Biomedical subjects

E P Bruggemann

Publications and source records attributed to E P Bruggemann.

12 recordsLinked to original sources

PubMed1998

Characterization of an unstable allele of the Arabidopsis HY4 locus.

The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144(Delta), contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously converts to a nonfunctional hy4 allele. In addition, HY4-B144* is lethal in homozygotes and suppresses local recombination. We discuss genetic and epigenetic mechanisms that may account for the unusual behavior of the HY4-B144* allele.

Alleles↗

PubMed1994

Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin.

An iodinated derivative of forskolin, 6-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]forskolin ([125I]6-AIPP-Fsk), photolabels the multidrug efflux pump P-glycoprotein in membranes prepared from the multidrug-resistant cell lines KB-V1 and KB-C1. The labeling site for [125I]6-AIPP-Fsk was localized by immunoprecipitation of tryptic fragments of P-glycoprotein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fragment was immunoprecipitated by antiserum raised against residues 348-419 of P-glycoprotein, PEPG9, but not by antisera raised against flanking regions PEPG7 and PEPG11. A peptide that corresponds to residues 343-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fragment by antiserum against PEPG9 but had no effect on the immunoprecipitation of photolabeled fragments by antiserum against PEPG7. A second peptide, corresponding to residues 360-376, had no effect on the immunoprecipitation by antiserum against PEPG9. [125I]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular mass tryptic fragments were immunoprecipitated by three carboxyl-terminal antisera. Therefore, [125I]6-AIPP-Fsk labels both halves of P-glycoprotein, and labeling in the amino-terminal half can be localized to residues 291-359, which span proposed transmembrane regions 5 and 6. KB-V1 membranes photolabeled with [125I]6-AIPP-Fsk and [125I]iodoarylazidoprazosin were digested with either Staphylococcus aureus V8 protease or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Environmental safety issues for genetically modified animals.

Organisms modified by the techniques of modern biotechnology may differ significantly from normal organisms or organisms modified by other methods. Before transgenic organisms are introduced into the environment, the potential environmental effects should be assessed. In general, modification of ecologically important traits in undomesticated species presents the greatest environmental risk. Transgenic livestock probably pose low risk to the environment. Transgenic fish and live virus-based vaccines pose greater risks and present challenging questions for environmental risk assessment.

Animals↗

PubMed1992

Characterization of the azidopine and vinblastine binding site of P-glycoprotein.

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Pseudomonas exotoxin fusion proteins are potent immunogens for raising antibodies against P-glycoprotein.

Antibodies to specific regions of human P-glycoprotein have been difficult to obtain. We developed a method to express in E. coli fusions between Pseudomonas exotoxin and specific regions of human P-glycoprotein. We used the polymerase chain reaction to amplify the desired regions of MDR1 cDNA and to introduce appropriate restriction sites. These fragments were cloned into the 3' end of the Pseudomonas exotoxin gene. With this system we produced large amounts of fusion proteins for immunizations, and we obtained positive rabbit antiserum against P-glycoprotein with most of these antigens. We now have a comprehensive panel of polyclonal antibodies against P-glycoprotein. This system should be generally useful to raise antibodies against other eukaryotic proteins that are difficult to prepare in large quantities.

ADP Ribose Transferases↗

PubMed1990

Cellular and biochemical characterization of thioxanthenes for reversal of multidrug resistance in human and murine cell lines.

We have previously shown that phenothiazines sensitize multidrug resistant (MDR) cells to chemotherapeutic drugs in a manner related to specific structural features, and have identified structurally related thioxanthenes with increased anti-MDR activity. We have now studied the structure-activity relationships of 16 thioxanthenes in the human breast cancer line MCF-7 AdrR. trans-Thioxanthene stereoisomers were 2- to 7-fold more potent than cis-thioxanthenes for antagonizing MDR. The most potent thioxanthenes possessed a halogenated tricyclic ring connected by a 3-carbon alkyl bridge to a piperazinyl or piperadinyl side group. The chemosensitizing effects of the lead compound, trans-flupenthixol, its stereoisomer cis-flupenthixol, its phenothiazine homologue fluphenazine, and the calcium channel blocker verapamil, were further studied in a series of sensitive and MDR cell lines. trans-Flupenthixol caused a greater reversal of cellular resistance to doxorubicin, vinblastine, vincristine, and colchicine in MCF-7 AdrR, KB-V1, and P388/DOX MDR cells than the other chemosensitizers. In particular, trans-flupenthixol was 2- to 3-fold more potent for reversing MDR than equimolar concentrations of verapamil. Furthermore, trans-flupenthixol fully reversed resistance to doxorubicin, vincristine, and colchicine in MDR MCF-7 and NIH 3T3 cells transfected with the mdr1 gene. None of these agents altered MDR in a non-P-glycoprotein expressing MCF-7 cell line selected with mitoxantrone, nor in any of the parental cell lines. The stereoselective antagonism of the flupenthixol isomers on several putative cellular targets was studied to explore the mechanism of their chemosensitizing activity. cis- and trans-flupenthixol were equally active inhibitors of protein kinase C and calmodulin. Both cis- and trans-flupenthixol were also potent inhibitors of [3H]azidopine binding to P-glycoprotein. The apparent lack of clinical toxicity of trans-flupenthixol makes it an attractive drug for possible use in the modulation of tumor resistance in vivo if appropriate tissue concentrations can be achieved.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells.

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Use of recombinant P-glycoprotein fragments to produce antibodies to the multidrug transporter.

Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine.

Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1986

Determination of the molecularity of the colicin E1 channel by stopped-flow ion flux kinetics.

A fluorescence technique that measures fast ion fluxes across liposome membranes was used to determine the molecularity of the colicin E1 channel. The rate of flux of Tl+ (used as a K+ analogue) into large unilamellar vesicles was measured by its ability to quench the fluorescence of a membrane-impermeable fluorophore entrapped in the vesicles. The dependence of Tl+ flux rate on the concentration of ionophore in the vesicle suspension reveals the molecularity of the ionophore. The method is demonstrated with two ionophores, valinomycin and gramicidin, whose molecularities are known to be one and two, respectively. The molecularity of the colicin E1 channel was determined to be one. This method can be used to study the properties of any ionophore that mediates K+ flux.

Colicins↗

PubMed1983

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol.

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.

Animals↗

PubMed1982

The physical state of quick-frozen membranes and lipids.

Lipid bilayers and biomembranes produce nearly identical calorimeter scans regardless of whether they are slowly cooled under near-equilibrium conditions or rapidly frozen at rates used in freeze-fracture electron microscopy. Except for the melting of ice at 273 K, for both cooling regimens no significant thermal events occur from 100 K to the usual gel to liquid crystal transition. The gel to liquid crystal transition itself is somewhat altered by rapid cooling when bilayers contain mixed lipid species. Combined with X-ray diffraction studies, the results indicate that quickly frozen bilayers are crystalline, but that the crystalline domains are quite small or otherwise disordered. In contrast to the behavior of lipids in bilayers, hexagonal-phase calcium cardiolipin easily forms a glass upon cooling.

Animals↗