scholarly journals In Situ Biochemical Demonstration That P-Glycoprotein Is a Drug Efflux Pump with Broad Specificity

2000 ◽  
Vol 148 (5) ◽  
pp. 863-870 ◽  
Author(s):  
Yu Chen ◽  
Sanford M. Simon
Keyword(s):  
Fluorescent Protein ◽  
Efflux Pump ◽  
Multiple Drug Resistance ◽  
Living Cells ◽  
Drug Accumulation ◽  
Multiple Drug ◽  
Gfp Expression ◽  
Active Efflux ◽  
P Glycoprotein ◽  
Wide Range

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.

scholarly journals DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression.

1982 ◽  
Vol 2 (8) ◽  
pp. 881-889 ◽  
Author(s):  
P G Debenham ◽  
N Kartner ◽  
L Siminovitch ◽  
J R Riordan ◽  
V Ling
Keyword(s):  
Drug Resistance ◽  
Plasma Membranes ◽  
Multiple Drug Resistance ◽  
Chinese Hamster ◽  
Surface Glycoprotein ◽  
Multiple Drug ◽  
Cell Surface Glycoprotein ◽  
Resistance Phenotype ◽  
P Glycoprotein ◽  
Wide Range

Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.

DNA-mediated transfer of multiple drug resistance and plasma membrane glycoprotein expression

1982 ◽  
Vol 2 (8) ◽  
pp. 881-889
Author(s):  
P G Debenham ◽  
N Kartner ◽  
L Siminovitch ◽  
J R Riordan ◽  
V Ling
Keyword(s):  
Drug Resistance ◽  
Plasma Membranes ◽  
Multiple Drug Resistance ◽  
Chinese Hamster ◽  
Surface Glycoprotein ◽  
Multiple Drug ◽  
Cell Surface Glycoprotein ◽  
Resistance Phenotype ◽  
P Glycoprotein ◽  
Wide Range

Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals Chemosensitizing multiple drug resistance of human carcinoma by bicyclol involves attenuated P-glycoprotein, GST-P and Bcl-2

2006 ◽  
Vol 5 (5) ◽  
pp. 536-543 ◽  
Author(s):  
Bing Zhu ◽  
Geng Tao Liu ◽  
Yong Mei Zhao ◽  
Ruo Su Wu ◽  
Samuel J Strada
Keyword(s):  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
Human Carcinoma ◽  
P Glycoprotein

scholarly journals Efflux‐Pump–Derived Multiple Drug Resistance to Ethambutol Monotherapy inMycobacterium tuberculosisand the Pharmacokinetics and Pharmacodynamics of Ethambutol

2010 ◽  
Vol 201 (8) ◽  
pp. 1225-1231 ◽  
Author(s):  
Shashikant Srivastava ◽  
Sandirai Musuka ◽  
Carleton Sherman ◽  
Claudia Meek ◽  
Richard Leff ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Efflux Pump ◽  
Multiple Drug Resistance ◽  
Multiple Drug ◽  
Pharmacokinetics And Pharmacodynamics

Co-amplification of double minute chromosomes, multiple drug resistance, and cell surface P-glycoprotein in DNA-mediated transformants of mouse cells

1984 ◽  
Vol 4 (3) ◽  
pp. 500-506
Author(s):  
S M Robertson ◽  
V Ling ◽  
C P Stanners
Keyword(s):  
Drug Resistance ◽  
Multiple Drug Resistance ◽  
Resistant Mutant ◽  
Genetic System ◽  
Cell Membrane Permeability ◽  
Multiple Drug ◽  
P Glycoprotein ◽  
Double Minute ◽  
Mouse Cells ◽  
Double Minute Chromosomes

A genetic system comprised of mammalian cell mutants which demonstrate concomitant resistance to a number of unrelated drugs has been described previously. The resistance is due to reduced cell membrane permeability and is correlated with the presence of large amounts of a plasma membrane glycoprotein termed P-glycoprotein. This system could represent a model for multiple drug resistance which develops in cancer patients treated with chemotherapeutic drugs. We demonstrate here that the multiple drug resistance phenotype can be transferred to mouse cells with DNA from a drug-resistant mutant and then amplified quantitatively by culture in media containing increasing concentrations of drug. The amount of P-glycoprotein was correlated directly with the degree of drug resistance in the transformants and amplified transformants. In addition, the drug resistance and expression of P-glycoprotein of the transformants were unstable and associated quantitatively with the number of double minute chromosomes. We suggest that the gene for multiple drug resistance and P-glycoprotein is contained in these extrachromosomal particles and is amplified by increases in double minute chromosome number. The potential use of this system for manipulation of mammalian genes in general is discussed.

Spectrum and resistance determinants of oral streptococci clinical isolates

2020 ◽  
Vol 65 (10) ◽  
pp. 632-637
Author(s):  
Nataliia Valerievna Davidovich ◽  
A. S. Galieva ◽  
N. G. Davydova ◽  
O. G. Malygina ◽  
N. N. Kukalevskaya ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Multiple Drug Resistance ◽  
Antibiotic Sensitivity ◽  
Periodontal Pocket ◽  
Multiple Drug ◽  
Oral Streptococci ◽  
Antibiotic Resistant ◽  
Resistance Determinants ◽  
Wide Range ◽  
Clinically Significant

The profiles of oral streptococci sensitivity to antibacterial drugs may reflect information about the presence of macroorganism resistance determinants. The aim of the work was to isolate the spectrum of oral streptococci from the microbiota of the oral cavity of patients and to determine their sensitivity to a wide range of antibiotics. A total of 342 microbial streptococcal isolates were isolated from saliva samples and a periodontal pocket and tested for antibiotic sensitivity. Species identification of streptococci was carried out using biochemical API test systems. Evaluation of antibiotic resistance was performed using E-tests. Real-time PCR was used to identify the presence of tetracycline and macrolide resistance genes. The study identified six types of oral streptococci: S. oralis, S. salivarius, S. mitis, S. sanguinis, S. anginosus and S. mutans. All streptococci were sensitive to linezolid and meropenem. The proportion of penicillin-resistant streptococci in the subgroup S. oralis / mitis / mutans was 47,8% versus 23,5% in the subgroup S. salivarius / sanguinis / anginosus (p = 0.020). Significant levels of resistance were revealed to macrolides (erythromycin) - 47,9%, tetracyclines (tetracycline) - 44,4% and quinolones (ofloxacin) - 41%. Multiple drug resistance (MDR) was detected in 31,9% of oral streptococcal isolates, a combination of erythromycin, tetracycline and ofloxacin resistance was prevalent in 79 isolates (23,1%). The most common genotypes of macrolides and tetracycline resistant oral streptococci (in 127 streptococcal isolates with combined resistance) were ermB-mefE + and tetM + tetQ-, respectively. Thus, S. oralis / mitis / mutans group streptococci predominated in the structure of antibiotic-resistant oral streptococci, including MDR. So, being in one of the most densely populated biotopes of a macroorganism, oral streptococci can mediate the transfer of resistance determinants to more pathogenic and clinically significant microorganisms, which requires careful monitoring of their level of susceptibility to antimicrobial agents.

scholarly journals Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport

1996 ◽  
Vol 320 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Frances J. SHAROM ◽  
Xiaohong YU ◽  
Giulio DiDIODATO ◽  
Joseph W. K. CHU
Keyword(s):  
Drug Transport ◽  
Efflux Pump ◽  
Membrane Vesicles ◽  
Multidrug Resistant ◽  
Drug Uptake ◽  
Peptide Transport ◽  
Active Efflux ◽  
Hydrophobic Peptides ◽  
P Glycoprotein ◽  
Drug Studies

P-Glycoprotein functions as an ATP-driven active efflux pump for many natural products and chemotherapeutic drugs. Hydrophobic peptides have been shown to block drug uptake by P-glycoprotein, indicating that they might be transport substrates. The present study examines the interaction of the synthetic peptide series NAc-LnY-amide with the multidrug transporter. Several peptides in this series caused up to 3.5-fold enhancement of colchicine accumulation in membrane vesicles from multidrug resistant (MDR) cells, which suggests the existence of novel interactions between the binding sites for peptides and drug. Peptides did not stimulate vinblastine transport, which was inhibited as expected for competing substrates. These peptides displayed modest stimulatory effects on the ATPase activity of P-glycoprotein. None blocked azidopine photoaffinity labelling, showing that they probably occupy a binding site separate from that for the drug. Studies with 125I-labelled NAc-LLY-amide showed that it was transported by P-glycoprotein in both membrane vesicles and reconstituted proteoliposomes. Uptake of the peptide was rapid, saturable, osmotically sensitive and occurred against a concentration gradient. The enhancing effect of NAc-LLY-amide on colchicine transport was reciprocated, i.e. colchicine greatly increased the transport of labelled peptide by P-glycoprotein. Peptide transport was also modulated, both positively and negatively, by other MDR spectrum drugs. It is concluded that linear hydrophobic peptides are indeed transported by P-glycoprotein, and some have interactions with drug substrates that result in mutual stimulation of transport.

Sign in / Sign up

Export Citation Format

Share Document