Molecular analysis of the multidrug transporter

The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold. P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump. The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold. This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds. These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel. With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions. Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established. The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.     

X-inactivation pattern in carriers of X-linked retinitis pigmentosa: A valuable means of prognostic evaluation?

In a large family with X-linked retinitis pigmentosa 2 (XLRP2), we reexamined 7 obligate carrier females and 6 daughters of obligate carriers, whose linkage relationships suggested that they carried the XLRP2 gene. The phenotype varied from totally normal eyes through mild retinal changes to complete loss of vision. The X-inactivation analysis was carried out with the highly informative probe M27 on DNA from blood lymphocytes. This probe detects a locus DXS 255 that is differentially methylated on the active and inactive X chromosomes. In 5 blind heterozygotes (aged 43 to 68 years), we found that the X chromosome carrying the RP2 gene was methylated and active in nearly all their cells. The opposite X inactivation pattern was found in a carrier female (aged 45 years) who gave normal findings on eye examination. Carriers with less skewed X inactivation had a less severe clinical outcome. However, we found little or no correlation between their phenotypes and the methylation status of their X chromosomes. Our results suggest that it may be possible to develop a predictive test that could identify cases with severe outcome and perhaps cases with normal outcome.     

CYTOCHALASIN B INFLUENCES DICTYOSOMAL VESICLE PRODUCTION AND MORPHOGENESIS IN THE DESMID EUASTRUM1

Cytochalasin B (CB) applied to young developing cells of the desmid Euastrum oblongum Ralfs ex Ralfs, at concentrations that do not entirely inhibit cytoplasmic streaming, retarded cell growth and caused malformations of cell shape. While the basic symmetry of the cell was maintained, only the first indentations were formed and the cell body appeared to be swollen. Electron microscopic investigations revealed that vesicle production at the dictyosomes was disturbed by cytochalasin. In contrast to untreated control cells, where vesicles with electron-dense contents (“dark vesicles”) were formed during primary wall formation, vesicles pinched off by the dictyosomes during CB treatment exhibited an “empty” appearance. These vesicles, which correspond to the “dark vesicles” in size, were accumulated around the dictyosomes without being transported to the plasma membrane and were frequently connected to the trans-cisternae of the Golgi bodies. We speculate that CB may influence the transfer of products from the endoplasmic reticulum (ER) to the dictyosomes via transition vesicles, which results in a disturbed vesicle production at the Golgi bodies. CB also causes a shift in ER and dictyosome distribution. Moreover, a cortical actin system appears to be involved in the cell shaping of Euastrum. The arrangement of microtubules around the nucleus is not affected by the drug.     

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme fromHalobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.Presented at the Session Water in the Solar System and Its Role in Exobiology during the 26th General Assembly of the European Geophysical Society, 22–26 April 1991 in Wiesbaden, Germany.     

Binding of Insecticidal Crystal Proteins of Bacillus thuringiensis to the Midgut Brush Border of the Cabbage Looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Selection for Resistance to One of the Crystal Proteins

The susceptibility of Trichoplusia ni larvae to several Bacillus thuringiensis insecticidal crystal proteins (ICPs) was tested. Neonatal larvae proved to be susceptible to solubilized trypsin-treated CryIA(a), CryIA(b), and CryIA(c) (50% lethal concentrations [LC₅₀s], 570, 480, and 320 ng/cm², respectively) but showed little susceptibility to CryIB and CryID (LC₅₀s, 5,640 and 2,530 ng/cm², respectively). The toxicity of ICPs was correlated to binding to the epithelial brush border of the midgut, as revealed by immunocytochemical staining with monoclonal antibodies. In vitro binding experiments with iodinated ICPs and brush border membrane vesicles indicated that CryIA(b) and CryIA(c) share the same high-affinity binding site, whereas CryIA(a) binds to a different one. The affinities of CryIA(b) and CryIA(c) for the binding site were similar (K_d = 3.6 and 4.7 nM, respectively), and the mean binding-site concentration was 0.71 pmol/mg of vesicle protein. Selection of a population with increasing concentrations of CryIA(b) produced 31-fold resistance in seven generations. The realized heritability (h²) was 0.19. The increase of homozygosity (for resistance factors) as selection proceeded was reflected in the increase in the slopes of the dose-mortality curves. Resistance was specific for CryIA(b) and did not extend to CryIA(a) or even to CryIA(c). This result was not predicted by the binding-site model, in which CryIA(b) and CryIA(c) bind to the same high-affinity binding site. This result may suggest a more complicated relationship between in vitro binding of ICPs to specific sites in the epithelial membrane of the midgut and the in vivo toxic effect.     

Response of chrysanthemum to uniconazole and daminozide applied as dip to cuttings or as foliar spray

Uniconazole and daminozide were used as dip on unrooted cuttings or as foliar spray on pinched Dendranthema grandiflora Tzvelev. Dalvina to control height. Stem elongation was determined on cuttings dipped in solutions of 0, 1.25, 2.5, 5, or 10 mg/L uniconazole or cuttings were dipped and later treated with foliar sprays in concentrations of 1.25/5, 1.25/10, 2.5/10, and 5/5 mg/L uniconazole, respectively. Other plants were sprayed once or twice with uniconazole at 10 mg/L. Daminozide treatments included a pre-plant dip/foliar spray application of 1000/2000 mg/L, respectively, or two foliar sprays of 2,000 mg/L. Uniconazole dip alone retarded stem elongation linearly up to 8 weeks after propagation, 5 weeks after pinching, but was not discernible from the control treatment 8 weeks after pinching. Uniconazole at 2.5/10 and 5/5 mg/L as a dip/spray combination resulted in plants 33% shorter than the control at the end of the production. Doubling uniconazole dip or spray treatments from 5 to 10 mg/L provided no additional reduction of stem elongation. The single uniconazole spray and both daminozide treatments had no effect on final height, although daminozide treatments reduced stem dry weight compared to the control. Stem dry weight was reduced by uniconazole dip/spray combinations compared to dip treatments alone. Similarly, inflorescence and root dry weights were also reduced by the highest uniconazole concentrations. Higher concentrations of uniconazole reduced transpiration on a per leaf area basis up to 47% compared to the control at the end of production. In contrast to previous work, leaf area and leaf thickness increased with some uniconazole treatments, while time to anthesis was not affected by any of the treatments.