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S B Levy

Publications and source records attributed to S B Levy.

At least 73 records · Page 4Linked to original sources
PubMed1997

Differential changes in genome structure and expression of the mdr gene family in multidrug-resistant murine erythroleukemia cell lines.

The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Increased efflux of vincristine, but not of daunorubicin, associated with the murine multidrug resistance protein (MRP).

The multidrug resistance protein (MRP) is a membrane protein that mediates altered transport of cytotoxic drugs. Although MRP overexpression has been described in doxorubicin-selected human tumor cell lines, the murine PC-V10 and PC-V40 cell lines are members of the only reported series of vincristine-selected cell lines that overexpress mrp. Western blotting, using an antiserum developed against human MRP, demonstrated high-level expression of murine MRP primarily in the plasma membranes in each of the vincristine-selected cell lines. Only PC-V160, selected for high level resistance, demonstrated concomitant overexpression of the P-glycoprotein. As compared with parental cells, each of the drug-selected cell lines demonstrated an energy-dependent, decreased net accumulation of vincristine without any changes in the initial rates of vincristine influx. However, there was an enhanced rate of vincristine loss, 2.3-fold from the PC-V40 cell line and 3.9-fold from the PC-V160 cell line. Selective plasma membrane permeabilization with digitonin equalized vincristine accumulation among the parental, the PC-V40, and the PC-V160 cell lines. No intracellular pH differences were detected among the cell lines. Despite high-level MRP expression, daunorubicin accumulation and the rate of daunorubicin loss in the PC-V40 cells were the same as that observed in parental PC4 cells. Fluorescence microscopy demonstrated no difference in the pattern of subcellular daunorubicin accumulation between parental and PC-V40 cells. These studies demonstrate that murine MRP, overexpressed and found predominantly in the plasma membrane of vincristine-selected PC-V40 cells, is associated with an energy-dependent increased efflux of vincristine, but not with efflux or altered distribution of daunorubicin.

2,4-Dinitrophenol↗
PubMed1996

Expression of c-fos precedes MDR3 in vincristine and adriamycin selected multidrug resistant murine erythroleukemia cells.

Expression of murine P-glycoprotein (P-gp), encoded by mdrl or mdri3, confers a multidrug resistance phenotype. Higher expression of c-fos and c-jun has also been demonstrated in multidrug resistant human and murine cells. We detected increased expression of c-fos early in the derivation of two series of murine erythroleukemia sublines selected for resistance to vincristine or adriamycin which eventually overexpress mdr3. We speculate that early expression of c-fos prepares cells for overexpression of other genes, such as mdr3, that contribute to the multidrug resistance phenotype.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1996

Untranslated sequence upstream of MarA in the multiple antibiotic resistance locus of Escherichia coli is related to the effector-binding domain of the XylS transcriptional activator.

MarA, the 129-amino-acid (aa) protein which plays a crucial role in the multiple antibiotic resistance (Mar) phenotype in Escherichia coli, shows homology to members of the XylS/AraC family of transcriptional regulators. Although these proteins vary in size from around 100 to 350 aa they all contain a DNA-binding domain with a helix-turn-helix motif. The larger ones, e.g., XylS, AraC, and Rob, contain an additional domain either at their amino- or at their carboxyterminus. This domain is important for effector-binding or dimerization or of unknown function. MarA consists only of the DNA-binding component. Nevertheless, a sequence with a coding potential of 141 aa upstream of its ATG start-codon showed 20.5-26.9% aa identity with the corresponding section within the effector-binding domain of XylS from the TOL plasmid of Pseudomonas putida when translated in the same reading frame as MarA. However, the reading frame was interrupted by 11 translational stops. In another frame, this upstream sequence actually codes for a real protein, MarR, that is completely unrelated to XylS. Implications for the putative evolution of regulatory proteins through translocation of domains followed by adaptation are discussed.

Amino Acid Sequence↗
PubMed1996

Purification of the Tn10-specified tetracycline efflux antiporter TetA in a native state as a polyhistidine fusion protein.

The bacterial tetracycline-resistance determinant from Tn10 encodes a 43 kDa membrane protein, TetA, responsible for active efflux of tetracyclines. The tetA gene was cloned behind a T7 promoter/lac operator in a plasmid that provided fusion of TetA to a polyhistidine-carboxy terminal tail. A second plasmid provided a regulated T7 RNA polymerase. The specific activity of the TetA fusion protein was between 10-40% that of the wild-type protein as assayed by tetracycline resistance in cells and by transport in membrane vesicles. The fusion protein, overproduced approximately 3-13-fold, was purified by nickel chelation chromatography. Calculations from circular dichroism spectra of the purified protein solubilized in dodecylmaltoside gave an alpha-helix content of 54-64%, close to the 68% predicted from the amino acid sequence by hydropathy analysis (12 membrane-spanning helices) for the native protein in the membrane bilayer. Fluorescence studies showed binding activity of the purified protein to its substrate, the tetracycline analogue 13-(cyclopentylthio)-5-hydroxy-6-alpha-deoxytetracycline. These findings suggested that the purified protein was in a native state.

Antiporters↗
PubMed1996

An alpha hydroxy acid derivative suitable for sensitive skin.

BACKGROUND: Individuals with sensitive skin may not tolerate some products formulated with alpha hydroxy acids (AHAs) due to unacceptable levels of stinging and irritation. OBJECTIVE: To develop a neutral molecule that is well tolerated by individuals with sensitive skin without stinging or irritation, yet retains the beneficial effects of AHAs. METHODS: A neutral amide derivative, methoxypropylgluconamide (MPG), was invented. Formulations containing MPG (15%) were tested by cummulative irritation, repeat insult patch testing, and lactic acid stinging assay. Bilateral comparison clinical and histological studies of xerosis and photoaging assessments were performed. RESULTS: MPG was nonirritating and nonsensitizing. Clinically significant improvements in xerosis and photoaging including uneven pigmentation were seen. Improvement in the morphology of the stratum corneum epidermis and in the amount of glycosaminoglycans in the epidermis and dermis were seen histologically. CONCLUSIONS: A molecule was invented (MPG) that could be formulated in a neutral vehicle to avoid skin irritation and produced improvements in xerosis and photoaging.

Biopsy↗
PubMed1996

Multiple antibiotic resistance (mar) locus protects Escherichia coli from rapid cell killing by fluoroquinolones.

The multiple antibiotic resistance (mar) locus in Escherichia coli consists of two divergently expressed operons (marC and marRAB), both of which contribute to the Mar phenotype. Overexpression of the marRAB operon protected E. coli against rapid cell killing by fluoroquinolones. Inactivation of the operon in mar mutants restored a wild-type bactericidal susceptibility. Both operons of the locus were required for protection from the quinolone-mediated bactericidal activity in mar locus deletion mutants. The effect was lost at high concentrations of fluoroquinolones, unlike the case for the previously described genes hipA and hipQ. The inducible mar locus appears to specify a novel antibactericidal mechanism which may play a role in the emergence of fluoroquinolone-resistant clinical E. coli isolates.

Anti-Infective Agents↗
PubMed1996

Identification for mar mutants among quinolone-resistant clinical isolates of Escherichia coli.

Quinolone-resistant clinical Escherichia coli isolates were examined for mutations in the marRAB operon of the multiple antibiotic resistance (mar) locus. Among 23 strains evaluated, 8 were chosen for further study: 3 that showed relatively high levels of uninduced, i.e., constitutive, expression of the operon and 5 with variable responses to induction by salicylate or tetracyclines. The marR genes, specifying the repressor of the operon, cloned from the three strains constitutively expressing the operon did not reduce the level of expression of beta-galactosidase from a marO::lacZ transcriptional fusion and were therefore mutant; however, marR genes cloned from the five other clinical strains repressed LacZ expression and were wild type. All three mutant marR genes contained more than one mutation: a deletion and a point mutation. Inactivation of the mar locus in the three known marR mutant strains with a kanamycin resistance cassette introduced by homologous recombination reduced resistance to quinolones and multiple antibiotics. These findings indicate that mar operon mutations exist in quinolone-resistant clinical E. coli isolates and contribute to quinolone and multidrug resistance.

4-Quinolones↗
PubMed1995

The NH2-terminal half of the Tn10-specified tetracycline efflux protein TetA contains a dimerization domain.

The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented.

Antiporters↗
PubMed1995

Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli.

The marRAB operon is one of two operons in the mar locus of Escherichia coli that are divergently transcribed from a central regulatory region, marO. The marRAB operon, transcribed from marOII, controls intrinsic resistance or susceptibility to multiple antibiotics and is inducible by structurally unrelated compounds such as tetracycline and chloramphenicol (S. P. Cohen, H. Hachler, and S. B. Levy, J. Bacteriol. 175:1484-1492, 1993). To clarify the role of the operon in response to environmental signals, its transcription was studied under different conditions, using a marOII-lacZ transcriptional fusion introduced into the chromosome of wild-type or mar-deleted cells. In wild-type cells, uncoupling agents (such as carbonyl cyanide m-chlorophenylhydrazone) and different redox-cycling compounds (e.g., menadione and plumbagin) induced expression from the marOII-lacZ fusion two- to sevenfold. In the mar-deleted strain, LacZ expression from the fusion was 10-fold higher than in wild-type cells. This activity was temperature sensitive (3-fold lower at 42 than at 30 degrees C) and decreased 20-fold with the introduction of the gene for MarR. Structurally different compounds which induce the mar operon in wild-type cells reversed the MarR repression of marOII-lacZ expression. To determine the size of MarR, it was fused to MalE as a MarR fusion protein of 144 amino acids [MarR(144)] or of 125 amino acids (deleted of 19 amino acids at the N terminus) [MarR(125)]. Only the MarR(144) fusion showed repressor ability. The purified MarR(144) fusion, but not the MarR(125) fusion, bound specifically to marO in vitro, as revealed by gel retardation, with an apparent dissociation constant of 5 x 10(-9) M. MarR, therefore, controls expression of the marRAB operon presumably by binding to marO. MarR repression in cells can be reversed by different compounds, facilitating the response of bacteria to multiple environmental stress conditions.

Bacterial Proteins↗
PubMed1995

Identification of new genes regulated by the marRAB operon in Escherichia coli.

Random TnphoA and TnlacZ translational fusions were introduced into an Escherichia coli strain with a deletion of the multiple antibiotic resistance (mar) locus, complemented in trans by a temperature-sensitive plasmid bearing the mar locus with a constitutively expressed mar operon. Five gene fusions (two with lacZ and three with phoA) regulated by the mar operon were identified by increased or decreased marker enzyme activity following loss of the complementary plasmid at the restrictive temperature. Expression of LacZ from both lacZ fusions increased in the presence of the mar operon; expression from the three phoA fusions was represented by the mar operon. The lacZ fusions were mapped at 31.5 and 14 min on the Escherichia coli chromosome. One of the phoA fusions was located at 51.6 min while the two others mapped at 77 min. Cloning and sequencing of a portion of the fused genes showed all of them to be different. The phoA fusions at 77 min were located in a recently identified gene, slp, a lipoprotein of unknown function (D.M. Alexander and A. C. St. John, Mol. Microb. 11:1059-1071, 1994). The others showed no homology with any known genes of E. coli. The insertions caused small but reproducible changes in the antibiotic susceptibility profile. This approach has enabled the identification of new genes in E. coli which are regulated by the marRAB operon and involved in the Mar phenotype.

Chromosome Mapping↗
PubMed1994

Overexpression of the multidrug resistance-associated protein (MRP) gene in vincristine but not doxorubicin-selected multidrug-resistant murine erythroleukemia cells.

Multidrug-resistant sublines of the murine erythroleukemia cell line PC4 were sequentially selected in increasing vincristine concentrations (5-160 ng/ml). The low- and intermediate-level resistant cell lines, selected in < or = 40 ng/ml of vincristine, demonstrated resistance to Vinca alkaloids and to an epipodophyllotoxin but little or none to an anthracycline. The expression of murine mdr genes, as analyzed by Northern blotting, revealed a baseline expression of murine mdr2 in parental cells that was unchanged in the drug-resistant cell lines. Overexpression of mdr3 was observed only in the highest-level resistant cell line, PC-V160, whereas mdr1 mRNA was not detected in any of the cell lines. The polymerase chain reaction, using mdr3-specific primers, excluded the possibility that low levels of P-glycoprotein expression contributed to the resistance phenotype in the low and intermediate-level resistant cell lines. Northern blot analysis using a human complementary DNA probe for the multidrug resistance-associated protein (MRP) demonstrated overexpression of murine mrp in each of the vincristine-selected sublines. Genomic amplification of the mrp gene was coincident with mrp overexpression. The expression of mrp was also examined in two series of previously characterized doxorubicin-selected cell lines derived from parental PC4 and C7D murine erythroleukemia cells. In contrast to the vincristine-selected cell lines, overexpression of mrp was not detected. These studies demonstrate that, in murine erythroleukemia cells selected for vincristine resistance, overexpression of murine mrp occurred prior to that for murine mdr. In contrast to human MRP, selection for vincristine, but not doxorubicin resistance, resulted in the overexpression of murine mrp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Molecular requirements for the inhibition of the tetracycline antiport protein and the effect of potent inhibitors on the growth of tetracycline-resistant bacteria.

Forty-seven compounds and tetracycline (Tc) structural analogues were tested for their ability to interfere with [3H]Tc uptake in everted inner membrane vesicles derived from Tc-resistant Escherichia coli D1-209, bearing the class B tetracycline resistance efflux protein (Tet protein). For effective inhibition of Tc uptake, the molecule had to have an intact ABCD tetracyclic carbon skeleton and a conjugated phenolic beta-diketone substructure at positions 10-12a with the subsequent development of keto-enol tautomerization. Molecular variations at carbon positions 2, 4, 5, 6, 7, 8, and 9 did not decrease, and some increased, the inhibitory activity as compared to that of Tc. Among these compounds, the highest inhibition of uptake occurred with certain position 6 and 13 derivatives of 5-hydroxytetracycline. In a group of 13-(propylthio) derivatives of 5-OH-Tc [13-propyl, 13-(3-chloropropyl), and 13-(2-carboxyethyl)] there was a correlation between uptake inhibitory activity and antibacterial activity. The 13-(3-chloropropyl) derivative, with the best efflux inhibitory activity, exhibited synergistic activity when tested in combination with doxycycline against Tc-resistant E. coli bearing the class A or B determinant, against Staphylococcus aureus bearing class K, and against Enterococcus faecalis bearing the class L determinant. The 13-propyl analogue also showed high transport blocking activity and showed synergistic antibacterial activity against E. coli bearing the class A determinant and additive activity against the other Tc-resistant bacteria. The synergistic antibacterial activity of these compounds was not shown by the 13-[(2-carboxyethyl)thio] homologue, whose efflux blocking activity was 70-fold less. These findings suggest that multiple sites on the Tc molecule are available for synthetic modification toward the development of an effective Tc blocking agent. Such compounds, used alone or in combination with a standard tetracycline, show improved antibacterial activity.

Antiporters↗
PubMed1994

Distribution of globin genes and histone variants in micrococcal nuclease-generated subfractions of chromatin from Friend erythroleukemia cells at different malignant states.

The distribution of the alpha and beta-globin genes and histone variants was examined in micrococcal nuclease-generated chromatin fractions of three Friend murine erythroleukemia cell types differing in malignant potential and inducibility to erythroid differentiation. A preferential concentration of globin gene sequences, as compared to satellite DNA, was noted in a physiological salt-soluble, histone H1-depleted, mononucleosomal chromatin fraction (Sup 120) in all Friend cell types, even those in which the globin gene was not capable of transcriptional activation by chemical induction. The level of globin gene enrichment in the Sup 120 fraction was highest in the most malignant and inducible cell type. The chemical induction of erythroid differentiation in this cell line did not change the distribution of globin genes in the chromatin fractions. The Sup 120 chromatin fraction prepared from mouse brain nuclei was not enriched in globin genes. Besides the previously reported low H2A. 1/H2A.2 ratio [Blankstein and Levy: Nature 260:638-640, 1976], chromatin from the most tumorigenic cell type showed the lowest H2B.2 to H2B.1 ratio, highest levels of histone H4 acetylation, and the most pronounced change in relative amounts of two major electrophoretic bands of histone H1 variants as compared to the less malignant cell types. The histone variant content of the micrococcal nuclease-generated chromatin fractions from the three Friend cell types reflected the core histone variant differences for the respective intact nuclei. However, the electrophoretic separation of mononucleosomes by size revealed several classes with different H2A variant ratios. The results demonstrate the existence of structural differences in globin gene and histone variants in erythroleukemia cell chromatin associated with distinguishable phenotypes during malignant cell progression.

Acetylation↗