Clin Infect Dis, 2004 Sep 15, 39(6), e53 - 5 Epub 2004 Aug 27. Prolonged survival of an HIV-infected patient with multidrug-resistant Mycobacterium bovis infection treated with surgical resection; Ramos A et al.; We describe a case of multidrug-resistant tuberculosis due to Mycobacterium bovis in a human immunodeficiency virus-infected woman with good immunologic status . The patient presented with a hard mass measuring 10 cm in diameter on the lower left ribs and a lung nodule measuring 3 cm in diameter in the left superior lobe . No adequate pharmacological treatment was available . Both lesions were surgically resected . The patient has remained asymptomatic (without fever, cough, lymphadenopathy, or cutaneous masses) for 20 months, after discharge from the hospital. J Clin Microbiol, 2004 Oct, 42(10), 4432 - 7 Microscopic observation drug susceptibility assay, a rapid, reliable diagnostic test for multidrug-resistant tuberculosis suitable for use in resource-poor settings; Moore DA et al.; There is an urgent need for new tools to improve our ability to diagnose tuberculosis (TB) and multidrug-resistant TB (MDR-TB) in resource-poor settings . In a retrospective analysis undertaken in a region with a high incidence of TB, we evaluated the performance of the microscopic observation drug susceptibility assay (MODS), a novel assay developed in Peru which uses an inverted light microscope and culture in Middlebrook 7H9 broth to detect mycobacterial growth . MODS detected 94.0% of 1,908 positive sputum cultures, whereas Lowenstein-Jensen (LJ) culture detected only 86.9% (P < 0.001) . The median time to culture positivity was 8 days (compared to 16 days for the same 208 samples by LJ culture; P < 0.001, Wilcoxon signed rank test) . The results obtained by direct susceptibility testing using MODS demonstrated excellent concordance for isoniazid and rifampin and the detection of multidrug resistance with those obtained by indirect colorimetric methods: the microplate Alamar Blue assay (MABA) and the tetrazolium microplate assay (TEMA) (agreement, 95, 98, and 94%; kappa values, 0.8, 0.7, and 0.7, respectively) . The concordance of the susceptibility testing results for ethambutol and streptomycin was poor . MODS is a novel assay which can detect the organisms responsible for TB and MDR-TB directly from sputum inexpensively, rapidly, and effectively . A comprehensive prospective evaluation of MODS is under way in Peru, and independent validation in nonresearch laboratories should be undertaken at the earliest opportunity. Biochim Biophys Acta, 2004 Oct 11, 1665(1-2), 111 - 7 Impact of the growth phase on the activity of multidrug resistance pumps and membrane potential of S . cerevisiae: effect of pump overproduction and carbon source; Cadek R et al.; The potentiometric fluorescence probe diS-C3(3) is expelled from S . cerevisiae by ABC pumps Pdr5 and Snq2 and can conveniently be used for studying their performance . The activity of these pumps in a strain with wild-type PDR1 allele was shown to drop sharply on glucose depletion from the medium and then again at the end of the diauxic shift when the cells are adapted to growth on respiratory substrates . The presence of the PDR1-3 allele causing pump overproduction prevented this second drop and the pump activity typical for diauxic cells was largely retained . Growth phase-dependent changes of membrane potential measured by the same probe in pump-free mutants included a Deltapsi drop in the late exponential and diauxic growth phase, indicating lowered activity of H+ -ATPase . Suppression of activity of both ABC pumps and H+ -ATPase obviously signifies cell transition to an energy-saving mode . Challenging respiration-adapted cells with glucose showed a novel feature of yeast ABC pumps--a strong dependence of pump activity on the type of the carbon source. Wien Klin Wochenschr, 2004 Aug 31, 116(15-16), 561 - 4 Highly refractory acute myeloid leukemia; Fureder W et al.; In this study we evaluated 103 patients suffering from acute myeloid leukemia (AML) who did not respond to induction chemotherapy and defined a sub-group of patients with highly refractory disease characterized by a persistence of more than 1 G/L blast cells in the peripheral blood between days 12 and 16 of the first induction cycle . Only seven patients (one female, six males) met these criteria . Their median age was 65 years (range 41-82 years) . Four had de novo AML and three secondary AML . Cytogenetic analysis was performed in six patients: complex aberrations were detected in four patients and, unexpectedly, normal karyotypes were found in the other two . Analysis of multidrug-resistance factors revealed high co-expression of P-glycoprotein (P-gp) and lung resistance protein (LRP) in all four patients with highly refractory disease tested a finding in only 6% of patients with refractory disease and 3% of patients who achieved complete remission (CR) of disease . Furthermore, patients with highly refractory AML had substantially higher leukocyte counts than patients with refractory AML or CR, although this was not significant statistically . Overall, patients with highly refractory AML are characterized by a high incidence of complex cytogenetic aberrations and marked expression of drug transporters. Proc Natl Acad Sci U S A, 2004 Oct 12, 101(41), 14925 - 30 Epub 2004 Oct 04. The complete genomic sequence of Nocardia farcinica IFM 10152; Ishikawa J et al.; We determined the genomic sequence of Nocardia farcinica IFM 10152, a clinical isolate, and revealed the molecular basis of its versatility . The genome consists of a single circular chromosome of 6,021,225 bp with an average G+C content of 70.8% and two plasmids of 184,027 (pNF1) and 87,093 (pNF2) bp with average G+C contents of 67.2% and 68.4%, respectively . The chromosome encoded 5,674 putative protein-coding sequences, including many candidate genes for virulence and multidrug resistance as well as secondary metabolism . Analyses of paralogous protein families suggest that gene duplications have resulted in a bacterium that can survive not only in soil environments but also in animal tissues, resulting in disease. Drug Metab Dispos, 2005 Jan, 33(1), 55 - 9 Epub 2004 Oct 04. Bisphenol a glucuronidation and excretion in liver of pregnant and nonpregnant female rats; Inoue H et al.; In male rats challenged with the environmental estrogen bisphenol A, the compound is highly glucuronidated in the liver and is excreted largely into the bile . Given that in pregnancy the microsomal glucuronidation toward bisphenol A is attenuated, we hypothesized that elimination of bisphenol A from the liver may be reduced in pregnancy . This study was conducted to trace the elimination of bisphenol A in female rats, especially in pregnancy . In Sprague-Dawley rats, 1.5 mumol of bisphenol A was perfused into the liver via the portal vein . In both the male and the nonpregnant female, the infused bisphenol A was glucuronidated, then the resultant glucuronide was excreted mainly into the bile . In pregnant rats, however, bilious excretion of bisphenol A glucuronide was 60% of that observed in nonpregnant rats, and venous excretion increased reciprocally . During 1-h perfusion, total excretion of the glucuronide from the liver of male, nonpregnant female, and pregnant rats was 889.5 +/- 69.6, 1256.7 +/- 54.8, and 1038.8 +/- 33.3 nmoles, respectively . In Eisai hyperbilirubinemic rats (EHBR), perfusion of the liver with bisphenol A enabled us to determine that multidrug resistance-associated protein (MRP)2-mediating transport is the mechanism behind excretion of the glucuronide into the bile . The expression of MRP2 has been reported to be noticeably reduced in pregnancy . These results suggest that bisphenol A elimination by hepatic glucuronidation is slightly less in pregnancy than in non-pregnancy and that in pregnancy, more bisphenol A glucuronide is eliminated to the vein because of reduced MRP2 expression. Cancer Res, 2004 Oct 1, 64(19), 7130 - 8 Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance; Munoz-Martinez F et al.; Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer . Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains . However, their molecular mechanism of reversion has not been characterized . In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action . Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine . Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters . Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of {(3)H}colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target . Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein . Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations . Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein. Drug Metab Dispos, 2005 Jan, 33(1), 1 - 9 Epub 2004 Oct 01. The roles of transporters and enzymes in hepatic drug processing; Liu L et al.; A simple, physiological model was used to illustrate the competing nature of transporters and metabolic enzymes in hepatic drug processing . Enalapril, a drug whose basolateral influx and canalicular efflux are mediated by rat organic anion-transporting polypeptide 1 (Oatp1) and rat multidrug resistance-associated protein 2 (Mrp2), respectively, and metabolism by the carboxylesterases, was enlisted as the example to illustrate how the transport and intrinsic clearances are inter-related in the estimation of the hepatic and metabolic, and excretion clearances . Moreover, simulations were performed to explore the effects of inhibitors or inducers of transporters/enzymes to unravel the compensatory changes of alternate pathways . Generally speaking, inhibition of one pathway led to an apparent increase in the alternate (competing) pathway and total hepatic clearance was decreased; induction would lead to an apparent decrease in the alternate pathway and an increase in total hepatic clearance . A reduction in influx clearance brought about parallel decreases in the biliary and metabolic clearances, whereas a reduction in efflux basolateral clearance evoked similar increases in biliary and metabolic clearances . However, the steady-state tissue concentration (C(L,ss)) or area under the tissue concentration-time curve (AUC(L)) was reliant only on the unbound fraction in liver, and the secretory and metabolic intrinsic clearances and not the influx and efflux clearances . Variations in the influx and efflux intrinsic clearances evoked temporal changes in the tissue concentration-time profile but not the AUC(L) or C(L,ss) . The pharmacokinetic theory developed offers data interpretation from literature reports on P-glycoprotein and cytochrome P450 substrates with mdr1a/1b knockout versus wild-type mice, and rat liver perfusion studies, with and without the use of inhibitors . In some cases, critiques on data interpretation were made. Bioorg Med Chem, 2004 Nov 1, 12(21), 5553 - 62 Synthesis of new 6-alkylvinyl/arylalkylvinyl substituted 1,2,4-trioxanes active against multidrug-resistant malaria in mice; Singh C et al.; 3-Alkyl/arylalkyl substituted 2-butenols 9, 10, 23a-d undergo regiospecific photooxygenation to furnish beta-hydroxyhydroperoxides 11, 12, 24a-d, respectively, in reasonable yields . Acid catalyzed condensation of 11, 12, 24a-d with various ketones furnish new 1,2,4-trioxanes 13-18, 25a-d, 26a-d, 27a-d in good yields . Several of these trioxanes show promising antimalarial activity against multidrug-resistant Plasmodium yoelii in mice by oral and intramuscular routes. Biochem Biophys Res Commun, 2004 Nov 5, 324(1), 365 - 71 Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells; Celius T et al.; Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp) . Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells . Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time . RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured . Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells . This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions. Parasitol Today, 1991, 7(4), 70 - 6 The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance? Cowman AF. Chloroquine has been the mainstay of antimalarial chemotherapy but the rapid spread of resistance to this important drug has now compromised its efficacy . The mechanism of chloroquine resistance has not been known but recent evidence from Plasmodium falciparum, the causative agent of the most severe form of human malaria, suggested similarities to the multidrug resistance phenotype (MDR) of mammalian tumour cells which is mediated by a protein molecule termed P-glycoprotein . Two mdr genes (pfmdr1 and pfmdr2) encoding P-glycoprotein homologues have been identified in P . falciparum and one of these (pfmdr1) has several alleles that have been linked to the chloroquine resistance phenotype . In contrast analysis of a genetic cross between chloroquine-resistant and -sensitive P . falciparum has suggested that the genes encoding the known P-glycoprotein homologues are not linked . This review outlines the similarities of the chloroquine resistance phenotype with the MDR phenotype of mammalian tumour cells and explores the possible role of the pfmdr genes. Parasitol Today, 1986 Sep, 2(9), 250 - 3 Current approaches to malaria chemotherapy and prophylaxis; Wernsdorfer WH; Malaria continues to be one of the most serious and widespread parasitic diseases, still occurring in over 100 countries despite concentrated efforts to eradicate it from many regions . Sixty-one countries now report their malaria cases to the WHO, and the latest analysis of these figures' shows little improvement in the overall problem during the last 15 years . Some countries, notably India and China, continue to report downward trends, but the problem continues to deteriorate in rural areas where intense economic development is taking place, particularly in Asia and the Americas . In 1984, 5.3 million cases of malaria were reported to the WHO . This is believed to represent but a small fraction of the total number because, for example, 38 of the tropical African countries do not report their malaria cases . Estimates based on the degree of malaria endemicity suggest a total incidence o f around 100 million cases annually . Chloroquine-resistant falciporum malaria has been confirmed in more than 40 countries, often showing cross-resistance to other drugs, and attempts to combat resistance using combination drugs have led to disturbing reports of side-effects as well as multidrug resistance . Vector control is also impaired in many areas due to insecticide resistance . Faced with these problems, we asked Dr Walther Wernsdorfer, head of the WHO Malaria Action Programme, what is the current WHO philosophy of malaria chemotherapy and prophylaxis? J Liposome Res, 2004, 14(1-2), 61 - 76 Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes; De Carlo S et al.; The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes . Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure . It is used in drug delivery studies in order to treat systemic hypertension . We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications . We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles . The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions . The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification . The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids . This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles . The overall reaction is slow, with the process taking almost two hours to be completed . The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension . When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine. J Med Invest, 2004 Aug, 51(3-4), 194 - 201 Reduction of expression of the multidrug resistance protein (MRP)1 in glioma cells by antisense phosphorothioate oligonucleotides; Matsumoto Y et al.; The tumor cells' acquisition of resistance to multiple drugs due to overexpression of the multidrug resistance protein (MPRP)1 gene is one of major obstacles in cancer chemotherapy . We have attempted to reverse the multidrug resistance (MDR) phenotype by treating etoposide resistant glioma cell lines (T98G-VP and Gli36-VP) with RP1 antisense oligonucleotides . 20-mer phosphorothioate oligodeoxynucleotide (0.3 microM), complementary to the coding region in the MRP cDNA sequence, could significantly inhibit the growth of multidrug resistant cell lines, T98G-VP and Gli36-VP, cultured in etoposide containing medium . No such effect was observed for the parental T98G and Gli36 cell lines . Further investigations by the reverse transcription-polymerase chain reaction and immunoblotting revealed that antisense oligomer could result in a reduction in the level of MRP1 mRNA, probably through hindering MRP1 gene transcription . This study demonstrates that the antisense oligonucleotides can increase the sensitivity of the tumor cells to the anticancer drug by decreasing the expression of the MRP gene . This strategy may be applicable to cure cancer patients with MRP mediated MDR phenotype. Hum Mutat, 2004 Nov, 24(5), 438 - 9 ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE); Gheduzzi D et al.; Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter . The ABCC6 gene was sequenced in 38 unrelated PXE Italian families . The mutation detection rate was 82.9% . Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes . Twenty-three different mutations were identified, among which 11 were new . Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%) . The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected . Among the others, exons 9 and 12 were particularly involved . Almost all mutations were located in the intracellular site of MRP6 . A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations . The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder . J Biol Chem, 2004 Dec 17, 279(51), 53571 - 83 Epub 2004 Sep 30. Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region; Westlake CJ et al.; The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents . In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking . The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined . In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes . However, the truncated protein is inactive . Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity . Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure . Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally . Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity . We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2 . Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains. Biofizika, 2004 Jul-Aug, 49(4), 685 - 91 {Changes in lipid asymmetry and transport of glutathione-S-conjugates under the influence of calcium ions in human erythrocytes}; Nude mice model of human hepatocellular carcinoma via orthotopic implantation of histologically intact tissue; Hepatic Surgery Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, ChinaAIM: To establish a nude mice model of human hepatocellular carcinoma (HCC) via orthotopic implantation of histologically intact tissue, in order to study biologic features of HCC in vivo and to direct clinical treatment respectively . METHODS: Histologically intact fresh specimens of HCC were orthotopically implanted in nude mice (BALB/c, nu/nu) . Survival rate and growth curve were investigated with B-ultrasound . Morphological characteristics of pathology and spontaneous metastatic rates were detected with microscopy . Expression of multidrug resistance genes studied with immunohistochemical method and RT-PCR, and other biologic features of implanted tumor were observed and compared with human HCC specimens . RESULTS: Out of the specimens from two patients with HCC, only one specimen survived in nude mice . The orthotopic implantation tumor survival rate, spontaneous intrahepatic metastatic rate, pulmonary metastatic rate and bone metastases rate were 100%, 75.0%, 37.5% and 37.5% respectively in the first passage . AFP was kept on secreting and increasing with the size of the tumor . The morphological characteristics and biologic features were similar to the donor's, the protein and mRNA of MDR1 and LRP were expressed in tumors of the model and the donor, and there was no significant difference between them (P>0.05) . CONCLUSION: The model of nude mice with orthotopic implantation of histologically intact HCC tissue is an ideal model to study biologic features of HCC in vivo and to direct clinical treatment. J Thorac Cardiovasc Surg, 2004 Oct, 128(4), 523 - 8 Resectional surgery combined with chemotherapy remains the treatment of choice for multidrug-resistant tuberculosis; Shiraishi Y et al.; OBJECTIVE: Multidrug-resistant tuberculosis remains a significant health problem . The best available treatment for multidrug-resistant tuberculosis is the combination of pulmonary resection and antituberculous chemotherapy . We herein report the results of pulmonary resection combined with chemotherapy for multidrug-resistant tuberculosis at our institution during the years 2000 through 2002 . METHODS: Between 1983 and 2002, 87 patients underwent 95 pulmonary resections for multidrug-resistant tuberculosis . Of these, the 30 (34%) patients operated on from January 1, 2000, to December 31, 2002, are reviewed in the present study . All patients were maintained on multidrug regimens preoperatively and postoperatively . Indications for surgical intervention included persistently positive sputum and a high risk of relapse . Thirty-three pulmonary resections were performed, consisting of pneumonectomy (n = 12), lobectomy (n = 17), and segmentectomy (n = 4) . The bronchial stump was reinforced with a latissimus dorsi muscle flap in 29 resections . RESULTS: There was no operative mortality . Bronchopleural fistulas occurred in 2 patients . Five patients had a space problem . All patients attained sputum-negative status after the operation . Relapse occurred in 3 patients: 2 had a relapse at the bronchial stump, and the remaining patient had a relapse in the postlobectomy space . One late death occurred . Of the 29 survivors, 27 (93%) were free from disease, with a median follow-up of 24 months (range, 8-47 months) . CONCLUSIONS: An increasing number of patients with multidrug-resistant tuberculosis are requiring resectional surgery in the 21st century . Pulmonary resection combined with chemotherapy achieves high cure rates with acceptable morbidity and remains the treatment of choice for multidrug-resistant tuberculosis. Antivir Ther, 2004 Aug, 9(4), 519 - 28 ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages; Jorajuria S et al.; OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro . DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes . METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR . MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs . We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor . Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS . P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp . RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment . In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir . AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors . Indinavir (10 nM) gave 14% inhibition alone and 81% in combination . The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration . However, unlike PIs, neither AZT nor its metabolites interacted with P-gp . CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity . The cellular efflux of AZT probably involves MRP4 or MRP5 . In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1. Int J Tuberc Lung Dis, 2004 Sep, 8(9), 1114 - 9 Simple, phage-based (FASTPplaque) technology to determine rifampicin resistance of Mycobacterium tuberculosis directly from sputum; Albert H et al.; SETTING: Cape Town, South Africa . OBJECTIVE: To evaluate the performance of a simple, manual, phage-based test for determining rifampicin (RMP) resistance of Mycobacterium tuberculosis directly from smear-positive sputum specimens . DESIGN: A comparative study of the performance of the FASTPlaque (phage amplification) technology to determine RMP resistance directly from smear-positive sputum compared with isolation and the conventional indirect Middlebrook 7H11 agar proportion method . RESULTS: The FASTPlaque direct RMP test achieved sensitivity, specificity and overall accuracy of 100% (11/11), 100% (134/134) and 100% (145/145), respectively, compared with the conventional indirect susceptibility test method (resolved data) . The FASTPlaque direct RMP test reported results within 2 days from receipt of the specimen, while the conventional method took between 27 and 103 days (mean +/- SD 33.2 +/- 7.2 days) . CONCLUSION: FASTPlaque technology applied directly to smear-positive sputum offers performance comparable to conventional methods, with results available in 2 days instead of weeks to months . The test may form a useful part of DOTS-Plus programmes to combat multidrug-resistant tuberculosis, improving patient prognosis and reducing ongoing transmission of disease . It does not require specialised equipment, making it appropriate for high-burden countries. Int J Tuberc Lung Dis, 2004 Sep, 8(9), 1107 - 13 The Beijing genotype is emerging among multidrug-resistant Mycobacterium tuberculosis strains from Germany; Kubica T et al.; SETTING: Germany, 1995 to 2001 . OBJECTIVE: To determine the genetic relationship of 451 multidrug-resistant (MDR) Mycobacterium tuberculosis strains from Germany and to identify strains of the Beijing genotype . DESIGN: All strains were analysed using IS6110 fingerprinting and a cluster analysis was performed . Clustering of isolates was used as a measure for recent transmission . RESULTS: Two hundred and fourteen of 433 strains (49.4%) with more than four IS6110 copies formed 46 fingerprint clusters comprising two to 32 patients . Transmission links based on classical epidemiological data could be established for 39 cases (18.2%) and in 14 clusters (30.4%), and included three cases of exogenous reinfection with MDR strains . One hundred and seventy-five strains (38.8%) were of the Beijing genotype with an increasing annual proportion from 19.2% in 1995 to 58.3% in 2001 . About 70% of these patients had an indication of foreign birth, mainly the former Soviet Union . CONCLUSION: Transmission of MDR strains seems to be contributing to the spread of MDR-TB in Germany, and exogenous reinfection with MDR strains must be considered as a possible cause of treatment failure . A high proportion of these MDR strains is probably carried over from the former Soviet Union, and strains of the Beijing genotype represent an increasing cause of MDR-TB in Germany. Cancer Chemother Pharmacol, 2005 Feb, 55(2), 159 - 69 Epub 2004 Sep 29. Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model; Chen J et al.; The purposes of this study were to determine the effect of structural change on the intestinal disposition of isoflavones and to elucidate the mechanisms responsible for transport of phase II isoflavone conjugates . Transport and metabolism of six isoflavones (i.e., genistein, daidzein, glycitein, formononetin, biochanin A, and prunetin) were studied in the human intestinal Caco-2 model and mature Caco-2 cell lysate . Glucuronides were the main metabolites in intact Caco-2 cells for all isoflavones except prunetin, which was mainly sulfated . In addition, the 7-hydroxy group was the main site for glucuronidation whereas the 4'-hydroxy group was only one of the possible sites for sulfation . Glucuronidated isoflavones (except biochanin A) were preferably excreted to the basolateral side, whereas sulfated metabolites (except genistein and glycitein) were mainly excreted into the apical side . Polarized excretion of most isoflavone conjugates was inhibited by the multidrug resistance-related protein (MRP) inhibitor leukotriene C(4) (0.1 micro M) and the organic anion transporter (OAT) inhibitor estrone sulfate (10 micro M) . When formation and excretion rates of isoflavones were determined simultaneously, the results showed that formation served as the rate-limiting step for all isoflavone conjugates (both glucuronides and sulfates) except for genistein glucuronide, which had comparable excretion and formation rates . In conclusion, the intestinal disposition of isoflavones was structurally dependent, polarized, and mediated by MRP and OAT . Formation generally served as the rate-limiting step in the cellular excretion of conjugated isoflavones in the Caco-2 cell culture model. Cancer Biother Radiopharm, 2004 Aug, 19(4), 457 - 65 Tracers to monitor the response to chemotherapy: in vitro screening of four radiopharmaceuticals; de Geus-Oei LF et al.; OBJECTIVES: It has been postulated that radiopharmaceuticals can be used to predict the therapeutic response to (chemo)therapy, which could lead to individualized treatment regimens . In this study, 18F-deoxyglucose, 99mTc-tetrofosmin, 125I-deoxyuridineribose, and 125I-methyltyrosine were tested for this purpose . METHODS: The uterine sarcoma cell line MES-SA (MDR-) and its multidrug resistant variant, MES-SA/Dx5 (MDR+), were used . The MDR+ cells express high levels of P-glycoprotein, which makes them relatively resistant to various chemotherapeutic agents . Cells were cultured in the presence of escalating concentrations of doxorubicin, and the cellular uptake of the radiopharmaceuticals was determined . RESULTS: Decreasing 18F-deoxyglucose uptake at escalating doxorubicin concentrations reflected the chemosensitivity of the cells: 18F-deoxyglucose uptake in the MDR- cells was reduced to 40% of the baseline level in the presence of 1 microM of doxorubicin, compared to 74% in the MDR+ cells . The 125I-deoxyuridineribose uptake in MDR- cells was reduced to 2% of the baseline level when cultured at a concentration of 1 microM of doxorubicin, while this was 79% in the MDR+ cells . The same trend was observed with 125I-methyltyrosine . The enhanced doxorubicin chemosensitivity of MDR+ cells in the presence of verapamil, a modulator of P-glycoprotein, was reflected by the reduced uptake of 18F-deoxyglucose, 125I-deoxyuridineribose, and 125I-methyltyrosine . Furthermore, baseline 99mTc-tetrofosmin uptake in MDR+ cells was more than six-fold lower than in MDR- cells . CONCLUSION: In the presence of doxorubicin, the uptake of 18F-deoxyglucose, 125I-deoxyuridineribose and, to a lesser extent, 125I-methyltyrosine is more pronouncedly reduced in MDR- cells than in MDR+ cells . The reversal of doxorubicin-resistance of MDR+ cells by verapamil was also reflected by the uptake of 18F-deoxyglucose, 125I-deoxyuridineribose, and 125I-methyltyrosine . 99mTc-tetrofosmin uptake reflected P-glycoprotein expression without exposure to doxorubicin . Copyright Mary Ann Liebert, Inc. Cancer Biother Radiopharm, 2004 Aug, 19(4), 411 - 21 Influence of glutathione depletion on plasma membrane cholesterol esterification and on Tc-99m-sestamibi and Tc-99m-tetrofosmin uptakes: a comparative study in sensitive U-87-MG and multidrug-resistant MRP1 human glioma cells; Le Jeune N et al.; In our previous studies, we demonstrated a possible effect of cellular glutathione (GSH) depletion on plasma-membrane permeability and fluidity in glioma-cell lines . We therefore investigated the effect of GSH modulation on accumulation of two radiotracers, Tc-99m-sestamibi (MIBI) and Tc-99m-tetrofosmin (TFOS), and on plasma-membrane cholesterol content in sensitive U-87-MG and resistant U-87-MG-CIS and U-87-MG-MEL (MRP1 positive) human glioma-cell lines . GSH depletion was mediated by BSO pretreatment and addition of N-acetylcysteine reversed the effect . MIBI and TFOS uptakes, total cholesterol, and cholesteryl-ester contents were evaluated under each condition . In contrast with TFOS, MIBI accumulation was inversely proportional to the cell multidrug resistance phenotype . Similar cholesterol contents were observed in all cell lines, demonstrating that MRP1 did not modify lipid membrane composition . A decrease of intracellular GSH allows an increase of plasma-membrane cholesterol and a decrease of cholesteryl-ester content, which in turn results in spectacular TFOS uptake . The GSH status of the cells plays an important role in the plasma membrane cholesterol composition and TFOS uptake, which appears to be particularly sensitive to this modification . In contrast with MIBI, TFOS is not an MRP1 probe in glioma cells, and therefore appears to be a suitable tracer in this indication . Copyright Mary Ann Liebert, Inc. Neurology, 2004 Sep 28, 63(6), 1090 - 2 Failure to confirm association of a polymorphism in ABCB1 with multidrug-resistant epilepsy; Tan NC et al.; Alteration of ATP-binding cassette subfamily B member 1 transporter (ABCB1) can plausibly cause drug-resistant epilepsy as it influences brain penetration of drugs . The CC genotype at the ABCB1 C3435T polymorphism was reported to be associated with multidrug resistance . A replication study in 401 drug-resistant and 208 drug-responsive subjects with epilepsy showed no significant association between the CC genotype and drug-resistant epilepsy . The authors suggest the initial association may have arisen by chance. Nucleic Acids Res, 2004 Sep 27, 32(17), 5066 - 75 Print 2004. Pdr3 is required for DNA damage induction of MAG1 and DDI1 via a bi-directional promoter element; Zhu Y et al.; In order to understand how gene regulation is achieved in eukaryotes in response to DNA damage, we used budding yeast as a model lower eukaryotic organism and investigated the molecular events leading to the expression of two closely clustered damage-inducible genes, MAG1 and DDI1 . MAG1 and DDI1 are co-activated by a shared 8 bp repeat sequence, UAS(DM) . In this study, we screened a yeast genomic library, identified Pdr3 as the transcriptional activator and demonstrated in vivo and in vitro that Pdr3 binds UAS(DM) . Pdr3 is required for the activation of a number of genes encoding membrane efflux pumps and deletion of PDR3 results in reduced basal-level expression and loss of DNA damage induction of MAG1 and DDI1 . Interestingly, Pdr1, another transcriptional activator homologous to Pdr3 that is also required for the activation of multidrug-resistance genes, is not involved in the regulation of MAG1 and DDI1 expression, although it may also bind to UAS(DM) . Deletion of PDR3 does not affect the expression of other well-documented DNA damage-inducible genes; hence, yeast DNA damage-inducible genes appear to have distinct effectors although to a certain extent they share a common regulatory pathway mediated by DNA damage checkpoints. Eur J Pharm Sci, 2004 Oct, 23(2), 181 - 8 Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity; Vellonen KS et al.; The MTT (3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells . These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1) . MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay . Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay . Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability . This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity . The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line . This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay . Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial . Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies. Biochem Pharmacol, 2004 Nov 1, 68(9), 1815 - 23 The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells; Tarasiuk J et al.; The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer . Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient . Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs) . The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line . The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells . It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC) . The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake . In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein . Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR. Gastric Cancer, 2004, 7(3), 160 - 6 Regulation of drug sensitivity of gastric cancer cells by human calcyclin-binding protein (CacyBP); Shi Y et al.; BACKGROUND: Calcyclin-binding protein (CacyBP) was previously identified as an upregulated gene in a multidrug-resistant gastric cancer cell line, SGC7901/ADR, compared to its parental cells, SGC7901, by subtractive hybridization . The aim of this study was to explore the role of CacyBP in multidrug resistance (MDR) in gastric cancer cells . METHODS: The cDNA encoding CacyBP was generated by reverse-transcription-polymerase chain reaction (RT-PCR), and mouse antisera against CacyBP was raised using recombinant CacyBP as the immunogen . The expression of CacyBP in gastric cancer cells was determined by Northern and Western blots . Sense and antisense vectors for CacyBP were introduced into SGC7901 and SGC7901/ADR cells, respectively . The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to evaluate the drug sensitivity of gastric cancer cells . Flow cytometry was employed to determine adriamycin accumulation and retention in gastric cancer cells . RESULTS: Northern and Western blots demonstrated upregulation of CacyBP in SGC7901/ADR cells compared to SGC7901 cells . SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells were prepared, in which CacyBP was genetically increased and decreased, respectively . As compared with SGC7901, SGC7901-CacyBP cells exhibited significantly increased ( P < 0.01) IC(50) values for vincristine, adriamycin, and 5-fluorouracil . Meanwhile, as compared with SGC7901/ADR, SGC7901/ADR-anCacyBP cells exhibited significantly decreased ( P < 0.01) IC(50) values for these three drugs . SGC7901-CacyBP and SGC7901/ADR-anCacyBP cells displayed no obvious difference ( P > 0.05) in intracellular adriamycin content compared to their corresponding parental cells . CONCLUSIONS: Upregulation of CacyBP is associated with MDR in gastric cancer cells . CacyBP could regulate the responses of gastric cancer cells to chemotherapy . But the underlying mechanisms of CacyBP-related MDR need further identification. Eur J Nucl Med Mol Imaging, 2004 Nov, 31(11), 1523 - 1529 Epub 2004 Sep 21. In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide; Bai J et al.; PURPOSE: The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R . METHODS: The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) . Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99mTc-sestamibi (MIBI), a known substrate for P-glycoprotein . A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9 . The DTPA-ODN complexes at concentrations of 0.1-17.4 microM were reacted with 111InCl3 at pH 5 for 1 h . The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence . Cellular uptake of labeled ODN was examined in vitro . Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed . RESULTS: P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells . 99mTc-MIBI uptake in P388/S cells was higher than that in P388/R cells . Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount . The hybridization affinity of antisense 111In-ODN was preserved at approximately 85% irrespective of specific activity . Cellular uptake of antisense 111In-ODN did not differ from that of sense 111In-ODN in either P388/S cells or P388/R cells . However, lipid carrier incorporation significantly increased transmembrane delivery of 111In-ODN; moreover, specific uptake of antisense 111In-ODN was demonstrated in P388/R cells . CONCLUSION: Radiolabeling of ODN at high specific radioactivity and specific uptake of antisense 111In-ODN in drug-resistant cells may facilitate future gene imaging of mdr1 mRNA. Pathol Oncol Res, 2004, 10(3), 133 - 41 Epub 2004 Sep 25. Human osteosarcoma xenografts and their sensitivity to chemotherapy; Bruheim S et al.; Despite the increased survival rates of osteosarcoma patients attributed to adjuvant chemotherapy, at least one third of the patients still die due to their disease . Further improvements in the management of osteosarcoma may rely on a more individualised treatment strategy, as well as on the introduction of new drugs . To aid in the preclinical evaluation of new candidate substances against osteosarcoma, we have established 11 human osteosarcoma xenograft lines and characterised them with regard to response to five different reference drugs . Doxorubicin, cisplatin methotrexate, ifosfamide and lomustine were effective in 3/11, 3/11, 1/10, 5/11 and 4/11 of the xenografts, respectively . Five xenografts were resistant to all compounds tested . We also assessed the mRNA expression levels of the xenografts for the O(6)-Methylguanine DNA Methyltransferase (MGMT), DNA topoisomerase II- (Topo II)-alpha, Gluthathione-S-transferase (GST)-pi, Multidrug-resistance related protein (MRP) 1 and Multidrug-resistance (MDR) 1 genes . There was an inverse correlation between the transcript levels of GST-pi and doxorubicin growth inhibition (r=-0.66; p<0.05), and between the transcript levels of MGMT and the effect of lomustine (r=-0.72; p<0.01), whereas the expression of MRP1 and cisplatin growth inhibition was positively correlated (r=0.82; p<0.005) . This panel of xenografts should constitute a good tool for pharmacological and molecular studies in osteosarcoma. J Cell Physiol, 2004 Dec, 201(3), 409 - 19 FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line; Israeli D et al.; Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration . We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro . FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes . Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc . These effects were reversed by FGF inhibitors . Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD . We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold . Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle. Int J Cancer, 2005 Jan 10, 113(2), 229 - 40 Regulation of the multidrug resistance transporter P-glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species; Wartenberg M et al.; Hyperthermia is an important component of many cancer treatment protocols . In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids . Hyperthermia treatment of small (50-100 microm) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42 degrees C, whereas only moderate elevation of P-glycoprotein was found in large (350-450 microm) tumor spheroids . Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS) . Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment . This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability . Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment . Furthermore, upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) was observed . Elevation of HIF-1alpha and P-glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580 . In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1alpha and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1alpha and P-glycoprotein expression by ERK1,2 and JNK signaling cascades . In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1alpha is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1alpha under control conditions. Can J Physiol Pharmacol, 2004 Jul, 82(7), 431 - 7 Rh2, a compound extracted from ginseng, hypersensitizes multidrug-resistant tumor cells to chemotherapy; Jia WW et al.; Rh2 is a ginsenoside extracted from ginseng that has drawn attention in a few laboratories in Asian countries because of its potential tumor-inhibitory effect . In the present study, we tested Rh2 on many tumor-cell lines for its effects on cell proliferation, induction of apoptosis, and potential interaction with conventional chemotherapy agents . Our results showed that Rh2 inhibited cell growth by G1 arrest at low concentrations and induced apoptosis at high concentrations in a variety of tumor-cell lines, possibly through activation of caspases . The growth arrest and apoptosis may be mediated by 2 separate mechanisms . Apoptosis is not dependent on expression of the wild-type p53 nor the caspase 3 . In addition, the apoptosis induced by Rh2 was mediated through glucocorticoid receptors . Most interestingly, Rh2 can act either additively or synergistically with chemotherapy drugs on cancer cells . Particularly, it hypersensitized multidrug-resistant breast cancer cells to paclitaxel . These results suggest that Rh2 possesses strong tumor-inhibiting properties, and potentially can be used in treatments for multidrug-resistant cancers, especially when it is used in combination with conventional chemotherapy agents. Cells Tissues Organs, 2004, 177(3), 151 - 9 Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo; Pfutzner W et al.; In gene therapy, a clinically relevant therapeutic effect requires long-term expression of the desired gene at a level sufficient to correct or at least alleviate the underlying gene defect . One approach to achieve persistent as well as high-level transgene expression in a significant percentage of target cells would be to select cells expressing both the desired transgene and a linked selectable gene--such as the human multi-drug resistance (MDR1) gene--in a bicistronic vector . Because of its accessibility, the skin is a very attractive target tissue to select genetically modified cells, allowing topical application of a selecting agent, thus minimizing potential toxic side effects . Among the potential selecting drugs, agents that block cell division, such as colchicine, are of particular interest because the use of anti-mitotic drugs takes advantage of the rapid keratinocyte (KC) turnover in the epidermis and the need for continued proliferation to substitute the KC lost due to selection . Before assessing the therapeutic benefit of such an approach, several key questions need to be answered in preclinical models: (1) Does topical colchicine application achieve the desired in vivo effect by blocking KC mitosis without eliciting unwanted toxic side effects? (2) Are MDR-transduced (MDR+) human KC still able to proliferate and differentiate when treated with colchicine? (3) Can MDR+ KC be enriched by topical selection? (4) Does topical selection result in persistent transgene expression by selecting KC stem cells expressing MDR? To answer these questions and to test the feasibility of such an approach both an in vitro skin equivalent and an in vivo human skin graft model were developed in which MDR+ KC were treated with different dosages of colchicine . Quantitative and qualitative analyses of MDR expression in human KC showed that topical colchicine treatment selects high-level transgene expression in a high percentage of KC . Moreover, determination of transgene copy numbers demonstrated that MDR+ KC progenitor cells were enriched by topical selection resulting in long-term expression of the transgene in the skin . Thus, in summary, these models demonstrate that topical selection of MDR+ KC is a safe approach to efficiently enhance long-term gene expression in the skin and holds future promise for clinical gene therapy applications . Antimicrob Agents Chemother, 2004 Oct, 48(10), 3758 - 64 Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada; Boyd DA et al.; A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002 . We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain . Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions . The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10 . The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26 . All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla(CTX-M-15), bla(OXA-1), and bla(TEM-1), the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6')-Ib and aac(3)-II, are located in the MDR . The bla(CTX-M-15) gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence . Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada . Comparison of pC15-1a and pCTX15, found in an E . coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla(CTX-M-15), bla(TEM-1), bla(OXA-1), tetA, and aac(6')-Ib. Int J Cancer, 2005 Jan 10, 113(2), 213 - 20 Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADR; Du J et al.; In our previous work, cellular prion protein (PrPc) was identified as an upregulated gene in adriamycin-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901 . Here we investigate the expression of PrPc in gastric cancer and whether it was involved in multidrug resistance (MDR) of gastric cancer . We demonstrated that PrPc was ubiquitously expressed in gastric cancer cell lines and tissues . PrPc conferred resistance of both P-glycoprotein (P-gp)-related and P-gp-nonrelated drugs on SGC7901, which was accompanied by decreased accumulation and increased releasing amount of adriamycin in PrPc-overexpressing cell line . Inhibition of PrPc expression by antisense or RNAi technology could partially reverse multidrug-resistant phenotype of SGC7901/ADR . PrPc significantly upregulated the expression of the classical MDR-related molecule P-gp but not multidrug resistance associated protein and glutathione S-transferase pi . The PrPc-induced MDR could be partially reversed by P-gp inhibitor verapamil . PrPc could also suppress adriamycin-induced apoptosis and alter the expression of Bcl-2 and Bax, which might be another pathway contributing to PrPc-related MDR . The further study of the biological functions of PrPc may be helpful for understanding the mechanisms of occurrence and development of clinical gastric carcinoma and PrPc-related MDR and developing possible strategies to treat gastric cancer. Int J Cancer, 2005 Jan 1, 113(1), 158 - 65 Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from calophyllum brasiliense that acts by induction of apoptosis; Kimura S et al.; During our search for cancer chemopreventing compounds derived from plant sources, we discovered that the natural product GUT-70, isolated from the stem bark of Calophyllum brasiliense collected in Brazil, significantly inhibits the growth of leukemic cells . GUT-70, characterized as a tricyclic coumarin, 5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo{1,2-b;3,4-b'}dipyran-8-one (C(23)H(26)O(5)), inhibited all 6 human leukemic cell lines evaluated, including the P-glycoprotein overexpressing cell line, in a concentration and time-dependent manner with IC(50) values from 2-5 microM . Furthermore, GUT-70 did not inhibit colony formation by normal hematopoietic progenitors up to 30 microM and also did not inhibit the proliferation of normal human hepatocytes up to 30 microM . GUT-70 activated the caspase 2, 3, 8 and 9, and induced the apoptosis in leukemic cells, which was inhibited by caspase inhibitors . GUT-70 induced anti-leukemic effects independent of the p53-p2l(WAFl/CIP1) pathway and increased the overall expression of p27(KIP1) and p57(KIP2), to stop the cell cycle at the G(1)/S transition . Thus, a novel anti-cancer drug, GUT-70 isolated from the stem bark of C . brasiliense induces caspase-mediated and p53-independent apoptosis to overcome multidrug resistance and may become a potent leukemia therapeutics. Int J Cancer, 2004 Dec 20, 112(6), 934 - 42 RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance; Sharma R et al.; Increased active transport of LTC(4) observed frequently in multidrug-resistant cancer cells have been attributed to ABC-transporter proteins particularly, MRP1 . We have demonstrated recently that a novel non-ABC transporter, RLIP76 (RALBP1) can also mediate ATP-dependent transport of GSH-conjugates (GS-E) as well as doxorubicin (DOX) . We demonstrate RLIP76 reconstituted in artificial liposomes can catalyze ATP-dependent transport of LTC(4), which can be modulated by PKC-alpha . The ATPase activity of E . coli expressed homogenous RLIP76 was stimulated in a saturable fashion by LTC(4) with half maximal stimulation at 130 nM . Proteoliposomes reconstituted with RLIP76 catalyzed temperature and osmolar sensitive ATP-dependent transport of LTC(4) with K(m) values of 5.1 mM and 210 nM for ATP and LTC(4), respectively . V(max) for transport was found to be 3.2 nmol/min/mg . Colchicine inhibited LTC(4) transport to 50% at 5.8 microM . PKC-alpha catalyzed phosphorylation of RLIP76 and increased its transport activity by 2-3-fold . Membrane vesicles prepared from the small (SCLC) and non-small (NSCLC) lung cancer cell lines as well as HL-60 (leukemia) and U937 (lymphoma) cell lines exhibited ATP-dependent transport of LTC(4), which was inhibited by anti-RLIP76 antibodies . The rate of transport of LTC(4) in SCLC (H69, H378) was half of that observed in NSCLC cell lines but after transfection with RLIP76, the transport rate of LTC(4) in H69 became comparable to that in NSCLC cell lines . Anti-RLIP76 antibodies inhibited LTC(4) transport by 67-81% in all 8 cell lines examined, whereas N-19 anti-MRP1 antibodies inhibited transport of LTC(4) by only 11-26% . These results suggest that RLIP76 is the major LTC(4) transporter in cancer cells and that its transport activity is regulated by PKC-alpha-mediated phosphorylation . (c) 2004 Wiley-Liss, Inc Mol Pharmacol, 2004 Oct, 66(4), 1004 - 10 Modulation of multidrug resistance-associated protein 2 (Mrp2) and Mrp3 expression and function with small interfering RNA in sandwich-cultured rat hepatocytes; Tian X et al.; Canalicular multidrug resistance-associated protein 2 (Mrp2) and basolateral Mrp3 mediate the excretion of organic anions, including conjugated and unconjugated xenobiotics and bile acids, from the liver . The utility of RNA interference to specifically knock down the expression and function of transport proteins was demonstrated in sandwich-cultured rat hepatocytes, which exhibit functional and properly localized Mrp2 and Mrp3 over time in culture . Specific knockdown of Mrp2 (approximately 50% decrease in expression) resulted in an approximately 45% decrease in the biliary excretion index of carboxydichlorofluorescein (CDF) (9.3% versus 16.5%), but did not affect Mrp3 or radixin expression . Specific Mrp3 knockdown (approximately 50% decrease in expression) resulted in significantly higher accumulation of CDF in cells + bile canaliculi (32.3 +/- 2.5 versus 24.4 +/- 4.3 pmol/mg of protein/10 min), but no change in cellular accumulation (13.7 +/- 2.2 versus 15.6 +/- 4.0 pmol/mg of protein/10 min), consistent with an approximately 60% increase in the biliary excretion index of carboxydichlorofluorescein . The extent of protein knockdown was in good agreement with changes in carboxydichlorofluorescein disposition . In conclusion, modulation of drug transporters in sandwich-cultured rat hepatocytes by small interfering RNA treatment is a feasible in vitro approach to study the expression and function of drug transport proteins. Nucl Med Commun, 2004 Oct, 25(10), 1039 - 48 Optimization of technetium-99m Sestamibi single-photon emission tomography to define multidrug resistance with confidence; Moorin RE et al.; BACKGROUND: The efflux rate of technetium-99m Sestamibi (99mTc-Sestamibi) is a kinetic phenomenon related to the response of cancer cells to chemotherapy, and may be used to determine drug resistance . Measurement of the efflux rate requires accurate quantitative single-photon emission tomography (SPET) imaging within the time constraints imposed by the kinetics of the process . METHODS: A phantom study, at activity concentrations typically found with 99mTc-Sestamibi in vivo, was undertaken to optimize the SPET parameters and, in particular, to determine whether 180 degrees acquisition arcs with heads in 'L' configuration could be used for accurate quantification . Following the development of the most appropriate SPET protocol, a small patient pilot study was undertaken . RESULTS: Studies designed to evaluate statistical uncertainty (noise), contrast restitution and spatial resolution of the data sets, using different acquisition and reconstruction parameters, showed that 180 degrees SPET using a 64 x 64 matrix, 6 degrees angular sampling and iterative reconstruction was optimal . Finer linear and/or angular sampling afforded negligible improvement in resolution, but markedly increased the statistical uncertainty . Comparison of 360 degrees and 180 degrees acquisitions, utilizing conventional filtered backprojection and iterative reconstruction algorithms, demonstrated that the statistical uncertainty was reduced to a greater extent for 180 degrees data collection . For 360 degrees (64 x 64) data acquisition, statistical uncertainty decreased from 15% to 11% using the iterative algorithm, whilst the 180 degrees (64 x 64) data showed a reduction from 20% to 7%, and approached values obtained by planar imaging . The efflux measurements obtained in the patient pilot study were consistent with the observed chemotherapy response . CONCLUSION: Our study shows that 180 degrees acquisition arcs are a practical option for accurate quantitative SPET kinetic imaging for potential studies of chemotherapy response in patients with lung cancer. Adv Drug Deliv Rev, 2004 Oct 14, 56(12), 1793 - 809 Potential role of ABC transporters as a detoxification system at the blood-CSF barrier; de Lange EC; Exchange of compounds between blood and brain occurs at two barriers, the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) . The barrier function is mainly a result of the functionality of the cerebral endothelial cells and choroidal epithelial cells, respectively . These cell types have restricted permeability due to the presence of tight junctions between the cells . Furthermore, these cells express a broad range of transporters . So far, the BBB has been viewed as the most important barrier, especially as its surface is about 3 orders of magnitude larger than that of the BCSFB . Today, there is a shift in the appreciation of the contribution of the BCSFB . In a few recent studies, it has been shown that the BCSFB expresses two types of ATP-binding cassette (ABC) transporters, being the multidrug transporters P-glycoprotein (P-gp) and the multidrug resistance-related protein 1 (MRP1) . The knowledge on the function of these transporters in the BCSFB is relatively scarce, but in general, it seems that MRP1 transport is directed towards the blood side, which makes this transporter helpful in elimination of harmful compounds from the CSF . Thereby MRP1 potentially contributes to detoxification of the brain, as a whole, as it is also expressed at the level of the BBB . P-gp, however, while also functional as an efflux pump at the BBB, has an opposite transport direction at the level of the BCSFB, towards the CSF . P-gp may therefore raise the concentration of neurotoxic P-gp substrates in the CSF . Whether this will have a significant contribution to the toxicity in the regions directly exposed to the CSF (periventricular organs) remains to be determined . Specifically, in the epithelial cells of the choroid plexus of the BCSFB, P-gp and MRP1 together serve a protective role by preventing the accumulation of their overlapping and often toxic substrates . A concerted action of P-gp and MRP1 at the choroid plexus might contribute to the maintenance of the role of the BCSFB in brain homeostasis. Epilepsy Res, 2004 Jul-Aug, 60(2-3), 203 - 13 Selective and persistent upregulation of mdr1b mRNA and P-glycoprotein in the parahippocampal cortex of chronic epileptic rats; van Vliet E et al.; There is recent evidence that increased expression of multidrug transporters, such as P-glycoprotein (P-gp), may lead to reduced antiepileptic drug (AED) concentrations in the brain, shortly after status epilepticus (SE), thereby suggesting a possible mechanism for drug-resistance . To get insights on whether increased P-gp expression is a consequence of the initial insult, or evolves more gradually as a result of recurrent spontaneous seizures, we used a rat model of temporal lobe epilepsy in which spontaneous seizures develop after an electrically induced SE . We investigated the temporal and region-specific expression of two isoforms of the multidrug resistance gene (mdr1a and mdr1b, both encoding for P-gp) in two regions within the temporal lobe (the dentate gyrus (DG) and the parahippocampal cortex (PHC)) . Using real-time PCR, we found that the mdr1b isoform was increased in the temporal lobe, 1 week after SE; however, this increase was reversible in dentate gyrus while it persisted in the parahippocampal cortex of chronic epileptic rats . Mdr1b upregulation was related to the occurrence of spontaneous seizures, since this isoform was unchanged in rats that were stimulated, but that did not develop SE (non-SE) . The mdr1a isoform was transiently upregulated in the dentate gyrus . P-gp immunostaining was enhanced in endothelial and glia-like cells, 1 week after SE . In chronic epileptic rats, the number of strongly P-gp positive glia-like cells was much lower than 1 week after SE, and it was mainly present in the most ventral part of the temporal lobe . These cells were in close apposition to strongly stained blood vessels . These findings show that both mdr1a and mdr1b are induced by SE, although the increase in mdr1b isoform was more persistent . More importantly, increased P-gp expression is still present in chronic epileptic rats. Int J Antimicrob Agents, 2004 Oct, 24(4), 386 - 92 Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance; Kozovska Z et al.; Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors . To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor . Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance . The basic components of this system, the P(GAL1)-PDR3 and P(PDR5)-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S . cerevisiae strain deleted in the PDR1 and PDR3 genes . This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously . In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance. Int J Urol, 2004 Sep, 11(9), 721 - 7 Increased expression of lung resistance-related protein in lower grade urothelial carcinoma of the renal pelvis and ureter; Kong CZ et al.; BACKGROUND: Lung resistance-related protein (LRP), like multidrug resistance gene 1 (MDR1) and multidrug resistance-associated proteins (MRP), has been associated with intrinsic therapeutic resistance in various malignancies . To date, there has been no study on the expression of LRP in urothelial carcinomas of the renal pelvis and ureter . We investigated the protein and mRNA expression levels of LRP, MDR1 and MRP1 in this malignancy and the clinical significance of their expression was evaluated . METHODS: Forty urothelial carcinomas of the renal pelvis and ureter and 31 normal upper urothelial samples were examined by immunohistochemistry and reverse transcription polymerase chain reaction to determine the protein and mRNA levels of the multidrug resistance-related genes, respectively . RESULTS: The positive staining rates and mRNA levels of LRP were the highest among these multidrug resistance-related genes in both normal urothelium and carcinoma examinations . In contrast to the up-regulated expression of MDR1, the expression of LRP tended to be down-regulated in carcinomas . Moreover, the expression of LRP inversely correlated with tumor grades, but this correlation was not found for the other two genes . However, there was no correlation among the expression of the three genes observed . CONCLUSION: Lung resistance-related protein was strongly expressed in urothelial carcinomas of the renal pelvis and ureter, particularly in well-differentiated carcinomas. Curr Pharm Des, 2004, 10(24), 2965 - 79 Imaging drug resistance with radiolabeled molecules; Vaidyanathan G et al.; A major obstacle to successful cancer chemotherapy is drug resistance . Multidrug resistance (MDR) is often seen with chemotherapeutic agents such as anthracycline derivatives, vinca alkaloids and taxanes . Multiple aspects of cellular biochemistry have been implicated in the MDR process . Cellular mechanisms of resistance are due to the presence of efflux pumps, P-glycoprotein (P-gp) and multiple resistance-associated protein (MRP), which belong to the ATP-binding cassette (ABC) family of transporters . Another form of drug resistance is involved in the chemotherapy of cancers with alkylating agents such as nitrosourea derivatives and nitrogen mustards . The cytotoxicity of these agents is primarily due to alkylation of the DNA guanine residues at their O6-position, which leads, via a cascade of events, to DNA strand breaks . The DNA repair protein, alkylguanine-DNA alkyl transferase (AGT) removes the alkyl groups from the lesions stoichiometrically to a cysteine in its active site . This process is irreversible and results in the degradation of the protein and its recovery is entirely from de novo synthesis . Noninvasive methodologies for monitoring the transport activity of these efflux pumps and determining tumor content of AGT could serve as critical tools for optimizing chemotherapeutic protocols on a patient-specific basis and gaining an understanding of the dynamics of resistance in living patients . In this review, we will describe the efforts made to date to synthesize radioactive probes of chemotherapy resistance and their use to quantitate these transporters and DNA repair protein by radionuclide imaging. Curr Top Med Chem, 2004, 4(13), 1385 - 98 Pharmacogenetics of drug transporters and its impact on the pharmacotherapy; Sakaeda T et al.; Most drug responses are determined by the interplay of several gene products that influence pharmacokinetics and pharmacodynamics, i.e., drug metabolizing enzymes, drug transporters, and drug targets . With the sequencing of the human genome, it has been estimated that approximately 500-1200 genes code for drug transporters . Concerning the effects of genetic polymorphisms on pharmacotherapy, the best characterized drug transporter is the multidrug resistant transporter P-glycoprotein/MDR1, the gene product of MDR1 . Little such information is available on other drug transporters . MDR1 is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily, and is expressed mainly in intestines, liver, kidneys and brain . A number of various types of structurally unrelated drugs are substrates for MDR1, and their intestinal absorption, hepatobiliary secretion, renal secretion and brain transport are regulated by MDR1 . The first investigation on the effects of MDR1 genotypes on pharmacotherapy was reported in 2000: a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration . At present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene . Clinical investigations on the association of MDR1 genotypes with the expression and function of MDR1 in tissues, and with pharmacokinetics and pharmacodynamics have mainly focused on C3435T; however, there are still discrepancies in the results, suggesting that the haplotype of the gene should be analyzed instead of a SNP . C3435T is also reported to be a risk factor for a certain class of diseases including the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this also might be explained by the effects on MDR1 expression and function . In this review, the latest reports on the effects of genetic polymorphisms of MDR1 on pharmacotherapy are summarized, and the pharmacogenetics of other transporters is briefly introduced. Biochemistry, 2004 Sep 28, 43(38), 12081 - 9 The drug-binding pocket of the human multidrug resistance P-glycoprotein is accessible to the aqueous medium; Loo TW et al.; P-Glycoprotein (P-gp) is an ATP-dependent drug pump that transports a broad range of compounds out of the cell . Cross-linking studies have shown that the drug-binding pocket is at the interface between the transmembrane (TM) domains and can simultaneously bind two different drug substrates . Here, we determined whether cysteine residues within the drug-binding pocket were accessible to the aqueous medium . Cysteine mutants were tested for their reactivity with the charged thiol-reactive compounds sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) and {2-(trimethylammonium)ethyl)}methanethiosulfonate (MTSET) . Residue Ile-306(TM5) is close to the verapamil-binding site . It was changed to cysteine, reacted with MTSES or MTSET, and assayed for verapamil-stimulated ATPase activity . Reaction of mutant I306C(TM5) with either compound reduced its affinity for verapamil . We confirmed that the reduced affinity for verapamil was indeed due to introduction of a charge at position 306 by demonstrating that similar effects were observed when Ile-306 was replaced with arginine or glutamic acid . Mutant I306R showed a 50-fold reduction in affinity for verapamil and very little change in the affinity for rhodamine B or colchicine . MTSES or MTSET modification also affected the cross-linking pattern between pairs of cysteines in the drug-binding pocket . For example, both MTSES and MTSET inhibited cross-linking between I306C(TM5) and I868C(TM10) . Inhibition was enhanced by ATP hydrolysis . By contrast, cross-linking of cysteine residues located outside the drug-binding pocket (such as G300C(TM5)/F770C(TM8)) was not affected by MTSES or MTSET . These results indicate that the drug-binding pocket is accessible to water. Probl Tuberk Bolezn Legk, 2004, (7), 32 - 5 {Changes in drug resistance of Mycobacteria in the simultaneous use of chemotherapy and intravenous infusions of dissolved ozone}; Belianin II et al.; The outcomes of treatment were analyzed in 56 patients with ever-progressive multidrug-resistant pulmonary tuberculosis who had been long isolating Mycobacterium tuberculosis (MBT) . The patients were divided into 2 groups . In the study group (n = 36), 75% isolated MBT resistant to streptomycin (S), isoniazid (I), rifampicin (R), and kanamycin (K) . In this connection, 41.7% of them received only 2 second-line antituberculous drugs and 27.8% took 3 drugs . The control group (n = 20) was comparable with the study group in the rate of bacterial isolation and in the drug resistance of the causative agent . In addition to chemotherapy (CT), dissolved ozone (pO3) was intravenously injected to the patients of the study group twice a week . They received a total of 12 to 55 infusions . Four-month addition of pO3 infusions to CT eliminated the resistance of isolated MBT to I and/or R . MBT became susceptible to I in 38.9% of the patients, R in 16.7%, and to K in 11.2% . By month 4, the isolated MBT became susceptible to I, R, and K in 47.2% . The mechanisms responsible for lowering drug resistance in MBT are discussed . The clinical example shows that patients with multidrug-resistant tuberculosis may be treated with first-line drugs provided that systemic intravenous injection of pO3 is performed. World J Gastroenterol, 2004 Oct 15, 10(20), 3062 - 4 Multidrug resistance reversal in human gastric carcinoma cells by neferine; Cao JG et al.; AIM: To investigate the reversal effect of neferine on multidrug resistance in human gastric carcinoma cell line . METHODS: Cells of a human gastric cancer cells line, SGC7901, and its vincristine (VCR) -resistant variant, SGC7901/VCR, were cultivated with or without neferine and/or VCR . The cytotoxic effect of VCR was evaluated by the MTT assay . Cell apoptosis induced by VCR was determined by flow cytometry (FCM) . The expression of P-glycoprotein (P-gp) and a multidrug-resistance-associated protein (MRP) in cells was examined by immunofluorescence and FCM . RESULTS: Neferine at the concentration from 2.5 micromol/L to 10 micromol/L had no cytotoxicity to SGC7901 cells, and its variant SGC7901/VCR cells . The IC50 of VCR against SGC7901 and SGC7901/VCR cells was 0.059 microg/mL and 2.32 microg/mL, respectively, indicating that SGC7901/VCR cells were 39 times more resistant to VCR than its parent SGC7901 cells . After treatment with neferine at concentrations of 2.5, 5 and 10 micromol/L, the IC(50) of VCR to SGC7901/VCR cell line decreased to 0.340, 0.128 and 0.053 microg/mL, respectively, thus, increased the chemosensitivity by 6.8-, 18.1- and 43.8-fold, respectively . SGC7901/VCR cells were apoptosis resistant to VCR . Neferine (2.5, 5 and 10 micromol/L) promoted the VCR-induced apoptosis of SGC7901/VCR cells in a dose-dependent manner . The expressions of P-gp and MRP were strongly positive in SGC7901/VCR cells, which were significantly down-regulated after treatment with neferine (10 micromol/L) for 24 h . CONCLUSION: Neferine reverses multidrug resistance of human gastric carcinoma SGC7901/VCR cells, which may be associated with the down-regulations of P-gp and MRP expression in SGC701/VCR cells. J Infect Dis, 2004 Oct 15, 190(8), 1456 - 63 Epub 2004 Sep 20. Efficacy of monthly tafenoquine for prophylaxis of Plasmodium vivax and multidrug-resistant P . falciparum malaria; Walsh DS et al.; We assessed monthly doses of tafenoquine for preventing Plasmodium vivax and multidrug-resistant P . falciparum malaria . In a randomized, double-blind, placebo-controlled study, 205 Thai soldiers received either a loading dose of tafenoquine 400 mg (base) daily for 3 days, followed by single monthly 400-mg doses (n = 104), or placebo (n = 101), for up to 5 consecutive months . In volunteers completing follow-up (96 tafenoquine and 91 placebo recipients), there were 22 P . vivax, 8 P . falciparum, and 1 mixed infection . All infections except 1 P . vivax occurred in placebo recipients, giving tafenoquine a protective efficacy of 97% for all malaria (95% confidence interval {CI}, 82%-99%), 96% for P . vivax malaria (95% CI, 76%-99%), and 100% for P . falciparum malaria (95% CI, 60%-100%) . Monthly tafenoquine was safe, well tolerated, and highly effective in preventing P . vivax and multidrug-resistant P . falciparum malaria in Thai soldiers during 6 months of prophylaxis . Cancer Chemother Pharmacol, 2005 Feb, 55(2), 179 - 88 Epub 2004 Sep 16. Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine; Jin J et al.; PURPOSE: The present study aimed to evaluate the MDR reversal activity of bromotetrandrine (BrTet), a bromized derivative of tetrandrine (Tet), in vitro and in vivo . METHODS: Drug sensitivity was determined using the MTT assay . The in vivo effect of Tet was investigated using nude mice grafted with sensitive and resistant KB human epidermoid cancer cells . Doxorubicin (Dox) accumulation was analyzed by fluorospectrophotometry and the protein and mRNA levels of P-glycoprotein (P-gp) were determined by immunocytochemistry and RT-PCR, respectively . RESULTS: BrTet at 0.25, 0.5 and 1 micro M reversed Dox resistance in MDR human breast cancer MCF-7/Dox cells dose-dependently and its potency was greater than that of Tet at the same concentrations . BrTet reversed vincristine (VCR), Dox and paclitaxel resistance in MDR human oral epidermoid carcinoma KBv200 cells as well as innate VCR and Dox resistance in human hepatocellular carcinoma Bel(7402) cells . However, BrTet showed no effect on the IC(50) values of the above-mentioned anticancer drugs in sensitive MCF-7 and KB cells . No reversal effect of BrTet on the cytotoxicity of 5-fluorouracil and cisplatin, non-P-gp substrates, was observed . In nude mice bearing KBv200 xenografts on the left flank and KB xenografts on the right flank, i.p . injection of 5 mg/kg and 10 mg/kg BrTet significantly enhanced the antitumor activity of Dox against KBv200 xenografts with inhibitory rates of 33.0% and 39.2%, while Dox alone inhibited the growth of KBv200 xenografts by only 11.6% . No enhancement by BrTet was seen in KB xenografts . Moreover, BrTet at 5 mg/kg reversed paclitaxel resistance in KBv200 xenografts . Fluorospectrophotometric assay showed that BrTet significantly increased the intracellular accumulation of Dox in MCF-7/Dox cells in a dose-dependent manner . BrTet also inhibited the overexpression of P-gp in MCF-7/Dox cells, but had no effect on mdr1 expression . CONCLUSIONS: BrTet showed significant MDR reversal activity in vitro and in vivo . Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs . BrTet may be a promising MDR modulator for eventual assessment in the clinic. Nat Med, 2004 Oct, 10(10), 1117 - 21 Epub 2004 Sep 19. Modeling epidemics of multidrug-resistant M . tuberculosis of heterogeneous fitness; Cohen T et al.; Mathematical models have recently been used to predict the future burden of multidrug-resistant tuberculosis (MDRTB) . These models suggest the threat of multidrug resistance to TB control will depend on the relative 'fitness' of MDR strains and imply that if the average fitness of MDR strains is considerably less than that of drug-sensitive strains, the emergence of resistance will not jeopardize the success of tuberculosis control efforts . Multidrug resistance in M . tuberculosis is conferred by the sequential acquisition of a number of different single-locus mutations that have been shown to have heterogeneous phenotypic effects . Here we model the impact of initial fitness estimates on the emergence of MDRTB assuming that the relative fitness of MDR strains is heterogeneous . We find that even when the average relative fitness of MDR strains is low and a well-functioning control program is in place, a small subpopulation of a relatively fit MDR strain may eventually outcompete both the drug-sensitive strains and the less fit MDR strains . These results imply that current epidemiological measures and short-term trends in the burden of MDRTB do not provide evidence that MDRTB strains can be contained in the absence of specific efforts to limit transmission from those with MDR disease. Nat Med, 2004 Oct, 10(10), 1111 - 6 Epub 2004 Sep 19. Modeling the emergence of the 'hot zones': tuberculosis and the amplification dynamics of drug resistance; Blower SM et al.; 'Hot zones' are areas that have >5% prevalence (or incidence) of multidrug-resistant tuberculosis (MDRTB) . We present a new mathematical model (the amplifier model) that tracks the emergence and evolution of multiple (pre-MDR, MDR and post-MDR) strains of drug-resistant Mycobacterium tuberculosis . We reconstruct possible evolutionary trajectories that generated hot zones over the past three decades, and identify the key causal factors . Results are consistent with recently reported World Health Organization (WHO) data . Our analyses yield three important insights . First, paradoxically we found that areas with programs that successfully reduced wild-type pansensitive strains often evolved into hot zones . Second, some hot zones emerged even when MDR strains were substantially less fit (and thus less transmissible) than wild-type pansensitive strains . Third, levels of MDR are driven by case-finding rates, cure rates and amplification probabilities . To effectively control MDRTB in the hot zones, it is essential that the WHO specify a goal for minimizing the amplification probability. Chin Med J (Engl), 2004 Sep, 117(9), 1358 - 63 Expression of multidrug resistance-related markers in primary neuroblastoma; Lu QJ et al.; BACKGROUND: Multidrug resistance is associated with a poor prognosis in various human cancers . However, the clinical significance of the expression of multidrug resistance-related markers in neuroblastoma is still on debate . In this study, the effect of the expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and lung resistance protein (LRP) in neuroblastoma was evaluated . METHODS: The streptavidin-biotin immunoperoxidase (SP) technique was used to evaluate the expression of P-gp, MRP, and LRP in 70 cases of untreated primary neuroblastoma . RESULTS: The frequencies of the expression of P-gp, MRP, and LRP were 61.4%, 38.6%, and 24.3%, respectively . A significant positive correlation was observed between P-gp and MRP expression (P=0.001), as well as between LRP and MRP expression (P=0.01) . The rates of expression of P-gp and MRP were higher in tumors from patients aged greater than one year old than in tumors from patients aged less than 1 year old at time of diagnosis (P=0.01 and 0.018, respectively) . MRP expression in tumors that had metastasized was significantly more frequent than in tumors that had not metastasized (P=0.015) . The expression of all tested proteins showed a significant relationship with whether or not the tumor had differentiated (P=0.006, 0.000 or 0.001, respectively) . MRP expression was significantly associated with a reduction in both median survival time and 2-year cumulative survival (P=0.02) . By contrast, P-gp and MRP expression did not correlate with survival . According to Cox regression analysis, only the co-expression of P-gp and MRP had significant prognostic value (relative hazard, 3.513, P=0.033) . CONCLUSIONS: The intrinsic, multidrug resistance of neuroblastoma involves the combined effects of P-gp, MRP, and LRP . MRP expression may be an important factor determining prognosis in neuroblastoma. Chin Med J (Engl), 2004 Sep, 117(9), 1348 - 52 Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA; Li HF et al.; BACKGROUND: Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to eradicate metastatic malignant cells . So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells . METHODS: CIK cells were obtained from peripheral blood and induced by IFN-gamma, anti-CD3 monoclonal antibody, IL-2 and IL-1 . CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation . RT-PCR was used to detect mdr1 mRNA in transfected CIK cells . P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry . Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method . RESULTS: mdr1 mRNA was detected in transfected CIK cells . P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21% - 37% of the total CIK cells after transfection . The IC50 to doxorubicin increased to 22.3 - 45.8 times, and that to colchicines to 6.7 - 11.35 times, as compared to those of untransfected CIK cells . However, the cytotoxic activity to MCF7 cell line remained unaltered . CONCLUSIONS: CIK cells were successfully transfected with mdr1 cDNA by using electroporation . The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells. Int J Oncol, 2004 Oct, 25(4), 1031 - 7 Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells; Okamura H et al.; In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated . RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells . With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells . Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel . Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells . The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells . The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdr1 promoter activity . NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells . Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells. Cancer Gene Ther, 2004 Nov, 11(11), 699 - 706 Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference; Stege A et al.; Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms . The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp) . For stable reversal of "classical" MDR by RNA interference (RNAi) technology, an H1-RNA gene promoter-driven expression vector encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules was constructed . By introduction of anti-MDR1/P-gp shRNA expression vectors into the extremely high drug-resistant human gastric carcinoma cell line EPG85-257RDB, the MDR phenotype was completely reversed . The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells . The data indicate that stable shRNA-mediated RNAi can be tremendously effective in reversing MDR1/P-gp-mediated MDR and is therefore a promising strategy for overcoming MDR by gene therapeutic applications. Cancer Gene Ther, 2004 Nov, 11(11), 707 - 12 Reversal of P-glycoprotein-mediated multidrug resistance with small interference RNA (siRNA) in leukemia cells; Peng Z et al.; The multidrug resistance (MDR) mediated by P-glycoprotein (P-gp), the MDR1 gene product, is one of the major obstacles in leukemia treatment . The present study was designed to explore a MDR1-targeted small interfering RNA (si-MDR1) approach for reversal of P-gp-mediated MDR in the MDR human leukemia cell line k562/A02 . It was found that si-MDR1 significantly inhibited MDR1 expression at both mRNA and protein levels . Depletion of MDR1 by si-MDR1 correlated with the increased sensitivity of the cells to cytotoxic agents and with the enhanced intracellular retention of daunorubicin (DNR) . One base-pair mutated control (si-MDR1-Mut) lost the effect of si-MDR1 on both the degradation of mdr1 mRNA and the reduction of P-gp expression . These findings indicate that siRNA specifically and efficiently interferes with the expression of mdr1 and could be used as a molecularly defined therapeutic approach for MDR in the treatment of leukemia. J Antimicrob Chemother, 2004 Oct, 54(4), 809 - 17 Epub 2004 Sep 16. Prospective audit of bacteraemia management in a university hospital ICU using a general strategy of short-course monotherapy; Corona A et al.; OBJECTIVE: As optimal antibiotic therapy for bacteraemia remains unknown, different strategies have evolved . Routine practice in the University College London Hospitals intensive care unit (ICU) is to use short-course (5-6 days) monotherapy, unless specifically indicated (e.g . endocarditis, osteomyelitis) . We decided to assess this approach for treating community-, hospital-, and ICU-acquired bacteraemia by monitoring clinical response, relapse rate and patient outcome . DESIGN: Six-month prospective observational study from February to July 2000 . SETTING: Mixed medical-surgical tertiary referral ICU . PATIENTS: All 713 patients admitted to the ICU over the study period . MEASUREMENTS AND RESULTS: In total, 102 bacteraemic episodes occurred in 84 patients . Eight (57%) of 14 community-acquired bacteraemias, 22 (79%) of 28 hospital-acquired bacteraemias, and 48 (80%) of 60 ICU-acquired bacteraemias (in 49 patients) were treated with short-course monotherapy . Compared with previous reported studies, these patients had a low rate (23.8%) of death directly attributable to the bacteraemia and a satisfactory clinical response in 72% . Of six relapses (all Gram-negative), four had received combination therapy for severe deep-seated infections . ICU-acquired multidrug-resistant Gram-negative bacteraemias (6.5%) and fungaemias (3%) were also uncommon . No patient discharged from ICU subsequently developed a new bacteraemia relapse, or any long-term complication such as osteomyelitis . CONCLUSIONS: Our general strategy of short-course antibiotic monotherapy for treating bacteraemia in the critically ill appears to provide a satisfactory clinical response, low relapse rate and no long-term complications in a well-defined group of patients . Multicentre studies are warranted to compare short versus long course therapy, and monotherapy versus combination therapy. Am J Physiol Gastrointest Liver Physiol, 2005 Feb, 288(2), G327 - 36 Epub 2004 Sep 16. Role of microtubules in estradiol-17{beta}-D-glucuronide-induced alteration of canalicular Mrp2 localization and activity; Mottino AD et al.; Estradiol-17beta-d-glucuronide (E(2)-17G) induces a marked but reversible inhibition of bile flow in the rat together with endocytic retrieval of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane to intracellular structures . We analyzed the effect of pretreatment (100 min) with the microtubule inhibitor colchicine or lumicholchicine, its inactive isomer (1 mumol/kg iv), on changes in bile flow and localization and function of Mrp2 induced by E(2)-17G (15 mumol/kg iv) . Bile flow and biliary excretion of bilirubin, an endogenous Mrp2 substrate, were measured throughout, whereas Mrp2 localization was examined at 20 and 120 min after E(2)-17G by confocal immunofluorescence microscopy and Western analysis . Colchicine pretreatment alone did not affect bile flow or Mrp2 localization and activity over the short time scale examined (3-4 h) . Administration of E(2)-17G to colchicine-pretreated rats induced a marked decrease (85%) in bile flow and biliary excretion of bilirubin as well as internalization of Mrp2 at 20 min . These alterations were of a similar magnitude as in rats pretreated with lumicolchicine followed by E(2)-17G . Bile flow and Mrp2 localization and activity were restored to control levels within 120 min of E(2)-17G in animals pretreated with lumicolchicine . In contrast, in colchicine-pretreated rats followed by E(2)-17G, bile flow and Mrp2 activity remained significantly inhibited by 60%, and confocal and Western studies revealed sustained internalization of Mrp2 120 min after E(2)-17G . We conclude that recovery from E(2)-17G cholestasis, associated with exocytic insertion of Mrp2 in the canalicular membrane, but not its initial E(2)-17G-induced endocytosis, is a microtubule-dependent process. Insect Mol Biol, 2004 Oct, 13(5), 539 - 48 The dMRP/CG6214 gene of Drosophila is evolutionarily and functionally related to the human multidrug resistance-associated protein family; Tarnay JN et al.; ATP-binding cassette (ABC) transporters are involved in the transport of substrates across biological membranes and are essential for many cellular processes . Of the fifty-six Drosophila ABC transporter genes only white, brown, scarlet, E23 and Atet have been studied in detail . Phylogenetic analyses identify the Drosophila gene dMRP/CG6214 as an orthologue to the human multidrug-resistance associated proteins MRP1, MRP2, MRP3 and MRP6 . To study evolutionarily conserved roles of MRPs we have initiated a characterization of dMRP . In situ hybridization and Northern analysis indicate that dMRP is expressed throughout development and appears to be head enriched in adults . Functional studies indicate that DMRP is capable of transporting a known MRP1 substrate and establishes DMRP as a high capacity ATP-dependent, vanadate-sensitive organic anion transporter. Cas Lek Cesk, 2004, 143(7), 471 - 5 {Multidrug resistance markers monitoring in acute myeloid leukemia cells}; Jankovicova K et al.; BACKGROUND: P-gp, MRP, LRP, Bcl-2, and Bax proteins play an important role in the multidrug resistance of leukemic cells . P-gp, MRP, and LRP proteins decrease the intracellular drug concentration, Bcl-2 and Bax proteins influence the apoptosis process . The overexpression of some of these proteins is associated with poor prognosis of leukemia . The aim of this study was to find the relationship between some multidrug resistance markers and some clinical and laboratory parameters . We compare also P-gp expression between acute myeloid leukemia (AML) FAB subgroups M0-M6 . METHODS AND RESULTS: With use of flow cytometry we measured the expression of P-gp, MRP, LRP, Bcl-2, and Bax proteins in acute myeloid leukemia patients cells . The study proved the association between blast percentage and P-gp protein expression (p=0.015) and the association between blast percentage and Bcl-2 protein expression (p=0.041) . It was also shown the tendency of the LRP protein expression to associate with higher age of patients (p=0.062) . Another correlation between MRP and Bax expression (p=0.006), as well as between LRP and Bax expression was found (p=0.034) . In AML M0 FAB subgroup of patients the trend to higher P-gp protein expression was observed . CONCLUSIONS: The laboratory methodology to multidrug resistance markers detection was introduced . Some relations between multidrug resistance markers playing role in acute myeloid leukemia patients prognosis were suggested. Cell Oncol, 2004, 26(1-2), 3 - 11 Bodipy-FL-verapamil: a fluorescent probe for the study of multidrug resistance proteins; Rosati A et al.; Most of the substances used as fluorescent probes to study drug transport and the effect of efflux blockers in multidrug resistant cells have many drawbacks, such as toxicity, unspecific background, accumulation in mitochondria . New fluorescent compounds, among which Bodipy-FL-verapamil (BV), have been therefore proposed as more useful tools . The uptake of BV has been evaluated by cytofluorimetry and fluorescence microscopy using cell lines that overexpress P-glycoprotein (P388/ADR and LLC-PK(1)/ADR) or MRP (multidrug resistance-related protein) (PANC-1) and clinical specimens from patients . The effect of specific inhibitors for P-glycoprotein (verapamil and vinblastine) or MRP (MK571 and probenecid) has been also studied . BV intracellular concentrations were significantly lower in the two P-glycoprotein overexpressing cell lines in comparison with the parental lines . In addition, verapamil and vinblastine increased the intracellular concentrations of the dye; MK571 and probenecid, two MRP inhibitors, increased BV levels in PANC-1 cells, that express this protein . These findings were confirmed in clinical specimens from patients . Fluorescence microscopy revealed a faint fluorescence emission in P-glycoprotein or MRP expressing cell lines; however, treatment with specific inhibitors significantly increased the fluorescence . BV is a useful tool for studying multidrug resistance proteins with different techniques such as cytofluorimetry and fluorescence microscopy, but does not discriminate between P-glycoprotein and MRP . In comparison with other classic fluorescent probes, the assay with this dye is extremely rapid, simple, not toxic for cells, devoid of fluorescent background, and can be useful in the clinical settings. Proc Natl Acad Sci U S A, 2004 Sep 28, 101(39), 14073 - 8 Epub 2004 Sep 15. Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis; Lewinson O et al.; MdfA is an Escherichia coli multidrug-resistance transporter . Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds . In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH . MdfA-deleted E . coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid . This activity of MdfA requires Na(+) or K(+) . Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na(+)- or K(+)-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon . Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related . Together, the results demonstrate that a single protein has an unprecedented capacity to turn E . coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis. J Biol Chem, 2004 Nov 19, 279(47), 48787 - 93 Epub 2004 Sep 15. EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry; Rotem D et al.; Multidrug transporters recognize and transport substrates with apparently little common structural features . At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges . Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells . Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins . Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein . The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e . electrogenic), the transport of divalent substrate does not (i.e . electroneutral) . Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle . In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones . In the presence of proton gradients, resistance to both types of substrates is detected . The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance. IUBMB Life, 2004 Jun, 56(6), 355 - 9 Induction of drug-metabolizing enzymes and transporters in human bronchial epithelial cells by beclomethasone dipropionate; Kuzuya Y et al.; Inhaled steroids are the most potent anti-inflammatory therapy commonly used in bronchial asthma . There are, however, a small number of asthmatic patients who do not respond to inhaled steroid-treatment . The stimulation of metabolism and excretion of inhaled drugs at bronchial tissues might lead to a decrease in the effect of the drugs, although the molecular mechanism of this resistance is unclear . In this study, we found that beclomethasone dipropionate (BDP) stimulated the expression of mRNAs for uridine 5'-diphosphate glucuronosyl transferase 2B4 and 2B11, and transporters such as multidrug resistance P-glycoprotein, multidrug resistance-associated protein 1 and 2 in cultured bro