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1

ATP-binding cassette transporters in liver.  

PubMed

The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed. PMID:24105869

Wlcek, Katrin; Stieger, Bruno

2014-01-01

2

ATP Binding Cassette A1 Transporter Function and Tangier Disease  

Microsoft Academic Search

\\u000a The ATP binding cassette A1 (ABCA1) transporter facilitates the efflux of free cholesterol and phospholipid onto high density\\u000a lipoprotein (HDL) particles, mainly very small discoidal precursor HDL particles, known as prebeta 1 HDL. Once these particles\\u000a pick up these lipids, they are converted to small discoidal alpha 4 migrating HDL and with cholesterol esterification mature\\u000a into larger spherical alpha 3

Ernst J. Schaefer; H. Bryan Brewer

3

The ATP-binding cassette family: a structural perspective  

Microsoft Academic Search

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes,\\u000a using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes,\\u000a these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of\\u000a the

Veronica Kos; Robert Curtis Ford

2009-01-01

4

ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei  

Microsoft Academic Search

BACKGROUND: ATP binding cassette (ABC) systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243

David N Harland; Elie Dassa; Richard W Titball; Katherine A Brown; Helen S Atkins

2007-01-01

5

ATP-Binding Cassette Efflux Transporters in Human Placenta  

PubMed Central

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

6

ATP Binding Cassette Modulators Control Abscisic AcidRegulated Slow Anion Channels in Guard Cells  

Microsoft Academic Search

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea re- ceptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is

Nathalie Leonhardt; Alain Vavasseur; Cyrille Forestier

1999-01-01

7

Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview  

Microsoft Academic Search

Active drug efflux transporters of the ATP binding cassette (ABC)-containing family of proteins have a major impact on the pharmacological behavior of most of the drugs in use today. Pharmacological properties affected by ABC transporters include the oral bioavailability, hepatobiliary, direct intestinal, and urinary excretion of drugs and drug-metabolites and -conjugates. Moreover, the penetration of drugs into a range of

Alfred H Schinkel; Johan W Jonker

2003-01-01

8

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans  

E-print Network

that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans gene haf-6 is required for efficient RNAi. HAF-6 is a member of the ATP-binding cassette (ABC...

Timmons, Lisa; Hull, Dawn; Han, Wang; Echalier, B.; Sundaram, P.

2006-08-01

9

The role of Brugia malayi ATP-binding cassette (ABC) transporters in potentiating drug sensitivity  

Microsoft Academic Search

ATP binding cassette (ABC) systems are a diverse group of proteins that have been identified in every organism, from bacteria\\u000a to humans. Analysis of nematode genomes indicates that the number and arrangement of ABC systems are similar to other organisms,\\u000a with the majority being ABC transporters. There are few functional studies of ABC transporters in parasitic nematodes; most\\u000a reports have

Jeffrey B. Tompkins; Laurel E. Stitt; Alana M. Morrissette; Bernadette F. Ardelli

10

Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters  

Microsoft Academic Search

A group of ATP-binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance-associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP-related proteins are also characterized by a special, common membrane topology pattern.

Gábor E Tusnády; Éva Bakos; András Váradi; Balázs Sarkadi

1997-01-01

11

The Repertoire and Evolution of ATP-Binding Cassette Systems in Synechococcus and Prochlorococcus  

Microsoft Academic Search

Synechococcus and Prochlorococcus have made great contributions to earth’s photosynthetic biomass. ATP-binding cassette (ABC) protein systems have been characterized\\u000a to play important roles in various physiological functions, including carbon fixation, phosphate assimilation, and vitamin\\u000a B12 metabolism. In this study, the repertoire and domain architectures of ABC systems in Synechococcus and Prochlorococcus, as well as their potential evolutionary mechanism, have been

Lijing Bu; Jian Xiao; Lijun Lu; Gang Xu; Jinsong Li; Fangqing Zhao; Xiaokun Li; Jinyu Wu

2009-01-01

12

Cooperative, ATP-dependent association of the nucleotide binding cassettes during the catalytic cycle of ATP-binding cassette transporters.  

PubMed

ATP-binding cassette (ABC) transporters harvest the energy present in cellular ATP to drive the translocation of a structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria, and eukaryotes. The positively cooperative ATPase activity (Hill coefficient, 1.7) of a model soluble cassette of known structure, MJ0796, from Methanococcus jannaschii indicates that at least two binding sites participate in the catalytic reaction. Mutation of the catalytic base in MJ0796, E171Q, produced a cassette that can bind but not efficiently hydrolyze ATP. The equivalent mutation (E179Q) in a homologous cassette, MJ1267, had an identical effect. Both mutant cassettes formed dimers in the presence of ATP but not ADP, indicating that the energy of ATP binding is first coupled to the transport cycle through a domain association reaction. The non-hydrolyzable nucleotides adenosine 5'-(beta,gamma-imino)triphosphate and adenosine 5'-3-O-(thio)triphosphate were poor analogues of ATP in terms of their ability to promote dimerization. Moreover, inclusion of MgCl2, substitution of KCl for NaCl, or alterations in the polarity of the side chain at the catalytic base all weakened the ATP-dependent dimer, suggesting that electrostatic interactions are critical for the association reaction. Thus, upon hydrolysis of bound ATP and the release of product, both electrostatic and conformational changes drive the cassettes apart, providing a second opportunity to couple free energy changes to the transport reaction. PMID:11964392

Moody, Jonathan E; Millen, Linda; Binns, Derk; Hunt, John F; Thomas, Philip J

2002-06-14

13

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

PubMed Central

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. PMID:23324493

2013-01-01

14

Phylogenetic and functional classification of ATP-binding cassette (ABC) systems.  

PubMed

ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases. PMID:12370001

Bouige, Philippe; Laurent, David; Piloyan, Linda; Dassa, Elie

2002-10-01

15

ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism  

NASA Astrophysics Data System (ADS)

Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

Liao, Jielou

2004-03-01

16

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems  

PubMed Central

Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases. PMID:18535149

Davidson, Amy L.; Dassa, Elie; Orelle, Cedric; Chen, Jue

2008-01-01

17

Influence of ATP-Binding Cassette Polymorphisms on Neurological Outcome After Traumatic Brain Injury  

PubMed Central

Background As important mediators of solute transport at the blood–brain and blood–cerebrospinal fluid barriers, ATP-binding cassette (ABC) transporters (including ABCB1, ABCC1, and ABCC2), impact the bioavailability of drugs and endogenous substrates in the brain. While several ABCB1, ABCC1, and ABCC2 single nucleotide polymorphisms (SNPs) have been identified, their impact on outcome after traumatic brain injury (TBI) is unknown. Hypothesis ABCB1, ABCC1, and ABCC2 SNPs are associated with Glasgow Outcome Scale (GOS) score after TBI. Methods DNA samples from 305 adult patients with severe TBI (Glasgow Coma Scale, GCS score ? 8) were genotyped for tagging SNPs of ABCB1 (rs1045642; rs1128503), ABCC1 (rs212093; rs35621; rs4148382), and ABCC2 (rs2273697). For each SNP, patients were dichotomized based on presence of variant allele for multivariate analysis to determine associations with GOS assigned at 6 months adjusting for GCS, Injury Severity score, age, and patient sex. Results For ABCB1 rs1045642, patients homozygous for the T allele were less likely to be assigned poor outcome versus those possessing the C allele [CT/CC; odds of unfavorable GOS = 0.71(0.55?0.92)]. For ABCC1 rs4148382, patients homozygous for the G allele were less likely to be assigned poor outcome versus those possessing the A allele [AG/AA; odds of unfavorable GOS = 0.73(0.55?0.98)]. Conclusions In this single-center study, patients homozygous for the T allele of ABCB1 rs1045642 or the G allele of ABCC1 rs4148382 were found to have better outcome after severe TBI. Further study is necessary to replicate these very preliminary findings and to determine whether these associations are due to central nervous system bioavailability of ABC transporter drug substrates commonly used in the management of TBI, brain efflux of endogenous solutes, or both. PMID:23896815

Cousar, J'mir L.; Conley, Yvette P.; Willyerd, F. Anthony; Sarnaik, Ajit A.; Puccio, Ava M.; Okonkwo, David O.; Empey, Philip E.; Kochanek, Patrick M.; Bell, Michael J.

2014-01-01

18

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

PubMed

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

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

2013-11-01

19

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

PubMed

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. PMID:25156004

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

2014-09-01

20

NO1886 upregulates ATP binding cassette transporter A1 and inhibits diet-induced atherosclerosis in Chinese Bama minipigs  

Microsoft Academic Search

It is widely believed that high density lipoprotein- cholesterol (HDL-C) functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport (RCT), a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP binding cassette transporter called ABCA1 mediates the first step of RCT. NO-1886 has been proven to be highly

Chi Zhang; Weidong Yin; Duanfang Liao; Liang Huang; Chaoke Tang; Kazuhiko Tsutsumi; Zongbao Wang; Yi Liu; Qinkai Li; Hongjie Hou; Manbo Cai; Junxia Xiao

2006-01-01

21

Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection  

E-print Network

of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry

Paris-Sud XI, Université de

22

Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2  

Microsoft Academic Search

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (Her-1 or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor

Chun-ling Dai; Amit K. Tiwari; Chung-Pu Wu; Xiao-dong Su; Si-Rong Wang; Dong-geng Liu; Yan Huang; Robert W. Robey; Yong-ju Liang; Li-ming Chen; Cheng-Jun Shi; Suresh V. Ambudkar; Zhe-Sheng Chen

2008-01-01

23

Vacuolar import of phosphatidylcholine requires the ATP-binding cassette transporter Ybt1  

PubMed Central

ATP-binding cassette (ABC) transporters are well-known for their roles as multidrug resistance determinants but also play important roles in regulation of lipid levels. In the yeast Saccharomyces cerevisiae, the plasma membrane ABC transporter proteins Pdr5 and Yor1 are required for normal rates of transport of phosphatidyethanolamine to the surface of the cell. Loss of these ABC transporters causes a defect in phospholipid asymmetry across the plasma membrane and has bee linked with slowed rates of trafficking of other membrane proteins. Four ABC transporter proteins are found on the limiting membrane of the yeast vacuole and loss of one of these vacuolar ABC transporters, Ybt1, caused a major defect in the normal delivery of the phosphatidylcholine (PC) analogue NBD-PC (7-nitro-2,1,3-benzoxadiazol-PC) to the lumen of the vacuole. NBD-PC accumulates on cytosolic membranes in a ybt1? strain. We demonstrated that Ybt1 is required to import NBD-PC into vacuoles in the presence of ATP in vitro. Loss of Ybt1 prevented vacuolar remodeling of PC analogues. Turnover of Ybt1 was reduced under conditions in which function of this vacuolar remodeling pathway was required. Our data describe a novel vacuolar route for lipid remodeling and reutilization in addition to previously described enzymatic avenues in the cytoplasm. PMID:21649806

Gulshan, Kailash; Moye-Rowley, W. Scott

2011-01-01

24

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

PubMed

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. PMID:25006243

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

2014-08-22

25

Cyclic Nucleotide Compartmentalization: Contributions of Phosphodiesterases and ATP-Binding Cassette Transporters  

PubMed Central

Cyclic nucleotides [e.g., cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)] are ubiquitous second messengers that affect multiple cell functions from maturation of the egg to cell division, growth, differentiation, and death. The concentration of cAMP can be regulated by processes within membrane domains (local regulation) as well as throughout a cell (global regulation). The phosphodiesterases (PDEs) that degrade cAMP have well-known roles in both these processes. It has recently been discovered that ATP-binding cassette (ABC) transporters contribute to both local and global regulation of cAMP. This regulation may require the formation of macromolecular complexes. Some of these transporters are ubiquitously expressed, whereas others are more tissue restricted. Because some PDE inhibitors are also ABC transporter inhibitors, it is conceivable that the therapeutic benefits of their use result from the combined inhibition of both PDEs and ABC transporters. Deciphering the individual contributions of PDEs and ABC transporters to such drug effects may lead to improved therapeutic benefits. PMID:23072381

Cheepala, Satish; Hulot, Jean-Sebastien; Morgan, Jessica A.; Sassi, Yassine; Zhang, Weiqiang; Naren, Anjaparavanda P.; Schuetz, John D.

2015-01-01

26

ATP binding cassette transporter A-I and HDL metabolism: Effects of fatty acids  

PubMed Central

Ample evidence indicates that dietary fatty acids alters the plasma levels of HDL-C. However, the mechanisms underlying the effects of fatty acids still remain elusive. Recent advances in our understanding of ATP binding cassette transporter A1 (ABCA1) function and regulation have provided a valuable insight into the mechanisms by which fatty acids may affect plasma HDL-C levels. ABCA1 mediates the assembly of phospholipids and free cholesterol with apolipoprotein A-I, which is a critical step for HDL biogenesis. Studies have shown that unsaturated fatty acids, but not saturated fatty acids, repress the expression of ABCA1 in vitro. Although information on mechanisms for the fatty acid-mediated regulation of ABCA1 expression is still limited and controversial, recent evidence suggests that unsaturated fatty acids inhibit the expression of ABCA1 at the transcriptional and posttranscriptional levels. The transcriptional repression of ABCA1 expression by unsaturated fatty acids is likely LXR-dependent. Evidence also suggests that histone deacetylation may play a role in the repression. Posttranscriptionally, unsaturated fatty acids may facilitate ABCA1 protein degradation, which may involve phosphorylation of ABCA1 by protein kinases. Further studies are warranted to better understand the role of dietary fatty acids in HDL metabolism and their effects on cardiovascular health. PMID:21684139

Lee, Jiyoung; Park, Youngki; Koo, Sung I.

2011-01-01

27

Rice shoot branching requires an ATP-binding cassette subfamily G protein.  

PubMed

* Shoot branching is important for the establishment of plant architecture and productivity. * Here, characterization of rice (Oryza sativa) reduced culm number 1 (rcn1) mutants revealed that Rcn1 positively controls shoot branching by promoting the outgrowth of lateral shoots. Molecular studies revealed that Rcn1 encodes a novel member of ATP-binding cassette protein subfamily G (ABCG subfamily), also known as the white-brown complex (WBC) subfamily, and is designated OsABCG5. * Rcn1 is expressed in leaf primordia of main and axillary shoots, and in the vascular cells and leaf epidermis of older leaves. In addition, Rcn1 is expressed in the crown root primordia, endodermis, pericycle and stele in the root. No effect on Rcn1 expression in shoots or roots was seen when the roots were treated with auxins. Phenotypic analyses of rcn1 and tillering dwarf 3 (d3) double mutants at the seedling stage clarified that Rcn1 works independently of D3 in the branching inhibitor pathway. * Rcn1 is the first functionally defined plant ABCG protein gene that controls shoot branching and could thus be significant in future breeding for high-yielding rice. PMID:19140940

Yasuno, Naoko; Takamure, Itsuro; Kidou, Shin-ichiro; Tokuji, Yoshihiko; Ureshi, An-na; Funabiki, Atsushi; Ashikaga, Kazunori; Yamanouchi, Utako; Yano, Masahiro; Kato, Kiyoaki

2009-01-01

28

ATP-binding cassette transporter A1: From metabolism to neurodegeneration.  

PubMed

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoprotein A-I (apoA-I) and apolipoprotein E (apoE). ABCA1 is an essential regulator of high density lipoproteins (HDL) and reverse cholesterol transport - a role that determines its importance for atherosclerosis. Over the last 10years studies have provided convincing evidence that ABCA1, via its control of apoE lipidation, also has a role in Alzheimer's disease (AD). A series of reports have revealed a significant impact of ABCA1 on A? deposition and clearance in AD model mice, as well as an association of common and rare ABCA1 gene variants with the risk for AD. Since APOE is the major genetic risk factor for late onset AD, the regulation of apoE level or its functionality by ABCA1 may prove significant for AD pathogenesis. ABCA1 is transcriptionally regulated by Liver X Receptors (LXR) and Retinoic X Receptors (RXR) which provides a starting point for drug discovery and development of synthetic LXR and RXR agonists for treatment of metabolic and neurodegenerative disorders. This review summarizes the recent results of research on ABCA1, particularly relevant to atherosclerosis and AD. PMID:24844148

Koldamova, Radosveta; Fitz, Nicholas F; Lefterov, Iliya

2014-12-01

29

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly*?  

PubMed Central

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, Fe2+ 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. PMID:25006243

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

2014-01-01

30

High density lipoprotein deficiency and foam cell accumulation in mice with targeted disruption of ATP-binding cassette transporter-1  

Microsoft Academic Search

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting

John McNeish; Robert J. Aiello; Deborah Guyot; Tom Turi; Christopher Gabel; Charles Aldinger; Kenneth L. Hoppe; Marsha L. Roach; Lori J. Royer; Jeffery de Wet; Cyril Broccardo; Giovanna Chimini; Omar L. Francone

2000-01-01

31

Expression and localization of ATP binding cassette transporter A1 (ABCA1) in first trimester and term human placenta  

Microsoft Academic Search

ATP binding cassette transporter A1 (ABCA1) is a membrane transporter which performs cellular efflux of cholesterol and phospholipid. ABCA1's cholesterol transporting role in human placenta appears to be crucial for normal fetal development. Despite the critical importance of cholesterol in fetal development, expression of ABCA1 in the human placenta throughout gestation and its specific cellular localization have not been known

J. Bhattacharjee; F. Ietta; E. Giacomello; N. Bechi; R. Romagnoli; A. Fava; L. Paulesu

2010-01-01

32

The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial  

Microsoft Academic Search

The ABCR gene encodes a rod photoreceptor specific ATP-binding cassette transporter. Mutations in ABCR are associated with at least four inherited retinal dystrophies: Stargardt disease, Fundus Flavimaculatus, cone-rod dystrophy, and retinitis pigmentosa. A statistically significant increase in heterozygous ABCR alterations has been identified in patients with age-related macular degeneration (AMD). A pedigree is described which manifests both Stargardt disease and

Noah F Shroyer; Richard Alan Lewis; Rando Allikmets; Nanda Singh; Michael Dean; Mark Leppert; James R Lupski

1999-01-01

33

Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters  

Microsoft Academic Search

.   ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate\\u000a in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called\\u000a here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first

William Saurin; Maurice Hofnung; Elie Dassa

1999-01-01

34

Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state  

PubMed Central

Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors’ knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3–6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters. PMID:24920594

Choudhury, Hassanul G.; Tong, Zhen; Mathavan, Indran; Li, Yanyan; Iwata, So; Zirah, Séverine; Rebuffat, Sylvie; van Veen, Hendrik W.; Beis, Konstantinos

2014-01-01

35

Small Substrate Transport and Mechanism of a Molybdate ATP Binding Cassette Transporter in a Lipid Environment*  

PubMed Central

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. PMID:24722984

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

2014-01-01

36

Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia  

PubMed Central

ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor ?B (NF-?B) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-?B signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

Gibson, Christopher J.; Hossain, Muhammad M.; Richardson, Jason R.

2012-01-01

37

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

SciTech Connect

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 HisP, the proteins suspected to energize these transporters, shared as much as 32% identity in amino acid residues when their sequences were aligned (171). Later, it was found that several bacterial proteins involved in uptake of nutrients, export of toxins, cell division, bacterial nodulation of plants, and DNA repair displayed the same similarity in their sequences (127, 196). This led to the notion that the conserved protein, which had been shown to bind ATP (198, 201), would probably energize the systems mentioned above by coupling the energy of ATP hydrolysis to transport. The latter was demonstrated with the maltose and histidine transporters by use of isolated membrane vesicles (105, 379) and purified transporters reconstituted into proteoliposomes (30, 98). The determination of the sequence of the first eukaryotic protein strongly similar to these bacterial transporters (the P-glycoprotein, involved in resistance of cancer cells to multiple drugs) (169, 179) demonstrated that these proteins were not restricted to prokaryotes. Two names, 'traffic ATPases' (15) and the more accepted name 'ABC transporters' (193, 218), were proposed for members of this new superfamily. ABC systems can be divided into three main functional categories, as follows. Importers mediate the uptake of nutrients in prokaryotes. The nature of the substrates that are transported is very wide, including mono- and oligosaccharides, organic and inorganic ions, amino acids, peptides, ironsiderophores, metals, polyamine cations, opines, and vitamins. Exporters are involved in the secretion of various molecules, such as peptides, lipids, hydrophobic drugs, polysaccharides, and proteins, including toxins such as hemolysin. The third category of systems is apparently not involved in transport, with some members being involved in translation of mRNA and in DNA repair. Despite the large, diverse population of substrates handled and the difference in the polarity of transport, importers and exporters share a common organization made of two hydrophobic membrane-spanning or integral membrane (IM)

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

2010-07-27

38

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

PubMed Central

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. PMID:22906146

2012-01-01

39

Duplication of genes in an ATP-binding cassette transport system increases dynamic range while maintaining ligand specificity.  

PubMed

Many bacteria exist in a state of feast or famine where high nutrient availability leads to periods of growth followed by nutrient scarcity and growth stagnation. To adapt to the constantly changing nutrient flux, metabolite acquisition systems must be able to function over a broad range. This, however, creates difficulties as nutrient concentrations vary over many orders of magnitude, requiring metabolite acquisition systems to simultaneously balance ligand specificity and the dynamic range in which a response to a metabolite is elicited. Here we present how a gene duplication of a periplasmic binding protein in a mannose ATP-binding cassette transport system potentially resolves this dilemma through gene functionalization. Determination of ligand binding affinities and specificities of the gene duplicates with fluorescence and circular dichroism demonstrates that although the binding specificity is maintained the Kd values for the same ligand differ over three orders of magnitude. These results suggest that this metabolite acquisition system can transport ligand at both low and high environmental concentrations while preventing saturation with related and less preferentially metabolized compounds. The x-ray crystal structures of the ?-mannose-bound proteins help clarify the structural basis of gene functionalization and reveal that affinity and specificity are potentially encoded in different regions of the binding site. These studies suggest a possible functional role and adaptive advantage for the presence of two periplasmic-binding proteins in ATP-binding cassette transport systems and a way bacteria can adapt to varying nutrient flux through functionalization of gene duplicates. PMID:25210043

Ghimire-Rijal, Sudipa; Lu, Xun; Myles, Dean A; Cuneo, Matthew J

2014-10-24

40

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

PubMed Central

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. PMID:25268163

Wang, Yi-Jun; Zhang, Yun-Kai; Kathawala, Rishil J.; Chen, Zhe-Sheng

2014-01-01

41

AST1306, a potent EGFR inhibitor, antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance.  

PubMed

AST1306, an inhibitor of EGFR and ErbB2, is currently in phase I of clinical trials. We evaluated the effect of AST306 on the reversal of multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters. We found that AST1306 significantly sensitized the ABC subfamily G member 2 (ABCG2)-overexpressing cells to ABCG2 substrate chemotherapeutics. AST1306 significantly increased intracellular accumulation of [(3)H]-mitoxantrone in ABCG2-overexpressing cells by blocking ABCG2 efflux function. Moreover, AST1306 stimulated the ATPase activity of ABCG2. Homology modeling predicted the binding conformation of AST1306 to be within the transmembrane region of ABCG2. In conclusion, AST1306 could notably reverse ABCG2-mediated MDR. PMID:24747122

Zhang, Hui; Wang, Yi-Jun; Zhang, Yun-Kai; Wang, De-Shen; Kathawala, Rishil J; Patel, Atish; Talele, Tanaji T; Chen, Zhe-Sheng; Fu, Li-Wu

2014-08-01

42

Quantitation of ATP-binding cassette subfamily-A transporter gene expression in primary human brain cells.  

PubMed

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively. PMID:16738483

Kim, Woojin S; Guillemin, Gilles J; Glaros, Elias N; Lim, Chai K; Garner, Brett

2006-06-26

43

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

44

Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240  

Microsoft Academic Search

A single gene cluster encoding components of a putative ATP-binding cassette (ABC) transporter for basic amino acids was identified in the incomplete genome sequence of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus by BLAST searches. The cluster comprises three genes, and these were amplified from chromosomal DNA of G. stearothermophilus, ligated into plasmid vectors and expressed in Escherichia coli. The purified

Rebecca Fleischer; Antje Wengner; Frank Scheffel; Heidi Landmesser; Erwin Schneider

2005-01-01

45

The COMATOSE ATP-Binding Cassette Transporter Is Required for Full Fertility in Arabidopsis1[W][OA  

PubMed Central

COMATOSE (CTS) encodes a peroxisomal ATP-binding cassette transporter required not only for ?-oxidation of storage lipids during germination and establishment, but also for biosynthesis of jasmonic acid and conversion of indole butyric acid to indole acetic acid. cts mutants exhibited reduced fertilization, which was rescued by genetic complementation, but not by exogenous application of jasmonic acid or indole acetic acid. Reduced fertilization was also observed in thiolase (kat2-1) and peroxisomal acyl-Coenzyme A synthetase mutants (lacs6-1,lacs7-1), indicating a general role for ?-oxidation in fertility. Genetic analysis revealed reduced male transmission of cts alleles and both cts pollen germination and tube growth in vitro were impaired in the absence of an exogenous carbon source. Aniline blue staining of pollinated pistils demonstrated that pollen tube growth was affected only when both parents bore the cts mutation, indicating that expression of CTS in either male or female tissues was sufficient to support pollen tube growth in vivo. Accordingly, abundant peroxisomes were detected in a range of maternal tissues. Although ?-aminobutyric acid levels were reduced in flowers of cts mutants, they were unchanged in kat2-1, suggesting that alterations in ?-aminobutyric acid catabolism do not contribute to the reduced fertility phenotype through altered pollen tube targeting. Taken together, our data support an important role for ?-oxidation in fertility in Arabidopsis (Arabidopsis thaliana) and suggest that this pathway could play a role in the mobilization of lipids in both pollen and female tissues. PMID:17468211

Footitt, Steven; Dietrich, Daniela; Fait, Aaron; Fernie, Alisdair R.; Holdsworth, Michael J.; Baker, Alison; Theodoulou, Frederica L.

2007-01-01

46

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

PubMed Central

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. PMID:24883056

Word, Beverly; Wang, Honggang; Huang, Shiew-Mei; Lyn-Cook, Beverly

2014-01-01

47

Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters  

PubMed Central

Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL. PMID:25120277

Uto-Kondo, Harumi; Ayaori, Makoto; Nakaya, Kazuhiro; Takiguchi, Shunichi; Yakushiji, Emi; Ogura, Masatsune; Terao, Yoshio; Ozasa, Hideki; Sasaki, Makoto; Komatsu, Tomohiro; Sotherden, Grace Megumi; Hosoai, Tamaki; Sakurada, Masami; Ikewaki, Katsunori

2014-01-01

48

Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo  

PubMed Central

Multidrug resistance (MDR) to chemotherapeutic drugs is a formidable barrier to the success of cancer chemotherapy. Expressions of ATP-binding cassette (ABC) transporters contribute to clinical MDR phenotype. In this study, we found that afatinib, a small molecule tyrosine kinase inhibitor (TKI) targeting EGFR, HER-2 and HER-4, reversed the chemoresistance mediated by ABCG2 in vitro, but had no effect on that mediated by multidrug resistance protein ABCB1 and ABCC1. In addition, afatinib, in combination with topotecan, significantly inhibited the growth of ABCG2-overexpressing cell xenograft tumors in vivo. Mechanistic investigations exhibited that afatinib significantly inhibited ATPase activity of ABCG2 and downregulated expression level of ABCG2, which resulted in the suppression of efflux activity of ABCG2 in parallel to the increase of intracellular accumulation of ABCG2 substrate anticancer agents. Taken together, our findings may provide a new and useful combinational therapeutic strategy of afatinib with chemotherapeutical drug for the patients with ABCG2 overexpressing cancer cells. PMID:25436978

Wang, Xiao-kun; Kin Wah To, Kenneth; Huang, Li-yan; Xu, Jing-hong; Yang, Ke; Wang, Fang; Huang, Zhen-cong; Ye, Sheng; Fu, Li-wu

2014-01-01

49

Physicochemical factors controlling the activity and energy coupling of an ionic strength-gated ATP-binding cassette (ABC) transporter.  

PubMed

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects. PMID:23979139

Karasawa, Akira; Swier, Lotteke J Y M; Stuart, Marc C A; Brouwers, Jos; Helms, Bernd; Poolman, Bert

2013-10-11

50

Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters.  

PubMed

ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems. PMID:9873074

Saurin, W; Hofnung, M; Dassa, E

1999-01-01

51

ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides[W][OA  

PubMed Central

Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate. PMID:23723325

Francisco, Rita Maria; Regalado, Ana; Ageorges, Agnès; Burla, Bo J.; Bassin, Barbara; Eisenach, Cornelia; Zarrouk, Olfa; Vialet, Sandrine; Marlin, Thérèse; Chaves, Maria Manuela; Martinoia, Enrico; Nagy, Réka

2013-01-01

52

Potential role of ATP-binding cassette transporters against acaricides in the brown dog tick Rhipicephalus sanguineus sensu lato.  

PubMed

ATP-binding cassette (ABC) transporters have been shown to be involved in pesticide detoxification in arthropod vectors and are thought to contribute to the development of drug resistance. Little is currently known about the role they play in ticks, which are among the more important vectors of human and animal pathogens. Here, the role of ABC transporters in the transport of fipronil and ivermectin acaricides in the tick Rhipicephalus sanguineus (Ixodida: Ixodidae) was investigated. Larvae were treated with acaricide alone and acaricide in combination with a sub-lethal dose of the ABC transporter inhibitor cyclosporine A. The LC50 doses and 95% confidence intervals (CIs) estimated by mortality data using probit analysis were 67.930?p.p.m. (95% CI 53.780-90.861) for fipronil and 3741?p.p.m. (95% CI 2857-4647) for ivermectin. The pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC50 dose of fipronil to 4.808?p.p.m. (95% CI 0.715-9.527) and that of ivermectin to 167?p.p.m. (95% CI 15-449), which increased toxicity by about 14- and 22-fold, respectively. The comparison of mortality data for each separate acaricide concentration showed the synergic effect of cyclosporine A to be reduced at higher concentrations of acaricide. These results show for the first time a strong association between ABC transporters and acaricide detoxification in R.sanguineus s.l. PMID:25530472

Cafarchia, C; Porretta, D; Mastrantonio, V; Epis, S; Sassera, D; Iatta, R; Immediato, D; Ramos, R A N; Lia, R P; Dantas-Torres, F; Kramer, L; Urbanelli, S; Otranto, D

2015-03-01

53

Caveolin-1 interacts with ATP binding cassette transporter G1 (ABCG1) and regulates ABCG1-mediated cholesterol efflux.  

PubMed

ATP-binding cassette transporter G1 (ABCG1) plays an important role in macrophage reverse cholesterol transport in vivo by promoting cholesterol efflux onto lipidated apoA-I. However, the underlying mechanism is unclear. Here, we found that ABCG1 co-immunoprecipitated with caveolin-1 (CAV1) but not with flotillin-1 and -2. Knockdown of CAV1 expression using siRNAs significantly reduced ABCG1-mediated cholesterol efflux without detectable effect on ABCA1-mediated cholesterol efflux. Disruption of the putative CAV1 binding site in ABCG1, through replacement of tyrosine residues at positions 487 and 489 or at positions 494 and 495 with alanine (Y487AY489A and Y494AY495A), impaired the interaction of ABCG1 with CAV1 and significantly decreased ABCG1-mediated cholesterol efflux. The substitution of Tyr494 and Tyr495 with Phe or Trp that resulted in an intact CAV1 binding site had no effect. Furthermore, Y494AY495A affected trafficking of ABCG1 to the cell surface. The mutant protein is mainly located intracellularly. Finally, we found that CAV1 co-immunoprecipitated with ABCG1 and regulated cholesterol efflux to reconstituted HDL in THP-1-derived macrophages upon the liver X receptor agonist treatment. These findings indicate that CAV1 interacts with ABCG1 and regulates ABCG1-mediated cholesterol efflux. PMID:24576892

Gu, Hong-Mei; Wang, Fa-Qi; Zhang, Da-Wei

2014-06-01

54

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export  

PubMed Central

Mitochondrial iron levels are tightly regulated, as iron is essential for the synthesis of Fe/S clusters and heme in the mitochondria, but high levels can cause oxidative stress. The ATP-binding cassette (ABC) transporter ABCB8 is a mitochondrial inner membrane protein with an unknown function. Here, we show that ABCB8 is involved in mitochondrial iron export and is essential for baseline cardiac function. Induced genetic deletion of ABCB8 in mouse hearts resulted in mitochondrial iron accumulation and cardiomyopathy, as assessed by echocardiography and invasive hemodynamics. Mice with ABCB8 deletion in the heart also displayed mitochondrial damage, and higher levels of reactive oxygen species and cell death. Down-regulation of ABCB8 in vitro resulted in decreased iron export from isolated mitochondria, whereas its overexpression had the opposite effect. Furthermore, ABCB8 is needed for the maturation of the cytosolic Fe/S proteins, as its deletion in vitro and in vivo led to decreased activity of cytosolic, but not mitochondrial, iron–sulfur-containing enzymes. These results indicate that ABCB8 is essential for normal cardiac function, maintenance of mitochondrial iron homeostasis and maturation of cytosolic Fe/S proteins. In summary, this report provides characterization of a protein involved in mitochondrial iron export. PMID:22375032

Ichikawa, Yoshihiko; Bayeva, Marina; Ghanefar, Mohsen; Potini, Vishnu; Sun, Lin; Mutharasan, R. Kannan; Wu, Rongxue; Khechaduri, Arineh; Jairaj Naik, Tejaswitha; Ardehali, Hossein

2012-01-01

55

The Conformational Transition Pathways of ATP-Binding Cassette Transporter BtuCD Revealed by Targeted Molecular Dynamics Simulation  

PubMed Central

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B12 into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the “alternating access” mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O?I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I?O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process. PMID:22272354

Weng, Jingwei; Fan, Kangnian; Wang, Wenning

2012-01-01

56

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies*  

PubMed Central

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 ?m) and an ATPase activity with a Km of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6. PMID:23792964

Chavan, Hemantkumar; Taimur Khan, Mohiuddin Md.; Tegos, George; Krishnamurthy, Partha

2013-01-01

57

The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter.  

PubMed

Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B12 is bound in the "base-on" conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes. PMID:12475936

Borths, Elizabeth L; Locher, Kaspar P; Lee, Allen T; Rees, Douglas C

2002-12-24

58

Influence of ATP-Binding Cassette Transporters in Root Exudation of Phytoalexins, Signals, and in Disease Resistance  

PubMed Central

The roots of plants secrete compounds as a way to exchange information with organisms living in the soil. Here, we report the involvement of seven root-expressed ATP-binding cassette (ABC) transporters corresponding to both full and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3, Atnap5, and Atath10) in root exudation processes using Arabidopsis thaliana. Root exuded phytochemicals were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), and it was determined that some of the root exudates from the corresponding ABC transporter mutants were significantly different compared to the wild type. For example, Atabcg37 and Atabcc5 secreted higher levels of the phytoalexin camalexin, and Atabcg36 secreted higher levels of organic acids, specifically salicylic acid (SA). Furthermore, we analyzed the root tissue metabolites of these seven ABC transporter mutants and found that the levels of SA, quercetin, and kaempferol glucosides were higher in Atabcg36, which was correlated with higher expression levels of defense genes in the root tissues compared with the wild type. We did not observe significant changes in the root exudates of the half-size transporters except for Atabcf1 that showed lower levels of few organic acids. In summary, full-size transporters are involved in root secretion of phytochemicals. PMID:22783269

Badri, Dayakar V.; Chaparro, Jacqueline M.; Manter, Daniel K.; Martinoia, Enrico; Vivanco, Jorge M.

2012-01-01

59

PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana RootsW?  

PubMed Central

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid–reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells. PMID:16243904

Terasaka, Kazuyoshi; Blakeslee, Joshua J.; Titapiwatanakun, Boosaree; Peer, Wendy A.; Bandyopadhyay, Anindita; Makam, Srinivas N.; Lee, Ok Ran; Richards, Elizabeth L.; Murphy, Angus S.; Sato, Fumihiko; Yazaki, Kazufumi

2005-01-01

60

HDAC inhibitor-induced drug resistance involving ATP-binding cassette transporters (Review)  

PubMed Central

Histone deacetylase (HDAC) inhibitors are becoming a novel and promising class of antineoplastic agents that have been used for cancer therapy in the clinic. Two HDAC inhibitors, vorinostat and romidepsin, have been approved by the Food and Drug Administration to treat T-cell lymphoma. Nevertheless, similar to common anticancer drugs, HDAC inhibitors have been found to induce multidrug resistance (MDR), which is an obstacle for the success of chemotherapy. The most common cause of MDR is considered to be the increased expression of adenosine triphosphate binding cassette (ABC) transporters. Numerous studies have identified that the upregulation of ABC transporters is often observed following treatment with HDAC inhibitors, particularly the increased expression of P-glycoprotein, which leads to drug efflux, reduces intracellular drug concentration and induces MDR. The present review summarizes the key ABC transporters involved in MDR following various HDAC inhibitor treatments in a range of cancer cell lines and also explored the potential mechanisms that result in MDR, including the effect of nuclear receptors, which are the upstream regulatory factors of ABC transporters.

NI, XUAN; LI, LI; PAN, GUOYU

2015-01-01

61

Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR?–LXR?–ABCA1 pathway  

Microsoft Academic Search

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression

Yanni Xu; Fangfang Lai; Yang Xu; Yexiang Wu; Qi Liu; Ni Li; Yuzhen Wei; Tingting Feng; Zhihui Zheng; Wei Jiang; Liyan Yu; Bin Hong; Shuyi Si

2011-01-01

62

ATP-binding cassette transporter A1 locus is not a major determinant of HDLC levels in a population at high risk for coronary heart disease  

Microsoft Academic Search

ATP-binding cassette transporter A1 (ABCA1) transports cellular cholesterol to lipid-poor apolipoproteins. Mutations in the ABCA1 gene are linked to rare phenotypes, familial hypoalphalipoproteinemia (FHA) and Tangier disease (TD), characterized by markedly decreased plasma high-density lipoprotein cholesterol (HDL-C) levels. The aim was to test if the ABCA1 locus is a major locus regulating HDL-C levels in the homogenous Finnish population with

Sakari Kakko; Jani Kelloniemi; Peter von Rohr; Ina Hoeschele; Minna Tamminen; Margaret E. Brousseau; Y. Antero Kesäniemi; Markku J. Savolainen

2003-01-01

63

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium  

Microsoft Academic Search

Aims:Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI).Methods:ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and

K G Duncan; K Hosseini; K R Bailey; H Yang; R J Lowe; M T Matthes; J P Kane; M M LaVail; D M Schwartz; J L Duncan

2009-01-01

64

Differential expression and functionality of ATP binding cassette transporters in the human hair follicle.  

PubMed

Adenosine triphosphate binding cassette (ABC) transporters are involved in the active transport of an extremely diverse range of substrates across biological membranes. These transporters are commonly implicated in the development of multidrug resistance and are also involved in numerous physiological and homeostatic processes, including lipid transport, cell migration and differentiation. Since the expression of ABC transporters in the human hair follicle (HF) is as yet unclear, this study aimed to close this knowledge gap. By qPCR analysis, numerous members of the ABC transporter superfamily, such as ABCB1, G2 and A12, were found to be transcribed in full-length human scalp HFs. Immunofluorescence microscopy demonstrated that the intrafollicular protein expression of different xenobiotic ABC transporters (ABCB1, ABCC1, ABCC4, ABCG2) varies greatly, with ABCG2 expression primarily restricted to the epithelial stem cell region of the outer root sheath (bulge), whereas both ABCB1, ABCC1 and ABCC4 expression was more widespread. Lipid transporters ABCA1, ABCA12 and ABCA4 were almost uniformly expressed throughout the HF epithelium. Functional ABCB1/G2 activity was demonstrated by exclusion of the substrate dye, Hoechst 33342. In the bulge, this was reversed by ABCB1 and ABCG2 inhibition. These data encourage one to further investigate ABC transporters as potentially important regulators of HF epithelial biology. Clinically, pharmacological modulation of the activity of selected intrafollicular ABC transporters may permit novel therapeutic interventions, such as protecting HF stem cells from chemotherapy-induced damage, counteracting cholesterol-associated hypertrichosis, and manipulating the intrafollicular prostaglandin balance in androgenetic alopecia. This article is protected by copyright. All rights reserved. PMID:25418064

Haslam, I S; El-Chami, C; Faruqi, H; Shahmalak, A; O'Neill, C A; Paus, R

2014-11-21

65

Gene expression profiling reveals defined functions of the ATP-binding cassette transporter COMATOSE late in phase II of germination.  

PubMed

Phase II of germination represents a key developmental stage of plant growth during which imbibed seeds either enter stage III of germination, completing the germination process via radicle protrusion, or remain dormant. In this study, we analyzed the influence of the peroxisomal ATP-binding cassette transporter COMATOSE (CTS) on the postimbibition seed transcriptome of Arabidopsis (Arabidopsis thaliana) and also investigated interactions between gibberellin (GA) and CTS function. A novel method for analysis of transcriptome datasets allowed visualization of developmental signatures of seeds, showing that cts-1 retains the capacity to after ripen, indicating a germination block late in phase II. Expression of the key GA biosynthetic genes GA3ox1 and 2 was greatly reduced in cts seeds and genetic analysis suggested that CTS was epistatic to RGL2, a germination-repressing DELLA protein that is degraded by GA. Comparative analysis of seed transcriptome datasets indicated that specific cohorts of genes were influenced by GA and CTS. CTS function was required for expression of the flavonoid biosynthetic pathway. Confocal imaging demonstrated the exclusive accumulation of flavonoids in the epidermis of wild-type seeds. In contrast, flavonoids were absent from cts and kat2-1 mutant seeds, but accumulated following the application of sucrose, indicating an essential role for beta-oxidation in inducing flavonoid biosynthetic genes. These results demonstrate that CTS functions very late in phase II of germination and that its function is required for the expression of specific gene sets related to an important biochemical pathway associated with seedling establishment and survival. PMID:17322332

Carrera, Esther; Holman, Tara; Medhurst, Anne; Peer, Wendy; Schmuths, Heike; Footitt, Steven; Theodoulou, Frederica L; Holdsworth, Michael J

2007-04-01

66

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

PubMed Central

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

Aberuyi, N; Rahgozar, S; Moafi, A

2014-01-01

67

Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).  

PubMed

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway. PMID:22303012

Wang, Wuyang; Linsdell, Paul

2012-03-23

68

CEP-33779 antagonizes ATP-binding cassette subfamily B member 1 mediated multidrug resistance by inhibiting its transport function.  

PubMed

The overexpression of ATP-binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR), which is the major factor contributing to the failure of chemotherapy. The objective of this study was to investigate the enhancement of CEP-33779, a small-molecule inhibitor of Janus kinase 2 (JAK2), on the efficacy of conventional chemotherapeutic agents in MDR cells with overexpression of P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Our results showed that CEP-33779, at nontoxic concentrations, significantly sensitized ABCB1 overexpressing MDR cells to its anticancer substrates. CEP-33779 significantly increased intracellular accumulation and decreased the efflux of doxorubicin by inhibiting the ABCB1 transport function. Furthermore, CEP-33779 did not alter the expression of ABCB1 both at protein and mRNA levels but did stimulate the activity of ABCB1 ATPase. CEP-33779 was predicted to bind within the large hydrophobic cavity of homology modeled ABCB1. In addition, the down-regulation of JAK2 by shRNA altered neither the expression of ABCB1 nor the cytotoxic effect of chemotherapeutic agents in ABCB1-overexpressing cells. Significantly, CEP-33779 enhanced the efficacy of vincristine against the ABCB1-overexpressing and drug resistant KBv200 cell xenograft in nude mice. In conclusion, we conclude that CEP-33779 enhances the efficacy of substrate drugs in ABCB1-overexpressing cells by directly inhibiting ABCB1 transport function. The findings encouraged to further study on the combination therapy of CEP-33779 with conventional chemotherapeutic agents in ABCB1 mediated-MDR cancer patients. PMID:25058526

Tang, Shang-jun; Chen, Li-kun; Wang, Fang; Zhang, Yun-kai; Huang, Zhen-cong; To, Kenneth Kin Wah; Wang, Xiao-kun; Talele, Tanaji T; Chen, Zhe-sheng; Chen, Wei-qiang; Fu, Li-wu

2014-09-15

69

HIV-1 protein Nef inhibits activity of ATP-binding cassette transporter A1 by targeting endoplasmic reticulum chaperone calnexin.  

PubMed

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

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

2014-10-17

70

ATP-Binding Cassette Transporter Expression in Human Placenta as a Function of Pregnancy ConditionS?  

PubMed Central

Fetal drug exposure is determined by the type and concentration of placental transporters, and their regulation is central to the development of new treatments and delivery strategies for pregnant women and their fetuses. We tested the expression of several clinically important transporters in the human placenta associated with various pregnancy conditions (i.e., labor, preeclampsia, and preterm labor-inflammation). Placentas were obtained from five groups of women at the time of primary cesarean section: 1) term no labor; 2) term labor; 3) preterm no labor (delivered for severe preeclampsia); 4) preterm labor without inflammation (PTLNI); and 5) preterm labor with inflammation (PTLI). Samples were analyzed by Western blot and immunohistochemistry to identify changes in protein expression. Relative mRNA expression was determined by quantitative real-time polymerase chain reaction. A functional genomic approach was used to identify placental gene expression and elucidate molecular events that underlie the given condition. Placental expression of ATP-binding cassette transporters from women in labor and women with preeclampsia was unaltered. Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) and mRNA expression increased in placentas of women with preterm labor with inflammation. Molecular pathways of genes up-regulated in PTLI samples included cytokine-cytokine receptor interactions and inflammatory response compared with those in the PTLNI group. The mRNA expression of MDR1 and BCRP was correlated with that of interleukin-8, which also increased significantly in PTLI samples. These data suggest that the transfer of drugs across the placenta may be altered in preterm pregnancy conditions associated with inflammation through changes in MDR1 and BCRP. PMID:21430233

Mason, Cifford W.; Buhimschi, Irina A.; Buhimschi, Catalin S.; Dong, Yafeng; Weiner, Carl P.

2011-01-01

71

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains*  

PubMed Central

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter. PMID:23824183

Rawal, Manpreet Kaur; Khan, Mohammad Firoz; Kapoor, Khyati; Goyal, Neha; Sen, Sobhan; Saxena, Ajay Kumar; Lynn, Andrew M.; Tyndall, Joel D. A.; Monk, Brian C.; Cannon, Richard D.; Komath, Sneha Sudha; Prasad, Rajendra

2013-01-01

72

Phenotype prediction of non-synonymous single-nucleotide polymorphisms in human ATP-binding cassette transporter genes.  

PubMed

A large number of non-synonymous single-nucleotide polymorphisms (nsSNPs) have been found in human genome, but there is poor knowledge on the relationship between the genotype and phenotype of these nsSNPs. Human ATP-binding cassette (ABC) transporters are able to transport a number of important substrates including endogenous and exogenous compounds. This study aimed to predict the phenotypical impact of nsSNPs of human ABC transporter genes, and the predicted results were further validated by reported phenotypical data from site-directed mutagenesis and clinical genetic studies. One thousand and six hundred thirty-two nsSNPs were found from 49 human ABC transporter genes. Using the PolyPhen and SIFT algorithms, 41.8-53.6% of nsSNPs in ABC transporter genes were predicted to have an impact on protein function. The prediction accuracy was up to 63-85% when compared with known phenotypical data from in vivo and in vitro studies. There was a significant concordance between the prediction results using SIFT and PolyPhen. Of nsSNPs predicted as deleterious, the prediction scores by SIFT and PolyPhen were significantly related to the number of nsSNPs with known phenotypes confirmed by experimental and human studies. The amino acid substitution variants are supposed to be the pathogenetic basis of increased susceptibility to certain diseases with Mendelian or complex inheritance, altered drug resistance and altered drug clearance and response. Predicting the phenotypic consequence of nsSNPs using computational algorithms may provide a better understanding of genetic differences in susceptibility to diseases and drug response. The prediction of nsSNPs in human ABC transporter genes would be useful hints for further genotype-phenotype studies. PMID:20849526

Wang, Lin-Lin; Liu, Ya-He; Meng, Lu-Lu; Li, Chun Guang; Zhou, Shu-Feng

2011-02-01

73

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

PubMed Central

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 ?M). The best result in the series was obtained with the addition of verapamil (40 ?M), 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.

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

2014-01-01

74

Prognostic Significance and Molecular Mechanism of ATP-Binding Cassette Subfamily C Member 4 in Resistance to Neoadjuvant Radiotherapy of Locally Advanced Rectal Carcinoma  

PubMed Central

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. PMID:24454870

Gao, Xianhua; Xing, Junjie; Yu, Enda; Zhang, Wei; Zhang, Xiaoqing; Cao, Guangwen; Fu, Chuangang

2014-01-01

75

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

PubMed

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. PMID:24021237

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

2014-01-01

76

Ethanolic extract of propolis promotes reverse cholesterol transport and the expression of ATP-binding cassette transporter A1 and G1 in mice.  

PubMed

The ethanolic extract of propolis (EEP) is beneficial in increasing high density lipoprotein (HDL) cholesterol (HDL-C) and diminishing risks of atherosclerosis. In this study, we examined the effects of EEP on reverse cholesterol transport in mice. (3)H -cholesterol laden macrophage was injected intraperitoneally into mice fed by gastric gavage with EEP. Plasma lipid level was determined and (3)H-cholesterol was traced in plasma, liver and feces. The effects of EEP on ATP-binding cassette transporter A1 and G1 (ABCA1 and ABCG1) and scavenger receptor BI (SR-BI) in mice liver and in cultured cells were also investigated. EEP administration led to a significant increase in HDL-C and peritoneal macrophage-original (3)H-cholesterol in plasma, liver and feces. Liver protein expressions of ABCA1 and ABCG1 were increased but SR-B1 was not. In vitro experiments with HepG2 and Raw264.7 cell lines confirmed the above results. The finding of these studies shows that EEP-enhanced reverse cholesterol transport may have resulted from EEP stimulated plasma HDL level and hepatic ABCA1 and ABCG1 expression. PMID:21638064

Yu, Yang; Si, Yanhong; Song, Guohua; Luo, Tian; Wang, Jiafu; Qin, Shucun

2011-09-01

77

Vascular and extravascular distribution of the ATP-binding cassette transporters ABCB1 and ABCC1 in aged human brain and pituitary.  

PubMed

ATP-binding cassette (ABC) transporters play an increasing role in the understanding of pathologic peptide deposition in neurodegenerative diseases (NDs), such as Alzheimer's and Parkinson's. To describe the location of the most important ABC transporters for NDs in human brain tissue, we investigated ABCB1 and ABCC1 immunohistologically in the adult human brain and pituitary. Both transporters have similar but not identical expression patterns. In brain regions with an established blood-brain barrier (BBB), ABCB1 and ABCC1 were ubiquitously expressed in endothelial cells of the microvasculature and in a subset of larger blood vessels (mostly venules). Remarkably, both transporters were also found in fenestrated capillaries in circumventricular organs where the BBB is absent. Moreover, ABCB1 and ABCC1 were also expressed in various non-endothelia cells such as pericytes, astrocytes, choroid plexus epithelia, ventricle ependymal cells, and neurons. With regard to their neuronal expression it was shown that both transporters are located in specific nerve cell populations, which are also immunopositive for three putative cell markers of purinergic cell signalling, namely 5'-nucleotidase, adenosine deaminase and nucleoside triphosphate diphosphohydrolase-2. Therefore, we speculate that neuronal expression of ABCB1 and ABCC1 might be linked to adenosinergic/purinergic neuromodulation. Lastly, both transporters were observed in multiple adenohypophyseal cells. PMID:25218792

Bernstein, Hans-Gert; Hölzl, Gloria; Dobrowolny, Henrik; Hildebrandt, Jens; Trübner, Kurt; Krohn, Markus; Bogerts, Bernhard; Pahnke, Jens

2014-09-10

78

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

PubMed

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, ?-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. PMID:25461681

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

2015-01-01

79

Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1.  

PubMed

Members of the genus Rhodococcus are specialists in the biosynthesis and accumulation of triacylglycerols (TAGs). As no transport protein related to TAG metabolism has yet been characterized in these bacteria, we used the available genomic information of Rhodococcus jostii RHA1 to perform a broad survey of genes coding for putative lipid transporter proteins in this oleaginous micro-organism. Among the seven genes encoding putative lipid transporters, ro05645 (now called ltp1: lipid transporter protein) coding for an ATP-binding cassette protein was found clustered with others genes encoding enzymes catalysing the three putative acylation reactions of the Kennedy pathway for TAG synthesis. Overexpression of ltp1 in the RHA1 strain led to an increase of approximately sixfold and threefold in biomass and TAG production, respectively, when cells were cultivated on palmitic acid and oleic acid. Moreover, overexpression of ltp1 also promoted a significant increase in the uptake of a fluorescently labelled long-chain fatty acid (LCFA), as compared with the WT strain RHA1, and its further incorporation into the TAG fraction. Gluconate-grown cells showed increasing amounts of intracellular free fatty acids, but not of TAG, after overexpressing ltp1. Thus, for the first time to our knowledge, a transporter functionally related to TAG metabolism was identified in oleaginous rhodococci. Our results suggested that Ltp1 is an importer of LCFAs that plays a functional role in lipid homeostasis of R. jostii RHA1. PMID:24739215

Villalba, María S; Alvarez, Héctor M

2014-07-01

80

Ht31, a Protein Kinase A Anchoring Inhibitor, Induces Robust Cholesterol Efflux and Reverses Macrophage Foam Cell Formation through ATP-binding Cassette Transporter A1*  

PubMed Central

Macrophage foam cell is the predominant cell type in atherosclerotic lesions. Removal of excess cholesterol from macrophages thus offers effective protection against atherosclerosis. Here we report that a protein kinase A (PKA)-anchoring inhibitor, st-Ht31, induces robust cholesterol/phospholipid efflux, and ATP-binding cassette transporter A1 (ABCA1) greatly facilitates this process. Remarkably, we found that st-Ht31 completely reverses foam cell formation, and this process is ABCA1-dependent. The reversal is also accompanied by the restoration of well modulated inflammatory response to LPS. There is no detectable toxicity associated with st-Ht31, even when cells export up to 20% cellular cholesterol per hour. Using FRET-based PKA biosensors in live cells, we provide evidence that st-Ht31 drives cholesterol efflux by elevating PKA activity specifically in the cytoplasm. Furthermore, ABCA1 facilitates st-Ht31 uptake. This allows st-Ht31 to effectively remove cholesterol from ABCA1-expressing cells. We speculate that de-anchoring of PKA offers a novel therapeutic strategy to remove excess cholesterol from lipid-laden lesion macrophages. PMID:21106522

Ma, Loretta; Dong, Fumin; Denis, Maxime; Feng, Ying; Wang, Ming-Dong; Zha, Xiaohui

2011-01-01

81

Whole-Transcriptome Survey of the Putative ATP-Binding Cassette (ABC) Transporter Family Genes in the Latex-Producing Laticifers of Hevea brasiliensis.  

PubMed

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. PMID:25615936

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

2015-01-01

82

?-Lipoic acid ameliorates foam cell formation via liver X receptor ?-dependent upregulation of ATP-binding cassette transporters A1 and G1.  

PubMed

?-Lipoic acid (?-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether ?-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with ?-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, ?-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, ?-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by ?-LA depended on liver X receptor ? (LXR?), as evidenced by an increase in the nuclear levels of LXR? and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXR? activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXR? expression with small interfering RNA (siRNA). Consistently, ?-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXR? siRNA treatment. In conclusion, LXR?-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of ?-LA on foam cell formation. PMID:21034810

Cheng, Li-Ching; Su, Kuo-Hui; Kou, Yu Ru; Shyue, Song-Kun; Ching, Li-Chieh; Yu, Yuan-Bin; Wu, Yuh-Lin; Pan, Ching-Chian; Lee, Tzong-Shyuan

2011-01-01

83

Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.  

PubMed Central

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole. PMID:7565740

Egner, R; Mahé, Y; Pandjaitan, R; Kuchler, K

1995-01-01

84

Peroxisomal ATP-Binding Cassette Transporter COMATOSE and the Multifunctional Protein ABNORMAL INFLORESCENCE MERISTEM Are Required for the Production of Benzoylated Metabolites in Arabidopsis Seeds1[W  

PubMed Central

Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal ?-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of ?-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid. PMID:24254312

Bussell, John D.; Reichelt, Michael; Wiszniewski, Andrew A.G.; Gershenzon, Jonathan; Smith, Steven M.

2014-01-01

85

Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*  

PubMed Central

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-? (PPAR?) and carnitine palmitoyltransferase 1? (CPT1?). 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. PMID:24019513

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

2013-01-01

86

An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice  

PubMed Central

Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named “eibi1.c,” along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants. PMID:21737747

Chen, Guoxiong; Komatsuda, Takao; Ma, Jian Feng; Nawrath, Christiane; Pourkheirandish, Mohammad; Tagiri, Akemi; Hu, Yin-Gang; Sameri, Mohammad; Li, Xinrong; Zhao, Xin; Liu, Yubing; Li, Chao; Ma, Xiaoying; Wang, Aidong; Nair, Sudha; Wang, Ning; Miyao, Akio; Sakuma, Shun; Yamaji, Naoki; Zheng, Xiuting; Nevo, Eviatar

2011-01-01

87

Cloning of ABCA17, a novel rodent sperm-specific ABC (ATP-binding cassette) transporter that regulates intracellular lipid metabolism.  

PubMed

The A subclass of the ABC (ATP-binding cassette) transporter superfamily has a structural feature that distinguishes it from other ABC transporters, and is proposed to be involved in the transmembrane transport of endogenous lipids. Here we have cloned mouse and rat full-length cDNAs of ABCA17, a novel ABC transporter belonging to the A subclass. Mouse and rat ABCA17 proteins comprise 1733 and 1773 amino acid residues respectively, having 87.3% amino acid identity; mouse ABCA17 has amino acid identities of 55.3% and 36.7% with mouse ABCA3 and sea urchin ABCA respectively. RNA blot and quantitative real-time PCR analyses showed that ABCA17 mRNA is expressed exclusively in the testis. Examination of testis by in situ hybridization showed that ABCA17 mRNA is expressed in germ cells, mainly spermatocytes, in the seminiferous tubule. Immunoblot analysis using a specific antibody showed that ABCA17 is a protein of 200 kDa, and immunohistochemical analysis demonstrated that the protein is detected in the anterior head of sperm and elongated spermatids. ABCA17 was localized in the endoplasmic reticulum in transiently transfected HEK293 cells. Metabolic labelling analysis showed that intracellular esterified lipids, including cholesteryl esters, fatty acid esters and triacylglycerols, were significantly decreased in HEK293 cells stably expressing ABCA17 compared with untransfected cells. These results suggest that ABCA17 may play a role in regulating lipid composition in sperm. PMID:15810880

Ban, Nobuhiro; Sasaki, Mayumi; Sakai, Hiromichi; Ueda, Kazumitsu; Inagaki, Nobuya

2005-07-15

88

Paeonol suppresses lipid accumulation in macrophages via upregulation of the ATP?binding cassette transporter A1 and downregulation of the cluster of differentiation 36.  

PubMed

Paeonol, a potent antioxidant isolated from cortex moutan, possesses athero?protective activity, yet the detailed mechanisms are not fully investigated. This study was conducted to explore the role of paeonol and its underlying mechanisms in RAW264.7 macrophages and apolipoprotein E?deficient (ApoE?/?) mice. Paeonol treatment significantly attenuated intracellular lipid accumulation in macrophages, which may be the result of decreased oxidized low?density lipopro-tein (ox?LDL) uptake and increased cholesterol efflux. Additionally, paeonol markedly inhibited the mRNA and protein expression of the cluster of differentiation 36 (CD36) by decreasing nuclear translocation of c?Jun [a subunit of activator protein?1 (AP?1)]. Moreover, paeonol upregulated the protein stability of ATP?binding cassette transporter A1 (ABCA1) by inhibiting calpain activity, while ABCA1 mRNA expression was not altered. Furthermore, small hairpin RNA (shRNA) targeting haem oxygenase?1 (HO?1) inhibited the paeonol?mediated beneficial effects on the expression of c?Jun, CD36, ABCA1, calpain activity and lipid accumulation in macrophages. Accordingly, paeonol retarded the progress of atherosclerosis in ApoE?/? mice and modulated the expression of CD36 and ABCA1 in aortas similarly to that observed in macrophages. These results indicate that paeonol provides protective effects on foam cell formation by a novel HO?1?dependent mediation of cholesterol efflux and lipid accumulation in macrophages. PMID:25405950

Li, Xiuying; Zhou, Yuanda; Yu, Chao; Yang, Hui; Zhang, Chengzhi; Ye, Yun; Xiao, Shunlin

2015-02-01

89

Arabidopsis PEN3/PDR8, an ATP Binding Cassette Transporter, Contributes to Nonhost Resistance to Inappropriate Pathogens That Enter by Direct Penetration[W][OA  

PubMed Central

Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid–dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway. PMID:16473969

Stein, Mónica; Dittgen, Jan; Sánchez-Rodríguez, Clara; Hou, Bi-Huei; Molina, Antonio; Schulze-Lefert, Paul; Lipka, Volker; Somerville, Shauna

2006-01-01

90

Involvement of a soybean ATP-binding cassette-type transporter in the secretion of genistein, a signal flavonoid in legume-Rhizobium symbiosis.  

PubMed

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots. PMID:17556512

Sugiyama, Akifumi; Shitan, Nobukazu; Yazaki, Kazufumi

2007-08-01

91

Cooperative Interaction between Hepatocyte Nuclear Factor 4? and GATA Transcription Factors Regulates ATP-Binding Cassette Sterol Transporters ABCG5 and ABCG8?  

PubMed Central

Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4? (HNF4?) in the ABCG5/ABCG8 intergenic promoter, through which HNF4? strongly activated the expression of a reporter gene in both directions. The HNF4?-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4?. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4? leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4? and GATA were essential for maximal synergism. We also show that HNF4?, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4? and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4? acts synergistically with GATA factors to activate gene expression in a bidirectional fashion. PMID:17403900

Sumi, Koichi; Tanaka, Toshiya; Uchida, Aoi; Magoori, Kenta; Urashima, Yasuyo; Ohashi, Riuko; Ohguchi, Hiroto; Okamura, Masashi; Kudo, Hiromi; Daigo, Kenji; Maejima, Takashi; Kojima, Noriaki; Sakakibara, Iori; Jiang, Shuying; Hasegawa, Go; Kim, Insook; Osborne, Timothy F.; Naito, Makoto; Gonzalez, Frank J.; Hamakubo, Takao; Kodama, Tatsuhiko; Sakai, Juro

2007-01-01

92

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

PubMed Central

The ATP-binding cassette transporters ABCG5 and ABCG8 form heterodimers that limit absorption of dietary sterols in the intestine and promote cholesterol elimination from the body through hepatobiliary secretion. To identify cis-regulatory elements of the two genes, we have cloned and analyzed twenty-three evolutionary conserved region (ECR) fragments using the CMV-luciferase reporter system in HepG2 cells. Two ECRs were found to be responsive to the Liver-X-Receptor (LXR). Through elaborate deletion studies, regions containing putative LXREs were identified and the binding of LXR? was demonstrated by EMSA and ChIP assay. When the LXREs were inserted upstream of the intergenic promoter, synergistic activation by LXR?/RXR? in combination with GATA4, HNF4?, and LRH-1, which had been shown to bind to the intergenic region, was observed. In conclusion, we have identified two LXREs in ABCG5/ABCG8 genes for the first time and propose that these LXREs, especially in the ECR20, play major roles in regulating these genes. [BMB Reports 2013; 46(6): 322-327] PMID:23790976

Back, Su Sun; Kim, Jinsu; Choi, Daehyung; Lee, Eui Sup; Choi, Soo Young; Han, Kyuhyung

2013-01-01

93

Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis1[W][OPEN  

PubMed Central

Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar ?-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar Km values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites. PMID:24028845

Burla, Bo; Pfrunder, Stefanie; Nagy, Réka; Francisco, Rita Maria; Lee, Youngsook; Martinoia, Enrico

2013-01-01

94

Whole-Transcriptome Survey of the Putative ATP-Binding Cassette (ABC) Transporter Family Genes in the Latex-Producing Laticifers of Hevea brasiliensis  

PubMed Central

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. PMID:25615936

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

2015-01-01

95

Variation in the ATP-binding cassette transporter 2 gene is a separate risk factor for Systemic Lupus Erythematosus within the MHC  

PubMed Central

The ATP-binding cassette transporter (TAP) proteins are functionally relevant candidates for predisposition to Systemic Lupus Erythematosus (SLE) by virtue of their role in autoantigen presentation and location in the MHC. We tested if variation in the TAP genes (TAP1 and TAP2) is associated with SLE. We genotyped tag single nucleotide polymorphisms (SNPs) and performed family-based association analysis on 390 Caucasian pedigrees. We found significant evidence of association between TAP2 and SLE (rs241453, P = 1.33 × 10-6). Conditional logistic regression analysis suggests that this TAP2 effect is separate from the HLA-DRB1 alleles. Our analyses show that both rs241453 (P = 1.6 × 10-4) and HLA-DRB1*03xx (P = 2.3 × 10-4) have significant autonomous effects not due to linkage disequilibrium. Moreover, these loci exhibit a significant statistical interaction (P < 1.0 × 10-6), demonstrated by an increase in the odds ratio for the TAP2 association from OR = 2.00 (CI=1.17-3.42) in HLA-DRB1*03xx-negative subjects to OR = 4.29 (CI=1.88-9.76) in the subjects with at least one HLA-DRB1*03xx allele group. We report the largest association study of the TAP genes with SLE to date, and the first to test for its separate effect and interaction with the HLA alleles consistently associated with SLE. PMID:19387463

Ramos, Paula S.; Langefeld, Carl D.; Bera, LeeAnn; Gaffney, Patrick M.; Noble, Janelle A.; Moser, Kathy L.

2010-01-01

96

Molecular cDNA cloning and tissue distribution of mRNA encoding a novel ATP-binding cassette (ABC) half-transporter.  

PubMed

The majority of proteins belonging to the ATP-binding cassette (ABC) superfamily catalyzes translocation of substrates across biological membranes. Employing a reverse transcription-PCR approach with degenerate primers, we have identified a full-length cDNA from rat hepatocytes encoding a novel ABC transporter termed umat (ubiquitously expressed mammalian ABC half-transporter). The deduced sequence of 836 amino acids comprises an N-terminal membrane anchor domain and a single conserved C-terminal nucleotide binding fold, specifying umat as an ABC half-transporter. While the first 250 amino acid positions are highly divergent from other ABC transporters, clusters of conserved residues are evident along the rest of the protein. The greatest sequence similarity was observed with the fission yeast heavy metal tolerance protein hmt1 (44.5% identity in a 626-amino-acid overlap). Umat mRNA, expressed in all tissues analyzed, was most abundant in testis. Substantial umat mRNA expression in cultured primary rat hepatocytes suggests that hepatocyte cultures should represent an adequate model for investigation of umat function and regulation. PMID:9705847

Hirsch-Ernst, K I; Gaini-Rahimi, S; Ernst, B P; Schmitz-Salue, C; Blume, S; Kahl, G F

1998-08-10

97

ATP-binding cassette transporter A1 (ABCA1) deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-? peptide (1–40) at the blood–brain barrier  

Microsoft Academic Search

ATP-binding cassette transporter A1 (ABCA1) mediates apolipoprotein-dependent cholesterol release from cellular membranes. Recent studies using ABCA1 knockout mice have demonstrated that ABCA1 affects amyloid-? peptide (A?) levels in the brain and the production of senile plaque. Cerebral A?(1–40) was eliminated from the brain to the circulating blood via the blood–brain barrier (BBB), which expresses ABCA1. Therefore, in the present study,

Shin-ichi Akanuma; Sumio Ohtsuki; Youko Doi; Masanori Tachikawa; Shingo Ito; Satoko Hori; Tomoko Asashima; Tadafumi Hashimoto; Kaoru Yamada; Kazumitsu Ueda; Takeshi Iwatsubo; Tetsuya Terasaki

2008-01-01

98

Crystal Structures and Mutational Analysis of the Arginine, Lysine, Histidine-binding Protein ArtJ from Geobacillus stearothermophilus. Implications for Interactions of ArtJ with its Cognate ATP-binding Cassette Transporter, Art(MP) 2  

Microsoft Academic Search

ArtJ is the substrate-binding component (receptor) of the ATP-binding cassette (ABC) transport system ArtJ-(MP)2 from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine. The highest affinity is found for arginine (Kd=0.039(±0.014) ?M), while the affinities for lysine and histidine are about tenfold lower. We have determined the X-ray structures of ArtJ liganded with each of

Ardeschir Vahedi-Faridi; Viola Eckey; Frank Scheffel; Claudia Alings; Heidi Landmesser; Erwin Schneider; Wolfram Saenger

2008-01-01

99

Remote Communication through Solute Carriers and ATP Binding Cassette Drug Transporter Pathways: An Update on the Remote Sensing and Signaling Hypothesis  

PubMed Central

Recent data from knockouts, human disease, and transport studies suggest that solute carrier (SLC) and ATP binding cassette (ABC) multispecific “drug” transporters maintain effective organ and body fluid concentrations of key nutrients, signaling molecules, and antioxidants. These processes involve transcellular movement of solutes across epithelial barriers and fluid compartments (e.g., blood, cerebrospinal fluid, urine, bile) via “matching” or homologous sets of SLC (e.g., SLC21, SLC22, SLC47) and ABC transporters. As described in the “Remote Sensing and Signaling Hypothesis” (Biochem Biophys Res Commun 323:429–436, 2004; Biochem Biophys Res Commun 351:872–876, 2006; J Biol Chem 282:23841–23853, 2007; Nat Clin Pract Nephrol 3:443–448, 2007; Mol Pharmacol 76:481–490, 2009), highly regulated transporter networks with overlapping substrate preferences are involved in sensing and signaling to maintain homeostasis in response to environmental changes (e.g., substrate imbalance and injury). They function in parallel with (and interact with) the endocrine and autonomic systems. Uric acid (urate), carnitine, prostaglandins, conjugated sex steroids, cGMP, odorants, and enterobiome metabolites are discussed here as examples. Xenobiotics hitchhike on endogenous carrier systems, sometimes leading to toxicity and side effects. By regulation of the expression and/or function of various remote organ multispecific transporters after injury, the overall transport capacity of the remote organ to handle endogenous toxins, metabolites, and signaling molecules may change, aiding in recovery. Moreover, these transporters may play a role in communication between organisms. The specific cellular components involved in sensing and altering transporter abundance or functionality depend upon the metabolite in question and probably involve different types of sensors as well as epigenetic regulation. PMID:21325265

Wu, Wei; Dnyanmote, Ankur V.

2011-01-01

100

Stimulation of the liver X receptor pathway inhibits HIV-1 replication via induction of ATP-binding cassette transporter A1.  

PubMed

Cholesterol plays an important role in the HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits the activity of the major cellular cholesterol transporter ATP binding cassette transporter A1 (ABCA1), suggesting that the virus may use this mechanism to get access to cellular cholesterol. In this study, we investigated the effect on HIV infection of a synthetic liver X receptor (LXR) ligand, N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (TO-901317), which is a potent stimulator of ABCA1 expression. We demonstrate that TO-901317 restores cholesterol efflux from HIV-infected T lymphocytes and macrophages. TO-901317 potently suppressed HIV-1 replication in both cell types and inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in RAG-hu mice infected in vivo. This anti-HIV activity was dependent on ABCA1, because the effect of the drug was significantly reduced in ABCA1-defective T cells from a patient with Tangier disease, and RNA interference-mediated inhibition of ABCA1 expression eliminated the effect of TO-901317 on HIV-1 replication. TO-901317-mediated inhibition of HIV replication was due to reduced virus production and reduced infectivity of produced virions. The infectivity defect was in part due to reduced fusion activity of the virions, which was directly linked to reduced viral cholesterol. These results describe a novel approach to inhibiting HIV infection by stimulating ABCA1 expression. PMID:20479131

Morrow, Matthew P; Grant, Angela; Mujawar, Zahedi; Dubrovsky, Larisa; Pushkarsky, Tatiana; Kiselyeva, Yana; Jennelle, Lucas; Mukhamedova, Nigora; Remaley, Alan T; Kashanchi, Fatah; Sviridov, Dmitri; Bukrinsky, Michael

2010-08-01

101

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

PubMed

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 overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 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 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 in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol. PMID:11205902

Robey, R W; Medina-Pérez, W Y; Nishiyama, K; Lahusen, T; Miyake, K; Litman, T; Senderowicz, A M; Ross, D D; Bates, S E

2001-01-01

102

AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis  

PubMed Central

In bacteria, yeast, and mammals, iron-sulfur (Fe-S) cluster-containing proteins are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression. In humans, iron-storage diseases such as X-linked sideroblastic anemia and ataxia are caused by defects in Fe-S cluster availability. The biogenesis of Fe-S clusters involves several pathways, and in bacteria, the SufABCDSE operon has been shown to play a vital role in Fe-S biogenesis and repair during oxidative stress. Although Fe-S proteins play vital roles in plants, Fe-S cluster biogenesis and maintenance and physiological consequences of dysfunctional Fe-S cluster assembly remains obscure. Here we report that Arabidopsis plants deficient for the SufC homolog AtNAP7 show lethality at the globular stage of embryogenesis. AtNAP7 is expressed in developing embryos and in apical, root, and floral meristems and encodes an ATP-binding cassette/ATPase that can partially rescue growth defects in an Escherichia coli SufC mutant during oxidative stress. AtNAP7 is plastid-localized, and mutant embryos contain abnormal developing plastids with disorganized thylakoid structures. We found that AtNAP7 can interact with AtNAP6, a plastidic Arabidopsis SufD homolog, and because Arabidopsis plastids also harbor SufA, SufB, SufS, and SufE homologs, plastids probably contain a complete SUF system. Our results imply that AtNAP7 represents a conserved SufC protein involved in the biogenesis and/or repair of oxidatively damaged Fe-S clusters and suggest an important role for plastidic Fe-S cluster maintenance and repair during Arabidopsis embryogenesis. PMID:15184673

Xu, Xiang Ming; Møller, Simon Geir

2004-01-01

103

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).  

PubMed

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ?-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier. PMID:25041515

Shiono, Katsuhiro; Ando, Miho; Nishiuchi, Shunsaku; Takahashi, Hirokazu; Watanabe, Kohtaro; Nakamura, Motoaki; Matsuo, Yuichi; Yasuno, Naoko; Yamanouchi, Utako; Fujimoto, Masaru; Takanashi, Hideki; Ranathunge, Kosala; Franke, Rochus B; Shitan, Nobukazu; Nishizawa, Naoko K; Takamure, Itsuro; Yano, Masahiro; Tsutsumi, Nobuhiro; Schreiber, Lukas; Yazaki, Kazufumi; Nakazono, Mikio; Kato, Kiyoaki

2014-10-01

104

Nrf2 Upregulates ATP Binding Cassette Transporter Expression and Activity at the Blood–Brain and Blood–Spinal Cord Barriers  

PubMed Central

Activation of nuclear factor E2-related factor-2 (Nrf2), a sensor of oxidative stress, is neuroprotective in animal models of cerebral ischemia, traumatic brain injury, subarachnoid hemorrhage, and spinal cord injury. We show here that Nrf2 activation with sulforaphane (SFN) in vivo or in vitro increases expression and transport activity of three ATP-driven drug efflux pumps at the blood–brain barrier [P-glycoprotein, ATP binding cassette b1 (Abcb1); multidrug resistance-associated protein-2 (Mrp2), Abcc2; and breast cancer resistance protein (Bcrp), Abcg2]. Dosing rats with SFN increased protein expression of all three transporters in brain capillaries and decreased by 50% brain accumulation of the P-glycoprotein substrate verapamil. Exposing rat or mouse brain capillaries to SFN increased P-glycoprotein, Bcrp, and Mrp2 transport activity and protein expression; SFN increased P-glycoprotein activity in mouse spinal cord capillaries. Inhibiting transcription or translation abolished upregulation of P-glycoprotein activity. No such effects were seen in brain capillaries from Nrf2-null mice, indicating Nrf2 dependence. Nrf2 signaled indirectly to increase transporter activity/expression. The p53 inhibitor pifithrin abolished the SFN-induced increase in transporter activity/expression, and the p53-activator nutlin-3 increased P-glycoprotein activity. SFN did not alter P-glycoprotein transport activity in brain and spinal cord capillaries from p53-null mice. Inhibitors of p38 MAPK and nuclear factor ?B (NF-?B) blocked the effects of SFN and nutlin-3 on P-glycoprotein activity. These results implicate Nrf2, p53, and NF-?B in the upregulation of P-glycoprotein, Bcrp, and Mrp2 at blood–CNS barriers. They imply that the barriers are tightened selectively (efflux transporter upregulation) by oxidative stress, providing increased neuroprotection, but also reduced penetration of many therapeutic drugs. PMID:24948812

Wang, Xueqian; Campos, Christopher R.; Peart, John C.; Smith, Lindsay K.; Boni, Jessica L.; Cannon, Ronald E.

2014-01-01

105

Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress.  

PubMed

ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, 'ABCE' has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters. PMID:25531538

Saha, Jayita; Sengupta, Atreyee; Gupta, Kamala; Gupta, Bhaskar

2015-02-01

106

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

PubMed

Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM).10 subjects (4?F/6?M, 50-85?years, BMI 25-35?kg/m²) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5?mmol/L D-glucose (control) or additional 20?mmol/L D- or L-glucose and 25?ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined.Baseline ABCA1and ABCG1 expression was lower (>50%) in human monocytes and PBMC than leukocytes (p<0.05). 1?h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37% and 30%, respectively (p<0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10%; p<0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression.Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to diabetes-associated atherosclerosis. PMID:24838154

Spartano, N L; Lamon-Fava, S; Matthan, N R; Ronxhi, J; Greenberg, A S; Obin, M S; Lichtenstein, A H

2014-09-01

107

A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line  

PubMed Central

The importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called ‘Hospicells’ (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells. PMID:25095896

BENABOU, NADIA; MIRSHAHI, PEZHMAN; BORDU, CAMILE; FAUSSAT, ANNE-MARIE; TANG, RUOPING; THERWATH, AMU; SORIA, JEANETE; MARIE, JEAN-PIERE; MIRSHAHI, MASSOUD

2014-01-01

108

A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis.  

PubMed

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. PMID:25118291

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-10-01

109

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

PubMed

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. PMID:22761424

Chavan, Hemantkumar; Krishnamurthy, Partha

2012-09-14

110

Determinants of substrate specificity and biochemical properties of the sn-glycerol-3-phosphate ATP binding cassette transporter (UgpB-AEC2 ) of Escherichia coli.  

PubMed

Under phosphate starvation conditions, Escherichia coli can utilize sn-glycerol-3-phosphate (G3P) and G3P diesters as phosphate source when transported by an ATP binding cassette importer composed of the periplasmic binding protein, UgpB, the transmembrane subunits, UgpA and UgpE, and a homodimer of the nucleotide binding subunit, UgpC. The current knowledge on the Ugp transporter is solely based on genetic evidence and transport assays using intact cells. Thus, we set out to characterize its properties at the level of purified protein components. UgpB was demonstrated to bind G3P and glycerophosphocholine with dissociation constants of 0.68?±?0.02??M and 5.1?±?0.3??M, respectively, while glycerol-2-phosphate (G2P) is not a substrate. The crystal structure of UgpB in complex with G3P was solved at 1.8?Å resolution and revealed the interaction with two tryptophan residues as key to the preferential binding of linear G3P in contrast to the branched G2P. Mutational analysis validated the crucial role of Trp-169 for G3P binding. The purified UgpAEC2 complex displayed UgpB/G3P-stimulated ATPase activity in proteoliposomes that was neither inhibited by phosphate nor by the signal transducing protein PhoU or the phosphodiesterase UgpQ. Furthermore, a hybrid transporter composed of MalFG-UgpC could be functionally reconstituted while a UgpAE-MalK complex was unstable. PMID:23013274

Wuttge, Steven; Bommer, Martin; Jäger, Franziska; Martins, Berta M; Jacob, Sophie; Licht, Anke; Scheffel, Frank; Dobbek, Holger; Schneider, Erwin

2012-11-01

111

ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal  

PubMed Central

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration. PMID:24707049

Quazi, Faraz; Molday, Robert S.

2014-01-01

112

Unsaturated fatty acids repress expression of ATP binding cassette transporter A1 and G1 in RAW 264.7 macrophages.  

PubMed

Reverse cholesterol transport (RCT), a process to deliver excess cholesterol from the periphery to the liver for excretion from body, is a major atheroprotective property of high-density lipoproteins. As major transporters for cholesterol efflux in macrophages, ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) are critical for RCT. We investigated mechanisms for the regulation of ABCA1 and ABCG1 expression by fatty acids (FA) in RAW264.7 macrophages. Cells were incubated with 100 ?mol/L of palmitic, oleic, linoleic, linolenic or eicosapentaenoic acids in the absence or presence of T0901317, a liver X receptor (LXR) agonist. Unsaturated FA, but not saturated FA, significantly reduced ABCA1 and ABCG1 mRNA without the agonist. Trichostatin A (TSA), a histone deacetylase inhibitor, not only increased basal ABC transporter expression but abrogated the transcriptional repression by unsaturated FA. The increased basal ABCA1 and ABCG1 mRNA by TSA paralleled the increased peroxisome proliferator-activated receptor ? (PPAR?) and PPAR? coactivator 1? expression, whereas LXR? and PGC-1? expression was significantly lowered. Although the repressive effect of ABCA1 and ABCG1 mRNA by unsaturated FA was abolished by T0901317, protein levels remained diminished. Chemical and genetic deficiency of protein kinase C ? did not abolish the repressive effect of linoleic acid on ABCA1 and ABCG1. In conclusion, unsaturated FA repressed ABCA1 and ABCG1 expression by two distinct mechanisms in RAW 264.7 macrophages: LXR-dependent transcriptional repression possibly by modulating histone acetylation state and LXR-independent posttranslational inhibition. PMID:22209005

Ku, Chai Siah; Park, Youngki; Coleman, Sara L; Lee, Jiyoung

2012-10-01

113

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

PubMed

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

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

2013-09-01

114

Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages  

PubMed Central

Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to diabetes-associated atherosclerosis. PMID:24838154

Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

2014-01-01

115

Effect of triptolide on the regulation of ATP?binding cassette transporter A1 expression in lipopolysaccharide?induced acute lung injury of rats.  

PubMed

The aim of this study was to investigate the effect of triptolide on ATP?binding cassette transporter A1 (ABCA1) expression in lipopolysaccharide (LPS)?induced acute lung injury (ALI) in rats. Thirty male Sprague Dawley rats weighing 200?250 g were randomly divided into six groups: Normal (N, n=5), Control (C, n=5), LPS (L, n=5), Triptolide 25 µg (TP1, n=5), Triptolide 50 µg (TP2, n=5) and Triptolide 100 µg (TP3, n=5). The N group was not administered anything; the C group was administered 5 ml/kg normal saline intravenously and 7.5 ml/kg 1% dimethylsulfoxide (DMSO) intraperitoneally; the L group was administered 5 mg/kg 0.1% LPS and 1% DMSO; and the TP1, TP2 and TP3 groups were separately injected with 0.1% LPS and 25, 50 or 100 µg/kg triptolide, respectively. All groups had the same liquid?injection volume. Arterial blood gases, tumor necrosis factor?? (TNF??) and ABCA1 expression and general pathology were examined following the treatments. It was found that increasing the triptolide dose in the TP1?3 groups resulted in an increase in the expression of ABCA1 mRNA and protein. As compared with the L group, the ABCA1 expression showed a significant increase in TP2 and TP3 groups (P<0.05). In addition, the expression level of TNF?? was significantly increased in the L and TP1 groups, as compared with that in the N or C groups (P<0.05). Conversely, a marked decrease in TNF?? expression was detected in the TP2 and TP3 groups, as compared with the L or TP1 groups (P<0.05). In conclusion, this study found that triptolide could promote the expression of ABCA1 mRNA and protein and inhibit other inflammatory factors during LPS?induced ALI in rats. Regulating the expression of ABCA1 may be one of the protective mechanisms of triptolide. Furthermore, triptolide?induced increases in ABCA1 expression occurred in a dose?dependent manner between 25 and 100 µg/kg. PMID:25323823

Chen, Jun; Gao, Jianlin; Yang, Jianping; Zhang, Yukun; Wang, Lina

2014-12-01

116

Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

Xu, Yanni; Lai, Fangfang; Xu, Yang; Wu, Yexiang; Liu, Qi; Li, Ni; Wei, Yuzhen; Feng, Tingting; Zheng, Zhihui; Jiang, Wei; Yu, Liyan; Hong, Bin [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China)] [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China); Si, Shuyi, E-mail: sisyimb@hotmail.com [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China)] [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China)

2011-11-04

117

A Member of the PLEIOTROPIC DRUG RESISTANCE Family of ATP Binding Cassette Transporters Is Required for the Formation of a Functional Cuticle in Arabidopsis[W  

PubMed Central

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ?-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell. PMID:21628525

Bessire, Michael; Borel, Sandra; Fabre, Guillaume; Carraça, Luis; Efremova, Nadia; Yephremov, Alexander; Cao, Yan; Jetter, Reinhard; Jacquat, Anne-Claude; Métraux, Jean-Pierre; Nawrath, Christiane

2011-01-01

118

Genetic variant of V825I in the ATP-binding cassette transporter A1 gene and serum lipid levels in the Guangxi Bai Ku Yao and Han populations  

PubMed Central

Background Several genetic variants in the ATP-binding cassette transporter A1 (ABCA1) gene have associated with modifications of serum high-density lipoprotein cholesterol (HDL-C) levels and the susceptibility for coronary heart disease, but the findings are still controversial in diverse racial/ethnic groups. Bai Ku Yao is an isolated subgroup of the Yao minority in southern China. The present study was undertaken to detect the possible association of V825I (rs2066715) polymorphism in the ABCA1 gene and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 677 subjects of Bai Ku Yao and 646 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples. Polymerase chain reaction and restriction fragment length polymorphism assay combined with gel electrophoresis were performed for the genotyping of V825I variant, and then confirmed by direct sequencing. Results The levels of serum total cholesterol (TC), HDL-C, apolipoprotein (Apo) AI and ApoB were lower in Bai Ku Yao than in Han (P < 0.01 for all). The frequency of G and A alleles was 57.4% and 42.6% in Bai Ku Yao, and 57.7% and 42.3% in Han (P > 0.05); respectively. The frequency of GG, GA and AA genotypes was 33.7%, 47.4% and 18.9% in Bai Ku Yao, and 33.4%, 48.6% and 18.0% in Han (P > 0.05); respectively. There was no difference in the genotypic and allelic frequencies between males and females in the both ethnic groups. The subjects with AA genotype in Bai Ku Yao had higher serum TC levels than the subjects with GG and GA genotypes (P < 0.05). The participants with AA genotype in Han had lower serum HDL-C and ApoAI levels than the participants with GG and GA genotypes (P < 0.05 for each), but these results were found in males but not in females. Multivariate linear regression analysis showed that the levels of TC in Bai Ku Yao and HDL-C and ApoAI in male Han were correlated with genotypes (P < 0.05 for all). Serum lipid parameters were also correlated with sex, age, body mass index, alcohol consumption, and blood pressure in both ethnic groups (P < 0.05-0.001). Conclusion The present study suggests that the V825I polymorphism in the ABCA1 gene is associated with male serum HDL-C and ApoAI levels in the Han, and serum TC levels in the Bai Ku Yao populations. The difference in the association of V825I polymorphism and serum lipid levels between the two ethnic groups might partly result from different ABCA1 gene-enviromental interactions. PMID:21247457

2011-01-01

119

The R219K Polymorphism on ATP-Binding Cassette Transporter A1 Gene is Associated with Coronary Heart Disease Risk in Asia Population: Evidence from a Meta-Analysis.  

PubMed

A number of case-control studies have been conducted to investigate the relationship between the ATP-binding cassette transporter A1 (ABCA1) gene polymorphisms and risk of coronary heart disease (CHD). However, the results have been inconclusive. The purpose of the present study is to investigate whether this polymorphism confers significant susceptibility to CHD using a meta-analysis. We conducted searches of the published literature in PubMed, Embase, and CBM databases. 13 studies were included in our meta-analysis, involving a total of 11,678 individuals. Subgroup analyses were performed by ethnicity and cancer type. Statistically significant association between ABCA1 gene R219K polymorphism and increased CHD risk was found in total population analyses in all four genetic comparison models (ORC vs. T 1.19, 95 % CI 1.07-1.31; P = 0.001; ORHomozygote model 1.28, 95 % CI 1.07-1.52; P = 0.007; ORRecessive genetic model 1.22, 95 % CI 1.04-1.44, P = 0.015; ORDominant model 1.21, 95 % CI 1.07-1.35; P = 0.001). In subgroup analyses based on ethnicity, the association was still significant in Asians (All P values < 0.001), but not in Caucasians (All P values > 0.05). ABCA1 R219K polymorphism is associated with CHD susceptibility, and individuals with ABCA1 have a significantly higher risk of cancer particularly in Asians. PMID:25104170

Liu, Nannan; Hou, Mingxiao; Ren, Weidong; Cao, Junying; Wu, Hongli; Zhou, Weiwei

2015-01-01

120

Opposing Gatekeepers of Apical Sterol Transport: Niemann-Pick C1-Like 1 (NPC1L1) and ATP-Binding Cassette Transporters G5 and G8 (ABCG5/ABCG8)  

PubMed Central

Cholesterol is essential for the growth and function of all mammalian cells, but abnormally elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) are a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). For many years, statin drugs have been used to effectively lower LDL-C, but ASCVD still persists in most of the world. Hence, additional LDL-C lowering is now recommended, and the search for therapeutic strategies that work in synergy with statins has now begun. Intestinal absorption and biliary excretion of cholesterol represent two major pathways and continue to show promise as druggable processes. Importantly, both of these complex physiological pathways are tightly regulated by key proteins located at the apical surface of the small intestine and the liver. One of these proteins, the target of ezetimibe Niemann-Pick C1-Like 1 (NPC1L1), was recently identified to be essential for intestinal cholesterol absorption and protect against excessive biliary sterol loss. In direct opposition of NPC1L1, the heterodimer of ATP-binding cassette transporters G5 and G8 (ABCG5/ABCG8) has been shown to be critical for promoting biliary cholesterol secretion in the liver, and has also been proposed to play a direct role in intestinal disposal of sterols. The purpose of this review is to summarize the current state of knowledge regarding the function of these opposing apical cholesterol transporters, and provide a framework for future studies examining these proteins. PMID:20174593

Brown, J. Mark; Yu, Liqing

2010-01-01

121

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  

PubMed Central

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

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

2013-01-01

122

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.  

PubMed

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

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

2013-10-01

123

ATP-binding cassette transporter A1 is significantly involved in the intestinal absorption of a- and c-tocopherol but not in that of retinyl  

Microsoft Academic Search

Background: It has long been assumed that newly absorbed vita- min A and E enter the body only via enterocyte-produced chylomi- crons. However, recent results in cell cultures have shown that a fraction of a-tocopherol is secreted with intestinal HDL. Objectives: The aims of this study were to identify this transporter and to assess whether it is significantly implicated in

Emmanuelle Reboul; Doriane Trompier; Myriam Moussa; Alexis Klein; Patrick Borel

124

The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage*  

PubMed Central

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule. PMID:19797057

Nagy, Réka; Grob, Hanne; Weder, Barbara; Green, Porntip; Klein, Markus; Frelet-Barrand, Annie; Schjoerring, Jan K.; Brearley, Charles; Martinoia, Enrico

2009-01-01

125

Binding of PDZ-RhoGEF to ATP-binding Cassette Transporter A1 (ABCA1) Induces Cholesterol Efflux through RhoA Activation and Prevention of Transporter Degradation*  

PubMed Central

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 PPAR? 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. PMID:20348106

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

2010-01-01

126

Novel mechanism of transcriptional repression of the human ATP binding cassette transporter A1 gene in hepatic cells by the winged helix/forkhead box transcription factor A2.  

PubMed

ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXR?/RXR?. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene. PMID:24807696

Thymiakou, Efstathia; Kardassis, Dimitris

2014-06-01

127

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and HuH7 Cells  

PubMed Central

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPAR?) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPAR?-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPAR?- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters. PMID:23634230

Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Álvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

128

AtMRP6/AtABCC6, an ATP-Binding Cassette transporter gene expressed during early steps of seedling development and up-regulated by cadmium in Arabidopsis thaliana  

PubMed Central

Background ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described. Results This gene, located on chromosome 3, is bordered by AtMRP3 and AtMRP7. Using real-time quantitative PCR (RT-Q-PCR) and the GUS reporter gene, we found that this gene is essentially expressed during early seedling development, in the apical meristem and at initiation point of secondary roots, especially in xylem-opposite pericycle cells where lateral roots initiate. The level of expression of AtMRP6 in response to various stresses was explored and a significant up-regulation after cadmium (Cd) treatment was detected. Among the three T-DNA insertion lines available from the Salk Institute library, two knock-out mutants, Atmrp6.1 and Atmrp6.2 were invalidated for the AtMRP6 gene. In the presence of Cd, development of leaves was more affected in the mutants than wild-type plants, whereas root elongation and ramification was comparable. Conclusion The position of AtMRP6 on chromosome 3, flanked by two other MRP genes, (all of which being induced by Cd) suggests that AtMRP6 is part of a cluster involved in metal tolerance, although additional functions in planta cannot be discarded. PMID:18307782

Gaillard, Stéphane; Jacquet, Hélène; Vavasseur, Alain; Leonhardt, Nathalie; Forestier, Cyrille

2008-01-01

129

Structural model of ATP-binding proteing associated with cystic fibrosis, multidrug resistance and bacterial transport  

Microsoft Academic Search

THE ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization (reviewed in refs 1-3). This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export

Stephen C. Hyde; Paul Emsley; Michael J. Hartshorn; Michael M. Mimmack; Uzi Gileadi; Stephen R. Pearce; Maurice P. Gallagher; Deborah R. Gill; Roderick E. Hubbard; Christopher F. Higgins

1990-01-01

130

An ABC transporter complex containing S-adenosylmethionine (SAM)-induced ATP-binding protein is involved in antibiotics production and SAM signaling in Streptomyces coelicolor M145.  

PubMed

A sco3956-deletion mutant (?SCO3956) of Streptomyces coelicolor was generated to characterize the S-adenosylmethionine (SAM)-induced, ATP-binding cassette transporter (ABC transporter) ATP-binding protein, SCO3956. It produced actinorhodin (ACT) and undecylprodigiosin (RED) decreased by approx. 82 and 64 %, respectively. In addition, the effect of exogenous SAM was lost in the ?SCO3956. Plasmid-based complementation of sco3956 in ?SCO3956 restored ACT and RED levels of ?SCO3956 to wild-type levels (ACT: 20 ± 1.4 mg g(-1) DCW and RED: 5.3 ± 0.6 mg g(-1) DCW) and the exogenous effect significantly increased ACT and RED by approx. 129 and 135 %, respectively, when compared to the exogenous SAM non-treated sco3956 complementation strain. Thus, the ABC transporter ATP-binding protein, SCO3956, plays a critical role in ACT and RED production serving as a transducer of SAM signaling. PMID:22911564

Lee, Sung-Kwon; Mo, Sangjoon; Suh, Joo-Won

2012-10-01

131

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.  

PubMed

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ?abcAB and ?hmg strains). The B. ovis ?abcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the ?hmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ?abcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ?abcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo. PMID:21300772

Silva, Teane M A; Paixão, Tatiane A; Costa, Erica A; Xavier, Mariana N; Sá, Joicy Cortez; Moustacas, Valéria S; den Hartigh, Andreas B; Carvalho Neta, Alcina V; Oliveira, Sérgio C; Tsolis, Renée; Santos, Renato L

2011-04-01

132

Effect of ATP binding cassette/multidrug resistance proteins on ATP efflux of Saccharomyces cerevisiae.  

PubMed

Multidrug resistance (MDR) in mammalian tumors or tissues is often associated with the overexpression of the putative drug efflux pump P-glycoprotein (Pgp). One theory concerning the mechanism of Pgp activity is that efflux of ATP is coupled to drug efflux. Evidence in support of this theory has been observed in mammalian cells. Recently, the STS1 gene, which is a multidrug resistance gene related to the mammalian Pgp's, has been characterized in S. cerevisiae. Also, the mouse mdr3 Pgp has been functionally expressed in yeast cells. Therefore, it was of interest to determine whether the expression of these proteins affected ATP efflux from yeast. Although both genes were shown to confer MDR, thus confirming functional expression, the endogenous glucose-dependent, drug-stimulated ATP efflux activity of yeast was not affected by expression of STS1, and was decreased by the expression of mouse mdr3. PMID:9020050

Boyum, R; Guidotti, G

1997-01-01

133

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

PubMed

ABC transporters represent a large family of ATP-driven transmembrane transporters involved in uni- or bidirectional transfer of a large variety of substrates. Divided in seven families, they represent 48 transporter proteins, several of which have been associated with human disease. Among the latter is ABCC6, a unidirectional exporter protein primarily expressed in liver and kidney. ABCC6 deficiency has been shown to cause the ectopic mineralization disorder pseudoxanthoma elasticum (PXE), characterized by calcification and fragmentation of elastic fibers, resulting in oculocutaneous and cardiovascular symptoms. Unique in the group of connective tissue disorders, the pathophysiological 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 many ABC transporters, it is worthwhile to evaluate if and to what extent the knowledge on the physiology and pathophysiology of these other transporters may provide useful clues toward understanding the (patho)physiological role of ABCC6 and how its deficiency may be dealt with. PMID:24137173

Vanakker, Olivier M; Hosen, Mohammad J; Paepe, Anne De

2013-01-01

134

Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATP-binding cassette (ABC) transporter.  

PubMed

The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. PMID:25281745

Cole, Susan P C

2014-11-01

135

ATP-binding Cassette Transporters Are Required for Efficient RNA Interference in Caenorhabditis elegans  

Microsoft Academic Search

RNA interference (RNAi) is a conserved gene-silencing phenomenon that can be triggered by delivery of double-stranded RNA (dsRNA) to cells and is a widely exploited technology in analyses of gene function. Although a number of proteins that facilitate RNAi have been identified, current descriptions of RNAi and interrelated mechanisms are far from complete. Here, we report that the Caenorhabditis elegans

Prema Sundaram; Benjamin Echalier; Wang Han; Dawn Hull; Lisa Timmons

2006-01-01

136

ATP Binding to the Motor Domain from an ABC Transporter Drives Formation of a Nucleotide Sandwich Dimer  

PubMed Central

Summary It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps. PMID:12150914

Smith, Paul C.; Karpowich, Nathan; Millen, Linda; Moody, Jonathan E.; Rosen, Jane; Thomas, Philip J.; Hunt, John F.

2012-01-01

137

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein  

E-print Network

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein Sheref S. Mansy1 op- timization of a non-biological protein derived from a library of random amino acid sequences sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily

Heller, Eric

138

A Two-site Kinetic Mechanism for ATP Binding and Hydrolysis by E. coli Rep Helicase Dimer Bound to a  

E-print Network

A Two-site Kinetic Mechanism for ATP Binding and Hydrolysis by E. coli Rep Helicase Dimer Bound that are coupled to ATP binding and hydrolysis. We have investi- gated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep ``P2S'' dimer (formed

Lohman, Timothy M.

139

ATP binding turns plant cryptochrome into an efficient natural photoswitch.  

PubMed

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. PMID:24898692

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

2014-01-01

140

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch  

NASA Astrophysics Data System (ADS)

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.

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

2014-06-01

141

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch  

PubMed Central

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. PMID:24898692

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

2014-01-01

142

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

PubMed Central

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

2013-01-01

143

Cassette Books.  

ERIC Educational Resources Information Center

This catalog lists cassette books produced by the National Library Service for the Blind and Physically Handicapped during 1989. Books are listed alphabetically within subject categories under nonfiction and fiction headings. Nonfiction categories include: animals and wildlife, the arts, bestsellers, biography, blindness and physical handicaps,…

Library of Congress, Washington, DC. National Library Service for the Blind and Physically Handicapped.

144

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

SciTech Connect

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.

Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

2012-10-24

145

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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...

146

Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3  

PubMed Central

The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation. PMID:24142515

Beers, Michael F.; Zhao, Ming; Tomer, Yaniv; Russo, Scott J.; Zhang, Peggy; Gonzales, Linda W.; Guttentag, Susan H.

2013-01-01

147

Pseudoxanthoma elasticum: Mutations in the MRP6 gene encoding a transmembrane ATP-binding cassette (ABC) transporter  

PubMed Central

Pseudoxanthoma elasticum (PXE), the prototypic heritable connective tissue disorder affecting the elastic structures in the body, manifests with cutaneous, ophthalmologic, and cardiovascular findings, with considerable morbidity and mortality. The molecular basis of PXE has remained unknown, but the disease locus has recently been mapped to an ?500-kb interval on chromosome 16p13.1, without evidence for locus heterogeneity. In this study, we report pathogenetic mutations in MRP6, a member of the ABC transporter gene family, in eight kindreds with PXE. The mutation detection strategy consisted of heteroduplex scanning of coding sequences in the MRP6 gene, which were amplified by PCR by using genomic DNA as template, followed by direct nucleotide sequencing. A total of 13 mutant MRP6 alleles were disclosed in the eight probands with PXE. These genetic lesions consisted of either single base pair substitutions resulting in missense, nonsense, or splice site mutations, or large deletions resulting in allelic loss of the MRP6 locus. Examination of clinically unaffected family members in four multiplex families identified heterozygous carriers, consistent with an autosomal recessive inheritance pattern. Collectively, identification of mutations in the MRP6 gene provides the basis to examine the pathomechanisms of PXE and allows development of DNA-based carrier detection, prenatal testing, and preimplantation genetic diagnosis in families with a history of this disease. PMID:10811882

Ringpfeil, Franziska; Lebwohl, Mark G.; Christiano, Angela M.; Uitto, Jouni

2000-01-01

148

Characterization of Arabidopsis ABCG11/WBC11, an ATP binding cassette (ABC) transporter that is required for  

E-print Network

and was localized to the plasma membrane. These results show that WBC11 functions in secretion of surface waxes of Plant Biology, Michigan State University, East Lansing, MI 48824, USA, 3 Max-Planck-Institut fu¨ r Zu fusions, stunted growth and a reduction in cutin load on the plant surface. A mutant line previously

Kunst, Ljerka

149

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

PubMed

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 ?-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 ?-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. PMID:25543853

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

2015-02-01

150

Multidrug resistance protein 4/ ATP binding cassette transporter 4: a new potential therapeutic target for acute myeloid leukemia.  

PubMed

Less than a third of adults patients with acute myeloid leukemia (AML) are cured by current treatments, emphasizing the need for new approaches to therapy. We previously demonstrated that besides playing a role in drug-resistant leukemia cell lines, multidrug resistance protein 4 (MRP4/ABCC4) regulates leukemia cell proliferation and differentiation through the endogenous MRP4/ABCC4 substrate, cAMP. Here, we studied the role of MRP4/ABCC4 in tumor progression in a mouse xenograft model and in leukemic stem cells (LSCs) differentiation. We found a decrease in the mitotic index and an increase in the apoptotic index associated with the inhibition of tumor growth when mice were treated with rolipram (PDE4 inhibitor) and/or probenecid (MRPs inhibitor). Genetic silencing and pharmacologic inhibition of MRP4 reduced tumor growth. Furthermore, MRP4 knockdown induced cell cycle arrest and apoptosis in vivo. Interestingly, when LSC population was isolated, we observed that increased cAMP levels and MRP4/ABCC4 blockade resulted in LSCs differentiation. Taken together, our findings show that MRP4/ABCC4 has a relevant role in tumor growth and apoptosis and in the eradication of LSCs, providing the basis for a novel promising target in AML therapy. PMID:25301721

Copsel, Sabrina; Bruzzone, Ariana; May, Maria; Beyrath, Julien; Wargon, Victoria; Cany, Jeannette; Russel, Frans G M; Shayo, Carina; Davio, Carlos

2014-10-15

151

Differential Sensitivities of the Human ATP-Binding Cassette Transporters ABCG2 and P-Glycoprotein to Cyclosporin A  

E-print Network

with radiotherapy or surgery. Many cancers, however, are intrinsically resistant or become resistant to a variety called BCRP, MXR1, or ABCP, was first cloned from drug-resistant breast cancer and colon cancer cell-Glycoprotein to Cyclosporin A Karin F. K. Ejendal and Christine A. Hrycyna Department of Chemistry and the Purdue Cancer

Hrycyna, Christine A.

152

Regulation and Expression of the ATP-binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells  

PubMed Central

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker SSEA-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the down-regulation of two microRNAs (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of microRNA mimics and inhibitors of these two microRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by microRNAs in hESCs. PMID:22887864

Padmanabhan, Raji; Chen, Kevin G.; Gillet, Jean-Pierre; Handley, Misty; Mallon, Barbara S.; Hamilton, Rebecca S.; Park, Kyeyoon; Varma, Sudhir; Mehaffey, Michele G.; Robey, Pamela G.; McKay, Ronald D.G.; Gottesman, Michael M.

2012-01-01

153

Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams.  

PubMed

Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ?abcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2 mL of a suspension containing 1.2×10(9) CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50 ?L in each conjunctival sac of a suspension containing 1.2×10(10) CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ?abcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ?abcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ?abcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ?abcAB-infected ram had a positive iliac lymph node sample by PCR. PMID:24075357

Silva, Ana Patrícia C; Macêdo, Auricélio A; Costa, Luciana F; Turchetti, Andréia P; Bull, Valquíria; Pessoa, Moisés S; Araújo, Márcio S S; Nascimento, Ernane F; Martins-Filho, Olindo A; Paixão, Tatiane A; Santos, Renato L

2013-12-27

154

XENOBIOTIC REGULATION OF THE ATP BINDING CASSETTE TRANSPORTER ABCB6 AND ITS SIGNIFICANCE TO HEPATIC HEME HOMEOSTASIS  

E-print Network

porphyria Uroporphyrinogen III cosynthase Porphyria cutanea tarda Uroporphyrinogen decarboxylase Hepatoerythropoietic porphyria Uroporphyrinogen decarboxylase Hereditary coproporphyria Coproporphyrinogen oxidase Variegate porphyria Protoporphyrinogen...

Chavan, Hemantkumar Dilip

2013-12-31

155

Inactivation of Multiple Bacterial Histidine Kinases by Targeting the ATP-Binding Domain.  

PubMed

Antibacterial agents that exploit new targets will be required to combat the perpetual rise of bacterial resistance to current antibiotics. We are exploring the inhibition of histidine kinases, constituents of two-component systems. Two-component systems are the primary signaling pathways that bacteria utilize to respond to their environment. They are ubiquitous in bacteria and trigger various pathogenic mechanisms. To attenuate these signaling pathways, we sought to broadly target the histidine kinase family by focusing on their highly conserved ATP-binding domain. Development of a fluorescence polarization displacement assay facilitated high-throughput screening of ?53?000 diverse small molecules for binding to the ATP-binding pocket. Of these compounds, nine inhibited the catalytic activity of two or more histidine kinases. These scaffolds could provide valuable starting points for the design of broadly effective HK inhibitors, global reduction of bacterial signaling, and ultimately, a class of antibiotics that function by a new mechanism of action. PMID:25531939

Wilke, Kaelyn E; Francis, Samson; Carlson, Erin E

2015-01-16

156

Unique functional and structural properties of the LRRK2 protein ATP-binding pocket.  

PubMed

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult. PMID:25228699

Liu, Zhiyong; Galemmo, Robert A; Fraser, Kyle B; Moehle, Mark S; Sen, Saurabh; Volpicelli-Daley, Laura A; DeLucas, Lawrence J; Ross, Larry J; Valiyaveettil, Jacob; Moukha-Chafiq, Omar; Pathak, Ashish K; Ananthan, Subramaniam; Kezar, Hollis; White, E Lucile; Gupta, Vandana; Maddry, Joseph A; Suto, Mark J; West, Andrew B

2014-11-21

157

ATP Binding to Synaspsin IIa Regulates Usage and Clustering of Vesicles in Terminals of Hippocampal Neurons.  

PubMed

Synaptic transmission is expensive in terms of its energy demands and was recently shown to decrease the ATP concentration within presynaptic terminals transiently, an observation that we confirm. We hypothesized that, in addition to being an energy source, ATP may modulate the synapsins directly. Synapsins are abundant neuronal proteins that associate with the surface of synaptic vesicles and possess a well defined ATP-binding site of undetermined function. To examine our hypothesis, we produced a mutation (K270Q) in synapsin IIa that prevents ATP binding and reintroduced the mutant into cultured mouse hippocampal neurons devoid of all synapsins. Remarkably, staining for synaptic vesicle markers was enhanced in these neurons compared with neurons expressing wild-type synapsin IIa, suggesting overly efficient clustering of vesicles. In contrast, the mutation completely disrupted the capability of synapsin IIa to slow synaptic depression during sustained 10 Hz stimulation, indicating that it interfered with synapsin-dependent vesicle recruitment. Finally, we found that the K270Q mutation attenuated the phosphorylation of synapsin IIa on a distant PKA/CaMKI consensus site known to be essential for vesicle recruitment. We conclude that ATP binding to synapsin IIa plays a key role in modulating its function and in defining its contribution to hippocampal short-term synaptic plasticity. PMID:25609616

Shulman, Yoav; Stavsky, Alexandra; Fedorova, Tatiana; Mikulincer, Dan; Atias, Merav; Radinsky, Igal; Kahn, Joy; Slutsky, Inna; Gitler, Daniel

2015-01-21

158

Functional role of ATP binding to synapsin I in synaptic vesicle trafficking and release dynamics.  

PubMed

Synapsins (Syns) are synaptic vesicle (SV)-associated proteins involved in the regulation of synaptic transmission and plasticity, which display a highly conserved ATP binding site in the central C-domain, whose functional role is unknown. Using molecular dynamics simulations, we demonstrated that ATP binding to SynI is mediated by a conformational transition of a flexible loop that opens to make the binding site accessible; such transition, prevented in the K269Q mutant, is not significantly affected in the absence of Ca(2+) or by the E373K mutation that abolishes Ca(2+)-binding. Indeed, the ATP binding to SynI also occurred under Ca(2+)-free conditions and increased its association with purified rat SVs regardless of the presence of Ca(2+) and promoted SynI oligomerization. However, although under Ca(2+)-free conditions, SynI dimerization and SV clustering were enhanced, Ca(2+) favored the formation of tetramers at the expense of dimers and did not affect SV clustering, indicating a role of Ca(2+)-dependent dimer/tetramer transitions in the regulation of ATP-dependent SV clustering. To elucidate the role of ATP/SynI binding in synaptic physiology, mouse SynI knock-out hippocampal neurons were transduced with either wild-type or K269Q mutant SynI and inhibitory transmission was studied by patch-clamp and electron microscopy. K269Q-SynI expressing inhibitory synapses showed increased synaptic strength due to an increase in the release probability, an increased vulnerability to synaptic depression and a dysregulation of SV trafficking, when compared with wild-type SynI-expressing terminals. The results suggest that the ATP-SynI binding plays predocking and postdocking roles in the modulation of SV clustering and plasticity of inhibitory synapses. PMID:25355227

Orlando, Marta; Lignani, Gabriele; Maragliano, Luca; Fassio, Anna; Onofri, Franco; Baldelli, Pietro; Giovedí, Silvia; Benfenati, Fabio

2014-10-29

159

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.  

PubMed

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. PMID:23331996

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

160

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase  

PubMed Central

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

161

Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.  

PubMed

Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P; Diffley, John F X

2014-09-01

162

Critical role of ?-phosphate in structural transition of Na,K-ATPase upon ATP binding.  

PubMed

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that ?-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while ?-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the "E1-open" to "E1-closed" conformation ready for phosphorylation. PMID:24893715

Petrushanko, Irina Yu; Mitkevich, Vladimir A; Anashkina, Anastasia A; Klimanova, Elizaveta A; Dergousova, Elena A; Lopina, Olga D; Makarov, Alexander A

2014-01-01

163

Critical role of ?-phosphate in structural transition of Na,K-ATPase upon ATP binding  

NASA Astrophysics Data System (ADS)

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that ?-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while ?-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

2014-06-01

164

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.  

PubMed

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles--including their nucleotide-free states--at ? 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. PMID:25209998

Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

2014-01-01

165

A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.  

PubMed

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

2010-09-01

166

ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.  

PubMed Central

VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family. PMID:12747802

Zalk, Ran; Shoshan-Barmatz, Varda

2003-01-01

167

Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site  

PubMed Central

Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket. PMID:24900346

2011-01-01

168

Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.  

PubMed

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state. PMID:25193668

Clausen, Johannes D; Holdensen, Anne Nyholm; Andersen, Jens Peter

2014-10-17

169

A Chemical Proteomics Approach to Profiling the ATP-binding Proteome of Mycobacterium tuberculosis *  

PubMed Central

Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics. PMID:23462205

Wolfe, Lisa M.; Veeraraghavan, Usha; Idicula-Thomas, Susan; Schürer, Stephan; Wennerberg, Krister; Reynolds, Robert; Besra, Gurdyal S.; Dobos, Karen M.

2013-01-01

170

Video Cartridges and Cassettes.  

ERIC Educational Resources Information Center

The economic and social significance of video cassettes (viewer-controlled playback system) is explored in this report. The potential effect of video cassettes on industrial training, education, libraries, and television is analyzed in conjunction with the anticipated hardware developments. The entire video cassette industry is reviewed firm by…

Kletter, Richard C.; Hudson, Heather

171

Identification of Mutations in Regions Corresponding to the Two Putative Nucleotide (ATP)Binding Folds of the Cystic Fibrosis Gene  

Microsoft Academic Search

Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found

Bat-Sheva Kerem; Julian Zielenski; Danuta Markiewicz; Dominique Bozon; Ephraim Gazit; Jacob Yahav; Dara Kennedy; John R. Riordan; Francis S. Collins; Johanna M. Rommens; Lap-Chee Tsui

1990-01-01

172

Effect of Alanine-293 Replacement on the Activity, ATP Binding, and Editing of Escherichia coli Leucyl-tRNA Synthetase  

E-print Network

Effect of Alanine-293 Replacement on the Activity, ATP Binding, and Editing of Escherichia coli was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure (2). Based on the conserved amino acid sequences and crystal structures, the 20 aaRSs are divided

Tian, Weidong

173

ACID: annotation of cassette and integron data  

E-print Network

Background: Although integrons and their associated gene cassettes are present in ~10% of bacteria and can represent up to 3% of the genome in which they are found, very few have been properly identified and annotated in ...

Boucher, Yan

174

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes  

SciTech Connect

The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

C Simmons; C Magee; D Smith; L Lauman; J Chaput; J Allen

2011-12-31

175

Characterization and purification of a novel dATP-binding protein in eukaryotes.  

PubMed Central

We characterized and purified an acidic dATP-binding protein, which, in its active form, resides in the nuclear fraction of a range of cells from mammals (including pig liver) and baker's yeast (Saccharomyces cerevisiae). This protein exhibits a high degree of specificity for the deoxy form of the naturally occurring nucleoside triphosphates and shows a marked preference for the purine deoxynucleoside triphosphates dATP and dGTP. The protein cleaves the terminal phosphate of dATP and appears to retain the dADP moiety of the nucleotide in a reaction that is resistant to both SDS and 8 M-urea. Fractionation of the nuclear preparation followed by non-denaturing PAGE and SDS/PAGE electrophoresis was sufficient to produce pure protein. The occurrence of this activity in all nuclei tested suggests that it plays an important role in nuclear metabolism. The specificity of the enzyme for deoxynucleoside triphosphates further suggests a role for this enzyme in DNA replication or repair, but the acidity of the protein argues against a direct interaction with DNA, and, indeed, the catalytic activity is not modulated by the inclusion of DNA in a variety of physical forms. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10. Fig. 11. Fig. 12. PMID:1445246

Dalton, A; Hornby, D P; Langston, S P; Blackburn, G M

1992-01-01

176

Sequence-based predictor of ATP-binding residues using random forest and mRMR-IFS feature selection.  

PubMed

We develop a computational and statistical approach (ATPBR) for predicting ATP-binding residues in proteins from amino acid sequences by using random forests with a novel hybrid feature. The hybrid feature incorporates a new feature called PSSMPP, the predicted secondary structure and orthogonal binary vectors. The mRMR-IFS feature selection method is utilized to construct the best prediction model. At last, ATPBR achieves significantly improved performance over existing methods, with 87.53% accuracy and a Matthew?s correlation coefficient of 0.554. In addition, our further analysis demonstrates that PSSMPP distinguishes more effectively between ATP-binding and non-binding residues. Besides, the optimal features selected by the mRMR-IFS method improve the prediction performance and may provide useful insights for revealing the mechanisms of ATP and proteins interactions. PMID:25014477

Ma, Xin; Sun, Xiao

2014-11-01

177

Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.  

PubMed

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains. PMID:24762105

Zhang, Longmei; Xu, Huijuan; Jie, Yanling; Gao, Chao; Chen, Wanjuan; Yin, Shikui; Samways, Damien S K; Li, Zhiyuan

2014-05-13

178

Activation of ATP Binding for the Autophosphorylation of DosS, a Mycobacterium tuberculosis Histidine Kinase Lacking an ATP Lid Motif*  

PubMed Central

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg440 in the DHp domain and Glu537 in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg440 and Glu537 reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity. PMID:23486471

Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

2013-01-01

179

Effect of poly(ethylene glycol)-block-polylactide nanoparticles on hepatic cells of mouse: Low cytotoxicity, but efflux of the nanoparticles by ATP-binding cassette transporters  

Microsoft Academic Search

The objective of this paper is to study the effects of poly(ethylene glycol)-block-polylactide (PLA–PEG) nanoparticles on hepatic cells of mouse. Blank PLA–PEG nanoparticles have been successfully prepared and MTT assay suggested that the nanoparticles with HepG2 cell co-culture model did not cause significant changes in membrane integrity in controlled concentration range (0.001–0.1mg\\/ml). Immunohistochemical analysis demonstrated that large dose of PLA–PEG

Yangde Zhang; Zhiyuan Hu; Maoying Ye; Yifeng Pan; Jiji Chen; Yulin Luo; Yanqiong Zhang; Lianxiang He; Jiwei Wang

2007-01-01

180

Stimulation of CD36 and the key effector of reverse cholesterol transport ATP-binding cassette A1 in monocytoid cells by niacin  

Microsoft Academic Search

Niacin, the first lipid lowering drug shown to improve survival after myocardial infarction, decreases LDL and increases HDL cholesterol levels. These effects cannot fully be explained by its suspected mechanism of action, inhibition of lipolysis and hepatic VLDL synthesis. Niacin has also been shown to interfere with the cyclic AMP (cAMP)\\/protein kinase A (PKA) pathway and massively stimulate prostaglandin D2

Tina Rubic; Matthias Trottmann; Reinhard L Lorenz

2004-01-01

181

Identification and localization of a putative ATP-binding cassette transporter in sea lice (Lepeophtheirus salmonis) and host Atlantic salmon (Salmo salar).  

PubMed

Some members of the ABC-transporter superfamily, such as P-glycoprotein and the multidrug resistance associated protein, may confer resistance to the avermectin subclass of macrocyclic lactones. The aim of this study was to examine the presence of ABC transporters in both sea lice (Lepeophtheirus salmonis) and its Atlantic salmon host (Salmo salar) using monoclonal antibodies (C219 and JSB-1, with high selectivity for P-gp) and a new polyclonal antibody (SL0525) generated against a putative sea louse ABC transporter. The antibody raised to SL0525 did not react with rat P-gp, suggesting that an ABC transporter, not necessarily P-gp, was isolated. C219 was the only antibody to localize P-gp in all 3 salmon tissues (intestine, kidney and liver). American lobster (Homarus americanus) was used as a reference crustacean for L. salmonis immunostaining reactions and showed positive staining in the hepatopancreatic and intestinal tissues with all 3 antibodies. The L. salmonis showed positive staining in the intestinal epithelial lining with all antibodies. This report represents the first documented evidence for the expression of ABC transporters in L. salmonis, its Atlantic salmon host, and the American lobster. PMID:17961285

Tribble, N D; Burka, J F; Kibenge, F S B; Wright, G M

2008-02-01

182

Variants in the ATP-Binding Cassette Transporter (ABCA7), Apolipoprotein E ?4, and the Risk of Late-Onset Alzheimer Disease in African Americans  

PubMed Central

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

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

183

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL\\/6 and apoE-knockout mice  

Microsoft Academic Search

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in

C. W. Joyce; M J Amar; G Lambert; B L Vaisman; B Paigen; Fruchart J Najib; R F Hoyt; E D Neufeld; A T Remaley; D S Fredrickson; H B Brewer; Fojo S Santamarina

2002-01-01

184

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL\\/6 and apoE-knockout mice  

Microsoft Academic Search

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg × apoE-knockout (KO) mice. Overexpression of hABCA1 in

Charles W. Joyce; Marcelo J. A. Amar; Gilles Lambert; Boris L. Vaisman; Beverly Paigen; Jamila Najib-Fruchart; Robert F. Hoyt Jr.; Edward D. Neufeld; Alan T. Remaley; Donald S. Fredrickson; H. Bryan Brewer Jr.; Silvia Santamarina-Fojo

2002-01-01

185

Becatecarin (rebeccamycin analog, NSC 655649) is a transport substrate and induces expression of the ATP-binding cassette transporter, ABCG2, in lung carcinoma cells  

PubMed Central

Purpose ABCG2 overexpression has been linked to resistance to topoisomerase inhibitors, leading us to examine the potential interaction between ABCG2 and becatecarin. Methods Interaction with ABCG2 was determined by ATPase assay, competition of [125I]iodoarylazidoprazosin (IAAP) photolabeling and flow cytometry. Cellular resistance was measured in four-day cytotoxicity assays. ABCG2 expression was measured by fluorescent-substrate transport assays and immunoblot. Results Becatecarin competed [125I]-IAAP labeling of ABCG2, stimulated ATPase activity and, at concentrations greater than 10 ?M, inhibited ABCG2-mediated transport. Becatecarin-selected A549 Bec150 lung carcinoma cells were 3.1-fold, 15-fold, 8-fold, and 6.8-fold resistant to becatecarin, mitoxantrone, SN-38 and topotecan, respectively. A549 Bec150 cells transported the ABCG2 substrates pheophorbide a, mitoxantrone and BODIPY-prazosin and displayed increased staining with the anti-ABCG2 antibody 5D3 compared to parental cells. Increased ABCG2 expression was confirmed by immunoblot. Conclusions Our results suggest that becatecarin is transported by ABCG2 and can induce ABCG2 expression in cancer cells. PMID:19132374

Robey, Robert W.; Obrzut, Tomasz; Shukla, Suneet; Polgar, Orsolya; Macalou, Sira; Bahr, Julian C.; Di Pietro, Attilio; Ambudkar, Suresh V.; Bates, Susan E.

2008-01-01

186

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens.  

PubMed

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l(-1), 40 g Tween 80 l(-1) and 30 g peptone l(-1), TliA was produced at a level of 2,200 U ml(-1) in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml(-1)) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production. PMID:24737082

Eom, Gyeong Tae; Song, Jae Kwang

2014-08-01

187

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.  

PubMed

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. PMID:24586633

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

2014-01-01

188

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  

PubMed Central

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. PMID:24586633

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

2014-01-01

189

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

Microsoft Academic Search

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

Yan Boucher; Camilla L Nesbø; Michael J Joss; Andrew Robinson; Bridget C Mabbutt; Michael R Gillings; W Ford Doolittle; HW Stokes

2006-01-01

190

Automated cassette-to-cassette substrate handling system  

DOEpatents

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.

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

2014-03-18

191

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

SciTech Connect

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.

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

2009-01-01

192

ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast  

SciTech Connect

Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast.

Rhee, Dong Keun [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Cho, Bon A [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Hyong Bai [Department of Bioinformatics, Korea University, Yeongigun, Chungnam 339-700 (Korea, Republic of)]. E-mail: hbkim5212@hotmail.com

2005-06-03

193

Characterization of the ATP-Binding Domain of the Sarco(endo)plasmic Reticulum -ATPase: Probing Nucleotide Binding by Multidimensional NMR  

E-print Network

, Tokyo Metropolitan UniVersity, Hachioji, Tokyo 192-0397, Japan ReceiVed August 21, 2001; ReVised containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity- tracture due to a slow rate of removal of Ca2+ from the cytosol. The disruption of one copy of the ATP2A2

Ikura, Mitsuhiko

194

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation.  

PubMed

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced. It is assumed, that under these conditions Hsp90 is required to protect other proteins from aggregation. One property of Hsp90 is its ability to undergo autophosphorylation. The N-terminal domain of Hsp90 has been shown to contain an unusual ATP-binding site. A well-known inhibitor of Hsp90 function is geldanamycin binding to the N-terminal ATP-binding site with high affinity. Recently it was shown that Hsp90 possesses a second ATP-binding site in the C-terminal region, which can be competed with novobiocin. Autophosphorylation of Hsp90 was analysed by incubation with gamma(32)P-ATP. Addition of geldanamycin did not interfere with the capability for autophosphorylation, while novobiocin indeed did. These results suggest that the C-terminal ATP-binding site is required for autophosphorylation of Hsp90. PMID:12127946

Langer, T; Schlatter, H; Fasold, H

2002-01-01

195

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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 ...

196

Functionality maps of the ATP binding site of DNA gyrase B: generation of a consensus model of ligand binding.  

PubMed

The Multiple Copy Simultaneous Search method (MCSS) was used to construct consensus functionality maps for functional group binding in the ATP binding site of DNA gyrase B. To account for the conformational flexibility of the protein active site, which involves small side chain fluctuations as well as large-scale loop motions, the calculations were done for three different conformations of the 24 kDa subdomain of DNA gyrase B. A postprocessing procedure that employs a continuum dielectric model to include solvent effects was used to calculate the binding free energy for every functional group. These results were ranked according to their affinity for DNA gyrase B and clustered using a new procedure based on van der Waals contacts that is better adapted for cases where multiple conformations are being considered. A total of 23 different functional groups were tested. The results gave consensus maps that indicate those functional group binding sites that are insensitive to the specific protein conformation. The maps also demonstrate that functional groups other than those found in the known ligands may bind competitively in the binding sites of known ligands. This suggests numerous scaffolds that can be used in the development of new ligands for the ATP and coumarinic binding sites in DNA gyrase B. Finally, the calculations show the existence of alternative binding sites near the known binding sites that could be targeted in the rational design for new inhibitors. PMID:15317451

Schechner, Martina; Sirockin, Finton; Stote, Roland H; Dejaegere, Annick P

2004-08-26

197

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

SciTech Connect

Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation ({delta}F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-{delta}F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.

Kerem, B.; Zielenski, J.; Markiewicz, D.; Bozon, D.; Kennedy, D.; Rommens, J.M. (Hospital for Sick Children, Toronto, Ontario (Canada)); Gazit, E. (Tel Aviv Univ. (Israel)); Yahav, J. (Chaim Sheba Medical Center, Tel Hashomer (Israel)); Riordan, J.R. (Univ. of Toronto, Ontario (Canada)); Collins, F.S. (Univ. of Michigan, Ann Arbor (United States)); Tsui, Lapchee (Hospital for Sick Children, Toronto, Ontario (Canada) Univ. of Toronto, Ontario (Canada))

1990-11-01

198

Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition  

PubMed Central

TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase–controlled phosphorylation to generate physiologically significant changes in TOR signaling. PMID:24247430

Hálová, Lenka; Du, Wei; Kirkham, Sara; Smith, Duncan L.

2013-01-01

199

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding  

PubMed Central

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors. PMID:21078955

Diao, Yarui; Liu, Wei; Wong, Catherine C. L.; Wang, Xi; Lee, Kaman; Cheung, Po-yan; Pan, Lifeng; Xu, Tao; Han, Jiahuai; Yates, John R.; Zhang, Mingjie; Wu, Zhenguo

2010-01-01

200

The Deviant ATP-binding Site of the Multidrug Efflux Pump Pdr5 Plays an Active Role in the Transport Cycle*  

PubMed Central

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites. PMID:24019526

Furman, Christopher; Mehla, Jitender; Ananthaswamy, Neeti; Arya, Nidhi; Kulesh, Bridget; Kovach, Ildiko; Ambudkar, Suresh V.; Golin, John

2013-01-01

201

Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor  

PubMed Central

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in kcat and a 15-fold-lower Km ATP although Km peptide was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases. PMID:11390649

Frankel, Mark; Ablooglu, Ararat J.; Leone, Joseph W.; Rusinova, Elena; Ross, J. B. Alexander; Heinrikson, Robert L.; Kohanski, Ronald A.

2001-01-01

202

Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.  

PubMed

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases. PMID:11390649

Frankel, M; Ablooglu, A J; Leone, J W; Rusinova, E; Ross, J B; Heinrikson, R L; Kohanski, R A

2001-07-01

203

ATP sequestration by a synthetic ATP-binding protein leads to novel phenotypic changes in Escherichia coli.  

PubMed

Artificial proteins that bind key metabolites with high affinity and specificity hold great promise as new tools in synthetic biology, but little has been done to create such molecules and examine their effects on living cells. Experiments of this kind have the potential to expand our understanding of cellular systems, as certain phenotypes may be physically realistic but not yet observed in nature. Here, we examine the physiology and morphology of a population of Escherichia coli as they respond to a genetically encoded, non-biological ATP-binding protein. Unlike natural ATP-dependent proteins, which transiently bind ATP during metabolic transformations, the synthetic protein DX depletes the concentration of intracellular ATP and ADP by a mechanism of protein-mediated ligand sequestration. The resulting ATP/ADP imbalance leads to an adaptive response in which a large population of bacilli cells transition to a filamentous state with dense lipid structures that segregate the cells into compartmentalized units. A wide range of biochemical and microscopy techniques extensively characterized these novel lipid structures, which we have termed endoliposomes. We show that endoliposomes adopt well-defined box-like structures that span the full width of the cell but exclude the synthetic protein DX. We further show that prolonged DX exposure causes a large fraction of the population to enter a viable-but-non-culturable state that is not easily reversed. Both phenotypes correlate with strong intracellular changes in ATP and ADP concentration. We suggest that artificial proteins, such as DX, could be used to control and regulate specific targets in metabolic pathways. PMID:23181457

Korch, Shaleen B; Stomel, Joshua M; León, Megan A; Hamada, Matt A; Stevenson, Christine R; Simpson, Brent W; Gujulla, Sunil K; Chaput, John C

2013-02-15

204

DNA-dependent protein kinase (DNA-PK) inhibitors: Structure–activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain  

Microsoft Academic Search

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure–activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110?), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC50=8nM).

Kate M. Clapham; Julia Bardos; M. Raymond V. Finlay; Bernard T. Golding; Edward J. Griffen; Roger J. Griffin; Ian R. Hardcastle; Keith A. Menear; Attilla Ting; Paul Turner; Gail L. Young; Céline Cano

2011-01-01

205

Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation.  

PubMed

The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions. PMID:25202014

Salzer, Ralf; Herzberg, Martin; Nies, Dietrich H; Joos, Friederike; Rathmann, Barbara; Thielmann, Yvonne; Averhoff, Beate

2014-10-31

206

Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r  

SciTech Connect

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.

Knight, K.L.; Hess, R.M.; McEntee, K.

1988-06-01

207

Signatures of the ATP-binding pocket as a basis for structural classification of the serine/threonine protein kinases of gram-positive bacteria.  

PubMed

Eukaryotic-like serine/threonine protein kinases (ESTPKs) are widely spread throughout the bacterial genomes. These enzymes can be potential targets of new antibacterial drugs useful for the treatment of socially important diseases such as tuberculosis. In this study, ESTPKs of pathogenic, probiotic, and antibiotic-producing Gram-positive bacteria were classified according to the physicochemical properties of amino acid residues in the ATP-binding site of the enzyme. Nine residues were identified that line the surface of the adenine-binding pocket, and ESTPKs were classified based on these signatures. Twenty groups were discovered, five of them containing >10 representatives. The two most abundant groups contained >150 protein kinases that belong to the various branches of the phylogenetic tree, whereas certain groups are genus- or even species-specific. Homology modeling of the typical representatives of each group revealed that the classification is reliable, and the differences between the protein kinase ATP-binding pockets predicted based on their signatures are apparent in their structure. The classification is expected to be useful for the selection of targets for new anti-infective drugs. PMID:22275035

Zakharevich, Natalia V; Osolodkin, Dmitry I; Artamonova, Irena I; Palyulin, Vladimir A; Zefirov, Nikolay S; Danilenko, Valery N

2012-05-01

208

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

PubMed Central

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

Montelone, Beth A.; Aguilera, Andrés

2014-01-01

209

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

SciTech Connect

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

Ebina, Y.; Araki, E.; Taira, M.; Shimada, F.; Mori, M.; Craik, C.S.; Siddle, K.; Pierce, S.B.; Roth, R.A.; Rutter, W.J.

1987-02-01

210

Effects on maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo  

PubMed Central

Resistance to the experimental human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir has been mapped to UL97 mutations at codons 353, 397, 409 and 411, in the kinase ATP-binding region, and to mutations in the UL27 gene. We studied the maribavir susceptibility phenotypes of additional UL97 mutations observed in vitro and in clinical trials, and the effect of simultaneous mutation in both UL97 and UL27. In vitro selection under maribavir identified a new locus of UL97 mutation within the conserved kinase p-loop (L337M), which conferred low grade maribavir resistance (3.5-fold increased EC50) without ganciclovir cross-resistance. During maribavir Phase III CMV prevention clinical trials, 3 previously unknown UL97 sequence variants were detected in plasma samples after 27 to 98 days of drug exposure (I324V, S334G and S386L). These variants did not confer any drug resistance despite proximity to mutations that confer maribavir resistance. The UL27 resistance mutation R233S, when added to strains containing UL97 mutations L337M or V353A, doubled their maribavir EC50s. These results expand the range of UL97 maribavir-resistance mutations into another part of the kinase ATP-binding region, but offer no genotypic evidence that development of drug resistance affected the outcomes of Phase III maribavir clinical trials after drug exposure of up to 14 weeks. There is a potential for increased maribavir resistance in UL27–UL97 double mutants. PMID:22664236

Chou, Sunwen; Hakki, Morgan; Villano, Stephen

2012-01-01

211

The amino acid sequence 442GDASE446 in Na/K-ATPase is an important motif in forming the high and low affinity ATP binding pockets.  

PubMed

A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects. PMID:14522987

Imagawa, Toshiaki; Kaya, Shunji; Taniguchi, Kazuya

2003-12-12

212

Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter  

Microsoft Academic Search

The disease diabetes mellitus arises as a consequence of a failure of the ?-cells in the islets of Langerhans of the pancreas to produce insulin in the amounts required to meet the needs of the body. Whole pancreas or islet transplants in patients with severe diabetes effectively restore insulin production. A lack of availability of donor pancreata requires the development

Andreas Lechner; Colin A Leech; Elizabeth J Abraham; Anna L Nolan; Joel F Habener

2002-01-01

213

HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck  

PubMed Central

Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting. PMID:22420777

2012-01-01

214

The interaction of Pcf11 and Clp1 is needed for mRNA 3'-end formation and is modulated by amino acids in the ATP-binding site.  

PubMed

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1. PMID:21993299

Ghazy, Mohamed A; Gordon, James M B; Lee, Susan D; Singh, Badri Nath; Bohm, Andrew; Hampsey, Michael; Moore, Claire

2012-02-01

215

The three-dimensional structure of MAP kinase p38[beta]: different features of the ATP-binding site in p38[beta] compared with p38[alpha  

SciTech Connect

The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38{alpha} and p38{beta}) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38{alpha} isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38{beta} may not provide any additional benefit. In order to aid the development of p38{alpha}-selective compounds, the three-dimensional structure of p38{beta} was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38{beta} were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05 {angstrom} resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38{alpha} C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38{beta}. This difference in size between the two pockets could be exploited in order to achieve selectivity.

Patel, Sangita B.; Cameron, Patricia M.; O'Keefe, Stephen J.; Frantz-Wattley, Betsy; Thompson, Jed; O'Neill, Edward A.; Tennis, Trevor; Liu, Luping; Becker, Joseph W.; Scapin, Giovanna; Merck

2010-10-18

216

4-Hydroxy-trans-2-nonenal (4-HNE) induces neuronal SH-SY5Y cell death via hampering ATP binding at kinase domain of Akt1.  

PubMed

Inhibition mechanism(s) of protein kinase B/Akt1 and its consequences on related cell signaling were investigated in human neuroblastoma SH-SY5Y cells exposed to 4-hydroxy-trans-2-nonenal (4-HNE), one of the most reactive aldehyde by-products of lipid peroxidation. In silico data indicate that 4-HNE interacts with kinase domain of Akt1 with the total docking score of 6.0577 and also forms H-bond to Glu234 residue similar to highly potent Akt1 inhibitor imidazopiperidine analog 8b, in which the protonated imidazole nitrogen involves in two hydrogen bonds between Glu234 and Asp292. The strong hydrogen bonding with Glu234 and hydrophobic interactions with several residues, namely Leu156, Gly157, Val164, Ala177, Tyr229, Ala230, Met281 and Thr291, at the vicinity which is normally occupied by the ribose of ATP, appear to be the main causes of Akt1 inhibition and lead to the significant conformational change on this region of protein. Results of mutational docking prove that Glu234 plays a major role in 4-HNE-mediated Akt1 inhibition. In silico data on Akt inhibition were further validated by observing the down-regulated levels of phosphorylated (Thr308/Ser493) Akt1 as well as the altered levels of the downstream targets of pAkt, namely downregulated levels of pGSK3? (Ser9), ?-catenin, Bcl2 and upregulated levels of pro-apoptotic markers, namely Bad, Bax, P(53) and caspase-9/3. The cellular fate of such pAkt inhibition was evidenced by increased reactive oxygen species, degraded nuclei, transferase dUTP nick end labeling positive cells and upregulated levels of pJNK1/2. We identified that 4-HNE-mediated Akt1 inhibition was due to the competitive inhibition of ATP by 4-HNE at the kinase domain of ATP binding sites. PMID:24825450

Kashyap, Mahendra P; Singh, Abhishek K; Yadav, Dharmendra K; Siddiqui, Maqsood A; Srivastava, Ritesh K; Chaturvedi, Vishal; Rai, Navneet

2015-02-01

217

Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.  

PubMed Central

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases. PMID:10595540

Martin, G.; Jenö, P.; Keller, W.

1999-01-01

218

Molecular modelling of the nucleotide-binding domain of Wilson's disease protein: location of the ATP-binding site, domain dynamics and potential effects of the major disease mutations  

PubMed Central

WNDP (Wilson's disease protein) is a copper-transporting ATPase that plays an essential role in human physiology. Mutations in WNDP result in copper accumulation in tissues and cause a severe hepato-neurological disorder known as Wilson's disease. Several mutations were surmised to affect the nucleotide binding and hydrolysis by WNDP; however, how the nucleotides bind to normal and mutated WNDP remains unknown. To aid such studies, we performed the molecular modelling of the spatial structure and dynamics of the ATP-binding domain of WNDP and its interactions with ATP. The three-dimensional models of this domain in two conformations were built using the X-ray structures of the Ca2+-ATPase in the E1 and E2 states. To study the functional aspects of the models, they were subjected to long-term molecular dynamics simulations in an explicit solvent; similar calculations were performed for the ATP-binding domain of Ca2+-ATPase. In both cases, we found large-scale motions that lead to significant changes of distances between several functionally important residues. The ATP docking revealed two possible modes of ATP binding: via adenosine buried in the cleft near residues H1069, R1151 and D1164, and via phosphate moiety ‘anchored’ by H-bonds with residues in the vicinity of catalytic D1027. Furthermore, interaction of ATP with both sites occurs if they are spatially close to each other. This may be achieved after relative domain motions of the ‘closure’ type observed in molecular dynamics simulations. The results provide a framework for analysis of disease mutations and for future mutagenesis studies. PMID:15147237

2004-01-01

219

Antibiotic-resistance cassettes for Bacillus subtilis  

Microsoft Academic Search

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Anne-Marie Guérout-Fleury; Kamran Shazand; Niels Frandsen; Patrick Stragier

1995-01-01

220

Phosphate binding and ATP-binding sites coexist in Na+/K(+)-transporting ATPase, as demonstrated by the inactivating MgPO4 complex analogue Co(NH3)4PO4.  

PubMed

Tetrammine cobalt(III) phosphate [Co(NH3)4PO4] inactivates Na+/K(+)-ATPase in the E2 conformational state, dependent on time and concentration, according to Eqn (1): Co(NH3)4PO4 + E2 Kd in equilibrium E2.Co(NH3)4PO4k2----E'2.Co(NH3)4PO4. The inactivation rate constant k2 for the formation of a stable E'2.Co(NH3)4PO4 at 37 degrees C was 0.057 min-1; the dissociation constant, Kd = 300 microM. The activation energy for the inactivation process was 149 kJ/mol. ATP and the uncleavable adenosine 5'-[beta, gamma-methylene]triphosphate competed with Co(NH3)4PO4 for its binding site with Ks = 0.41 mM and 5 mM, respectively. MgPO4 competed with Co(NH3)4PO4 linearly, with Ks = 50 microM, as did phosphate (Ks = 16 mM) and Mg2+ (Ks = 160 microM). It is concluded that the MgPO4 analogue binds to the MgPO4-binding subsite of the low-affinity ATP-binding site (of the E2 conformation). Also, Na+ (Ks = 860 microM) protected the enzyme against inactivation in a competitive manner. From the intersecting (slope and intercept linear) noncompetitive effect of Na+ against the inactivation by Co(NH3)4PO4, apparent affinities of K+ for the free enzyme of 41 microM, and for the E.Co(NH3)4PO4 complex of 720 microM, were calculated. Binding of Co(NH3)4PO4 to the enzyme inactivated Na+/K(+)-ATPase and K(+)-activated phosphatase, and, moreover, prevented the occlusion of 86Rb+; however, the activity of the Na(+)-ATPase, the phosphorylation capacity of the high-affinity ATP-binding site and the ATP/ADP-exchange reaction remained unchanged. With Co(NH3)432PO4 a binding capacity of 135 pmol unit enzyme was found. Phosphorylation and complete inactivation of the enzyme with Co(NH3)432PO4 or the 32P-labelled tetramminecobalt ATP ([gamma-32P]Co(NH3)4ATP) at the low-affinity ATP-binding site, allowed (independent of the purity of the Na+/K(+)-ATPase preparation) a further incorporation of radioactivity from 32P-labelled tetraaquachromium(III) ATP ([gamma-32P]CrATP) to the high-affinity ATP-binding site with unchanged phosphorylation capacity. However, inactivation and phosphorylation of Na+/K(+)-ATPase by [gamma-32P]CrATP prevented the binding of Co(NH3)4 32PO4 or [gamma-32P]Co(NH3)4ATP to the enzyme. [gamma-32P]CO(NH3)4ATP and Co(NH3)432PO4 are mutually exclusive. The data are consistent with the assumption of a cooperation of catalytic subunits within an (alpha,beta)2-diprotomer, which change their interactions during the Na+/K(+)-pumping process. Our findings seem not to support a symmetrical Repke and Stein model of enzyme action. PMID:1847680

Buxbaum, E; Schoner, W

1991-01-30

221

A Cassette Based System for Hydrogen Storage and Delivery  

SciTech Connect

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.

Britton Wayne E.

2006-11-29

222

Finger-Actuated, Self-Contained Immunoassay Cassettes  

PubMed Central

The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity. PMID:19597994

Qiu, Xianbo; Thompson, Jason A.; Chen, Zongyuan; Liu, Changchun; Chen, Dafeng; Ramprasad, Sudhir; Mauk, Michael G.; Ongagna, Serge; Barber, Cheryl; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L.A.M.; Bau, Haim H.

2010-01-01

223

Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.  

PubMed

Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7?M), verapamil (0.6 vs. 0.7?M) and prazosin (0.099 vs. 0.033?M) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

2014-04-25

224

The contribution of cholesterol-7alpha-hydroxylase, paraoxonase-1, and thioredoxin-interacting protein in lipid metabolism  

E-print Network

et al. 2004. The ABCA1 transporter modulates late endocyticThe ABC transporter family members ABCA1 (16), ABCG5/ABCG8ABCA1 – ATP Binding Cassette Protein A1 ABCG1 – ATP Binding Cassette Protein G1 ABCG5 – ATP-Binding Cassette Transporter

Ratliff, Eric Pham

2009-01-01

225

Micro computer control of a video cassette lecture.  

PubMed

Circuits have been developed to allow the presentation of a video cassette lecture to be controlled by an inexpensive micro computer. The potential of both video cassette lecture and standard computer assisted learning techniques is extended, resulting in increased interaction between the student and the teaching material presented to him. PMID:6991894

Davis, P D; Kenny, G N

1980-05-01

226

Structural Diversity of Class 1 Integrons and Their Associated Gene Cassettes in Klebsiella pneumoniae Isolates from a Hospital in China  

PubMed Central

Background Klebsiella pneumoniae strains carrying class 1 integrons are becoming more common worldwide, and their role in the dissemination of drug resistance is significant. The aim of this study was to characterize the structural diversity of class 1 integrons and their associated gene cassettes in K. pneumoniae isolates from hospital settings. Methodology/Principal Findings We analyzed a total of 176 K. pneumoniae isolates in a tertiary-care hospital in Beijing, China for the period of November 1, 2010-October 31, 2011. The presence of class 1 integrons and gene cassettes was analyzed by PCR and sequencing. The prevalence of class 1 integrons was 51.1% (90/176). Fourteen different gene cassettes and 10 different gene cassette arrays were detected. dfrA and aadA cassettes were predominant and cassette combination dfrA1-orfC was most frequently found (13.6%, 24/176). Strong association between resistance to a variety of drugs (both phenotypes and the associated genes) and the presence of class 1 integrons was observed. In addition, we also identified an association between some previously identified prevalent sequence types (such as ST11, ST15, ST147, ST562, and ST716) and the presence of class 1 integrons. Conclusions/Significance Data from this study demonstrated that class 1 integrons are highly diverse and are associated with a variety of drug resistance phenotypes, drug resistance genes, as well as genotypes among K. pneumoniae isolates. Continuous monitoring of gene cassettes in class 1 integrons is warranted to improve the understanding and control of drug resistance among hospital settings. PMID:24098729

Yi, Yong; Woo, Patrick C. Y.; Jing, Hua; Zhu, Baoli; Liu, Cui Hua

2013-01-01

227

Apparatus and method for loading and unloading multiple digital tape cassettes utilizing a removable magazine  

DOEpatents

An apparatus and method to automate the handling of multiple digital tape cassettes for processing by commercially available cassette tape readers and recorders. A removable magazine rack stores a plurality of tape cassettes, and cooperates with a shuttle device that automatically inserts and removes cassettes from the magazine to the reader and vice-versa. Photocells are used to identify and index to the desired tape cassette. The apparatus allows digital information stored on multiple cassettes to be processed without significant operator intervention.

Lindenmeyer, C.W.

1993-01-26

228

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A...

2011-04-01

229

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2012-04-01

230

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2014 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2014-04-01

231

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2010 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2010-04-01

232

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2010 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A...

2010-04-01

233

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2011-04-01

234

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2014 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A...

2014-04-01

235

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A...

2013-04-01

236

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A...

2012-04-01

237

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2013-04-01

238

Replicative resolution of integron cassette insertion  

PubMed Central

Site-specific recombination catalyzed by tyrosine recombinases follows a common pathway consisting of two consecutive strand exchanges. The first strand exchange generates a Holliday junction (HJ), which is resolved by a second strand exchange. In integrons, attC sites recombine as folded single-stranded substrates. Only one of the two attC site strands, the bottom one, is efficiently bound and cleaved by the integrase during the insertion of gene cassettes at the double-stranded attI site. Due to the asymmetry of this complex, a second strand exchange on the attC bottom strand (bs) would form linearized abortive recombination products. We had proposed that HJ resolution would rely on an uncharacterized mechanism, probably replication. Using an attC site carried on a plasmid with each strand specifically tagged, we followed the destiny of each strand after recombination. We demonstrated that only one strand, the one carrying the attC bs, is exchanged. Furthermore, we show that the recombination products contain the attC site bs and its entire de novo synthesized complementary strand. Therefore, we demonstrate the replicative resolution of single-strand recombination in integrons and rule out the involvement of a second strand exchange of any kind in the attC?×?attI reaction. PMID:22740653

Loot, Céline; Ducos-Galand, Magaly; Escudero, José Antonio; Bouvier, Marie; Mazel, Didier

2012-01-01

239

Erythrosin 5?-isothiocyanate labels Cys 549 as part of the low-affinity ATP binding site of Na +\\/K +ATPase 1 This work is part of the Ph.D. thesis of H.L. at the Justus-Liebig University Giessen. 1  

Microsoft Academic Search

The high-affinity E1ATP site of Na+\\/K+-ATPase labeled with fluorescein 5?-isothiocyanate and its E2ATP site labeled with erythrosin 5?-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813–28821], reside on separate and adjacent catalytic ? subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the

Holger Linnertz; Holger Kost; Tomas Obsil; Arnost Kotyk; Evzen Amler; Wilhelm Schoner

1998-01-01

240

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae  

PubMed Central

Background Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. Results To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 ?m ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. Conclusions These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided. PMID:24161108

2013-01-01

241

The effect of ABCG5/G8 polymorphisms on plasma HDL cholesterol levels depends on the ABCA1 gene variation in the Boston Puerto Rican Health Study  

Technology Transfer Automated Retrieval System (TEKTRAN)

Background: ATP-binding cassette transporters G5/G8 have shown an association with HDL-C. One of the most likely mechanisms to explain those associations is through ABCA1. Objective: To assess whether the effect of ABCG5/G8 polymorphisms on HDL-C is dependent on ABCA1, we studied potential interacti...

242

The ABC transporter gene family of Daphnia pulex  

Microsoft Academic Search

BACKGROUND: The large gene superfamily of ABC (ATP-binding cassette) transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex, providing for the first time information on ABC proteins

Armin Sturm; Phil Cunningham; Michael Dean

2009-01-01

243

Mitoxantrone Targets the ATP-binding Site of FAK, Binds the FAK Kinase Domain and Decreases FAK, Pyk-2, c-Src, and IGF-1R, In Vitro Kinase Activities  

PubMed Central

Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors and plays a key role in cell adhesion, spreading, motility, proliferation, invasion, angiogenesis, and survival. Recently, FAK has been proposed as a target for cancer therapy, and we performed computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database to target the ATP-binding site of FAK, K454. More than 140,000 small molecule compounds were docked into the crystal structure of the kinase domain of FAK in 100 different orientations using DOCK5.1 that identified small molecule compounds, targeting the K454 site, called A-compounds. To find the therapeutic efficacy of these compounds, we examined the effect of twenty small molecule compounds on cell viability by MTT assays in different cancer cell lines. One compound, A18 (1,4-bis(diethylamino)-5,8-dihydroxy anthraquinon) was a mitoxantrone derivative and significantly decreased viability in most of the cells comparable to the to the level of FAK kinase inhibitors TAE-226 (Novartis, Inc) and PF-573,228 (Pfizer). The A18 compound specifically blocked autophosphorylation of FAK like TAE-226 and PF-228. ForteBio Octet Binding assay demonstrated that mitoxantrone (1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino) ethylamino] anthracene-9,10-dione directly binds the FAK-kinase domain. In addition, mitoxantrone significantly decreased the viability of breast cancer cells in a dose-dependent manner and inhibited the kinase activity of FAK and Y56/577 FAK phosphorylation at 10-20 ?M. Mitoxantrone did not affect phosphorylation of EGFR, but decreased Pyk-2, c-Src, and IGF-1R kinase activities. The data demonstrate that mitotraxone decreases cancer viability, binds FAK-Kinase domain, inhibits its kinase activity, and also inhibits in vitro kinase activities of Pyk-2 and IGF-1R. Thus, this novel function of the mitoxantrone drug can be critical for future development of anti-cancer agents and FAK-targeted therapy research. PMID:22292772

Golubovskaya, Vita; Ho, Baotran; Zheng, Min; Magis, Andrew; Ostrov, David; Cance, William

2013-01-01

244

Development and fabrication of ITER divertor cassette body  

Microsoft Academic Search

The ITER divertor cassette body is a large (>20 tonne) stainless steel structure that supports the plasma facing components (PFCs), delivers cooling water to them, and provides shielding for the vacuum vessel and magnets. It is also required to withstand electromagnetic loads and route cooling water through itself to remove nuclear heating. It is intended to be a full life

K. T. Slattery; D. E. Driemeyer; K. D. Foreman; J. M. Mackereth; K. G. Sandell

1997-01-01

245

Effects of Electrostatic Charge on Aerosol Collection with Polystyrene Filter Cassettes  

Microsoft Academic Search

Electrostatic fields are present on polystyrene cassette filter holders commonly used to measure the concentration of particles in air. Beryllium concentrations were determined at a beryllium refinery using neutral, and positively and negatively charged cassettes. In laboratory experiments, tobacco smoke, magnetite and polyvinyltoluene latex spheres were collected by both charged and neutral cassettes. No differences in concentration measurements were observed

SCOTT TURNER; BEVERLY S. COHEN

1984-01-01

246

Evaluation of the E-Z Cassette Player. Report No. 16-RD-83.  

ERIC Educational Resources Information Center

An evaluation of the E-Z cassette player was conducted for the National Library Service (NLS) for the Blind and Physically Handicapped to determine if the player is suitable for readers who either had no previous experience with cassettes or who cannot operate a standard NLS cassette player. Players were distributed to volunteer library patrons…

Crotty, Timothy J.

247

Obstacles to the production of protein microarray cassettes  

NASA Astrophysics Data System (ADS)

This paper examines the necessary technologies to be mastered in order to build a practical micro array-based immunoassay cassette and its processing station for protein analysis. The interdependence of surface-chemistry, dye stability and imaging are outlined showning why a treated 100 nm film of Nitrocellulose adhered by an intervening layer to glass offers an efficacious surface for immobilizing an array of protein probes. The properties of a storage surface to support in desiccated form, fluidize and transport additional reagents are outlined and a practical solution proposed. Wet and Dry imaging are compared. The steps and functions expected for an assay platform comprising processing station and biochip cassette are identified. The performance of a successful bench-top automated multiplex immunoassay system is briefly described

Montagu, Jean; DeWeerd, Herman; Tyburczy, Nathan; Rodionova, Natalia; Maimonis, Peter

2009-02-01

248

Cassette less SOFC stack and method of assembly  

DOEpatents

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.

Meinhardt, Kerry D

2014-11-18

249

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

DOEpatents

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.

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

2012-10-23

250

AC Electrokinetics Facilitated Biosensor Cassette for Rapid Pathogen Identification  

PubMed Central

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 achieve robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency though electrokinetic 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 electrokinetic for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future. PMID:23626988

Ouyang, Mengxing; Mohan, Ruchika; Lu, Yi; Liu, Tingting; Mach, Kathleen E.; Sin, Mandy L. Y.; McComb, Mason; Joshi, Janhvi; Gau, Vincent

2013-01-01

251

Motion Pictures and Video Cassettes 1971. AV-USA Supplement 2.  

ERIC Educational Resources Information Center

The financial status of the motion picture and of the video cassette industry in 1970 are reviewed. Based on production rates and income of these industries, trends are discovered. Figures on local origination of television programing and commercials are also included. The section on video cassettes includes the following information: the current…

Hope, Thomas W.

252

Expression of Antibiotic Resistance Genes in the Integrated Cassettes of Integrons  

Microsoft Academic Search

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 Pant, and alterations in the sequence of Pant affected the level of resistance

CHRISTINA M. COLLIS; ANDRUTH M. HALL

1995-01-01

253

Cassette Sound Filmstrip Viewers -- Evaluations of Nine Machines. An In Depth Report  

ERIC Educational Resources Information Center

In evaluating cassette sound filmstrip viewers, many criteria are the same as those applied to cassette recorders in Educational Product Report; v7 n5 Nov '73 and silent filmstrip viewers in Educational Product Report; v6 n5 Feb '73. These criteria cover output power; frequency response; tape speed accuracy including drift, wow, and flutter; and…

Educational Product Report, 1974

1974-01-01

254

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

PubMed Central

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:21390267

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

2011-01-01

255

Gate assembly for the opening of an X-ray apparatus for receiving an X-ray cassette  

Microsoft Academic Search

An apparatus is described for receiving an x-ray cassette having means for minimizing the entrance of a second cassette or other objects into the receiving area comprising: a housing having an opening for receiving an x-ray film cassette, a gate positioned at the opening, the gate having first and second leaves pivotably mounted about a common pin, spring means for

Dieterien

1988-01-01

256

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations  

PubMed Central

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4? which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain–domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy. PMID:23483032

Arányi, Tamás; Bacquet, Caroline; de Boussac, Hugues; Ratajewski, Marcin; Pomozi, Viola; Fülöp, Krisztina; Brampton, Christopher N.; Pulaski, Lukasz; Saux, Olivier Le; Váradi, András

2013-01-01

257

Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication  

PubMed Central

Background Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler’s diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model. Results HLHK9?ureA, HLHK9?ureC, HLHK9?ureD and HLHK9?ureE all exhibited no urease activity. HLHK9?arcA1 and HLHK9?arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9?arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9?arcA1/arcA2 and HLHK9?ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9?ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9?ureA/arcA1/arcA2 and HLHK9?arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9?ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9?ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9?arcA1/arcA2 and HLHK9?ureA/arcA1/arcA2 were markedly reduced (p < 0.01). Conclusions In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis. PMID:24533585

2014-01-01

258

A mouse model of sitosterolemia: absence of Abcg8\\/sterolin-2 results in failure to secrete biliary cholesterol  

Microsoft Academic Search

BACKGROUND: Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5\\/sterolin-1 and ABCG8\\/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet

Eric L Klett; Kangmo Lu; Astrid Kosters; Edwin Vink; Mi-Hye Lee; Michael Altenburg; Sarah Shefer; Ashok K Batta; Hongwei Yu; Jianliang Chen; Richard Klein; Norbert Looije; Ronald Oude-Elferink; Albert K Groen; Nobuyo Maeda; Gerald Salen; Shailendra B Patel

2004-01-01

259

Effect of BIBF 1120 on reversal of ABCB1-mediated multidrug resistance  

Microsoft Academic Search

Background:   The overexpression of ATP-binding cassette (ABC) transporters is one of the main causes of multi-drug resistance (MDR) which\\u000a represents a major obstacle to the success of cancer chemotherapy. In this study, we examined the effect of BIBF 1120, an\\u000a inhibitor of vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs) and\\u000a fibroblast growth factor receptors (FGFRs) tyrosine

Qing-feng Xiang; Fang Wang; Xiao-dong Su; Yong-ju Liang; Li-sheng Zheng; Yan-jun Mi; Wei-qiang Chen; Li-wu Fu

2011-01-01

260

In Vivo Reverse Cholesterol Transport From Macrophages Lacking ABCA1 Expression Is Impaired  

Microsoft Academic Search

Background—ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cholesterol efflux to apoA-I in cholesterol loaded macrophages, a first step of reverse cholesterol transport (RCT) in vivo. Macrophage specific abca1 inactivation or overexpression, respectively, accelerated or suppressed the development of atherosclerosis in mouse models. However, it is yet to be established that the ABCA1 effect is related to specific

Ming-Dong Wang; Vivian Franklin; Yves L. Marcel

2010-01-01

261

Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core.  

PubMed

To determine whether integrons are present in a submarine gas hydrate community, metagenomic DNA was extracted from a gas-hydrate-bearing core, 150 m below the seafloor, from the Cascadian Margin. Integrons and gene cassettes were recovered by PCR from metagenomic DNA and sequenced. Thirty-seven integron integrase phylotypes were identified. The phylotypes were diverse and included members with homology to integrases from Methylomonas methanica, Desulfuromonas acetoxidans, Thermodesulfatator indicus, and marine uncultured bacteria. The gene cassette composition, 153 gene cassettes, was dominated by two types of encoded putative proteins. The first of these was predicted oxidoreductases, such as iron/sulfur cluster-binding proteins. A second type was alkyl transferases. Some cassette proteins showed homologies with those from methane-related archaea. These observations suggest that integrons may assist in the adaptation of microbial communities in this environment. PMID:24117886

Elsaied, Hosam; Stokes, Hatch W; Yoshioka, Hideyoshi; Mitani, Yasuo; Maruyama, Akihiko

2014-02-01

262

COLLECTION EFFICIENCY OF FIELD SAMPLING CASSETTES: INTERAGENCY ENERGY/ENVIRONMENT R AND D PROGRAM REPORT  

EPA Science Inventory

Industrial hygiene particulate samples are often collected under anisokinetic sampling conditions and in crosswinds. Experiments were conducted to quantitate errors associated with sampling under these non-ideal conditions. Three types of field sampling cassetts were tested to de...

263

21 CFR 892.1870 - Radiographic film/cassette changer programmer.  

Code of Federal Regulations, 2014 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a)...

2014-04-01

264

21 CFR 892.1870 - Radiographic film/cassette changer programmer.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a)...

2013-04-01

265

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2014 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2014-04-01

266

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2010 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2010-04-01

267

21 CFR 892.1870 - Radiographic film/cassette changer programmer.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a)...

2011-04-01

268

21 CFR 892.1870 - Radiographic film/cassette changer programmer.  

Code of Federal Regulations, 2010 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a)...

2010-04-01

269

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2013-04-01

270

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2011-04-01

271

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2012-04-01

272

21 CFR 892.1870 - Radiographic film/cassette changer programmer.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a)...

2012-04-01

273

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

PubMed

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. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 84-91, 2015. PMID:24764314

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

2015-01-01

274

The function of integron-associated gene cassettes in Vibrio species: the tip of the iceberg  

PubMed Central

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 mobilized 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 Vibrio rotiferianus DAT722, new insight into 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 cassettes may be important in processes such as bacteriophage resistance, adhesion/biofilm formation, protection from grazers and bacterial aggregation. PMID:24367362

Rapa, Rita A.; Labbate, Maurizio

2013-01-01

275

Circ Res . Author manuscript Liver X receptor activation stimulates iron export in human alternative  

E-print Network

), resulting in the induction of ABCA1, ABCG1 and ApoE expression. Moreover, in iron-loaded M2 macrophages by LXR activation. MESH Keywords ATP Binding Cassette Transporter 1 ; metabolism ; ATP-Binding Cassette Transporters ; metabolism ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism

Paris-Sud XI, Université de

276

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.  

PubMed

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs. PMID:16547094

Bateman, Jack R; Lee, Anne M; Wu, C-ting

2006-06-01

277

Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids  

PubMed Central

Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. PMID:21276210

2011-01-01

278

Storing self-contained gel capillary cassettes for POC medical diagnostics.  

PubMed

For effective clinical uptake of the lab on a chip/point of care technology (LOC-POC), in addition to cost advantages LOC-POC devices should offer multiple patient screening panels for related diseases as well as cold-chain transportation and storage abilities. We recently described a device that performs polymerase chain reaction (PCR) to simultaneously screen raw clinical samples from up to 16 patients for multiple infectious agents (Manage et al., Lab Chip, 2013, 13, 2576-2584). This cassette contains glass capillaries with desiccated semi-solid acrylamide gels that include all the reagents except for the sample, with integrated quality control. Here we report the development of protocols to store assembled PCR cassettes at room temperature, 4 °C or -20 °C as well as at +40 °C. We show that our cassettes are stable, with no loss of activity for at least 3 months at RT and at least 7 months at 4 °C and -20 °C. However, the activity of desiccated cassettes degrades when stored for more than 2 weeks at 40 °C, insufficient time for post-manufacture delivery and use of cassette PCR. To address this, we have evaluated two stage storage protocols. PCR cassettes can initially be stored at 4 °C and -20 °C for prolonged periods of time and removed for shorter term storage at RT, retaining activity for at least a month, which would facilitate transport to remote areas for testing. Effective use of cassette PCR in high temperature regions of the world, for experimental purposes defined here as 40 °C, appears to be feasible only after a first stage storage in the cold, followed by no more than 1 week at 40 °C. This should allow sufficient time for delivery by the manufacturer to a central area well served by power and refrigeration, for later ambient temperature transport and use in under-resourced areas that lack refrigeration. PMID:23966212

Manage, Dammika P; Lauzon, Jana; Zahariadis, George; Pilarski, Linda M

2013-10-21

279

A Comparison of the Closed-Face Cassette at Different Orientations While Measuring Total Particles.  

PubMed

ABSTRACT The current method for sampling aerosols using the 37-mm closed-face cassette (CFC) sampler is based on the orientation of the cassette at ?45° from horizontal. There is some concern as to whether this method is appropriate and may be underestimating exposures. An alternative orientation at ?0° (horizontal) has been discussed. This research compared the CFC's orientation at 45° from horizontal to the proposed orientation at horizontal, 0° in a controlled laboratory setting. The particles used in this study were fused alumina oxide in four sizes, approximately 9.5 ?m, 12.8 ?m, 18 ?m, and 44.3 ?m in aerodynamic diameter. For each test, one aerosol was dispersed in a wind tunnel operating at 0.2 m/s with samplers mounted in the breathing zone of a rotating mannequin. A sampling event consisted of four pairs of samplers, placed side by side (one pair at 45° and another at 0° cassette orientation), and exposed for a period of 45 minutes. A total of twelve sampling events, three sample events per particle size, were conducted with a total of ninety-four samples collected. Mass concentration measurements were compared to assess the relationship between the sampler orientations of the cassettes. In addition, the relationship between the mass collected on the cassette filter and on the interior walls of the cassette was also assessed. The results indicated that there was no significant difference between the measured concentrations based on the orientation of the CFC's. The amount of mass collected on the interior walls of the cassettes were relatively low (<5%) compared to expected (up to 100%) wall losses for both orientations. PMID:25337937

Cook, David M; Sleeth, Darrah K; Thiese, Matthew S; Larson, Rodney R

2014-10-22

280

Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron.  

PubMed

The gene ere(A) of the plasmid pIP1100 is larger than originally reported and is organized as an integron gene cassette. The ere(A) gene cassette carries its own promoter and is propagated by a class 2 integron with an insertion sequence element, IS1, inserted upstream of the intI2 gene. The mobility of the ere(A) cassette has been demonstrated. PMID:14506050

Biskri, Latefa; Mazel, Didier

2003-10-01

281

Erythromycin Esterase Gene ere(A) Is Located in a Functional Gene Cassette in an Unusual Class 2 Integron  

PubMed Central

The gene ere(A) of the plasmid pIP1100 is larger than originally reported and is organized as an integron gene cassette. The ere(A) gene cassette carries its own promoter and is propagated by a class 2 integron with an insertion sequence element, IS1, inserted upstream of the intI2 gene. The mobility of the ere(A) cassette has been demonstrated. PMID:14506050

Biskri, Latefa; Mazel, Didier

2003-01-01

282

Significant Productivity Improvement of the Baculovirus Expression Vector System by Engineering a Novel Expression Cassette  

PubMed Central

Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS. PMID:24824596

Gómez-Sebastián, Silvia; López-Vidal, Javier; Escribano, José M.

2014-01-01

283

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India  

PubMed Central

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 (?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, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. Conclusions Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes. PMID:23951238

Chakraborty, Ranadhir; Kumar, Arvind; Bhowal, Suparna Saha; Mandal, Amit Kumar; Tiwary, Bipransh Kumar; Mukherjee, Shriparna

2013-01-01

284

Capture and quality control mechanisms for ATP binding  

PubMed Central

The catalytic events in members of the nucleotidylyl transferase superfamily are initiated by a millisecond binding of ATP in the active site. Through metadynamics simulations on a class I aminoacyl-tRNA synthetase (aaRSs), the largest group in the superfamily, we calculate the free energy landscape of ATP selection and binding. Mutagenesis studies and fluorescence spectroscopy validated the identification of the most populated intermediate states. The rapid first binding step involves formation of encounter complexes captured through a fly-casting mechanism that acts up on the triphosphate moiety of ATP. In the slower nucleoside binding step, a conserved histidine in the HxxH motif orients the incoming ATP through base-stacking interactions resulting in a deep minimum in the free energy surface. Mutation of this histidine significantly decreases the binding affinity measured experimentally and computationally. The metadynamics simulations further reveal an intermediate quality control state that the synthetases and most likely other members of the superfamily use to select ATP over other nucleoside triphosphates. PMID:23276298

Li, Li; Martinis, Susan A.

2013-01-01

285

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme  

NASA Astrophysics Data System (ADS)

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

Zhang, Yun-Xin

2014-10-01

286

Cassette Sync Recorders. Parameter for Evaluation. Laboratory Test Findings. EPIE Report Number 86e.  

ERIC Educational Resources Information Center

This quarterly report by the Educational Products Information Exchange (EPIE) analyzes cassette sync recorders, giving parameters for evaluation and laboratory test findings. The report offers evaluations of the compatibility of different models and the standards for audio and cue tones. It also discusses national and international standards, EPIE…

Educational Products Information Exchange Inst., Stony Brook, NY.

287

STS-37 crewmembers watch Pilot Cameron juggle cassettes on OV-104's middeck  

NASA Technical Reports Server (NTRS)

STS-37 crewmembers watch Pilot Kenneth D. Cameron juggle cassette tapes on the middeck of Atlantis, Orbiter Vehicle (OV) 104. Laughing at Cameron's stunt are Mission Specialist (MS) Linda M. Godwin (foreground), Commander Steven R. Nagel (behind Cameron), and MS Jerry L. Ross (at floor level). Ross snacks on chocolate candy during the performance.

1991-01-01

288

Design Implications of Audio and Video Cassettes. I.E.T. Paper on Broadcasting No. 222.  

ERIC Educational Resources Information Center

This discussion of the educational potential of audiotape and videotape cassettes in distance teaching considers each medium individually, with examples of how the Open University has tried to exploit them in its teaching, and comments on some major differences between the two media and their implication for education. Characteristics of…

Durbridge, Nicola

289

Original Article High prevalence of trimethoprim-resistance cassettes in class 1 and 2  

E-print Network

Original Article High prevalence of trimethoprim-resistance cassettes in class 1 and 2 integrons techniques. Results: Class 1 integrons were the most prevalent (92.8%); class 2 integrons were found in 16 genes); this combination of integrase genes was most prevalent in S. boydii 20 and S. dysenteriae

Boyer, Edmond

290

STS-29 MS Bagian juggles audio cassettes on Discovery's, OV-103's, middeck  

NASA Technical Reports Server (NTRS)

On aft middeck, STS-29 Mission Specialist (MS) James P. Bagian juggles TEAC audio cassettes freefloating above foam insert as he attempts to organize them. In front of Bagian are aft middeck lockers and part of the open airlock hatch. Behind him are the starboard wall-mounted sleep restraints.

1989-01-01

291

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

NASA Technical Reports Server (NTRS)

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 preserved before being returned to Earth for analyses. Our results suggest that with protocol modifications and future verification testing we can utilize the versatile EMCS to conduct tightly controlled experiments inside our culture cassettes for a wide variety of organisms. These physiological experiments would be designed to answer questions at the molecular level about the specific stress responses of space flight.

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

2014-01-01

292

Background Notes  

NSDL National Science Digital Library

Country Background Notes, distributed on the DOSBACK list, are updated periodically and include information on US bilateral relations with foreign countries and on their governments, political conditions, and foreign relations. You can expect the DOSBACK list to generate about 3-4 email messages per month. Via DOSBACK you will receive the full-text version of newly released Background Notes. Archives of these two lists are also available at the Department of State Foreign Affairs Network (DOSFAN) gopher at the University of Illinois-Chicago.

293

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids  

PubMed Central

A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

2010-01-01

294

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

NASA Technical Reports Server (NTRS)

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 the EMCS ARC Seed Cassettes.

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

2014-01-01

295

Background & Publications  

E-print Network

Home Background & Projects Calendar Publications Staff Directory Station Videos Links Search Northern Michigan FruitNet 2008 Weekly Update NW Michigan Horticultural Research Station Nikki Rothwell maggot from growers. European red mites and two-spotted spider mites have reached threshold levels

296

Overproduction and dissection of proteins by the expression-cassette polymerase chain reaction.  

PubMed Central

We report an efficient, general approach for the construction of protein-overproducing strains of Escherichia coli. The method, expression-cassette polymerase chain reaction (ECPCR), allows the insertion of virtually any contiguous coding sequence between sequences that direct high-level protein biosynthesis in E. coli. The gene expression cassettes obtained by ECPCR are inserted into a regulated overexpression plasmid, and the resulting construct is used to transform E. coli. By effecting simultaneous 5' and 3' modification of a coding sequence, ECPCR permits the facile generation of mutant proteins having N- and/or C-terminal truncations. The method is a highly efficient way to dissect a multidomain protein into its component domains. The efficiency of the ECPCR approach is demonstrated in this study by construction of permuted overexpression vectors for the first two extracellular domains of the human CD4 protein. Images PMID:2408046

MacFerrin, K D; Terranova, M P; Schreiber, S L; Verdine, G L

1990-01-01

297

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

NASA Astrophysics Data System (ADS)

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.

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

2011-06-01

298

SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette and casting stand  

E-print Network

SDS-PAGE (Sodium dodecyl sulphate Poly acrylamide gel electrophoresis) 1. Glass cassette (10 ml) using following reagents except 10% APS10 and TEMED15 . 2.4ml dH2O 5.0ml Acrylamide/Bis20 2 following reagents except 10% APS10 and TEMED15 . 5.7ml dH2O 1.7ml Acrylamide/Bis20 2.5ml stacking buffer40

Schmidt, Marius

299

A Precisely Regulated Gene Expression Cassette Potently Modulates Metastasis and Survival in Multiple Solid Cancers  

Microsoft Academic Search

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

Kun Yu; Kumaresan Ganesan; Lay Keng Tan; Mirtha Laban; Jeanie Wu; Xiao Dong Zhao; Hongmin Li; Carol Ho Wing Leung; Yansong Zhu; Chia Lin Wei; Shing Chuan Hooi; Lance Miller; Patrick Tan

2008-01-01

300

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.  

PubMed

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media. PMID:24061566

Lazar, Zbigniew; Rossignol, Tristan; Verbeke, Jonathan; Crutz-Le Coq, Anne-Marie; Nicaud, Jean-Marc; Robak, Ma?gorzata

2013-11-01

301

Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes.  

PubMed

A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was approximately 14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351]. PMID:10077473

Li, Y; Glazer, A N

1999-01-01

302

Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera)  

PubMed Central

Honeybees (Apis mellifera), which are important pollinators of plants, display remarkable individual behaviors that collectively contribute to the organization of a complex society. Advances in dissecting the complex processes of honeybee behavior have been limited in the recent past due to a lack of genetic manipulation tools. These tools are difficult to apply in honeybees because the unit of reproduction is the colony, and many interesting phenotypes are developmentally specified at later stages. Here, we report highly efficient integration and expression of piggyBac-derived cassettes in the honeybee. We demonstrate that 27 and 20% of queens stably transmitted two different expression cassettes to their offspring, which is a 6- to 30-fold increase in efficiency compared with those generally reported in other insect species. This high efficiency implies that an average beekeeping facility with a limited number of colonies can apply this tool. We demonstrated that the cassette stably and efficiently expressed marker genes in progeny under either an artificial or an endogenous promoter. This evidence of efficient expression encourages the use of this system to inhibit gene functions through RNAi in specific tissues and developmental stages by using various promoters. We also showed that the transgenic marker could be used to select transgenic offspring to be employed to facilitate the building of transgenic colonies via the haploid males. We present here the first to our knowledge genetic engineering tool that will efficiently allow for the systematic detection and better understanding of processes underlying the biology of honeybees. PMID:24821811

Schulte, Christina; Theilenberg, Eva; Müller-Borg, Marion; Gempe, Tanja; Beye, Martin

2014-01-01

303

Construction of an ori cassette for adapting shuttle vectors for use in Haemophilus influenzae.  

PubMed

An ori (origin of DNA replication) cassette, pORC, containing the P15a ori and the kanamycin-resistance-encoding gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which gene from Tn5, was constructed. The cassette was used to convert an Escherichia coli promoter selection vector, which contains a promoterless chloramphenicol (Cm) acetyltransferase-encoding gene (cat) downstream from a multiple cloning site (MCS) [Brosius and Lupski, Methods Enzymol. 153 (1987) 54-68], to an E. coli-Haemophilus influenzae shuttle vector. The shuttle vector, pQL1, was shown to transform E. coli and H. influenzae efficiently. H. influenzae promoters were cloned into pQL1 by ligation of Sau3A-digested H. influenzae chromosomal fragments. Selection and semiquantitative analysis of promoter strength were performed on agar plates containing different concentrations of Cm. With the use of pQL1, H. influenzae gene regulation can now be studied in either H. influenzae or E. coli. In addition, elements of pORC can be used to convert other specialized E. coli vectors to E. coli-H. influenzae shuttle vectors. PMID:7959040

Heidecker, G J; Pozsgay, J M; Stull, T L

1994-12-01

304

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

PubMed

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. PMID:25379615

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

2015-01-01

305

A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis.  

PubMed

There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes. PMID:23911571

Lin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongyou; Li, Weifen

2013-11-01

306

Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays  

PubMed Central

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5?kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like ? propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome. PMID:21248857

Elsaied, Hosam; Stokes, Hatch W; Kitamura, Keiko; Kurusu, Yasurou; Kamagata, Yoichi; Maruyama, Akihiko

2011-01-01

307

Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species  

PubMed Central

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed. PMID:22876180

Domingues, Sara; Harms, Klaus; Fricke, W. Florian; Johnsen, Pål J.; da Silva, Gabriela J.; Nielsen, Kaare Magne

2012-01-01

308

Hygromycin B and Apramycin Antibiotic Resistance Cassettes for Use in Campylobacter jejuni  

PubMed Central

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. PMID:24751825

Cameron, Andrew; Gaynor, Erin C.

2014-01-01

309

Molecular Microbiology (2001) 40(1), 257269 BldD is a direct regulator of key developmental genes in  

E-print Network

by mutations that caused the loss of white fuzzy aerial hyphae, resulting in colonies with a smooth, shiny al., 1987), an ATP-binding cassette (ABC) membrane-spanning transporter (bldK; Nodwell et al., 1996

Elliot, Marie A.

310

REGULATION OF ABCG5 AND ABCG8 STEROL TRANSPORTERS IN BILIARY CHOLESTEROL ELIMINATION, REVERSE CHOLESTEROL TRANSPORT AND DYSLIPIDEMIA.  

E-print Network

??ATP-binding cassette transporters ABCA1 and ABCG1 initiate reverse cholesterol transport generating HDL particles, whereas ABCG5/G8 promote biliary cholesterol secretion thereby facilitating the last step of… (more)

Sabeva, Nadezhda Steliyanova

2011-01-01

311

Characterization of a complex context containing mecA but lacking genes encoding cassette chromosome recombinases in Staphylococcus haemolyticus  

PubMed Central

Background Methicillin resistance determinant mecA is generally transferred by SCCmec elements. However, the mecA gene might not be carried by a SCCmec in a Staphylococcus haemolyticus clinical isolate, WCH1, as no cassette chromosome recombinase genes were detected. Therefore, the genetic context of mecA in WCH1 was investigated. Results A 40-kb region containing mecA was obtained from WCH1, bounded by orfX at one end and several orfs of S. haemolyticus core chromosome at the other. This 40-kb region was very complex in structure with multiple genetic components that appeared to have different origins. For instance, the 3.7-kb structure adjacent to orfX was almost identical to that on the chromosome of Staphylococcus epidermidis RP62a but was absent from S. haemolyticus JCSC1435. Terminal inverted repeats of SCC were found but no ccr genes could be detected. mecA was bracketed by two copies of IS431, which was flanked by 8-bp direct target repeat sequence (DR). Conclusions The presence of 8-bp DR suggests that the two copies of IS431 might have formed a composite transposon for mobilizing mecA. This finding is of significance as multiple copies of IS431 are commonly present in the contexts of mecA, which might have the potential to form various composite transposons that could mediate the mobilization of mecA. This study also provides an explanation for the absence of ccr in some staphylococci isolates carrying mecA. PMID:23521926

2013-01-01

312

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

NASA Astrophysics Data System (ADS)

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.

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

313

United States Geological Survey (USGS) FM cassette seismic-refraction recording system  

SciTech Connect

In this two chapter report, instrumentation used to collect seismic data is described. This data acquisition system has two parts: (1) portable anolog seismic recorders and related ``hand-held-testers`` (HHT) and (2) portable digitizing units. During the anolog recording process, ground motion is sensed by a 2-Hz vertical-component seismometer. The voltage output from the seismometer is split without amplification and sent to three parallel amplifier circuit boards. Each circuit board amplifiers the seismic signal in three stages and then frequency modulates the signal. Amplification at the last two stages can be set by the user. An internal precision clock signal is also frequency modulated. The three data carrier frequencies, the clock carrier frequency, and a tape-speed compensation carrier frequency are summed and recorded on a recorded on a cassette tape. During the digitizing process, the cassette tapes are played back and the signals are demultiplexed and demodulated. An anolog-to-digital converter converts the signals to digital data which are stored on 8-inch floppy disks. 7 refs., 19 figs., 6 tabs.

Murphy, J.M.

1988-12-31

314

Using phage integrases in a site-specific dual integrase cassette exchange strategy.  

PubMed

?C31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ?C31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites. Previously, we have used these integrases sequentially to integrate plasmid DNA into the genome. This approach relied on placing a landing pad attP for Bxb1 integrase in the genome by using phiC31 integrase-mediated recombination at a genomic pseudo attP site. In this chapter, we present a protocol for using these integrases simultaneously to facilitate cassette exchange at a predefined location. This approach permits greater control and accuracy over integration. We also present a general method for using polymerase chain reaction assays to verify that the desired cassette exchange occurred successfully. PMID:25408400

Geisinger, Jonathan M; Calos, Michele P

2015-01-01

315

Electricity generation from model organic wastewater in a cassette-electrode microbial fuel cell.  

PubMed

A new highly scalable microbial fuel cell (MFC) design, consisting of a series of cassette electrodes (CE), was examined for increasing power production from organic matter in wastewater. Each CE chamber was composed of a box-shaped flat cathode (two air cathodes on both sides) sandwiched in between two proton-exchange membranes and two graphite-felt anodes. Due to the simple design of the CE-MFC, multiple cassettes can be combined to form a single unit and inserted into a tank to treat wastewater. A 12-chamber CE-MFC was tested using a synthetic wastewater containing starch, peptone, and fish extract. Stable performance was obtained after 15 days of operation in fed-batch mode, with an organic removal efficiency of 95% at an organic loading rate of 2.9 kg chemical oxygen demand (COD) per cubic meter per day and an efficiency of 93% at 5.8 kg COD per cubic meter per day. Power production was stable during this period, reaching maximum power densities of 129 W m(-3) (anode volume) and 899 mW m(-2) (anode projected area). The internal resistance of CE-MFC decreased from 2.9 (day 4) to 0.64 Omega (day 25). These results demonstrate the usefulness of the CE-MFC design for energy production and organic wastewater treatment. PMID:18581110

Shimoyama, Takefumi; Komukai, Shoko; Yamazawa, Akira; Ueno, Yoshiyuki; Logan, Bruce E; Watanabe, Kazuya

2008-08-01

316

Predicting film dose to aid in cassette placement for radiation therapy portal verification film images.  

PubMed

EC film has improved portal localization images with better contrast and improved distinction of bony structures and air-tissue interfaces. A cassette with slower speed screens was used with EC film to image the treatment portal during the entire course of treatment (verification) instead of taking separate films after treatment. Measurements of film density vs source to film distance (SFD) were made using 15 and 25 cm thick water phantoms with both 6 and 18 MV photons from I to 40 cm past the phantom. A characteristic (H & D) curve was measured in air to compare dose to film density. Results show the reduction in radiation between patient and cassette more closely follows an "inverse cube law" rather than an inverse square law. Formulas to calculate radiation exposure to the film, and the desired SFD were based on patient tumor dose, calculation of the exit dose, and the inverse cube relationship. A table of exposure techniques based on the SFD for a given tumor dose was evaluated and compared to conventional techniques. Although the film has a high contrast, there is enough latitude that excellent films can be achieved using a fixed SFD based simply on the tumor dose and beam energy. Patient diameter has a smaller effect. The benefits of imaging portal films during the entire treatment are more reliability in the accuracy of the portal image, ability to detect patient motion, and reduction in the time it takes to take portal images. PMID:12512719

Keys, Richard A; Marks, James E; Haus, Arthur G

2002-12-01

317

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

PubMed

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. PMID:23764017

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

2013-11-01

318

Use of cassette-electrode microbial fuel cell for wastewater treatment.  

PubMed

Cassette-electrode microbial fuel cells (CE-MFCs) have been developed for the conversion of biomass wastes into electric energy. The present study modified CE-MFC for its application to wastewater treatment and examined its utility in a long-term (240 days) experiment to treat a synthetic wastewater, containing starch, yeast extract, peptone, plant oil, and a detergent (approximately 500 mg of total chemical oxygen demand [COD] per liter). A test MFC reactor (1 l in capacity) was equipped with 10 cassette electrodes with total anode and cathode projection areas of 1440 cm(2), and the operation was initiated by inoculating with rice paddy-field soil. It was demonstrated that CE-MFC achieved COD removal rates of 80% at hydraulic-retention times of 6 h or greater, and electricity was generated at a maximum power density of 150 mW m(-2) and Coulombic efficiency of 20%. Microbial communities established on anodes of CEs were analyzed by pyrosequencing of PCR-amplified 16S rRNA gene fragments, showing that Geobacter, Clostridium, and Geothrix were abundantly detected in anode biofilms. These results demonstrate the utility of CE-MFC for wastewater treatment, in which Geobacter and Geothrix would be involved in the electricity generation. PMID:23041137

Miyahara, Morio; Hashimoto, Kazuhito; Watanabe, Kazuya

2013-02-01

319

Role of ABCG1 and other ABCG family members in lipid metabolism  

Microsoft Academic Search

The molecular cloning and identification of mu- tations in ATP-binding cassette transporters in hereditary diseases have greatly expanded our knowledge of the nor- mal physiology of intracellular lipid transport processes. In addition to the well-known ATP-binding cassette transporter A1 (ABCA1) molecule, ABC transporters belonging to the ABCG (White) subfamily (ABCG1, ABCG5, and ABCG8) have been shown to be critically involved

Gerd Schmitz; Thomas Langmann; Susanne Heimerl

320

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

NASA Technical Reports Server (NTRS)

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.

Ashton, Gary

1993-01-01

321

Copiers and Duplicators for Audio Tape Cassettes: Criteria for Selection, Laboratory Test Findings, Manufacturers' Data Reports. EPIE Report: Number 84e.  

ERIC Educational Resources Information Center

Because needs for cassette duplication equipment differ, the user must match his requirements to the capabilities of manufactured products, and the essential product characteristics. To help educators in choosing cassette duplicators, EPIE has gathered data from manufacturers and tested 11 units. The first section of the report explains the…

Educational Products Information Exchange Inst., Stony Brook, NY.

322

Design, Syntheses and Biological Applications of Through-bond Energy Transfer Cassettes and Novel Non-covalently Cell Penetrating Peptides  

E-print Network

A xanthene-BODIPY cassette is used as a ratiometric intracellular pH reporter for imaging protein-dye conjugates in living cells. A model was hypothesized to explain the pH-dependent energy transfer efficiencies from the donor to the acceptor based...

Han, Junyan

2012-02-14

323

Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette  

Technology Transfer Automated Retrieval System (TEKTRAN)

Bacteriophage and reporter phage are used for typing and/or detection of pathogens. The temperate tailed phage fV10 has been utilized for phage-typing E. coli O157:H7. By modifying fV10 to transduce kanamycin resistance and the a luxCDABE cassette, we developed a reporter bacteriophage (fV10-lux) p...

324

Rapid genetic modification of mouse embryonic stem cells by inducible cassette exchange recombination  

PubMed Central

Summary Embryonic stem cell (ESC) differentiation is a useful tool by which to develop large quantities of cells in vitro representing early stages of embryonic development. A conditional gene expression system allows interrogation of factors at specific time points in the differentiation of ES cells to defined cell types. We have developed a method for rapidly generating conditional inducible murine ES cells by targeting genes into an Inducible Cassette Exchange (ICE) locus. The ICE locus encodes a doxycycline-inducible floxed Cre, which replaces itself with an incoming floxed gene of interest. The derivative cell lines, selected in G418, thus bear doxycycline-inducible transgenes. We provide detailed methods for performing ICE recombination and generating derivative doxycycline-inducible cell lines. PMID:24233789

Iacovino, Michelina; Roth, Megan E.; Kyba, Michael

2014-01-01

325

Bacterial resistance evolution by recruitment of super-integron gene cassettes.  

PubMed

The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events. PMID:11952913

Rowe-Magnus, Dean A; Guerout, Anne-Marie; Mazel, Didier

2002-03-01

326

Systematic Derivation of Marker Sets for Staphylococcal Cassette Chromosome mec Typing? †  

PubMed Central

The aim of this study was to identify optimized sets of genotyping targets for the staphylococcal cassette chromosome mec (SCCmec). We analyzed the gene contents of 46 SCCmec variants in order to identify minimal subsets of targets that provide useful resolution. This was achieved by firstly identifying and characterizing each available SCCmec element based on the presence or absence of 34 binary targets. This information was used as input for the software “Minimum SNPs,” which identifies the minimum number of targets required to differentiate a set of genotypes up to a predefined Simpson's index of diversity (D) value. It was determined that 22 of the 34 targets were required to genotype the 46 SCCmec variants to a D of 1. The first 6, 9, 12, and 15 targets were found to define 21, 29, 35, and 39 SCCmec variants, respectively. The genotypes defined by these marker subsets were largely consistent with the relationships between SCCmec variants and the accepted nomenclature. Consistency was made virtually complete by forcing the computer program to include ccr1 and ccr5 in the target set. An alternative target set biased towards discriminating abundant SCCmec variants was derived by analyzing an input file in which common SCCmec variants were repeated, thus ensuring that markers that discriminate abundant variants had a large effect on D. Finally, it was determined that mecA single nucleotide polymorphisms (SNPs) can increase the overall genotyping resolution, as different mecA alleles were found in otherwise identical SCCmec variants. PMID:17517844

Stephens, Alex J.; Huygens, Flavia; Giffard, Philip M.

2007-01-01

327

Transduction of staphylococcal cassette chromosome mec elements between strains of Staphylococcus aureus.  

PubMed

Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80? and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process. PMID:23939891

Scharn, Caitlyn R; Tenover, Fred C; Goering, Richard V

2013-11-01

328

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA  

PubMed Central

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

329

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

SciTech Connect

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.

Meier, Alan; Rosen, Karen

1999-03-01

330

Roles of two large serine recombinases in mobilizing the methicillin-resistance cassette SCCmec  

PubMed Central

Summary Methicillin-resistant Staphylococcus aureus (MRSA) emerged via acquisition of a mobile element, staphylococcal cassette chromosome mec (SCCmec). Integration and excision of SCCmec is mediated by an unusual site-specific recombination system. Most variants of SCCmec encode two recombinases, CcrA and CcrB, that belong to the large serine family. Since CcrA and CcrB are always found together, we sought to address their specific roles. We show here that CcrA and CcrB can carry out both excisive and integrative recombination in E. coli in the absence of any host-specific or SCCmec-encoded cofactors. CcrA and CcrB are promiscuous in their substrate choice: they act on many non-canonical pairs of recombination sites in addition to the canonical ones, which may explain tandem insertions into the SCCmec attachment site. Moreover, CcrB is always required, but CcrA is only required if one of the four half sites is present. Recombinational activity correlates with DNA-binding: CcrA recognizes only that half site, which overlaps a conserved coding frame on the host chromosome. Therefore, we propose that CcrA serves as a specificity factor that emerged through modular evolution to enable recognition of a bacterial recombination site that is not an inverted repeat. PMID:23651464

Misiura, Agnieszka; Pigli, Ying Z.; Boyle-Vavra, Susan; Daum, Robert S.; Boocock, Martin R.; Rice, Phoebe A.

2013-01-01

331

Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels.  

PubMed

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here, we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin (IL)-10) in large-vessel endothelial cells (ECs) at levels as high as obtained with the cytomegalovirus (CMV) immediate early promoter, while retaining EC specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely four copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining--and possibly enhancing--EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large-vessel EC, while minimizing systemic effects. PMID:21179172

Dronadula, N; Du, L; Flynn, R; Buckler, J; Kho, J; Jiang, Z; Tanaka, S; Dichek, D A

2011-05-01

332

Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant  

Microsoft Academic Search

Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal

Peter P. Cherepanov; Wilfried Wackernagel

1995-01-01

333

Multiplex Real-Time PCR for Rapid Staphylococcal Cassette Chromosome mec Typing ?  

PubMed Central

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the ?mecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the ?mecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates. PMID:19726600

Chen, Liang; Mediavilla, José R.; Oliveira, Duarte C.; Willey, Barbara M.; de Lencastre, Herminia; Kreiswirth, Barry N.

2009-01-01

334

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity.  

PubMed

dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage. PMID:18451045

Gil, Filipa; Catalão, Maria João; Moniz-Pereira, José; Leandro, Paula; McNeil, Michael; Pimentel, Madalena

2008-05-01

335

Structure of In31, a blaIMP-Containing Pseudomonas aeruginosa Integron Phyletically Related to In5, Which Carries an Unusual Array of Gene Cassettes  

PubMed Central

The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-?-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents. PMID:10103196

Laraki, Nezha; Galleni, Moreno; Thamm, Iris; Riccio, Maria Letizia; Amicosante, Gianfranco; Frère, Jean-Marie; Rossolini, Gian Maria

1999-01-01

336

Changes in expression of splice cassettes of NMDA receptor GluN1 subunits within the frontal lobe and memory in mice during aging  

PubMed Central

Age-related decline in memory has been associated with changes in mRNA and protein expression of different NMDA receptor subunits. The NMDA receptor GluN1 subunit appears to be necessary and sufficient for receptor function. There is evidence that the mRNA expressions of some splice forms of the subunit are influenced by aging and/or behavioral testing experience in old mice. The present study explored the relationships between behavioral testing experience and protein expression of different GluN1 subunit isoforms in the prefrontal/frontal cortex of the brain during aging. Aged C57BL/6 mice with behavioral testing experience showed declines in performance in both spatial working and reference memory tasks. Protein expression of GluN1 C-terminal cassettes C2 and C2?, but not the C1 or N1 cassettes, was observed to decline with increasing age, regardless of experience. In middle-age animals, higher expressions of the GluN1 subunit and C2? cassette proteins were associated with good reference memory on initial search. Aged animals with a higher protein expression of GluN1 subunits containing C1 cassettes and the whole population of GluN1 subunits exhibited a closer proximity to the former platform location within the final phase of probe trials. However, the old mice with high expression of the C1 cassette did not show an accurate search during this phase. The old mice with lower expression of the C1 cassette protein more closely mimicked the performances of the young and middle-aged mice. These results indicate that there was heterogeneity in the effect of aging on the expression of the GluN1 subunits containing different splice cassettes. It also suggests that the GluN1 subunit might be most important for good reference memory during middle age, but this relationship may not be maintained into old age. PMID:21443909

Das, Siba R.; Magnusson, Kathy R.

2011-01-01

337

Background Subtraction Techniques  

Microsoft Academic Search

Background subtraction is a commonly used class of techniques for segmenting out objects of interest in a scene for applications such as surveillance. This paper surveys a repre- sentative sample of the published techiques for background subtraction, and analyses them with respect to three important attributes: foreground detection; background maintenance; and postprocessing.

Alan M. McIvor

338

The Cosmic Background Explorer.  

ERIC Educational Resources Information Center

Outlines the Cosmic Background Explorer (COBE) mission to measure celestial radiation. Describes the instruments used and experiments involving differential microwave radiometers, and a far infrared absolute spectrophotometer. (YP)

Gulkis, Samuel; And Others

1990-01-01

339

Design and Construction of Two Yeast Shuttle Vectors Containing Human Procollagen Genes Expression Cassette for Expression in Yeast  

PubMed Central

Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic response in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction (PCR). Finally they were cloned in yeast vector pPICZ?A containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression. PMID:23407617

Abdemami, Baharak; Shokrgozar, Mohammad Ali; Shahreza, Hossein Khanahmad; Ghavami, Mehdi

2011-01-01

340

Background music reactive games  

Microsoft Academic Search

In this paper, we discuss the concept of games that react to their background music. Instead of limiting the player to a fixed set of songs, the background music can be any song chosen from the player's own music collection. Due to the relative simplicity of such existing game titles, we wanted to explore the potential of the concept more

Juha Arrasvuori; Jukka Holm

2010-01-01

341

Correlators in nontrivial backgrounds  

SciTech Connect

Operators in N=4 super Yang-Mills theory with an R-charge of O(N{sup 2}) are dual to backgrounds which are asymtotically AdS{sub 5}xS{sup 5}. In this article we develop efficient techniques that allow the computation of correlation functions in these backgrounds. We find that (i) contractions between fields in the string words and fields in the operator creating the background are the field theory accounting of the new geometry, (ii) correlation functions of probes in these backgrounds are given by the free field theory contractions but with rescaled propagators and (iii) in these backgrounds there are no open string excitations with their special end point interactions; we have only closed string excitations.

Mello Koch, Robert de [National Institute for Theoretical Physics, Department of Physics and Centre for Theoretical Physics, University of the Witwatersrand, Wits, 2050 (South Africa); Stellenbosch Institute for Advanced Studies, Stellenbosch (South Africa); Ives, Norman; Stephanou, Michael [National Institute for Theoretical Physics, Department of Physics and Centre for Theoretical Physics, University of the Witwatersrand, Wits, 2050 (South Africa)

2009-01-15

342

The cosmic neutrino background  

NASA Technical Reports Server (NTRS)

The cosmic neutrino background is expected to consist of relic neutrinos from the big bang, of neutrinos produced during nuclear burning in stars, of neutrinos released by gravitational stellar collapse, and of neutrinos produced by cosmic ray interactions with matter and radiation in the interstellar and intergalactic medium. Formation of baryonic dark matter in the early universe, matter-antimatter annihilation in a baryonic symmetric universe, and dark matter annihilation could have also contributed significantly to the cosmic neutrino background. The purpose of this paper is to review the properties of these cosmic neutrino backgrounds, the indirect evidence for their existence, and the prospects for their detection.

Dar, Arnon

1991-01-01

343

The GLAST Background Model  

NASA Astrophysics Data System (ADS)

In order to estimate the ability of the GLAST/LAT to reject unwanted background of charged particles, optimize the on-board processing, size the required telemetry and optimize the GLAST orbit, we developed a detailed model of the background particles that would affect the LAT. In addition to the well-known components of the cosmic radiation, we included splash and reentrant components of protons, electrons (e+ and e-) from 10 MeV and beyond as well as the albedo gamma rays produced by cosmic ray interactions with the atmosphere. We made estimates of the irreducible background components produced by positrons and hadrons interacting in the multilayered micrometeorite shield and spacecraft surrounding the LAT and note that because the orbital debris has increased, the shielding required and hence the background are larger than were present in EGRET. Improvements to the model are currently being made to include the east-west effect.

Ormes, J. F.; Atwood, W.; Burnett, T.; Grove, E.; Longo, F.; McEnery, J.; Mizuno, T.; Ritz, S.

2007-07-01

344

The Cosmic Background Radiation  

E-print Network

We summarise the current status of cosmic microwave background spectrum and anisotropy measurements, and their theoretical interpretation. This is the update of the mini-review for the 1997 web-version of the Review of Particle Properties.

George Smoot; Douglas Scott

1997-11-08

345

IR-background database  

Microsoft Academic Search

The Swedish Defence Research Agency (FOI) has recently performed systematic measurements in order to establish an IR-background database. It will be used for a wide range of applications and provide a basis for the modeling of IR-background properties of Swedish terrain. Experimental data like this is also necessary for the validation of methods and programs for synthetic IR-scene simulation. The

Claes Nelsson; Paer Nilsson; Roland Lindell; Emma Bernhardsson

2001-01-01

346

The Second Step of ATP Binding to DnaK Induces Peptide Release  

Microsoft Academic Search

The interaction of the nucleotide-free molecular chaperone DnaK (Hsp70) fromEscherichia coliwith nucleotides was studied under equilibrium and transient kinetic conditions. These studies used the intrinsic fluorescence signal of the single tryptophan residue (Trp102) of DnaK, or of novel fluorescent nucleotide analogs of ADP and ATP, N8-(4-N?-methylan thraniloylaminobutyl)-8-aminoadenosine 5?-di- or triphosphate (MABA-ADP and MABA-ATP) as spectroscopic probes. Titration of MABA-ADP with

Holger Theyssen; Hans-Peter Schuster; Lars Packschies; Bernd Bukau; Jochen Reinstein

1996-01-01

347

The oxidation-state-dependent ATP-binding site of cytochrome c. A possible physiological significance.  

PubMed Central

Cytochrome c binds certain physiological anions that are known to modulate the biological properties of the protein, although it is not known whether this effect is fortuitous or has physiological significance. We have examined the ability of the protein and its semisynthetic analogues to associate with certain of these anions, e.g. ATP, ADP, Pi and citrate. Our results show that specific residues or clusters of residues on the surface of horse heart cytochrome c are involved in the recognition sites for these anions. We also observed that binding at one site is linked to the oxidation state of the protein. PMID:3019313

Corthésy, B E; Wallace, C J

1986-01-01

348

Combination of Multiplex PCRs for Staphylococcal Cassette Chromosome mec Type Assignment: Rapid Identification System for mec, ccr, and Major Differences in Junkyard Regions  

Microsoft Academic Search

Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphy- lococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and

Yoko Kondo; Teruyo Ito; Xiao Xue Ma; Shinya Watanabe; Barry N. Kreiswirth; Jerome Etienne; Keiichi Hiramatsu

2007-01-01

349

PREVELANCE OF CLASS 1 INTEGRONS AND AMR GENE CASSETTES AMONG ENTERIC BACTERIA FOUND IN MULTI-SITE GROUP-LEVEL COHORTS OF HUMANS AND SWINE  

Technology Transfer Automated Retrieval System (TEKTRAN)

The prevalence of antimicrobial resistance (AMR) genotypic characteristics (Class 1 integron and AMR gene cassettes) in commensal Escherichia coli (EC) isolated from humans and swine in a semi-closed, integrated farrow-to-fork population were evaluated in a cross-sectional study. The objective of t...

350

Isolated Cerebellar Variant of Adrenoleukodystrophy with a de novo Adenosine Triphosphate-Binding Cassette D1 (ABCD1) Gene Mutation  

PubMed Central

X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms. PMID:24954351

Kang, Joon Won; Lee, Sang Mi; Koo, Kyo Yeon; Lee, Young-Mock; Nam, Hyo Suk; Quan, Zhejiu

2014-01-01

351

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

NASA Astrophysics Data System (ADS)

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 ?M 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.

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

2012-06-01

352

Cosmic Microwave Background  

NSDL National Science Digital Library

In this lesson, students explore the cosmic microwave background to understand why it permeates the universe and why it peaks as microwave radiation. Students should be able to explain that the origin of the background radiation is the uniform thermal radiation of the big bang and that the radiation produced was evenly distributed around the small early universe, causing it to permeate today's universe. This activity is part of the Cosmic Times teachers guide and is intended to be used in conjunction with the 1965 Cosmic Times Poster.

353

The Cosmic Background Explorer  

NASA Technical Reports Server (NTRS)

The Cosmic Background Explorer (CBE), NASA's cosmological satellite which will observe a radiative relic of the big bang, is discussed. The major questions connected to the big bang theory which may be clarified using the CBE are reviewed. The satellite instruments and experiments are described, including the Differential Microwave Radiometer, which measures the difference between microwave radiation emitted from two points on the sky, the Far-Infrared Absolute Spectrophotometer, which compares the spectrum of radiation from the sky at wavelengths from 100 microns to one cm with that from an internal blackbody, and the Diffuse Infrared Background Experiment, which searches for the radiation from the earliest generation of stars.

Gulkis, Samuel; Lubin, Philip M.; Meyer, Stephan S.; Silverberg, Robert F.

1990-01-01

354

The cosmic microwave background  

NASA Technical Reports Server (NTRS)

Recent limits on spectral distortions and angular anisotropies in the cosmic microwave background are reviewed. The various backgrounds are described, and the theoretical implications are assessed. Constraints on inflationary cosmology dominated by cold dark matter (CDM) and on open cosmological models dominated by baryonic dark matter (BDM), with, respectively, primordial random phase scale-invariant curvature fluctuations or non-gaussian isocurvature fluctuations are described. More exotic theories are addressed, and I conclude with the 'bottom line': what theorists expect experimentalists to be measuring within the next two to three years without having to abandon their most cherished theories.

Silk, Joseph

1991-01-01

355

Radioactive Decay 1. Background  

E-print Network

Radioactive Decay 1. Background It is well known that many nuclei are unstable and are transformed into other nuclear species by means of either alpha decay or beta decay. The rate at which those radioactive on the number N of radioactive nuclei in the sample and also on the probability for each nucleus to decay

Elster, Charlotte

356

Country background Forest history  

E-print Network

33 Country background Forest history During the Gallo-Roman period (1st­4th century AD), forests this proportion decreased dramatically to only 15­17 % of the land area. This residual forest was then severely Colbert's Forest Ordinance was instituted in 1669 a gradual restoration took place. High forests produced

Paris-Sud XI, Université de

357

David Smith Academic background  

E-print Network

David Smith Academic background Ph.D. in Mathematics (Algebra), Université de Sherbrooke, Canada project program (I. Assem, F. Bergeron, C. Reutenauer, D. Smith) $132,000 ($44,000 per year for 3 years. Schiffler and D. Smith, Friezes, strings and cluster variables, to appear in Glasgow Mathematcal Journal. 2

358

PANDEMIC INFLUENZA background briefing  

E-print Network

PANDEMIC INFLUENZA background briefing Biomedicine Forum 5 November 2008 compiled by David Evans, Dave Carr, David Lynn and Phil Green Transmission electron micrograph of Influenza A virus (Wellcome influenza!' Page 2 #12;Consequences of an influenza pandemic THE PANDEMIC THREAT DEATH If the next pandemic

Rambaut, Andrew

359

Local microwave background radiation  

E-print Network

An inquiry on a possible local origin for the Microwave Background Radiation is made. Thermal MBR photons are contained in a system called {\\it magnetic bottle} which is due to Earth magnetic field and solar wind particles, mostly electrons. Observational tests are anticipated.

Domingos Soares

2014-11-13

360

Cosmic Microwave Background Theory  

Microsoft Academic Search

A long-standing goal of theorists has been to constrain cosmological parameters that define the structure formation theory from cosmic microwave background (CMB) anisotropy experiments and large-scale structure (LSS) observations. The status and future promise of this enterprise is described. Current band-powers in ell -space are consistent with a Delta T flat in frequency and broadly follow inflation-based expectations. That the

J. Richard Bond

1998-01-01

361

The cosmic microwave background  

NASA Technical Reports Server (NTRS)

Recent observational and theoretical investigations of the cosmic microwave background radiation (CMBR) are reviewed. Particular attention is given to spectral distortions and CMBR temperature anisotropies at large, intermediate, and small angular scales. The implications of the observations for inflationary cosmological models with curvature fluctuation are explored, and it is shown that the limits determined for intermediate-scale CMBR anisotropy almost rule out a baryon-dominated cosmology.

Silk, Joseph

1989-01-01

362

Targets, backgrounds, and discrimination  

NASA Astrophysics Data System (ADS)

The present volume discusses a model-based aircraft identification technique, target intensity and angle scintillations, spatiotemporal nonstationary scene generation, an overview of the Strategic Scene Generation Model (SSGM), nuclear backgrounds for SSGM, and an atmospheric and transmittance code for 50-300 km altitudes. Also discussed are a data base for airborne target signatures, the auroral module of the Strategic High Altitude Radiance Code, and the 3D characteristics of underexpanded and overexpanded rectangular jets. (No individual items are abstracted in this volume)

Accetta, J. S.; Kelley, G. H.

363

Backgrounds Data Center  

NASA Astrophysics Data System (ADS)

The Backgrounds Data Center (BDC) is the designated archive for backgrounds data collected by Ballistic Missile Defense Organization (BMDO) programs, some of which include ultraviolet sensors. Currently, the BDC holds ultraviolet data from the IBSS, UVPI, UVLIM, and FUVCAM sensors. The BDC will also be the prime archive for Midcourse Space Experiment (MSX) data and is prepared to negotiate with program managers to handle other datasets. The purpose of the BDC is to make data accessible to users and to assist them in analyzing it. The BDC maintains the Science Catalog Information Exchange System (SCIES) allowing remote users to log in, read or post notices about current programs, search the catalogs for datasets of interest, and submit orders for data. On-site facilities are also available for the analysis of data, and consist of VMS and UNIX workstations with access to software analysis packages such as IDL, IRAF, and Khoros. Either on-site or remotely, users can employ the BDC-developed graphical user interface called the Visual Interface for Space and Terrestrial Analysis (VISTA) to generate catalog queries and to display and analyze data. SCIES and VISTA permit nearly complete access to BDC services and capabilities without the need to be physically present at the data center.

Snyder, William A.; Gursky, Herbert; Heckathorn, Harry M.; Lucke, Bob L.; Dorland, Bryan N.; Kessel, R. A.; Berg, S. L.; Dombrowski, E. G.

1994-09-01

364

Holography for Schrodinger backgrounds  

E-print Network

We discuss holography for Schrodinger solutions of both topologically massive gravity in three dimensions and massive vector theories in (d+1) dimensions. In both cases the dual field theory can be viewed as a d-dimensional conformal field theory (two dimensional in the case of TMG) deformed by certain operators that respect the Schrodinger symmetry. These operators are irrelevant from the viewpoint of the relativistic conformal group but they are exactly marginal with respect to the non-relativistic conformal group. The spectrum of linear fluctuations around the background solutions corresponds to operators that are labeled by their scaling dimension and the lightcone momentum k_v. We set up the holographic dictionary and compute 2-point functions of these operators both holographically and in field theory using conformal perturbation theory and find agreement. The counterterms needed for holographic renormalization are non-local in the v lightcone direction.

Monica Guica; Kostas Skenderis; Marika Taylor; Balt van Rees

2010-08-11

365

The diffuse UV background  

NASA Technical Reports Server (NTRS)

The diffuse radiation field in the UV (900-3,000 A) affects the structure of galactic molecular clouds and conveys important information concerning the physical characteristics and spatial distribution of gas and dust in the universe. Continuum emission in this range is probably dominated by interstellar dust scattering in our galaxy. For view directions and angular resolutions allowing observations in the rifts between galactic dust clouds, the background due to the integrated light of spiral galaxies may be detected, providing important information on their structure and evolution. The redshifted emission from an intergalactic medium may be observable in the regions between nearby bright galaxies. Present observations provide weak constraints on the radiation field required to ionize the intergalactic medium at the level required by the Gunn-Peterson test.

Paresce, F.; Jakobsen, P.

1980-01-01

366

Characterization of the gene cassette required for biosynthesis of the (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate capsule of serogroup A Neisseria meningitidis.  

PubMed

The (alpha1-->6)-linked N-acetyl-D-mannosamine-1-phosphate meningococcal capsule of serogroup A Neisseria meningitidis is biochemically distinct from the sialic acid-containing capsules produced by other disease-associated meningococcal serogroups (e.g., B, C, Y, and W-135). We defined the genetic cassette responsible for expression of the serogroup A capsule. The cassette comprised a 4,701-bp nucleotide sequence located between the outer membrane capsule transporter gene, ctrA, and galE, encoding the UDP-glucose-4-epimerase. Four open reading frames (ORFs) not found in the genomes of the other meningococcal serogroups were identified. The first serogroup A ORF was separated from ctrA by a 218-bp intergenic region. Reverse transcriptase (RT) PCR and primer extension studies of serogroup A mRNA showed that all four ORFs were cotranscribed in the opposite orientation to ctrA and that transcription of the ORFs was initiated from the intergenic region by a sigma-70-type promoter that overlapped the ctrA promoter. The first ORF exhibited 58% amino acid identity with the UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) 2-epimerase of Escherichia coli, which is responsible for the conversion of UDP-GlcNAc into UDP-N-acetyl-D-mannosamine. Polar or nonpolar mutagenesis of each of the ORFs resulted in an abrogation of serogroup A capsule production as determined by colony immunoblots and enzyme-linked immunosorbent assay. Replacement of the serogroup A biosynthetic gene cassette with a serogroup B cassette by transformation resulted in capsule switching from a serogroup A capsule to a serogroup B capsule. These data indicate that assembly of the serogroup A capsule likely begins with monomeric UDP-GlcNAc and requires proteins encoded by three other genes found in the serogroup A N. meningitidis-specific operon located between ctrA and galE. PMID:9515923

Swartley, J S; Liu, L J; Miller, Y K; Martin, L E; Edupuganti, S; Stephens, D S

1998-03-01

367

New insights into the role of the adenosine triphosphate–binding cassette transporters in high-density lipoprotein metabolism and reverse cholesterol transport  

Microsoft Academic Search

Four adenosine triphosphate–binding cassette (ABC) transporters—ABCA1, ABCG1, ABCG5, and ABCG8—have been identified and shown to modulate cholesterol and lipoprotein metabolism. Recent analyses of ABCA1 indicate that upregulation of ABCA1 in the liver and macrophages of transgenic mice is associated with increased plasma high-density lipoprotein (HDL) cholesterol levels, increased net flux of cholesterol to the liver, and reduced diet-induced atherosclerosis. In

H. Bryan Brewer; Silvia Santamarina-Fojo

2003-01-01

368

Vibrational spectroscopy to study degradation of natural dyes. Assessment of oxygen-free cassette for safe exposition of artefacts.  

PubMed

An important issue connected with conservation chemistry is how to improve the storage and exposure conditions in order to suppress the fading and degradation of dyes and other components of paintings. Although the oxygen-free exposure cassettes are commonly known in museums, there is still lack of information in the literature about the effect of anoxic conditions on the degradation of dyes. This study is an attempt to start a database formation on the dyes degradation. Five commercial dyes (indigo, dragon's blood, curcumin, madder, carminic acid) were submitted to accelerated ageing by exposure to intensive light in the visible range in both oxygen-free (anoxia) and -rich conditions. Degradation of the samples was investigated by several analytical techniques (attenuated total reflectance infrared spectroscopy, Raman spectroscopy, reflectance UV-Vis spectroscopy, X-ray fluorescence spectroscopy and optical microscopy). The conclusions are based on the estimators (derived from the determination of colour differences from Vis spectra and from the changes in FTIR and Raman vibrational bands intensity). According to them, only indigo, dragon's blood and curcumin show greater stability in anoxic conditions in comparison with oxygen-rich ones while madder, carminic acid undergo greater degradation. PMID:21165610

Koperska, Monika; ?ojewski, Tomasz; ?ojewska, Joanna

2011-03-01

369

Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus  

PubMed Central

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. PMID:24972080

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.

2014-01-01

370

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.  

PubMed

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. PMID:24972080

Hill-Cawthorne, Grant A; Hudson, Lyndsey O; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S; Clark, Taane G; Pain, Arnab

2014-01-01

371

Development of an on-line extraction turbulent flow chromatography tandem mass spectrometry method for cassette analysis of Caco-2 cell based bi-directional assay samples.  

PubMed

Caco-2 cells are frequently used for screening compounds for their permeability characteristics and P-glycoprotein (P-gp) interaction potential. Bi-directional permeability studies performed on Caco-2 cells followed by analysis by HPLC-UV or LC-MS method constitutes the "method of choice" for the functional assessment of efflux characteristics of a test compound. A high throughput LC-MS/MS method has been developed using on-line extraction turbulent flow chromatography coupled to tandem mass spectrometric detection to analyze multiple compounds present in Hanks balanced salt solution in a single analytical run. All standard curves (P-gp substrates: quinidine, etoposide, rhodamine 123, dexamethasone, and verapamil and non-substrates: metoprolol, sulfasalazine, propranolol, nadolol, and furosemide) were prepared in a cassette mode (ten-in-one) while Caco-2 cell incubations were performed both in discreet mode and in cassette mode. The standard curve range for most compounds was 10-2500 nM with regression coefficients (R(2)) greater than 0.99 for all compounds. The applicability and reliability of the analysis method was evaluated by successful demonstration of efflux ratio greater than 1 for the P-gp substrates studied in the Caco-2 cell model. The use of cassette mode analysis through selected reaction monitoring mass spectrometry presents an attractive option to increase the throughput, sensitivity, selectivity, and efficiency of the model over discreet mode UV detection. PMID:16307910

Smalley, James; Kadiyala, Pathanjali; Xin, Baomin; Balimane, Praveen; Olah, Timothy

2006-01-18

372

Efficient simultaneous excision of multiple selectable marker cassettes using I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae.  

PubMed

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. PMID:24833416

Solis-Escalante, Daniel; Kuijpers, Niels G A; van der Linden, Franka H; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

2014-08-01

373

Background sources in optical communications  

NASA Technical Reports Server (NTRS)

The characterization and measurement of background radiation relevant to optical communications system performance is addressed. The necessary optical receiver parameters are described, and radiometric concepts required for the calculation of collected background power are developed. The most important components of optical background power are discussed, and their contribution to the total collected background power in various communications scenarios is examined.

Vilnrotter, V. A.

1983-01-01

374

Cosmic Microwave Background  

NASA Astrophysics Data System (ADS)

The cosmic microwave background (CMB) radiation, the relic of the early phases of the expanding universe, is bright, full of information, and difficult to measure. Along with the recession of galaxies and the primordial nucleosynthesis, it is one of the strongest signs that the Hot Big Bang Model of the universe is correct. It is brightest around 2 mm wavelength, has a temperature of T_{cmb} = 2.72548 ± 0.00057 K, and has a blackbody spectrum within 50 parts per million. Its spatial fluctuations (around 0.01% on 1{}^{circ } scales) are possibly the relics of quantum mechanical processes in the early universe, modified by processes up to the decoupling at a redshift of about 1,000 (when the primordial plasma became mostly transparent). In the cold dark matter (DM) model with cosmic acceleration (? CDM), the fluctuation statistics are consistent with the model of inflation and can be used to determine other parameters within a few percent, including the Hubble constant, the ? constant, the densities of baryonic and dark matter, and the primordial fluctuation amplitude and power spectrum slope. In addition, the polarization of the fluctuations reveals the epoch of reionization at a redshift approximately twice that determined from the Gunn-Peterson trough due to optically thick Lyman ? absorption in QSO spectra. It is of historic importance, and a testament to the unity of theory and experiment, that we now have a standard model of cosmology that is consistent with all of the observations.Current observational challenges include (1) improvement of the spectrum distortion measurements, especially at long wavelengths, where the measured background is unexpectedly bright; (2) the search for the B-mode polarization (the divergence-free part of the polarization map), arising from propagating gravitational waves; and (3) the extension of fluctuation measurements to smaller angular scales. Much more precise spectrum observations near 2 mm are likely and would test some very interesting theories. Current theoretical challenges include explanation of the dark matter and dark energy; understanding, estimating, and removing the interference of foreground sources that limit the measurements of the CMB; detailed understanding of the influence of nonequilibrium processes on the decoupling and reionization phases; and searches for signs of the second order or exotic processes (e.g., isocurvature fluctuations, cosmic strings, non-Gaussian fluctuations). At this writing, we await the cosmological results of the Planck mission.

Mather, John; Hinshaw, Gary; Page, Lyman

375

Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium–bicarbonate cotransporter NBCn1 (SLC4A7)  

PubMed Central

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3? transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1. PMID:23959679

Liu, Ying; Qin, Xue; Wang, Deng-Ke; Guo, Yi-Min; Gill, Harindarpal S; Morris, Nathan; Parker, Mark D; Chen, Li-Ming; Boron, Walter F

2013-01-01

376

Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium-bicarbonate cotransporter NBCn1 (SLC4A7).  

PubMed

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus--MEAD in rat only, MERF in human only--as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3(-) transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1. PMID:23959679

Liu, Ying; Qin, Xue; Wang, Deng-Ke; Guo, Yi-Min; Gill, Harindarpal S; Morris, Nathan; Parker, Mark D; Chen, Li-Ming; Boron, Walter F

2013-10-15

377

Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2?-fucosyllactose  

PubMed Central

Background The trisaccharide 2?-fucosyllactose (2?-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2?-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2?-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2?-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate ?1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2?-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2?-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose. Results Here we report the construction of the first selection marker-free E. coli strain that produces 2?-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the ?-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2?-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2?-FL. Using an improved production strain, feasibility of large scale 2?-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2?-FL concentration of 20.28?±?0.83 g l-1 and a space-time-yield of 0.57 g l-1 h-1. Conclusions By chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2?-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2?-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2?-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2?-FL could be demonstrated. PMID:23635327

2013-01-01

378

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

PubMed Central

Members of the Plasmodium falciparum Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5, and show that two genetically different parasite lines expressing DC5-PfEMP1 bind PECAM1, and that anti-DC5-specific antibodies inhibit binding of DC5-PfEMP1-expressing parasites to transformed human bone marrow endothelial cells (TrHBMEC). We also show that antibodies against each of the four domains characteristic for DC5 react with native PfEMP1 expressed on the surface of infected erythrocytes, and that some of these antibodies are cross-reactive between the two DC5-containing PfEMP1 molecules tested. Finally, we confirm that anti-DC5 antibodies are acquired early in life by individuals living in malaria endemic areas, that individuals having high levels of these antibodies are less likely to develop febrile malaria episodes and that the antibody levels correlate positively with hemoglobin levels. PMID:23874884

Berger, Sanne S.; Turner, Louise; Wang, Christian W.; Petersen, Jens E. V.; Kraft, Maria; Lusingu, John P. A.; Mmbando, Bruno; Marquard, Andrea M.; Bengtsson, Dominique B. A. C.; Hviid, Lars; Nielsen, Morten A.; Theander, Thor G.; Lavstsen, Thomas

2013-01-01

379

Acid-Soluble Internal Capsules for Closed-Face Cassette Elemental Sampling and Analysis of Workplace Air  

PubMed Central

Airborne particles that are collected using closed-face filter cassettes (CFCs), which are used widely in the sampling of workplace aerosols, can deposit in places other than on the filter and thereby may not be included in the ensuing analysis. A technique for ensuring that internal non-filter deposits are included in the analysis is to collect airborne particles within an acid-soluble internal capsule that, following sampling, can be dissolved along with the filter for subsequent elemental analysis. An interlaboratory study (ILS) was carried out to evaluate the use of cellulosic CFC capsule inserts for their suitability in the determination of trace elements in airborne samples. The ILS was performed in accordance with an applicable ASTM International standard practice, ASTM E691, which describes statistical procedures for investigating interlaboratory precision. Performance evaluation materials consisted of prototype cellulose acetate capsules attached to mixed-cellulose ester filters. Batches of capsules were dosed with Pb-containing materials (standard aqueous solutions, and certified reference material soil and paint). Also, aerosol samples containing nine target analyte elements (As, Cd, Co, Cr, Cu, Fe, Pb, Mn, and Ni) were generated using a multiport sampler; various concentrations and sampling times were employed to yield samples fortified at desired loading levels. Triplicates of spiked capsules at three different loadings were conveyed to each volunteer laboratory; loading levels were unknown to the participants. The laboratories were asked to prepare the samples by acid dissolution and to analyze aliquots of extracted samples by atomic spectrometry in accordance with applicable ASTM International Standards. Participants were asked to report their results in units of ?g of each target element per sample. For the elements investigated, interlaboratory precision and recovery estimates from the participating laboratories demonstrated the utility of the cellulosic capsule inserts for the measurement of sampled trace elements. PMID:23548078

Harper, Martin; Ashley, Kevin

2013-01-01

380

Novel Pseudo-Staphylococcal Cassette Chromosome mec Element (?SCCmec57395) in Methicillin-Resistant Staphylococcus pseudintermedius CC45  

PubMed Central

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I–pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to ?-lactams (mecA; blaZ), aminoglycosides [aac(6?)-Ie–aph(2?)-Ia; aph(3?)-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (?SCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ?SCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ?SCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ?SCCmec57395 element amplified by long-range PCR revealed the presence of ?SCCmec57395 in the 33 additional isolates of MRSP CC45. The ?SCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand. PMID:23979735

Chanchaithong, Pattrarat; Prapasarakul, Nuvee; Rossano, Alexandra; Blum, Shlomo E.; Elad, Daniel; Schwendener, Sybille

2013-01-01

381

Sterolins ABCG5 and ABCG8: regulators of whole body dietary sterols  

PubMed Central

ABCG5 and ABCG8 are two ATP-binding cassette half-transporters that belong to the G family members. They were identified as proteins that are mutated in a rare human disorder, sitosterolemia, and their identification led to the completion of the physiological pathways by which dietary cholesterol, as well as noncholesterol sterols, traffics in the mammalian body. These proteins are likely to function as heterodimers, and current evidence suggests that these proteins are responsible for the majority of sterol secretions into bile, thus may define the long sought-after biliary sterol transporters. This review will focus on some of the backgrounds of this physiology, the genetics and regulation of these genes, as well as our current understanding of their functions. This review will also highlight the current limitations in our knowledge gap. PMID:16440216

Hazard, Starr E.

2006-01-01

382

Beam induced backgrounds: CDF experience  

SciTech Connect

We summarize the experiences of the Collider Detector at Fermilab (CDF) experiment in the presence of backgrounds originating from the counter circulating beams in the Fermilab Tevatron. These backgrounds are measured and their sources identified. Finally, we outline the strategies employed to reduce the effects of these backgrounds on the experiment.

Tesarek, R.J.; /Fermilab

2008-05-01

383

Background issues for defensive interceptors  

SciTech Connect

Mean nuclear backgrounds are large, but are arguably amenable to frame-to-frame subtraction. Striated backgrounds on the sensors for defensive interceptors could, however, cause clutter leak-through, which could make detection and track difficult. Nominal motions and backgrounds give signal to clutter ratios too low to be useful. Clutter leakage due to line-of-sight drift can be reduced by stabilizing the line of sight around the background clutter itself. Current interceptors have detector arrays large enough for operation independent of nuclear backgrounds in their fields of view. 6 refs., 2 figs.

Canavan, G.H.

1991-03-01

384

A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus.  

PubMed

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type. PMID:20616192

Ghaznavi-Rad, Ehsanollah; Nor Shamsudin, Mariana; Sekawi, Zamberi; van Belkum, Alex; Neela, Vasanthakumari

2010-10-01

385

Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5?-triphosphate-binding cassette transporters far beyond an efflux pump  

PubMed Central

This study examines adenosine 5?-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus?Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50): P-glycoprotein inhibition using valspodar (PSC833) 5 ?M, CAS 115104-28-4 (MK571) 50 ?M and probenecid 2·5 ?M], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC-transporters emerge as potential targets in immunosuppressive therapies interfering with DCs maturation, thereby abrogating innate immune response when it is activated after ischaemia. PMID:23600833

Lloberas, N; Rama, I; Llaudó, I; Torras, J; Cerezo, G; Cassis, L; Franquesa, M; Merino, A; Benitez-Ribas, D; Cruzado, J M; Herrero-Fresneda, I; Bestard, O; Grinyó, J M

2013-01-01

386

Synchronization of video recording and laser pulses including background light suppression  

NASA Technical Reports Server (NTRS)

An apparatus for and a method of triggering a pulsed light source, in particular a laser light source, for predictable capture of the source by video equipment. A frame synchronization signal is derived from the video signal of a camera to trigger the laser and position the resulting laser light pulse in the appropriate field of the video frame and during the opening of the electronic shutter, if such shutter is included in the camera. Positioning of the laser pulse in the proper video field allows, after recording, for the viewing of the laser light image with a video monitor using the pause mode on a standard cassette-type VCR. This invention also allows for fine positioning of the laser pulse to fall within the electronic shutter opening. For cameras with externally controllable electronic shutters, the invention provides for background light suppression by increasing shutter speed during the frame in which the laser light image is captured. This results in the laser light appearing in one frame in which the background scene is suppressed with the laser light being uneffected, while in all other frames, the shutter speed is slower, allowing for the normal recording of the background scene. This invention also allows for arbitrary (manual or external) triggering of the laser with full video synchronization and background light suppression.

Kalshoven, Jr., James E. (Inventor); Tierney, Jr., Michael (Inventor); Dabney, Philip W. (Inventor)

2004-01-01

387

Steps toward discovering the function and expression of multiple drug resistance genes in "Arabidopsis thaliana"  

Technology Transfer Automated Retrieval System (TEKTRAN)

The ATP-binding cassette (ABC) superfamily is the largest protein family identified in all organisms. It is a highly conserved domain responsible for the ATP-dependent transport of substances including ions, carbohydrates, xenobiotics, drugs, and peptides. Also, the subfamily, multidrug resistance...

388

Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens  

Technology Transfer Automated Retrieval System (TEKTRAN)

The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devastating fungal pathogens leaf rust, stripe rust, and powdery mildew. In previous studies, this broad-spectrum resistance was shown to be controlled by a single gene which encodes a putative ATP-binding cassette transport...

389

Evidence for the Existence of a Sulfonylurea-Receptor-Like Protein in Plants: Modulation of Stomatal Movements and Guard Cell Potassium Channels by Sulfonylureas and Potassium Channel Openers  

Microsoft Academic Search

Limitation of water loss and control of gas exchange is accomplished in plant leaves via stomatal guard cells. Stomata open in response to light when an increase in guard cell turgor is triggered by ions and water influx across the plasma membrane. Recent evidence demonstrating the existence of ATP-binding cassette proteins in plants led us to analyze the effect of

Nathalie Leonhardt; Elena Marin; Alain Vavasseur; Cyrille Forestier

1997-01-01

390

Correction of Apolipoprotein A-I-mediated Lipid Efflux and High Density Lipoprotein Particle Formation in Human Niemann-Pick Type C Disease Fibroblasts  

Technology Transfer Automated Retrieval System (TEKTRAN)

Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of lo...

391

A Genomic View of Sugar Transport in Mycobacterium smegmatis and Mycobacterium tuberculosis  

Microsoft Academic Search

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobac- terium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19\\/28) in M. smeg- matis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes

Fritz Titgemeyer; Johannes Amon; Stephan Parche; Maysa Mahfoud; Johannes Bail; Maximilian Schlicht; Nadine Rehm; Dietmar Hillmann; Joachim Stephan; Britta Walter; Andreas Burkovski; Michael Niederweis

2007-01-01

392

Selection and characterization of a high-activity ribozyme directed against the antineoplastic drug resistance–associated ABC transporter BCRP\\/MXR\\/ABCG2  

Microsoft Academic Search

Breast cancer resistance protein (BCRP) is a recently identified new member of the superfamily of ATP-binding cassette transporters. BCRP is a “half transporter” that may homo- or heterodimerize to form an active transport complex. A considerable overexpression of BCRP was reported from various atypical multidrug-resistant tumor cell lines, in particular from those which were established by treatment with mitoxantrone. Thus,

Petra Kowalski; Anke Wichert; Per Sonne Holm; Manfred Dietel; Hermann Lage

2001-01-01

393

Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment  

EPA Science Inventory

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

394

An inventory of the human ABC proteins  

Microsoft Academic Search

Currently 30 human ABC proteins are represented by full sequences in various databases, and this paper provides a brief overview of these proteins. ABC proteins are composed of transmembrane domains (TMDs), and nucleotide binding domains (NBDs, or ATP-binding cassettes, ABSs). The arrangement of these domains, together with available membrane topology models of the family members, are presented. Based on their

Izabella Klein; Balázs Sarkadi; András Váradi

1999-01-01

395

Common and Rare ABCA1 Variants Affecting Plasma HDL Cholesterol  

Microsoft Academic Search

Mutations in ABCA1, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of ABCA1 can affect plasma HDL cholesterol in the general

Jian Wang; John R. Burnett; Kue Young; Bernard Zinman; Anthony J. G. Hanley; Philip W. Connelly; Stewart B. Harris; Robert A. Hegele

2009-01-01

396

Bicalutamide failure in prostate cancer treatment: Involvement of Multi Drug Resistance proteins  

Microsoft Academic Search

Prolonged bicalutamide treatment induced pathology regression although relapses with a more aggressive form of prostate cancer have been observed. This failure could be due to androgen receptor mutation. In the present work we hypothesized an alternative mechanism responsible for bicalutamide failure involving activity of ATP-binding cassette (ABC) pumps such as P-glycoprotein, Breast Cancer Receptor Protein (BCRP), and Multi Resistant Proteins

Nicola Antonio Colabufo; Vincenzo Pagliarulo; Francesco Berardi; Marialessandra Contino; Carmela Inglese; Mauro Niso; Patrizia Ancona; Giancarlo Albo; Arcangelo Pagliarulo; Roberto Perrone

2008-01-01

397

Novel ABCC6 Mutations in Pseudoxanthoma Elasticum  

Microsoft Academic Search

Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in an ABC (ATP-Binding Cassette) transporter gene (ABCC6), which manifests with cutaneous, ophthalmologic, and cardiovascular findings. We studied a cohort of 19 families with PXE, and identified 16 different mutations, nine of which were novel variants. The mutation detection rate was about 77%. We found that arginine codon

Nicolas Chassaing; Ludovic Martin; Juliette Mazereeuw; Laurence Barrié; Sonia Nizard; Jean-Louis Bonafé; Patrick Calvas; Alain Hovnanian

2004-01-01

398

LXR-mediated activation of macrophage stearoyl-CoA desaturase generates unsaturated fatty acids that destabilize ABCA1  

Microsoft Academic Search

Abnormal HDL metabolism among patients with diabetes and insulin resistance may contribute to their in- creased risk of atherosclerosis. ATP binding cassette trans- porter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Because fatty acids are increased in diabetes, we examined their ef- fects on ABCA1 activity

Yutong Wang; Buran Kurdi-Haidar; John F. Oram

2004-01-01

399

The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil  

Microsoft Academic Search

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection

H. Schoonbeek; G. Del Sorbo; Waard De M. A

2001-01-01

400

Diminished Self-Chaperoning Activity of the DF508 Mutant of CFTR Results in Protein Misfolding  

E-print Network

Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Cystic Fibrosis Research Center The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF

Dokholyan, Nikolay V.

401

Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium.  

Technology Transfer Automated Retrieval System (TEKTRAN)

Half molecule ATP-binding cassette transporters of the HMT1(heavy metal tolerance factor 1)subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans and Chlamydomonas reinhardtii, and have homologs in other species, including plants and humans. Based on studies i...

402

MAR Background Report MAR Background Report: Indigenous Protest in Brazil  

E-print Network

MAR Background Report MAR Background Report: Indigenous Protest in Brazil Hundreds of indigenous people demonstrated at the National Congress in Brasilia, capital of Brazil, following the announcement in the 1990s in the midst of extensive protests in Brazil and around the world. On February 8, an indigenous

Milchberg, Howard

403

Background Check Summary Type Needs background check if began  

E-print Network

form. Background check is required if, 2) Upon receipt of form, HR BGC emails candidate a link Check Coordinator receives outcome; if candidate meets Yale policy, HR BGC notifies primary department; completes a Postdoc/Postgrad Background Check Request Form. 5) Upon receipt of this form, HR BGC emails

Haller, Gary L.

404

Background  

E-print Network

Chronic Obstructive Pulmonary Disease (COPD) is a lung disease characterized by chronic inflammation of the airways. The primary risk factor for COPD is cigarette smoking; other risk factors include long-term exposure to environmental lung irritants and certain genetic conditions. 1 COPD is also found to be associated with significant comorbidities, including heart disease, kidney disease, asthma, and arthritis, as well as various types of cancer. 2 A large-scale study, using electronic primary care records of more than 1.2 million patients, found that COPD was associated with significantly higher odds of cardiovascular disease, stroke, and diabetes mellitus. 3 There is growing evidence to suggest that systemic inflammation is potentially a common pathway for multiple chronic conditions found among adults with COPD. 4 COPD can adversely affect one’s quality of life (QoL). Depression has often been associated with COPD. In an observational study of 35,722 patients with COPD, the incidence rate of new-onset diagnoses of depression was significantly higher in the COPD group, compared to the COPD-free group. 5 Sleeping difficulties and physical inactivity are also common among those with COPD. 6,7 The aim of this report is to enumerate the prevalence and risk of secondary chronic diseases, and poor quality of life, among North Carolina adults with COPD.

unknown authors

2011-01-01

405

Background  

NASA Technical Reports Server (NTRS)

An analysis was made of the UF6 fueled gas core reactor as a function of cavity reactor criticality and fluid mechanics tests, investigations of uranium optical emission spectra, and radiant heat transfer power plant studies. Data are also given on nuclear and thermodynamic cycle analysis.

1976-01-01

406

Background  

E-print Network

• Requirement to control a team of 5-a-side soccer playing robots • Due to camera and communication time delays it is very difficult to control a mobile robot moving faster than 1 m/sec • Robots currently use an independent term PID controller • Investigate FLCs as an alternative

unknown authors

407

Background  

Cancer.gov

Extensive evidence has demonstrated that 24-hour dietary recalls provide the highest quality, least biased dietary data. Traditional 24-hour recalls, however, are expensive and impractical for large-scale research because they rely on trained interviewers and multiple administrations to estimate usual intakes. As a result, researchers often make use of food frequency questionnaires, which are less expensive but contain substantial error.

408

Strep-tag II and Twin-Strep based cassettes for protein tagging by homologous recombination and characterization of endogenous macromolecular assemblies in Saccharomyces cerevisiae.  

PubMed

Peptide sequences fused to a gene of interest facilitate the isolation of proteins or protein complexes from cell extracts. In the case of fluorescent protein tags, the tagged protein can be visually localized in living cells. To tag endogenous genes, PCR-based homologous recombination is a powerful approach used in the yeast Saccharomyces cerevisiae. This approach uses short, homologous DNA sequences that flank the tagging cassette to direct recombination. Here, we constructed a set of plasmids, whose sequences were optimized for codon usage in yeast, for Strep-tag II and Twin-Strep tagging in S. cerevisiae. Some plasmids also contain sequences encoding for a fluorescent protein followed by the purification tag. We demonstrate using the yeast pyruvate dehydrogenase (PDH) complex that these plasmids can be used to purify large protein complexes efficiently. We furthermore demonstrate that purification from the endogenous pool using the Strep-tag system results in functionally active complexes. Finally, using the fluorescent tags, we show that a kinase and a phosphatase involved in regulating the activity of the PDH complex localize in the cells' mitochondria. In conclusion, our cassettes can be used as tools for biochemical, functional, and structural analyses of endogenous multi-protein assemblies in yeast. PMID:24969434

Rai, Jay; Pemmasani, J Kalyani; Voronovsky, Andriy; Jensen, Ida S; Manavalan, Arulmani; Nyengaard, Jens R; Golas, Monika M; Sander, Bjoern

2014-11-01

409

Characterization of In40 of Enterobacter aerogenes BM2688, a Class 1 Integron with Two New Gene Cassettes, cmlA2 and qacF  

PubMed Central

Enterobacter aerogenes BM2688, which is resistant to multiple antibiotics, and its aminoglycoside-susceptible derivative BM2688-1 were isolated from the same clinical sample. Strain BM2688 harbored plasmid pIP833, which carries a class 1 integron, In40, containing (in addition to qacE?1 and sul1, which are characteristic of class 1 integrons) four gene cassettes: aac(6?)-Ib, qacF, cmlA2, and oxa-9. The cmlA2 gene had 83.7% identity with the previously described nonenzymatic chloramphenicol resistance cmlA1 gene. The qacF gene conferred resistance to quaternary ammonium compounds and displayed a high degree of similarity with qacE (67.8% identity) which, however, has been found as part of a cassette with a very different 59-base element. The oxa-9 gene was not expressed due to a lack of promoter sequences. Study of the antibiotic-susceptible derivative BM2688-1 indicated that a 3,148-bp deletion between the 3? end of the aac(6?)-Ib gene and the 3? conserved segment of In40 was responsible for the loss of resistance. The occurrence of this DNA rearrangement, which did not involve homologous sequences, suggests that the In40 integrase could promote recombination at secondary sites. PMID:9756755

Ploy, Marie-Cécile; Courvalin, Patrice; Lambert, Thierry

1998-01-01

410

Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.  

PubMed

In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

2014-05-01

411

Background: Extratropical Cyclones and NWP  

E-print Network

Chapter 2 Background: Extratropical Cyclones and NWP 2.1 Introduction This chapter provides background information about extratropical cyclones, numerical weather prediction (NWP) and ensemble prediction. It is organised as follows. Section 2.2 discusses extratropical cyclones and begins with a brief

Froude, Lizzie

412

Cosmic Superstring Scattering in Backgrounds  

E-print Network

We generalize the calculation of cosmic superstring reconnection probability to non-trivial backgrounds. This is done by modeling cosmic strings as wound tachyon modes in the 0B theory, and the spacetime effective action is then used to couple this to background fields. Simple examples are given including trivial and warped compactifications. Generalization to $(p,q)$ strings is discussed.

Mark G. Jackson

2006-08-22

413

Background reduction in cryogenic detectors  

SciTech Connect

This paper discusses the background reduction and rejection strategy of the Cryogenic Dark Matter Search (CDMS) experiment. Recent measurements of background levels from CDMS II at Soudan are presented, along with estimates for future improvements in sensitivity expected for a proposed SuperCDMS experiment at SNOLAB.

Bauer, Daniel A.; /Fermilab

2005-04-01

414

Background events in microchannel plates  

NASA Technical Reports Server (NTRS)

Measurements have been made to assess the characteristics and origins of background events in microchannel plates (MCPs). An overall background rate of about 0.4 events/sq cm persec has been achieved consistently for MCPs that have been baked and scrubbed. The temperature and gain of the MCPs are found to have no significant effect on the background rate. Detection of 1.46-MeV gamma rays from the MCP glass confirms the presence of K-40, with a concentration of 0.0007 percent, in MCP glass. It is shown that beta decay from K-40 is sufficient to cause the background rate and spectrum observed. Anticoincidence measurements indicate the the background rate caused by cosmic ray interactions is small (less than 0.016 events/sq cm per sec).

Siegmund, O. H. W.; Vallerga, J.; Wargelin, B.

1988-01-01

415

Use of a novel rapid and resource-efficient cassette dosing approach to determine the pharmacokinetics and CNS distribution of small molecule 7-transmembrane receptor allosteric modulators in rat  

PubMed Central

Approaches to efficiently and accurately define the pharmacokinetics (PK) of large sets of small molecules in rodents have been previously described. Likewise, a variety of methods for determining brain tissue distribution (BTD) have been reported for use in the discovery of therapeutics targeting the central nervous system (CNS). Herein we describe a novel cassette approach to efficiently obtain concurrent PK and BTD data from a dose of up to five compounds in one rat over 24 h. In conjunction with fraction unbound (fu) data obtained in plasma and brain homogenate, this approach serves as an efficient means to determine compound unbound brain:unbound plasma partition coefficients (Kp,uu), thereby providing insight to compounds bearing poor permeability and/or active transporter activity impacting their permeation of the blood–brain barrier (BBB). This integrated approach was utilized in a lead optimization effort towards the discovery of CNS-penetrant allosteric modulators of a seven-transmembrane (7TM) receptor target. Rat PK and brain distribution was rapidly obtained for 70 compounds and correlated to data obtained from in vitro assessments. Two compounds that were evaluated in cassette and discrete studies, displayed agreement in PK (compound 1: cassette CLp = 1.6 mL min?1 kg?1, discrete CLp = 1.6 mL min?1 kg?1; compound 2: cassette CLp = 11 mL min?1 kg?1, discrete CLp = 8.1 mL min?1 kg?1) and BTD (compound 1: cassette Kp = 0.11, discrete Kp = 0.09; compound 2: cassette Kp < 0.05, discrete Kp = 0.04). The resulting data were used to guide medicinal chemistry efforts and to enable the progression of optimized compounds to in vivo pharmacodynamic assessments. PMID:25505618

Bridges, Thomas M; Morrison, Ryan D; Byers, Frank W; Luo, Shuanghui; Scott Daniels, J

2014-01-01

416

Aluminum as a source of background in low background experiments  

E-print Network

Neutrinoless double beta decay would be a key to understanding the nature of neutrino masses. The next generation of High Purity Germanium experiments will have to be operated with a background rate of better than 10^-5 counts/(kg y keV) in the region of interest around the Q value of the decay. Therefore, so far irrelevant sources of background have to be considered. The metalization of the surface of germanium detectors is in general done with aluminum. The background from the decays of 22Na, 26Al, 226Ra and 228Th introduced by this metalization is discussed. It is shown that only a special selection of aluminum can keep these background contributions acceptable.

B. Majorovits; I. Abt; M. Laubenstein; O. Volynets

2011-05-18

417

Low background counting at the LBNL low background facility  

SciTech Connect

The Low Background Facility (LBF) at the Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California provides low background gamma spectroscopy services to end-users in two unique facilities: locally within a carefully-constructed, low background laboratory space; and a satellite underground station (600 m.w.e) in Oroville, CA. These facilities provide a variety of gamma spectroscopy services to low background experiments primarily in the form of passive material screening for primordial radioisotopes (U, Th, K) or common cosmogenic and anthropogenic products, as well as active screening via neutron activation analysis for specific applications. A general overview of the facilities, services, and capabilities will be discussed. Recent activities will also be presented, including the recent installation of a 3? muon veto at the surface facility, cosmogenic activation studies of TeO{sub 2} for CUORE, and environmental monitoring of Fukushima fallout.

Thomas, K. J.; Norman, E. B. [Department of Nuclear Engineering, University of California, Berkeley, CA 94720, United States and Nuclear Science Division, Lawrence Berkeley Laboratory, CA 94720 (United States)] [Department of Nuclear Engineering, University of California, Berkeley, CA 94720, United States and Nuclear Science Division, Lawrence Berkeley Laboratory, CA 94720 (United States); Smith, A. R.; Chan, Y. D.; Hurley, D. L. [Nuclear Science Division, Lawrence Berkeley Laboratory, CA 94720 (United States)] [Nuclear Science Division, Lawrence Berkeley Laboratory, CA 94720 (United States); Wang, B. S. [Department of Nuclear Engineering, University of California, Berkeley, CA 94720 (United States)] [Department of Nuclear Engineering, University of California, Berkeley, CA 94720 (United States)

2013-08-08

418

Stimulation of murine biliary cholesterol secretion by thyroid hormone is dependent on a functional ABCG5/G8 complex  

PubMed Central

Abstract Secretion of cholesterol into bile is important for the elimination of cholesterol from the body. Thyroid hormone (TH) increases biliary cholesterol secretion and hepatic gene expression of adenosine triphosphate (ATP)-binding cassette, subfamily G (WHITE), member 5 (ABCG5) and ATP-binding cassette, subfamily G (WHITE), member 8 (ABCG8), two half-transporters that act as a heterodimeric complex promoting sterol secretion. In addition, nuclear liver x receptor-alpha (LXRa), also regulated by TH, induces gene expression of ABCG5/G8. We here investigated if the TH-induced stimulation of biliary cholesterol secretion is mediated by the ABCG5/G8 complex in vivo, and if so, whether LXRa is involved. Mice homozygous for disruption of Abcg5 (Abcg5?/?) or Lxra (Lxra?/?) and their wild-type counterparts were treated with triiodothyronine (T3) for 14 days and compared to untreated mice of corresponding genetic backgrounds. Bile was collected by gallbladder cannulation, and liver samples were analyzed for gene expression levels. Basal biliary cholesterol secretion in Abcg5?/? mice was 72% lower than in Abcg5+/+ mice. T3 treatment increased cholesterol secretion 3.1-fold in Abcg5+/+ mice, whereas this response was severely blunted in Abcg5?/? mice. In contrast, biliary cholesterol secretion in T3-treated Lxra+/+ and Lxra?/? mice was increased 3.5- and 2.6-fold, respectively, and did not differ significantly. Conclusions: TH-induced secretion of cholesterol into bile is largely dependent on an intact ABCG5/G8 transporter complex, whereas LXRa is not critical for this effect. (HEPATOLOGY 2012;56:1828–1837) PMID:22829162

Bonde, Ylva; Plösch, Torsten; Kuipers, Folkert; Angelin, Bo; Rudling, Mats

2012-01-01

419

Darkling Beetle Life Cycle Background  

E-print Network

Darkling Beetle Life Cycle Background: Metamorphosis is a process the juvenile stage to the adult stage. The Darkling Beetle (Tenebrio molitor) undergoes a complete, or holometabolous, metamorphosis. Adult beetles reproduce sexually and lay

Rose, Michael R.

420

Analysis of the XRS background  

NASA Technical Reports Server (NTRS)

Background counts on the XRS Calorimeter spectrometer of Astro-E2 have several sources, including primary cosmic rays and secondary particles interacting with the pixels and with the silicon structure of the array. After rejecting events coincident between pixels or between a pixel and the anti-coincidence detector behind the calorimeter array, the residual background on the ground in the 0.1 - 10 keV band is 1e-3 counts/s (8e-3 counts/s/sq cm). We will present the details of the ground background events and the rejection criteria required lo remove them while minimizing deadtime. We will also present preliminary analysis of the in-orbit background.

Kilbourne, Caroline A.; Boyce, K. R.; Brown, G. V.; Cottam, J.; Fujimoto, R.; Furusho, T.; Ishisaki, Y.; Kelley, R. L.; McCammon, D.; Mitsuda, K.

2005-01-01

421

Low background techniques in XMASS  

SciTech Connect

The XMASS project aims to detect pp and {sup 7}Be solar neutrinos, neutrino-less double beta decay, and dark matter searches using ultra-pure liquid xenon. The first stage of XMASS project is concentrated on dark matter searches using 800 kg liquid xenon detector which requires low background and low threshold. Several techniques applied to XMASS detector for low background will be presented.

Takeda, Atsushi [Kamioka Observatory, ICRR, University of Tokyo, 456 Higashi-Mozumi, Kamioka-cho, Hida, Gifu, 506-1205 (Japan)

2011-04-27