism of hypertension disease. In this paper we apply a proposed two-stage bio-network construction approach that combines the power and computational efficiency of classification methods with the well-established predictive ability of Dynamic Bayesian Network. We demonstrate our method using the analysis of male young-onset hypertension microarray dataset. Four key genes were identified by the Least Angle Shrinkage and Selection Operator (LASSO) and three Support Vector Machine Recursive Feature Elimination (SVM-RFE) methods. Results show that cell regulation FOXQ1 may inhibit the expression of focusyltransferase-6 (FUT6) and that ABCG1 ATP-binding cassette sub-family G may also play inhibitory role against NR2E3 nuclear receptor sub-family 2 and CGB2 Chromatin Gonadotrophin.

  16. Constitutive expression of Wnt/β-catenin target genes promotes proliferation and invasion of liver cancer stem cells

    Science.gov (United States)

    CHEN, WEI; ZHANG, YU-WEI; LI, YANG; ZHANG, JIAN-WEN; ZHANG, TONG; FU, BIN-SHENG; ZHANG, QI; JIANG, NAN

    2016-01-01

    Wnt/β-catenin is an important signaling pathways involved in the tumorgenesis, progression and maintenance of cancer stem cells (CSCs). In the present study, the role of Wnt/β-catenin signaling in CSC-mediated tumorigenesis and invasion in liver CSCs was investigated. A small population of cancer stem-like side population (SP) cells (3.6%) from liver cancer samples were identified. The cells were highly resistant to drug treatment due to the enhanced expression of drug efflux pumps, such as ABC subfamily G member 2, multidrug resistance protein 1 and ATP-binding cassette subfamily B member 5. Furthermore, using TOPflash and reverse transcription-quantitative polymerase chain reaction analysis, Wnt/β-catenin signaling and the transcriptional regulation of Wnt/β-catenin target genes including dickkopf Wnt signaling pathway inhibitor 1, axis inhibition protein 2 and cyclin D1 were observed to be markedly upregulated in liver cancer SP cells. As a consequence, SP cells possessed infinite cell proliferation potential and the ability to generating tumor spheres. In addition, upon reducing Wnt/β-catenin signaling, the rates of proliferation, tumor sphere formation and tumor invasion of SP cells were markedly reduced. Therefore, these data suggest that Wnt/β-catenin signaling is a potential therapeutic target to reduce CSC-mediated tumorigenicity and invasion in liver cancer. PMID:26956539

  17. Identification of a novel variant of staphylococcal cassette chromosome mec, type II.5, and Its truncated form by insertion of putative conjugative transposon Tn6012.

    Science.gov (United States)

    Han, Xiao; Ito, Teruyo; Takeuchi, Fumihiko; Ma, Xiao Xue; Takasu, Michihiko; Uehara, Yoshio; Oliveira, Duarte C; de Lencastre, Hermínia; Hiramatsu, Keiichi

    2009-06-01

    We identified two novel staphylococcal cassette chromosome mec (SCCmec) elements in sequence type 8 methicillin-resistant Staphylococcus aureus strains isolated in Japan: type II.5 SCCmec, whose J1 region was highly homologous to that of type I.2 SCCmec of strain PL72 (previously isolated in Poland), and its J1 region variant caused by the deletion/insertion of putative conjugative transposon Tn6012, identified in four S. aureus genomes.

  18. Estrous Cycle and Gestational Age-Dependent Expression of Members of the Interleukin-36 Subfamily in a Semi-Allogeneic Model of Infected and Non-Infected Murine Pregnancy

    Science.gov (United States)

    Murrieta-Coxca, José Martin; Gómez-Chávez, Fernando; Baeza-Martínez, Damariz Adriana; Cancino-Diaz, Mario Eugenio; Cancino-Diaz, Juan Carlos; Pérez-Tapia, Sonia Mayra; Reyes-Maldonado, Elba; Rodríguez-Martínez, Sandra

    2016-01-01

    The IL-36 subfamily is a recently described group of cytokines with pro-inflammatory behavior, comprising three agonists (α, β, and γ), its receptor (R), and one antagonist (Ra). The expression and function of IL-36 subfamily members in the estrous cycle in healthy and infected pregnancy has not been described. We evaluated mRNA and protein expression of IL-36 family members during the estrous cycle, implantation, fetal development, and post-labor periods in a model of allogenic pregnancy in mice. We also explored the ability of Listeria monocytogenes to modulate the expression of IL-36 subfamily members during pregnancy. Expression of IL-36 subfamily members showed different expression during the estrous cycle and pregnancy but was induced at estrous, 16.5 days post coitum (dpc), 18.5 dpc, and labor. IL-36 subfamily members showed a characteristic distribution in the glandular epithelium, perimetrium, myometrium, and stratum vasculare. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members, an observation that correlated with an increasing prevalence of fetal loss. In conclusion, IL-36 agonists showed specific patterns of mRNA and protein expression that might suggest functional specialization or specific target cells. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members. PMID:27713746

  19. Estrous cycle and gestational age-dependent expression of members of the interleukin-36 subfamily in a semi-allogeneic model of infected and non-infected murine pregnancy

    Directory of Open Access Journals (Sweden)

    José Martin Murrieta Coxca

    2016-09-01

    Full Text Available The IL-36 subfamily is a recently described group of cytokines with pro-inflammatory behavior, comprising three agonists (α, β and γ, its receptor (R and one antagonist (Ra. The expression and function of IL-36 subfamily members in the estrous cycle in healthy and infected pregnancy have not been described. We evaluated mRNA and protein expression of IL-36 family members during the estrous cycle, implantation, fetal development and post-labor periods in a model of allogenic pregnancy in mice. We also explored the ability of Listeria monocytogenes to modulate expression of IL-36 subfamily members during pregnancy. Expression of IL-36 subfamily members showed different expression during the estrous cycle and pregnancy, but was induced at estrous, 16.5 days post coitum (dpc, 18.5 dpc and labor. IL-36 subfamily members showed a characteristic distribution in the glandular epithelium, perimetrium, myometrium, and stratum vasculare. Infection with Listeria monocytogenes during pregnancy induced strong production of IL-36 subfamily members, an observation that correlated with an increasing prevalence of fetal loss. Conclusions: IL-36 agonists showed specific patterns of mRNA and protein expression that might suggest functional specialization or specific target cells. Infection with Listeria monocytogenes during pregnancy induced strong production of IL-36 subfamily members.

  20. Efficient simultaneous excision of multiple selectable marker cassettes using I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae.

    Science.gov (United States)

    Solis-Escalante, Daniel; Kuijpers, Niels G A; van der Linden, Franka H; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

    2014-08-01

    Large strain construction programs and functional analysis studies are becoming commonplace in Saccharomyces cerevisiae and involve construction of strains that carry multiple selectable marker genes. Extensive strain engineering is, however, severely hampered by the limited number of recyclable marker genes and by the reduced genome stability that occurs upon repeated use of heterologous recombinase-based marker removal methods. The present study proposes an efficient method to recycle multiple markers in S. cerevisiae simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection-excision. This method relies on artificial generation of double-strand breaks around the selection marker cassette by the meganuclease I-SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%), suggesting that I-SceI-based marker removal has the potential to co-excise an even larger number of markers. This locus- and marker-independent method can be used for both dominant and auxotrophy-complementing marker genes. Seven pDS plasmids carrying various selectable markers, which can be used for PCR-based generation of deletion cassettes suited for I-SceI marker recycling, are described and made available to the scientific community.

  1. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Science.gov (United States)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  2. Checklist of the subfamily Adoncholaiminae Gerlach and Riemann, 1974 (Nematoda: Oncholaimida: Oncholaimidae) of the world: genera, species, distribution, and reference list for taxonomists and ecologists

    Science.gov (United States)

    2016-01-01

    Abstract Background Adoncholaiminae is one of the seven subfamilies in the free-living aquatic nematode family Oncholaimidae. Nematodes in Adoncholaiminae are found from various water environment of the world. However, a checklist of all Adoncholaiminae species including full literature, especially information of experimental (not taxonomic) works, has not been updated for more than 40 years. New information A revised checklist of the subfamily Adoncholaiminae of the world is provided. It contains 31 valid and 13 invalid species names in four genera with synonyms, collection records, and full literature from 1860's to 2015 for each species. A literature survey of total 477 previous papers was conducted in this work, and 362 of them are newly added to checklist. PMID:26929708

  3. The discovery of phiAGATE, a novel phage infecting Bacillus pumilus, leads to new insights into the phylogeny of the subfamily Spounavirinae.

    Directory of Open Access Journals (Sweden)

    Jakub Barylski

    Full Text Available The Bacillus phage phiAGATE is a novel myovirus isolated from the waters of Lake Góreckie (a eutrophic lake in western Poland. The bacteriophage infects Bacillus pumilus, a bacterium commonly observed in the mentioned reservoir. Analysis of the phiAGATE genome (149844 base pairs resulted in 204 predicted protein-coding sequences (CDSs, of which 53 could be functionally annotated. Further investigation revealed that the bacteriophage is a member of a previously undescribed cluster of phages (for the purposes of this study we refer to it as "Bastille group" within the Spounavirinae subfamily. Here we demonstrate that these viruses constitute a distinct branch of the Spounavirinae phylogenetic tree, with limited similarity to phages from the Twortlikevirus and Spounalikevirus genera. The classification of phages from the Bastille group into any currently accepted genus proved extremely difficult, prompting concerns about the validity of the present taxonomic arrangement of the subfamily.

  4. Comparative analysis of serine/arginine-rich proteins across 27 eukaryotes: insights into sub-family classification and extent of alternative splicing.

    Directory of Open Access Journals (Sweden)

    Dale N Richardson

    Full Text Available Alternative splicing (AS of pre-mRNA is a fundamental molecular process that generates diversity in the transcriptome and proteome of eukaryotic organisms. SR proteins, a family of splicing regulators with one or two RNA recognition motifs (RRMs at the N-terminus and an arg/ser-rich domain at the C-terminus, function in both constitutive and alternative splicing. We identified SR proteins in 27 eukaryotic species, which include plants, animals, fungi and "basal" eukaryotes that lie outside of these lineages. Using RNA recognition motifs (RRMs as a phylogenetic marker, we classified 272 SR genes into robust sub-families. The SR gene family can be split into five major groupings, which can be further separated into 11 distinct sub-families. Most flowering plants have double or nearly double the number of SR genes found in vertebrates. The majority of plant SR genes are under purifying selection. Moreover, in all paralogous SR genes in Arabidopsis, rice, soybean and maize, one of the two paralogs is preferentially expressed throughout plant development. We also assessed the extent of AS in SR genes based on a splice graph approach (http://combi.cs.colostate.edu/as/gmap_SRgenes. AS of SR genes is a widespread phenomenon throughout multiple lineages, with alternative 3' or 5' splicing events being the most prominent type of event. However, plant-enriched sub-families have 57%-88% of their SR genes experiencing some type of AS compared to the 40%-54% seen in other sub-families. The SR gene family is pervasive throughout multiple eukaryotic lineages, conserved in sequence and domain organization, but differs in gene number across lineages with an abundance of SR genes in flowering plants. The higher number of alternatively spliced SR genes in plants emphasizes the importance of AS in generating splice variants in these organisms.

  5. Two new mite species of the subfamily Harpirhynchinae Dubinin, 1957 (Acariformes: Harpirhynchidae), parasites of the passerine birds (Aves: Passeriformes) in Australia and South Asia.

    Science.gov (United States)

    Bochkov, Andre V; Klompen, Hans

    2015-09-01

    Two new mite species of the subfamily Harpirhynchinae Dubinin, 1957 (Acariformes: Harpirhynchidae) are described from passerine birds (Aves: Passeriformes): Harpirhynchoides artamus n. sp. from Artamus fuscus Vieillot (Artamidae) from an unknown locality in South Asia and Neharpyrhynchus domrowi n. sp. from three host species of the family Meliphagidae, Acanthorhynchus tenuirostris (Latham) (type-host) from Australia (New South Walles), Ptiloprora perstriata (De Vis) and Myzomela rosenbergii Schlegel from Papua New Guinea.

  6. The expression of T-cell receptor Vβ subfamily in hepatitis B virus-related acute-on-chronic liver failure patients and its clinical significance

    Institute of Scientific and Technical Information of China (English)

    施文娟

    2014-01-01

    Objective To investigate the expression and clinical significance of T-cell receptor(TCR)Vβsubfamily in hepatitis B virus(HBV)-related acute-on-chronic liverfailure(HBV-ACLF)patients.Methods Twenty-eight patients with HBV-ACLF(HBV-ACLF group)and 32patients with chronic hepatitis B flare(CHB-F group),who were treated in The Second People’s Hospital from

  7. Genetic association of aromatic hydrocarbon receptor (AHR) and cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) polymorphisms with dioxin blood concentrations among pregnant Japanese women

    OpenAIRE

    Kobayashi, Sumitaka; Sata, Fumihiro; Sasaki, Seiko; Ban, Susumu; Miyashita, Chihiro; Okada, Emiko; Limpar, Mariko; Yoshioka, Eiji; Kajiwara, Jumboku; TODAKA, Takashi; Saijo, Yasuaki; Kishi, Reiko

    2013-01-01

    Dioxins are metabolized by cytochrome P450, family 1 (CYP1) via the aromatic hydrocarbon receptor (AHR). We determined whether different blood dioxin concentrations are associated with polymorphisms in AHR (dbSNP ID: rs2066853), AHR repressor (AHRR; rs2292596), CYP1 subfamily A polypeptide 1 (CYP1A1; rs4646903 and rs1048963), CYP1 subfamily A polypeptide 2 (CYP1A2; rs762551), and CYP1 subfamily B polypeptide 1 (CYP1B1; rs1056836) in pregnant Japanese women. These six polymorphisms were detect...

  8. A review of the mite subfamily Harpirhynchinae (Acariformes: Harpirhynchidae)--parasites of New World birds (Aves: Neognathae).

    Science.gov (United States)

    Bochkov, Andre V; OConnor, Barry M; Klompen, Hans

    2015-09-30

    Mites of the subfamily Harpirhynchinae (Acariformes: Cheyletoidea: Harpirhynchidae) associated with neognathous birds (Aves: Neognathae) in the New World are revised. In all, 68 species in 8 genera are recorded. Among them, 27 new species and 1 new genus are described as new for science: Harpyrhynchoides gallowayi Bochkov, OConnor and Klompen sp. nov. from Columba livia (Columbiformes: Columbidae) from Canada (Manitoba), H. zenaida Bochkov, OConnor and Klompen sp. nov. from Zenaida macroura (Columbiformes: Columbidae) from USA (Michigan), H. calidris Bochkov, OConnor and Klompen sp. nov. from Calidris minutilla (Charadriiformes: Scolopacidae) from USA (Kansas), H. actitis Bochkov, OConnor and Klompen sp. nov. from Actitis macularius (Charadriiformes: Scolopacidae) from Canada (British Columbia), H. charadrius Bochkov, OConnor and Klompen sp. nov. from Charadrius vociferus (Charadriiformes: Charadriidae) from USA (Texas), H. pluvialis Bochkov, OConnor and Klompen sp. nov. from Pluvialis dominica (Charadriiformes: Charadriidae) from USA (Ohio), H. bubulcus Bochkov, OConnor and Klompen sp. nov. from Bubulcus ibis (Pelecaniformes: Ardeidae) from USA (Florida), H. ixobrychus Bochkov, OConnor and Klompen sp. nov. from Ixobrychus exilis (Pelecaniformes: Ardeidae) from USA (Michigan), H. puffinus Mertins sp. nov. from Puffinus gravis (Procellariformes: Procellariidae) from USA (Florida), H. megascops Bochkov, OConnor and Klompen sp. nov. from Megascops asio (Strigiformes: Strigidae) from USA (Michigan), H. athene Bochkov, OConnor and Klompen sp. nov. from Athene canicularia (Strigiformes: Strigidae) from USA (Texas), H. coccyzus Bochkov, OConnor and Klompen sp. nov. from Coccyzus americanus (Cuculiformes: Cuculidae) from USA (Michigan), H. crotophaga Bochkov, OConnor and Klompen sp. nov. from Crotophaga ani (Cuculiformes: Cuculidae) from Suriname; Crassacarus Bochkov, OConnor and Klompen, gen. nov.: Crassacarus alexfaini Bochkov, OConnor and Klompen sp. nov. (type of genus

  9. Crystal structure of a raw-starch-degrading bacterial α-amylase belonging to subfamily 37 of the glycoside hydrolase family GH13

    Science.gov (United States)

    Liu, Yanhong; Yu, Jigang; Li, Fudong; Peng, Hui; Zhang, Xuecheng; Xiao, Yazhong; He, Chao

    2017-01-01

    Subfamily 37 of the glycoside hydrolase family GH13 was recently established on the basis of the discovery of a novel α-amylase, designated AmyP, from a marine metagenomic library. AmyP exhibits raw-starch-degrading activity and consists of an N-terminal catalytic domain and a C-terminal starch-binding domain. To understand this newest subfamily, we determined the crystal structure of the catalytic domain of AmyP, named AmyPΔSBD, complexed with maltose, and the crystal structure of the E221Q mutant AmyPΔSBD complexed with maltotriose. Glu221 is one of the three conserved catalytic residues, and AmyP is inactivated by the E221Q mutation. Domain B of AmyPΔSBD forms a loop that protrudes from domain A, stabilizes the conformation of the active site and increases the thermostability of the enzyme. A new calcium ion is situated adjacent to the -3 subsite binding loop and may be responsible for the increased thermostability of the enzyme after the addition of calcium. Moreover, Tyr36 participates in both stacking and hydrogen bonding interactions with the sugar motif at subsite -3. This work provides the first insights into the structure of α-amylases belonging to subfamily 37 of GH13 and may contribute to the rational design of α-amylase mutants with enhanced performance in biotechnological applications. PMID:28303907

  10. Morphological "primary homology" and expression of AG-subfamily MADS-box genes in pines, podocarps, and yews.

    Science.gov (United States)

    Englund, Marie; Carlsbecker, Annelie; Engström, Peter; Vergara-Silva, Francisco

    2011-01-01

    The morphological variation among reproductive organs of extant gymnosperms is remarkable, especially among conifers. Several hypotheses concerning morphological homology between various conifer reproductive organs have been put forward, in particular in relation to the pine ovuliferous scale. Here, we use the expression patterns of orthologs of the ABC-model MADS-box gene AGAMOUS (AG) for testing morphological homology hypotheses related to organs of the conifer female cone. To this end, we first developed a tailored 3'RACE procedure that allows reliable amplification of partial sequences highly similar to gymnosperm-derived members of the AG-subfamily of MADS-box genes. Expression patterns of two novel conifer AG orthologs cloned with this procedure-namely PodAG and TgAG, obtained from the podocarp Podocarpus reichei and the yew Taxus globosa, respectively-are then further characterized in the morphologically divergent female cones of these species. The expression patterns of PodAG and TgAG are compared with those of DAL2, a previously discovered Picea abies (Pinaceae) AG ortholog. By treating the expression patterns of DAL2, PodAG, and TgAG as character states mapped onto currently accepted cladogram topologies, we suggest that the epimatium-that is, the podocarp female cone organ previously postulated as a "modified" ovuliferous scale-and the canonical Pinaceae ovuliferous scale can be legitimally conceptualized as "primary homologs." Character state mapping for TgAG suggests in turn that the aril of Taxaceae should be considered as a different type of organ. This work demonstrates how the interaction between developmental-genetic data and formal cladistic theory could fruitfully contribute to gymnosperm systematics.

  11. The ZNF75 zinc finger gene subfamily: Isolation and mapping of the four members in humans and great apes

    Energy Technology Data Exchange (ETDEWEB)

    Villa, A.; Strina, D.; Frattini, A. [Consiglio Nazionale delle Ricerche, Milan (Italy)] [and others

    1996-07-15

    We have previously reported the characterization of the human ZNF75 gene located on Xq26, which has only limited homology (less than 65%) to other ZF genes in the databases. Here, we describe three human zinc finger genes with 86 to 95% homology to ZNF75 at the nucleotide level, which represent all the members of the human ZNF75 subfamily. One of these, ZNF75B, is a pseudogene mapped to chromosome 12q13. The other two, ZNF75A and ZNF75C, maintain on ORF in the sequenced region, and at least the latter is expressed in the U937 cell line. They were mapped to chromosomes 16 and 11, respectively. All these genes are conserved in chimpanzees, gorillas, and orangutans. The ZNF75B homologue is a pseudogene in all three great apes, and in chimpanzee it is located on chromosome 10 (phylogenetic XII), at p13 (corresponding to the human 12q13). The chimpanzee homologue of ZNF75 is also located on the Xq26 chromosome, in the same region, as detected by in situ hybridization. As expected, nucleotide changes were clearly more abundant between human and organutan than between human and chimpanzee or gorilla homologues. Members of the same class were more similar to each other than to the other homologues within the same species. This suggests that the duplication and/or retrotranscription events occurred in a common ancestor long before great ape speciation. This, together with the existance of at least two genes in cows and horses, suggests a relatively high conservation of this gene family. 20 refs., 5 figs., 1 tab.

  12. A novel fractal approach for predicting G-protein-coupled receptors and their subfamilies with support vector machines.

    Science.gov (United States)

    Nie, Guoping; Li, Yong; Wang, Feichi; Wang, Siwen; Hu, Xuehai

    2015-01-01

    G-protein-coupled receptors (GPCRs) are seven membrane-spanning proteins and regulate many important physiological processes, such as vision, neurotransmission, immune response and so on. GPCRs-related pathways are the targets of a large number of marketed drugs. Therefore, the design of a reliable computational model for predicting GPCRs from amino acid sequence has long been a significant biomedical problem. Chaos game representation (CGR) reveals the fractal patterns hidden in protein sequences, and then fractal dimension (FD) is an important feature of these highly irregular geometries with concise mathematical expression. Here, in order to extract important features from GPCR protein sequences, CGR algorithm, fractal dimension and amino acid composition (AAC) are employed to formulate the numerical features of protein samples. Four groups of features are considered, and each group is evaluated by support vector machine (SVM) and 10-fold cross-validation test. To test the performance of the present method, a new non-redundant dataset was built based on latest GPCRDB database. Comparing the results of numerical experiments, the group of combined features with AAC and FD gets the best result, the accuracy is 99.22% and Matthew's correlation coefficient (MCC) is 0.9845 for identifying GPCRs from non-GPCRs. Moreover, if it is classified as a GPCR, it will be further put into the second level, which will classify a GPCR into one of the five main subfamilies. At this level, the group of combined features with AAC and FD also gets best accuracy 85.73%. Finally, the proposed predictor is also compared with existing methods and shows better performances.

  13. Stereochemistry of Fuscumol and Fuscumol Acetate Influences Attraction of Longhorned Beetles (Coleoptera: Cerambycidae) of the Subfamily Lamiinae.

    Science.gov (United States)

    Hughes, G P; Meier, L R; Zou, Y; Millar, J G; Hanks, L M; Ginzel, M D

    2016-10-01

    The chemical structures of aggregation-sex pheromones of lon