Multidrug resistance: Versatile drug sensors of bacterial cells.

One way that bacteria resist antimicrobial agents involves 'drug sensors', proteins that bind a range of structurally unrelated antimicrobial drugs and induce expression of multidrug resistance pumps. The basis for drug recognition is now becoming clear from the recently determined crystal structure of a drug sensor.     

Carbonyl group of aliphatic side chain of pentoxifylline does not play role for P-glycoprotein antagonizing effect of pentoxifylline

Previously we have found that pentoxifylline (PTX), but not caffeine, theophylline, or 1-methyl-3-isobutylxanthine, affects sensitivity of L1210/VCR cells, a line with multidrug resistance mediated by P-glycoprotein (P-gp) to vincristine (VCR) and doxorubicine. Comparison of chemical structure of PTX with other above xanthines has revealed only one marked difference. PTX contains extended aliphatic chain containing reactive electrophilic carbonyl group in the position N1. The investigation of possibility that this group is crucial for PTX-induced MDR reversal represents the aim of the current paper. To prove this hypothesis, we used the new synthesized PTX derivative in which the carbonyl group is modified by a substance containing amino-group and the product of reaction is the respective Schiff base (SB). Successful reaction was observed when PTX reacted with 3,5-diaminobenzenesulfonyl acid (DABS). The product of reaction of DABS with carbonyl group of aliphatic part of PTX was proved using NMR and IR spectroscopy. We found that the resulting PTX derivative PTX-SB revealed higher cytotoxicity on both sensitive L1210 and multidrug resistant L1210/VCR cells than PTX. Moreover, PTX-SB exerts more pronounced MDR reversal effect on L1210/VCR cells than PTX. These results indicate that electrophilic carbonyl group on aliphatic chain located in position N1 of PTX is not essential for MDR reversal effects of PTX.     

Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells

The multidrug-resistant cancer cell lines NCI/AdR(RES) and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.     

Multidrug resistance P-glycoprotein inhibits the antiapoptotic action of mitochondria-targeted antioxidant SkQR1

Mitochondria-targeted antioxidants of the SkQR1 family, being accumulated in energized mitochondria, protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing the cell-damaging effect induced by hydrogen peroxide. Using various human transformed cell lines and SkQR1 (a fluorescent member of the SkQ family), we show that SkQRI is ejected from chemotherapy-resistant cells by P-glycoprotein - one of the main transport proteins determining multidrug resistance typical for many neoplastic cells. It is also shown that SkQR1 ejection is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQRI from apoptotic action of hydrogen peroxide. Protection of the resistant subline occurs only after inhibition of P-glycoprotein.     

Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication.

The radioactively labelled product of RNA-dependent RNA polymerase+ from ribosomes of immature chicken erythrocytes was tested for the presence of newly replicated globin mRNA using unlabelled globin complementary DNA. No radioactively labelled globin mRNA sequences were found in the product, providing direct confirmation that this RNA-dependent RNA polymerase is not involved in globin mRNA amplification.     

A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors.

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.     

Glutathione S-transferase does not play a role in multidrug resistance of L1210/VCR cell line

Multidrug resistance of cancer cells is often accompanied by the (over)expression of integral plasma membrane P-glycoprotein, an ATP-dependent transport pump for diverse unrelated compounds. The glutathione detoxification system represents another mechanism that may be involved in multidrug resistance. In the multidrug-resistant L1210/VCR cell line obtained by long-term adaptation of parental L1210 cells to vincristine, an increased expression of P-glycoprotein has previously been established. In this paper, we investigated if the glutathione detoxification system is also involved in the multidrug resistance of these cells. L1210/VCR cells with resistance induced by adaptation to vincristine were also found to be cross-resistant to vinblastine, actinomycin D, mitomycin C, doxorubicin and cyclophosphamide. The resistance of the above cells to vincristine and doxorubicin was accompanied by a depression of drug accumulation (which has not yet been established for other drug). L1210/VCR cells are able to survive better than sensitive cells under conditions when glutathione was depleted by L-buthionine sulfoximine. Nevertheless, L-buthionine sulfoximine did not influence the resistance of L1210/VCR cells to vincristine. Moreover, the presence of sublethal concentrations of cytostatics neither changed the IC50 value of resistant cells to L-buthionine sulfoximine nor the cytoplasmic activity of glutathione S-transferase, the crucial enzyme of glutathione detoxification system. All the above findings indicate that the glutathione detoxification system is not involved in the mechanisms that ensure the multidrug resistance phenotype of L1210/VCR cells.     

Metabolomic approaches reveal that cell wall modifications play a major role in ethylene-mediated resistance against Botrytis cinerea

In Arabidopsis, resistance to the necrotrophic fungus Botrytis cinerea is conferred by ethylene via poorly understood mechanisms. Metabolomic approaches compared the responses of the wild‐type, the ethylene‐insensitive mutant etr1‐1, which showed increased susceptibility, and the constitutively active ethylene mutants ctr1‐1 and eto2 both exhibited decreased susceptibility to B. cinerea. Fourier transform–infrared (FT‐IR) spectroscopy demonstrated reproducible biochemical differences between treatments and genotypes. To identify discriminatory mass‐to‐charge ratios (m/z) associated with resistance, discriminant function analysis was employed on spectra derived from direct injection electrospray ionisation‐mass spectrometry on the derived principal components of these data. Ethylene‐modulated m/z were mapped onto Arabidopsis biochemical pathways and many were associated with hydroxycinnamate and monolignol biosynthesis, both linked to cell wall modification. A high‐resolution linear triple quadrupole‐Orbitrap hybrid system confirmed the identity of key metabolites in these pathways. The contribution of these pathways to defence against B. cinerea was validated through the use of multiple Arabidopsis mutants. The FT‐IR microspectroscopy indicated that spatial accumulation of hydroxycinnamates and monolignols at the cell wall to confine disease was linked ot ethylene. These data demonstrate the power of metabolomic approaches in elucidating novel biological phenomena, especially when coupled to validation steps exploiting relevant mutant genotypes.     

A major role for a set of non-active site mutations in the development of HIV-1 protease drug resistance

A major problem in the chemotherapy of HIV-1 infection is the appearance of drug resistance. In the case of HIV-1 protease inhibitors, resistance originates from mutations in the protease molecule that lower the affinity of inhibitors while still maintaining a viable enzymatic profile. Drug resistance mutations can be classified as active site or non-active site mutations depending on their location within the protease molecule. Active site mutations directly affect drug/target interactions, and their action can be readily understood in structural terms. Non-active site mutations influence binding from distal locations, and their mechanism of action is not immediately apparent. In this paper, we have characterized a mutant form of the HIV-1 protease, ANAM-11, identified in clinical isolates from HIV-1 infected patients treated with protease inhibitors. This mutant protease contains 11 mutations, 10 of which are located outside the active site (L10I/M36I/S37D/M46I/R57K/L63P/A71V/G73S/L90M/I93L) and 1 within the active site (I84V). ANAM-11 lowers the binding affinity of indinavir, nelfinavir, saquinavir, and ritonavir by factors of 4000, 3300, 5800, and 80000, respectively. Surprisingly, most of the loss in inhibitor affinity is due to the non-active site mutations as demonstrated by additional experiments performed with a protease containing only the 10 non-active site mutations (NAM-10) and another containing only the active site mutation (A-1). Kinetic analysis with two different substrates yielded comparable catalytic efficiencies for A-1, ANAM-11, NAM-10, and the wild-type protease. These studies demonstrate that non-active site mutations can be the primary source of resistance and that their role is not necessarily limited to compensate deleterious effects of active site mutations. Analysis of the structural stability of the proteases by differential scanning calorimetry reveals that ANAM-11 and NAM-10 are structurally more stable than the wild-type protease while A-1 is less stable. Together, the binding and structural thermodynamic results suggest that the non-active site mutants affect inhibitor binding by altering the geometry of the binding site cavity through the accumulation of mutations within the core of the protease molecule.     

h-ANF does not play a role in the regulation of myocardial force of contraction

In the present study, h-ANF failed to reduce myocardial force of contraction alone and in the presence of isoprenaline. This lack of effect was observed in electrically driven, isolated human papillary muscle strips and rat papillary muscles. In contrast, adenosine and carbachol exerted antiadrenergic actions in rat and human myocardium. It is concluded that h-ANF obviously does not play a role in the regulation of human and rat myocardial force of contraction, whereas adenosine and carbachol are capable to produce antiadrenergic effects in the human and rat myocardium.     

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells

Ca²⁺-permeable store-operated channels (SOCs) mediate Ca²⁺ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1^?/? mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1^?/? VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1^?/? VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1^?/? VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.     

Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.     

The alpha protein ICP0 does not appear to play a major role in the regulation of herpes simplex virus gene expression during infection in tissue culture.

The herpes simplex virus type 1 (HSV-1) alpha protein ICP0 trans-activates HSV-1 early genes in transient expression assays. To investigate the function of ICP0 during HSV-1 infection, we have lowered the level of ICP0 by use of a recombinant plasmid that has been engineered to express the antisense message. Cell lines were constructed which stably carry the antisense plasmid. Total protein profiles from infected antisense cell lines showed that the level of ICP0 was reduced to less than 10% of the wild type level in two of the cell lines. However, reducing the level of ICP0 did not have a significant effect on the expression of HSV-1 early or late genes. The polypeptide patterns for the remaining infected cell polypeptides were similar in that no bands were absent although there were some quantitative differences. The level of two early proteins, glycoprotein B and glycoprotein D was reduced in one of the cell lines, however, levels were nearly equivalent to the control infection for two other cell lines tested. Virus yields were the same for the antisense cell lines and for parent cells. Decreased ICP0 levels did not lead to more restrictive phenotypes for an alpha 4 or alpha 27 mutant as protein patterns were similar for these mutants in antisense and parent cells. Therefore, while ICP0 has been demonstrated to be a strong inducer of gene expression in transient expression assays, it does not appear to have a major role as an activator during the productive infection of tissue culture cells.     

Ring-like nucleoid does not play a key role in radioresistance of Deinococcus radiodurans

The conclusion based on transmission electron microscopy, "the tightly packed ring-like nucleoid of the Deinococcus radiodurans R1 is a key to radioresistance", has instigated lots of debates. In this study, according to the previous research of PprI’s crucial role in radioresistance of D. radiodurans, we have attempted to examine and compare the nucleoid morphology differences among wild-type D. ra-diodurans R1 strain, pprI function-deficient mutant (YR1), and pprI function-complementary strains (YR1001, YR1002, and YR1004) before and after exposure to ionizing irradiation. Fluorescence mi-croscopy images indicate: (1) the majority of nucleoid structures in radioresistant strain R1 cells ex-hibit the tightly packed ring-like morphology, while the pprI function-deficient mutant YR1 cells carrying predominate ring-like structure represent high sensitivity to irradiation; (2) as an extreme radioresistant strain similar to wild-type R1, pprI completely function-complementary strain YR1001 almost displays the loose and irregular nucleoid morphologies. On the other hand, another radioresistant pprI partly function-complementary strain YR1002’s nucleiods exhibit about 60% ring-like structure; (3) a PprI C-terminal deletion strain YR1004 consisting of approximately 60% of ring-like nucleoid is very sensi-tive to radiation. Therefore, our present experiments do not support the conclusion that the ring-like nucleoid of D. radiodurans does play a key role in radioresistance.     

Relevance of multidrug resistance 1 and P-glycoprotein to drug resistance in patients with systemic lupus erythematosus

Although corticosteroids and immunosuppressants are widely used for the treatments of various autoimmune diseases such as systemic lupus erythematosus (SLE), we often experience patients with SLE who are resistant to these treatments. P-glycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, is known to play a pivotal role in the acquisition of drug resistance to chemotherapies in malignancy. However, the relevance of MDR-1 and P-gp to resting and activated lymphocyte, major targets of the treatments in autoimmune diseases, remains unclear. We found that peripheral lymphocytes in patients with SLE express P-gp on the surface and its expression is highly correlated with disease activity. P-gp on lymphocytes is induced by not only genotoxic stresses but also activation stimuli such as cytokines, resulting in active efflux of corticosteroids from cytoplasm of lymphocytes, resulting in drug-resistance and high disease activity. However, the addition of P-gp antagonists such as ciclosporin A and inhibitors of P-gp synthesis successfully reduce efflux of corticosteroids from lymphocytes in vitro and these results imply that P-gp antagonists and P-gp synthesis inhibitors could work in order to overcome drug-resistance in vivo. Therefore, we propose that the measurement of P-gp on lymphocytes is a useful marker to indicate drug resistance and requirement of antagonists and/or intensive treatments to overcome drug resistance in active SLE patients.     

Angiotensin-converting enzyme gene I/D dimorphism does not play a major role in the susceptibility of Malaysian systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes systemic damage, involving auto-reactive antibodies and over-deposition of immune complexes. Susceptibility to SLE is believed to be multifactorial, and genetics is one of the proven etiological factors; it can affect SLE development, severity and prognosis. We investigated a possible association between the angiotensin-converting enzyme gene and susceptibility to SLE in the Malaysian population. PCR was employed for the determination of I/D dimorphism of this gene. The I allele was more frequent than the D allele in both the SLE patients (N = 170) and healthy controls (N = 190). However, there was no significant difference in the distribution of these two alleles between both groups studied (χ(2) = 0.284, P > 0.05). Interestingly, the DD homozygous genotype scored notably higher in the healthy control group (χ(2) = 7.568, P < 0.05), while the ID heterozygote was observed to be significantly associated with SLE (χ(2) = 11.143, P < 0.05). In conclusion, with respect to the Malaysian population, the DD genotype might play a protective role in the development of SLE while in contrast, those who carry the ID genotype might be at potential risk for onset of this disease.