Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.
Mol Cell Biol 1992 Jun
PMID:Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene. 135 Mar 25

Recent data concerning cloning and sequencing of mdr genes involved in multiple drug resistance in higher eukaryotes are reviewed. Structures of ABC-superfamily members, including the mdr products as well as mechanisms of their superproduction at various levels are considered. The possible role of MDR-transporter in normal tissues and various approaches to overcoming the MDR phenotype are discussed. Non-P-glycoprotein mechanisms of drug resistance capable to modify MDR phenotype and applications of mdr in biotechnology are provided.
Mol Biol (Mosk)
PMID:[Multiple resistance of eukaryotic cells caused by P-glycoprotein]. 135 45

Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis.
Mol Cell Biol 1991 Mar
PMID:Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis. 199 13

After acclimation to 100, 75 and 50% of Sea Water (SW) external salinities, a significant reduction in MET (Mean Epithelial Thickness) and MDR (Mean Diverticular Radius) indicates a decrease in the digestive cell volume dependant on the lowering of environmental salinity. The interstitial connective tissue seems to be unable to osmoregulate and hence stand severe changes in cell size depending on external salinity. 50% SW acclimated periwinkles show a general pattern of general stress response (decreasing MET and MDR, and increasing ND -Numerical Density of lysosomes- and lysosomal size). A reduction in number and size of digestive lysosomes in winkles acclimated to 75% of Sea Water evidences the functioning of regulatory mechanism of digestive cell volume.
Cell Mol Biol 1991
PMID:Responses of winkles digestive cells and their lysosomal system to environmental salinity changes. 205 84

The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragments of MDR1 cDNA and by cloning and sequencing of genomic fragments. We have found no evidence for any other cross-hybridizing MDR genes. The sequence of two exons of the MDR2 gene was determined from genomic clones. Hybridization with single-exon probes showed that the human MDR1 gene is closely related to two genes in mouse and hamster DNA, whereas MDR2 corresponds to one rodent gene. The human MDR locus was mapped by field-inversion gel electrophoresis, and both MDR genes were found to be linked within 330 kilobases. The expression patterns of the human MDR genes were examined by enzymatic amplification of cDNA. In multidrug-resistant cell lines, increased expression of MDR1 mRNA was paralleled by a smaller increase in the levels of MDR2 mRNA. In normal human tissues, MDR2 was coexpressed with MDR1 in the liver, kidney, adrenal gland, and spleen. MDR1 expression was also detected in colon, lung, stomach, esophagus, muscle, breast, and bladder.
Mol Cell Biol 1989 Sep
PMID:Structure and expression of the human MDR (P-glycoprotein) gene family. 257 Oct 78

According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase. This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR). We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro. The conditions for RNA IL-2 alpha detection were optimized. The maximum yield of the specific product was obtained at pH 8.5-9.0. The influence of bivalent cations on the efficiency of RT reaction of coupled RT/PCR can be expressed as: Mn2+ > or = Cu2+ > Mg2+ > Cd2+ >> Co2+. The optimal ratio is 1.25-1.88 for Mn2+/dNTPs and 1.88-2.5 for Cu2+/dNTPs and Cd2+/dNTPs. The maximum yield of the RT/PCR product is found at Mg2+/dNTPs = 3.75. When Mn2+ is used instead of Mg2+ in the PCR reaction the efficiency of RT/PCR decreases. The RT/PCR method embracing thermostable Tth DNA-polymerase provides detection of 10(3) copies of RNA IL-2 alpha. An efficient method of the express-diagnostics of MDR-1 gene expression by coupled RT/PCR using Tth DNA polymerase is described.
Mol Biol (Mosk)
PMID:[Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)]. 747 58

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alcohol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low Km substrate.
J Steroid Biochem Mol Biol 1995 Jun
PMID:Enzymes and binding proteins affecting retinoic acid concentrations. 762

We examined whether the arachidonic acid competitive antagonist, ETYA (5,8,11,14-eicosatetraynoic acid), modulated drug sensitivity in a cell line that over-expresses the multiple drug resistance protein, MDR1. ETYA was nontoxic to drug-sensitive parental KB3-1 cells or drug-resistant MDR KB8-5-11 cells, with an IC50 of 190 microM for both lines. ETYA (20 microM) increased rhodamine 123 accumulation in KB8-5-11 MDR cells but not in KB3-1 sensitive cells. Arachidonic acid at 20 microM did not alter rhodamine accumulation in either cell line. Increasing the concentration of ETYA from 40 to 160 microM or incubation beyond 30 min did not increase KB8-5-11 dye retention. Forty microM or more ETYA increased KB3-11 dye retention. In a 6 day proliferation assay of KB8-5-11 cells, a nontoxic (40 microM) concentration of ETYA reduced the IC50 for doxorubicin 4-fold, the IC50 for colchicine 2-fold, but had no effect on the IC50 for vinblastine. ETYA at 40 microM did not alter the IC50 for any drug tested with KB3-1 cells. Therefore: (a) ETYA (20 or 40 microM) modulated resistance of KB8-5-11 cells to several drugs to a limited extent, without potentiating toxicity in the parental line, while arachidonic acid did not. (b) Since cationic rhodamine 123 is concentrated in mitochondria, the extent is dependent upon the transmembrane potential, and increased dye retention due to ETYA may in part be related to altered ETYA-induced cell membrane potential.
Res Commun Mol Pathol Pharmacol 1994 Nov
PMID:Reduced drug resistance in a multidrug resistant cell line by 5,8,11,14-eicosatetraynoic acid. 788 68

A kinetic approach was used to analyze the mechanism by which a substitution of valine for glycine at position 185 in the multidrug transporter alters its substrate specificity so that colchicine and etoposide transport is increased, daunorubicin transport is unchanged, and vinblastine transport is decreased. Time courses for uptake and efflux of colchicine, vinblastine, etoposide, and daunorubicin for NIH/3T3 mouse cells transfected with wild-type (MDR1-G185) and mutant (MDR1-V185) strains of the human mdr1 gene were determined at room temperature in the presence and absence of an energy supply. The initial rate of vinblastine uptake was reduced approximately 5-fold by glucose feeding of ATP-depleted wild-type (MDR1-G185) cells but was only halved in MDR1-V185 transfectants. In contrast, glucose feeding decreased the initial rate of colchicine uptake approximately 4-fold in the MDR1-V185 (mutant) transfectant but not in the MDR1-G185 (wild-type) transfectant. Efflux of colchicine was accelerated > 5-fold in both the MDR1-V185 (mutant) and MDR1-G185 (wild-type) transfectants when glucose was given to raise ATP levels. The effects on initial rates of colchicine uptake accounted semiquantitatively for the increased colchicine resistance of MDR1-V185 (mutant) transfectants. Similar effects were found for etoposide in the MDR-V185 transfectants. Quinidine in the external medium greatly inhibited drug entry rates but had little effect on efflux, whereas verapamil inhibited both uptake and efflux. A possible interpretation of these data is that the multidrug transporter extracts drugs from the external and internal halves of the membrane bilayer by different paths, which are distinguishable by mutation and inhibitors.
Mol Pharmacol 1994 Apr
PMID:Kinetic evidence suggesting that the multidrug transporter differentially handles influx and efflux of its substrates. 791 Mar 72

Changes in the steady state level of retinols, retinaldehydes and retinyl esters in the trans and 11-cis forms and trans retinoic acid were measured in whole chicken eye during development from day 6 in ovo to day 3 post-hatch. These retinoids, quantified by different HPLC systems, were detected in this time sequence: trans-retinol and trans-retinyl esters in the first week in ovo, 11-cis-retinol in the second week. The highest level of 11-cis-retinaldehyde and 11-cis-retinyl esters was reached at the end of development in ovo; however, their levels increased further after hatching. The retinoic acid level decreased at the end of the first week, rising again at the end of the second week. The enzyme activities involved in the metabolism of these retinoids-acyl-CoA: retinol acyltransferase, trans-retinol dehydrogenase, 11-cis-retinol dehydrogenase, trans-retinyl ester hydrolase and trans: 11-cis-retinol isomerase were also estimated and they were detectable already in the first week of development in ovo. At day 6 of the biosynthesis of retinoic acid by the retinaldehyde dehydrogenase activity from retina cytosol was also shown.
Mol Cell Biochem 1994 Mar 16
PMID:Retinoid dynamics in chicken eye during pre- and postnatal development. 807 8


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