Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein

2013 ◽  
Vol 450 (2) ◽  
pp. 387-395 ◽  
Author(s):  
Ágnes Telbisz ◽  
Csilla Özvegy-Laczka ◽  
Tamás Hegedűs ◽  
András Váradi ◽  
Balázs Sarkadi
Keyword(s):  
Bile Acids ◽  
Multidrug Transporter ◽  
Wild Type ◽  
Functional Protein ◽  
Highly Active ◽  
Mutant Variant ◽  
Wide Range ◽  
In The Wild ◽  
Lipid Environment ◽  
Abcg2 Protein

The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.

scholarly journals Deciphering the evolution of microbial interactions: in silico studies of two-member microbial communities

2022 ◽  
Author(s):  
Gayathri Sambamoorthy ◽  
Karthik Raman
Keyword(s):  
Microbial Communities ◽  
In Silico ◽  
Microbial Interactions ◽  
Interaction Patterns ◽  
Wild Type ◽  
Community Function ◽  
Evolutionary Forces ◽  
In Silico Studies ◽  
Wide Range ◽  
In The Wild

Microbes thrive in communities, embedded in a complex web of interactions. These interactions, particularly metabolic interactions, play a crucial role in maintaining the community structure and function. As the organisms thrive and evolve, a variety of evolutionary processes alter the interactions among the organisms in the community, although the community function remains intact. In this work, we simulate the evolution of two-member microbial communities in silico to study how evolutionary forces can shape the interactions between organisms. We employ genomescale metabolic models of organisms from the human gut, which exhibit a range of interaction patterns, from mutualism to parasitism. We observe that the evolution of microbial interactions varies depending upon the starting interaction and also on the metabolic capabilities of the organisms in the community. We find that evolutionary constraints play a significant role in shaping the dependencies of organisms in the community. Evolution of microbial communities yields fitness benefits in only a small fraction of the communities, and is also dependent on the interaction type of the wild-type communities. The metabolites cross-fed in the wild-type communities appear in only less than 50% of the evolved communities. A wide range of new metabolites are cross-fed as the communities evolve. Further, the dynamics of microbial interactions are not specific to the interaction of the wild-type community but vary depending on the organisms present in the community. Our approach of evolving microbial communities in silico provides an exciting glimpse of the dynamics of microbial interactions and offers several avenues for future investigations.

scholarly journals ISOLATION AND STUDY OF MUTANTS LACKING A DEREPRESSIBLE PHOSPHATASE IN CHLAMYDOMONAS REINHARDI

Genetics ◽  
1975 ◽  
Vol 80 (2) ◽  
pp. 239-250
Author(s):  
R F Matagne ◽  
R Loppes
Keyword(s):  
Alkaline Phosphatase ◽  
Inorganic Phosphate ◽  
Double Mutant ◽  
Culture Medium ◽  
Acid Phosphatases ◽  
Wild Type ◽  
Optimal Activity ◽  
Wide Range ◽  
In The Wild ◽  
Definition Of

ABSTRACT In the green alga Chlamydomonas reinhardi, removal of inorganic phosphate from the culture medium results in the increase of phosphatase activity (derepression) in the wild-type (WT) strain as well as in a double mutant (P2Pa) lacking the two main constitutive acid phosphatases. Following treatment of WT and P2Pa with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), mutants were recovered which display very low phosphatase activities when grown in the absence of phosphate; as shown by electrophoresis, they lack one non-migrating phosphatase (PD mutants). This enzyme is active over a wide range of pH with an optimum at pH 7.5. The comparison of electropherograms from WT and mutants grown on media with or without phosphate allowed us to provide a tentative definition of the pool of derepressible phosphatases in Chlamydomonas : in addition to the neutral phosphatase lacking in PD mutants, Chlamydomonas produces two electrophoretic forms of alkaline phosphatase showing an optimal activity at pH 9.5.

scholarly journals Overexpression of a 3-Ketoacyl-CoA Thiolase in Leptosphaeria maculans Causes Reduced Pathogenicity on Brassica napus

2006 ◽  
Vol 19 (6) ◽  
pp. 588-596 ◽  
Author(s):  
Candace E. Elliott ◽  
Barbara J. Howlett
Keyword(s):  
Brassica Napus ◽  
Leptosphaeria Maculans ◽  
Ectopic Expression ◽  
Random Mutagenesis ◽  
Wild Type ◽  
In Planta ◽  
Functional Protein ◽  
In The Wild ◽  
Dna Insertion

Agrobacterium tumefaciens-mediated random mutagenesis was used to generate insertional mutants of the fungus Leptosphaeria maculans. Of 91 transformants screened, only one (A3) produced lesions of reduced size on cotyledons of canola (Brassica napus). Genes flanking the T-DNA insertion had the best matches to an alcohol dehydrogenase class 4 (ADH4)-like gene (Adh4L) and a 3-ketoacyl-CoA thiolase gene (Thiol) and were expressed in mutant A3 in vitro and in planta at significantly higher levels than in the wild type. This is the first report of a T-DNA insertion in fungi causing increased gene expression. Transformants of the wild-type isolate expressing both Adh4L and Thiol under the control of a heterologous promoter had similar pathogenicity to mutant A3. Ectopic expression of only thiolase resulted in loss of pathogenicity, suggesting that thiolase overexpression was primarily responsible for the reduced pathogenicity of the A3 isolate. The thiolase gene encoded a functional protein, as shown by assays in which a nontoxic substrate (2, 4 dichlorophenoxybutyric acid) was converted to a toxic product. The use of a translational fusion with a reporter gene showed thiolase expressed in organelles that are most likely peroxisomes.

Steric contributions to CO binding in heme proteins: a density functional analysis of FeCO vibrations and deformability

2001 ◽  
Vol 05 (03) ◽  
pp. 312-322 ◽  
Author(s):  
PAWEL M. KOZLOWSKI ◽  
KATHLEEN M. VOGEL ◽  
MAREK Z. ZGIERSKIA ◽  
THOMAS G. SPIRO
Keyword(s):  
Heme Proteins ◽  
Density Functional ◽  
Minimum Energy ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein ◽  
Wide Range ◽  
In The Wild ◽  
Phase Combination ◽  
Atom Displacement

Non-local Density Functional Theory (DFT) is applied to the calculation of geometry and vibrational frequencies of FeII (porphine)(imidazole)(CO), a model for CO adducts of heme proteins. Bond distances and angles are in agreement with crystallographic data, and frequencies are correctly calculated for C–O and Fe–C stretching and for Fe–C - O bending. This last mode is actually the out-of-phase combination of Fe–C–O bending and Fe–C tilting coordinates, which are heavily mixed because of a large bend–tilt interaction force constant. The in-phase combination is predicted at a very low frequency, 73 cm-1, and to have a low infrared intensity; attempts to detect it in far-IR spectra via 13 C 18 O isotope sensitivity have been unsuccessful. The stretch–bend interaction lowers the energy required for FeCO distortion. A soft potential may account for the wide range of crystallographically determined Fe–C–O displacements and orientations in myoglobin ( Mb ). The minimum energy path for displacement of the O atom from the heme normal was calculated by relaxing the structure while constraining only the O atom displacement from the heme normal. Energies of 0.2 to 3.5 kcal mol-1 are required for the range of reported displacement, 0.3–1.3 Å. However, vibrational spectroscopy limits the allowable displacement to the low end of this range. The O atom displacement is computed via DFT to be 0.6 Å for a 7 ° angle of the C–O stretching IR dipole relative to the heme normal, the maximum value compatible with IR polarization measurements on MbCO . FeCO distortion is predicted to diminish both ν CO and ν FeC , thereby producing deviations from the well-established backbonding correlation; the scatter of the data permits a maximum displacement of 0.5 Å. This displacement would cost about 1.6 kcal mol-1 of steric energy. A small distortion energy is consistent with the CO affinity changes produced by mutations of the distal histidine residue in Mb . Taking the leucine mutant as reference, we estimate the 1.6 kcal mol-1 affinity loss in the wild-type protein to be the resultant of a 0.0–1.6 kcal steric inhibition, a 0.5 kcal mol-1 attraction of the distal histidine sidechain for the bound CO [weak H -bond], and a 0.5–2.1 kcal mol-1 attraction of the same side-chain for a water molecule in the deoxy protein. The observed 2.3 kcal mol-1 O 2 affinity increase in the wild-type protein relative to the leucine mutant then implies a 2.8–4.4 kcal mol-1 attraction of the histidine sidechain for bound O 2, consistent with a substantial H -bond interaction with the distal histidine. Thus steric inhibition can account for only a minor fraction of the discrimination factor against CO and in favor of O 2 which is produced by the heme–myoglobin interaction.

Freeze-substituted fungal cells of Nectria haematococca: A comparison of the macroconidial walls of the adhesion-competent wild type with an adhesion-reduced mutant

1990 ◽  
Vol 48 (3) ◽  
pp. 688-689
Author(s):  
Thecan Caesar-Ton That ◽  
Lynn Epstein
Keyword(s):  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Wild Type ◽  
Nectria Haematococca ◽  
Fruit Extract ◽  
Dialysis Tubing ◽  
Tube Emergence ◽  
Contrast Microscopy ◽  
In The Wild ◽  
Freeze Damage

Nectria haematococca mating population I (anamorph, Fusarium solani) macroconidia attach to its host (squash) and non-host surfaces prior to germ tube emergence. The macroconidia become adhesive after a brief period of protein synthesis. Recently, Hickman et al. (1989) isolated N. haematococca adhesion-reduced mutants. Using freeze substitution, we compared the development of the macroconidial wall in the wild type in comparison to one of the mutants, LEI.Macroconidia were harvested at 1C, washed by centrifugation, resuspended in a dilute zucchini fruit extract and incubated from 0 - 5 h. During the incubation period, wild type macroconidia attached to uncoated dialysis tubing. Mutant macroconidia did not attach and were collected on poly-L-lysine coated dialysis tubing just prior to freezing. Conidia on the tubing were frozen in liquid propane at 191 - 193C, substituted in acetone with 2% OsO4 and 0.05% uranyl acetate, washed with acetone, and flat-embedded in Epon-Araldite. Using phase contrast microscopy at 1000X, cells without freeze damage were selected, remounted, sectioned and post-stained sequentially with 1% Ba(MnO4)2 2% uranyl acetate and Reynold’s lead citrate. At least 30 cells/treatment were examined.

Backpacking with the Saints

2015 ◽  
Author(s):  
Belden C. Lane
Keyword(s):  
American Southwest ◽  
Spiritual Practice ◽  
Spiritual Journey ◽  
Human Response ◽  
John Muir ◽  
Hildegard Of Bingen ◽  
Wide Range ◽  
Thich Nhat Hanh ◽  
In The Wild ◽  
John Of The Cross

Carrying only basic camping equipment and a collection of the world's great spiritual writings, Belden C. Lane embarks on solitary spiritual treks through the Ozarks and across the American Southwest. For companions, he has only such teachers as Rumi, John of the Cross, Hildegard of Bingen, Dag Hammarskjöld, and Thomas Merton, and as he walks, he engages their writings with the natural wonders he encounters--Bell Mountain Wilderness with Søren Kierkegaard, Moonshine Hollow with Thich Nhat Hanh--demonstrating how being alone in the wild opens a rare view onto one's interior landscape, and how the saints' writings reveal the divine in nature. The discipline of backpacking, Lane shows, is a metaphor for a spiritual journey. Just as the wilderness offered revelations to the early Desert Christians, backpacking hones crucial spiritual skills: paying attention, traveling light, practicing silence, and exercising wonder. Lane engages the practice not only with a wide range of spiritual writings--Celtic, Catholic, Protestant, Buddhist, Hindu, and Sufi Muslim--but with the fascination of other lovers of the backcountry, from John Muir and Ed Abbey to Bill Plotkin and Cheryl Strayed. In this intimate and down-to-earth narrative, backpacking is shown to be a spiritual practice that allows the discovery of God amidst the beauty and unexpected terrors of nature. Adoration, Lane suggests, is the most appropriate human response to what we cannot explain, but have nonetheless learned to love. An enchanting narrative for Christians of all denominations, Backpacking with the Saints is an inspiring exploration of how solitude, simplicity, and mindfulness are illuminated and encouraged by the discipline of backcountry wandering, and of how the wilderness itself becomes a way of knowing-an ecology of the soul.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 618
Author(s):  
Yue Jin ◽  
Shihao Li ◽  
Yang Yu ◽  
Chengsong Zhang ◽  
Xiaojun Zhang ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Wild Type ◽  
Expression Levels ◽  
In The Wild ◽  
Cuticle Proteins ◽  
Apolipoprotein D ◽  
High Level ◽  
Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

scholarly journals cot1: A Regulator of Arabidopsis Trichome Initiation

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 565-577
Author(s):  
Daniel B Szymanski ◽  
Daniel A Klis ◽  
John C Larkin ◽  
M David Marks
Keyword(s):  
Cell Fate ◽  
Gene Dosage ◽  
Signal Transduction Pathway ◽  
Spatial Arrangement ◽  
Complex Signal ◽  
Chromosome 2 ◽  
Wild Type ◽  
Trichome Formation ◽  
In The Wild ◽  
Leaf Trichome

Abstract In Arabidopsis, the timing and spatial arrangement of trichome initiation is tightly regulated and requires the activity of the GLABROUS1 (GL1) gene. The COTYLEDON TRICHOME 1 (COT1) gene affects trichome initiation during late stages of leaf development and is described in this article. In the wild-type background, cot1 has no observable effect on trichome initiation. GL1 overexpression in wild-type plants leads to a modest number of ectopic trichomes and to a decrease in trichome number on the adaxial leaf surface. The cot1 mutation enhances GL1-overexpression-dependent ectopic trichome formation and also induces increased leaf trichome initiation. The expressivity of the cot1 phenotype is sensitive to cot1 and 35S::GL1 gene dosage, and the most severe phenotypes are observed when cot1 and 35S::GL1 are homozygous. The COT1 locus is located on chromosome 2 15.3 cM north of er. Analysis of the interaction between cot1, try, and 35S::GL1 suggests that COT1 is part of a complex signal transduction pathway that regulates GL1-dependent adoption of the trichome cell fate.

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