P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

The dynamical attainability of ESS in evolutionary games

In this paper, the attainability of ESS of the evolutionary game among n players under the frequency-independent selection is studied by means of a mathematical model describing the dynamical development and a concept of stability (strongly determined stability). It is assumed that natural selection and small mutations cause the phenotype to change gradually in the direction of fitness increasing. It is shown that (1) the ESS solution is not always evolutionarily attainable in the evolutionary dynamics, (2) in the game where the interaction between two species is completely competitive, the Nash solution is always attainable, and (3) one of two species may attain the state of minimum fitness as a result of evolution. The attainability of ESS is also examined in two game models on the sex ratio of wasps and aphids in light of our criterion of the attainability of ESS.     

Translation and functional expression of cell-cell channel mRNA inXenopus oocytes

Summary mRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.     

Large scale selection of aluminum-resistant mutants from plant cell culture: expression and inheritance in seedlings

Summary A large number of aluminum-resistant variants, selected from non-mutagenized homozygous diploid cell cultures of Nicotiana plumbaginifolia Viv., are characterized. Of 115 variants cloned and reselected from single cells, 67 retained Al resistance in callus cultures after 6–9 months of growth in the absence of Al. There was no association between Al resistance and callus growth in the absence of Al, suggesting that the Al-resistant phenotype is not detrimental in the absence of Al challenge and that Al resistance is not the result of increased vigor. Plants regenerated from initially resistant callus lines that subsequently lost their resistance failed, with one exception, to transmit resistance to their seedling progeny. Fertile plants were regenerated from 40 of the 67 variants that retained stable Al resistance in callus culture. All 40 transmitted Al resistance to their seedling progeny (selfed and backcrossed) in segregation ratios expected for a single dominant mutation. The selfed progeny of many variants also segregated for recessive lethal mutations which were attributed to independent mutations that occurred during cell culture.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

A model of a myelinated nerve axon: threshold behaviour and propagation

A model of a myelinated nerve axon is developed on the basis of FitzHugh-Nagumo dynamics under the assumption that the nodes of Ranvier are of small but finite width. It is shown that a periodic excited state may not exist if the width of the nodes is too small and the leakage across the myelin sheath is too great. The propagation of a super threshold pulse is prevented in the absence of nodes. Global stability of the resting equilibrium state is investigated as well as the propagation of wave front, type solutions.     

Permanent coexistence in general models of three interacting species

We address the question of the long term coexistence of three interacting species whose dynamics are governed by the ordinary differential equations x _i = X _i f _i (i = 1, 2, 3). In order for any theory in this area to be useful in practice, it must utilize as little information as possible concerning the forms of the f _i , in view of the great difficulty of determining these experimentally. Here we obtain, under rather general conditions on the equations, a criterion for judging whether the species will coexist in a biologically realistic manner. This criterion depends only on the behaviour near the one or two species equilibria of the two dimensional subsystems, the behaviour there being relatively easy to examine experimentally. We show that with the exception of one class of cases, which is a generalization of a classical example of May and Leonard [21], invasibility at each such equilibrium suitably interpreted is both necessary and sufficient for a strong form of coexistence to hold. In the exceptional case, a single additional condition at the equilibria is enough to ensure coexistence.     

Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley

Diurnal variations of in vitro and in vivo (intact tissue assay) nitrate reductase (EC 1.6.6.1) activity and stability were examined in leaves of wheat ( Triticum aestivum L. cv. Runar), oat ( Avcna saliva L. cv. Mustang) and barley ( Hordeum vulgure L. cv. Agneta and cv. Gunillu). Nitrate reductase activity was generally higher for wheat than for oat and barley. However, the diurnal variations of nitrate reductase activity and stability were principally the same for all species, e.g. the high activity during the photoperiod was associated with low stability. All species showed a rapid (30-60 min) increase in the in vitro and in vivo activity when the light was switched on. When light was switched off the in vitro activity decreased rapidly whereas decrease in in vivo activity was slower. These experiments support the hypothesis that an activation/ deactivation mechanism is involved in the regulation of diurnal variations in nitrate reductase activity. Red light enhanced nitrate reductase activity in etiolated wheat and barley leaves. In green leaves, however, the daily increase in nitrate reductase activity was not induced by a brief red light treatment. Indications of different regulation mechanisms for the diurnal variations of nitrate reductase activity among the cereals were not found.     

Aspects of the mineral nutrition of some native British plants — inter-site variation

Fifteen common native British plants were each sampled at a range of sites in Great Britain and green tissues analysed for several inorganic nutrients. Sampling criteria are discussed. The inter-site variation of each element within a species is assessed as a frequency distribution of raw data. Sample values of parameters including arithmetic mean, variance (coefficient of variation), skewness and kurtosis are presented. Their stability is assessed from nitrogen in sub-samples of Pteridium. This suggested sample sizes were adequate but some distributions had sufficient kurtosis to affect the variance. These parameters showed distinctions between macro- and micro-elements and between species. Some mean values sharply distinguished between species and may help to assess current theories of strategy and adaptation but a wider range of species is needed to clarify trends. Coefficients of variation are discussed and were relatively low for a nation-wide survey after allowing for sampling constraints. Coefficients of skewness and kurtosis showed two-thirds of the sample distributions were non-normal. Ecological aspects of the distributions are discussed and are relevant to studies along environmental gradients. Published hypotheses linking positive skewness to stress in the field are considered and two other postulates discussed. Distribution bounds such as those confining 95% of the values are discussed in relation to possible critical levels of nutrients.Nomenclature follows Clapham et al. (1981), Excursion flora of the British Isles. 3rd ed. University Press, Cambridge, except Chamaenerion.