Comprehensive validation of a diagnostic strategy for sequencing genes with one or multiple pseudogenes using pseudoxanthoma elasticum as a model.

Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue. For this reason, their presence is often considered troublesome in molecular diagnostics. In pseudoxanthoma elasticum (PXE), a disease predominantly caused by mutations in ATP-binding cassette family C member 6 (ABCC6), the presence of two pseudogenes complicates the analysis of sequence data. With whole-exome sequencing (WES) becoming the standard of care in molecular diagnostics, we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6. We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities, contrary to WES. We propose a time- and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE, which is highly automatable.

Role of Membrane Lipid Rafts in MRP4 (ABCC4) Dependent Regulation of the cAMP Pathway in Blood Platelets.

BACKGROUND: Platelet cytosolic cyclic adenosine monophosphate (cAMP) levels are balanced by synthesis, degradation, and efflux. Efflux can occur via multidrug resistant protein-4 (MRP4; ABCC4) present on dense granule and/or plasma membranes. As lipid rafts have been shown to interfere on cAMP homeostasis, we evaluated the relationships between the distribution and activity of MRP4 in lipid rafts and cAMP efflux. METHODS: Platelet activation and cAMP homeostasis were analyzed in human and wild-type or MRP4-deleted mouse platelets in the presence of methyl-ß-cyclodextrin (MßCD) to disrupt lipid rafts, and of activators of the cAMP signalling pathways. Human platelet MRP4 and effector proteins of the cAMP pathway were analyzed by immunoblots in lipid rafts isolated by differential centrifugation. RESULTS: MßCD dose dependently inhibited human and mouse platelet aggregation without affecting per se cAMP levels. An additive inhibitory effect existed between the adenylate cyclase (AC) activator forskolin and MßCD that was accompanied by an overincrease of cAMP, and which was significantly enhanced upon MRP4 deletion. Finally, an efflux of cAMP out of resting platelets incubated with prostaglandin E1 (PGE1) was observed that was partly dependent on MRP4. Lipid rafts contained a small fraction (≈15%) of MRP4 and most of the inhibitory G-protein Gi, whereas Gs protein, AC3, and phosphodiesterases PDE2 and PDE3A were all present as only trace amounts. CONCLUSION: Our results are in favour of part of MRP4 present at the platelet surface, including in lipid rafts. Lipid raft integrity is necessary for cAMP signalling regulation, although MRP4 and most players of cAMP homeostasis are essentially located outside rafts.

Mesothelioma Biomarkers: Discovery in Search of Validation.

Malignant pleural mesothelioma (MPM) is an asbestos-related neoplasm that can only be treated successfully when correctly diagnosed and treated early. The asbestos-exposed population is a high-risk group that could benefit from sensitive and specific blood- or tissue-based biomarkers. We review recent work with biomarker development in MPM and literature of the last 20 years on the most promising blood- and tissue-based biomarkers. Proteomic, genomic, and epigenomic platforms are covered. SMRP is the only validated blood-based biomarker with diagnostic, monitoring and prognostic value. To strengthen development and testing of MPM biomarkers, cohorts for validation must be established by enlisting worldwide collaborations.

Increased expressions of cellular ATP-binding cassette transporters may be a promising diagnostic marker for colorectal cancer.

OBJECTIVES: To measure the blood expression levels of related drug-resistant ATP-binding cassette (ABC) transporters in colorectal cancer (CRC) patients and to assess these examined transporters for whether they present signi cant expression in connection with the tumor appearance of CRC. METHODS: In this case-control study, the messenger ribonucleic acids were isolated from the blood of 62 CRC patients who were recruited from King Abdulaziz University Hospital Oncology Clinic and 46 controls from King Fahad General Hospital Blood Bank (Jeddah, Saudi Arabia) from September 2016 to March 2017. The Biomedical Ethics Unit at King Abdulaziz University, Jeddah, Saudi Arabia approved this study. The expressions of ABC transporters were measured using quantitative polymerase chain reaction. GraphPad Prism 5 and REST 2009 Software were used to correlate the expressions with clinicopathological independent stages and body mass index. A p-value of less than 0.05 was considered significant. RESULTS: The results showed that the 3 ABC transporters, particularly ABCC1 (p less than 0.0001), were highly expressed in the blood of CRC patients compared with controls. However, none of the 3 transporters was related to the progression of CRC, age, gender, or body mass index. CONCLUSION: The expressions of ABC transporters were found to be significantly higher in CRC patients, and they may act as diagnostic markers and should potentially be tested for their contribution to drug sensitivity in CRC patients.

microRNA-139-5p confers sensitivity to antiepileptic drugs in refractory epilepsy by inhibition of MRP1.

AIM: Drug resistance is an intractable issue urgently needed to be overcome for improving efficiency of antiepileptic drugs in treating refractory epilepsy. microRNAs (miRNAs) have been proved as key regulators and therapeutic targets in epilepsy. Accordingly, the aim of the present study was to identify a novel differentially expressed miRNA which could improve the efficiency of antiepileptic drugs during the treatment of refractory epilepsy. METHODS AND RESULTS: Serum samples were collected from children with refractory epilepsy. An in vivo refractory epilepsy model was developed in SD rats by electrical amygdala kindling. We identified that miR-139-5p was decreased and multidrug resistance-associated protein 1 (MRP1) was remarkably upregulated in the serum samples from children with refractory epilepsy and the brain tissues from rat models of refractory epilepsy. After phenobarbitone injection in rat models of refractory epilepsy, the after discharging threshold in kindled amygdala was detected to screen out drug-resistant rats. Dual-luciferase reporter gene assay demonstrated that MRP1 was a target of miR-139-5p. In order to evaluate the effect of miR-139-5p/MRP1 axis on drug resistance of refractory epilepsy, we transfected plasmids into the hippocampus of drug-resistant rats to alter the expression of miR-139-5p and MRP1. TUNEL staining and Nissl staining showed that miR-139-5p overexpression or MRP1 downregulation could reduce the apoptosis and promote survival of neurons, accompanied by alleviated neuronal damage. CONCLUSION: Collectively, these results suggest an important role of miR-139-5p/MRP1 axis in reducing the resistance of refractory epilepsy to antiepileptic drugs.

Platelet miRNA-26b down-regulates multidrug resistance protein 4 in patients on chronic aspirin treatment.

BACKGROUND: Multidrug resistance protein-4 (MRP4) is an ATP binding cassette membrane transporter, actively involved in the efflux of important pharmacological and physiological molecules. Recently, its over-expression has been associated with reduced aspirin (ASA) efficacy after by-pass surgery. MicroRNAs (miRNAs) are small molecules of non-coding RNA involved in the regulation of many physiological and pathophysiological pathways, are abundant in platelets, and can be modulated by several drugs. In the present study, we assessed the role of platelet miRNAs in modulating MRP4 function in response to ASA. METHODS: MRP4 mRNA expression has been analyzed by RealTime PCR in platelets from patients on chronic ASA treatment versus a control group. A panel of miRNAs was run on the pool of each cohort. MiRNAs validation was performed by RealTime PCR. To verify whether MRP4 is the target of miR-26b also in platelets, miR-26b was transfected in platelet and DAMI cells with miRNA mimic technology. MRP4 expression was evaluated by flow cytometry and western blotting. RESULTS: We observed a higher MRP4 mRNA expression in platelets of patients under ASA treatment compared to the control group (p<0.005). MiR-26b was found significantly down-regulated in patients on ASA treatment as compared to control group (P < 0.005) and this was validated by RealTime PCR. MiR-26b transfection in platelets was associated to a significant down-regulation of MRP4 expression (p<0.005). MiR-26b transfection in DAMI cells was associated to a significant reduction of MRP4 mRNA and protein level (P < 0.05). CONCLUSION: We found that miR-26b is down-regulated in platelets in patients on chronic ASA treatment. Importantly, miR-26b can specifically downregulate MRP4. Thus, miR-26b seems to be involved in MRP4 modulation and may contribute to ASA resistance.

Pathogenic variants in the ABCC6 gene are associated with an increased risk for ischemic stroke.

Ischemic stroke causes a high mortality and morbidity worldwide. It results from a complex interplay of incompletely known environmental and genetic risk factors. We investigated the ABCC6 gene as a candidate risk factor for ischemic stroke because of the increased ischemic stroke incidence in the autosomal recessive disorder pseudoxanthoma elasticum, caused by biallelic pathogenic ABCC6 variants, the higher cardiovascular risk in heterozygous carriers and the established role of ABCC6 dysfunction in myocardial ischemia. We established segregation of a known pathogenic ABCC6 variant (p.[Arg1314Gln]) in 11/19 family members of an ischemic stroke patient in a large multigenerational family suffering from ischemic stroke and/or cardiovascular disease at a relatively young age. In an independent case-control study in 424 ischemic stroke patients and 250 healthy controls, pathogenic ABCC6 variants were 4.9 times more frequent (P = 0.036; 95% CI 1.11-21.33) in the ischemic stroke patient cohort. To study cellular consequences of ABCC6 deficiency in the brain, immunostaining of brain sections in Abcc6-deficient mice and wild-type controls were performed. An upregulation of Bmp4 and Eng and a downregulation of Alk2 was identified in Abcc6-/- mice, suggesting an increase in apoptosis and angiogenesis. As both of these processes are induced in ischemia, we propose that a pro-ischemic state may explain the higher risk to suffer from ischemic stroke in patients carrying a pathogenic ABCC6 variant, as this may lower the threshold to develop acute ischemic events in these patients. In conclusion, this study identified heterozygous ABCC6 variants as a risk factor for ischemic stroke. Further, dysregulation of Bmp (Bmp4, Alk2) and Tgfß (Eng) signaling in the brain of Abcc6-/- mice could lead to a pro-ischemic state, lowering the threshold to develop acute ischemic events. These data demonstrate the importance of a molecular analysis of the ABCC6 gene in patients diagnosed with cryptogenic ischemic stroke.

Myeloid related proteins are up-regulated in autoimmune thyroid diseases and activate toll-like receptor 4 and pro-inflammatory cytokines in vitro.

PURPOSE: Myeloid-related protein (MRP) family plays an important role in the promotion of cell proliferation and the production of inflammatory cytokines. We investigated the expression of MRP6, MRP8 and MRP14 in thyroid tissues, serum, and peripheral blood monocular cells (PBMCs) in patients with autoimmune thyroid diseases (AITD). METHOD: The expression of MRP6, MRP8, and MRP14 was investigated using immunohistochemical staining and quantitative real-time polymerase chain reaction in the thyroid glands of 7 patients with Graves' disease (GD), 8 with Hashimoto's thyroiditis (HT), and 7 healthy controls (HC). The serum levels of MRP8/MRP14 complex and MRP6 were investigated in 30 patients with GD, 36 with HT, and 30 with HC. The mRNA expression of MRP proteins in PBMCs was also explored. PBMCs from each group were incubated with MPRs and their effect on Toll-like receptor 4(TLR4) expression and their effect on the levels of the pro-inflammatory cytokines in supernatant were analyzed upon incubating with TLR4 and signaling pathways inhibitors. RESULTS: Serum levels of MRP8/MRP14 and MRP6 were up-regulated in patients with AITD. In addition, mRNA expression of MRP proteins in PBMCs and the thyroid gland was markedly elevated in AITD patients. MRP6 and MPR8 promoted the secretion of TNF-α and IL-6 in cultured PBMCs, and this elevation was more pronounced in AITD patients; we also found that this up-regulation was regulated by TLR4/phosphoinositide 3-kinase/nuclear factor-κB signaling pathway. CONCLUSION: The expression of MRP proteins was elevated in AITD patients. Therefore, an MRP-TLR4 dependent signaling may play an important role in the pathogenesis of AITD.

Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1ß). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1ß, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

MDR-1 and MRP-1 activity in peripheral blood leukocytes of rheumatoid arthritis patients.

BACKGROUND: Rheumatoid Arthritis is a chronic disease leading to decreased quality of life with a rather variable response rate to Disease Modifying Anti Rheumatic Drugs. Methotrexate (MTX) is the gold standard therapy in Rheumatoid Arthritis. The Multidrug resistance Related Protein and Multi Drug Resistance protein 1, also called P-glycoprotein-170 transporters can alter the intracellular concentration of different drugs. Methotrexate is an MRP1 substrate and thus the functional activity of MRP1 might have a clinical impact on the efficiency of the Methotrexate-therapy in Rheumatoid Arthritis. METHODS: We have compared the functional Multidrug Activity Factors (MAF) of the MDR1 and MRP1 transporters of Peripheral Blood Leukocytes of 59 Rheumatoid Arthritis patients with various response rate to MTX-therapy (MTX-responder, MTX-resistant and MTX-intolerant RA-groups) and 47 non-RA controls in six different leukocyte subpopulations (neutrophil leukocytes, monocytes, lymphocytes, CD4+, CD8+ and CD19+ cells). There was a decreased MAF of RA patients compared to non- Rheumatoid Arthritis patients and healthy controls in the leukocyte subpopulations. There was a significant difference between the MAF values of the MTX-responder and MTX intolerant groups. But we have not found significant differences between the MAF values of the MTX-responder and MTX-resistant Rheumatoid Arthritis -groups. RESULTS: Our results suggest that MDR1 and MRP1 functional activity does not seem to affect the response rate to MTX-therapy of Rheumatoid Arthritis-patients, but it might be useful in predicting MTX-side effects. We have demonstrated the decreased functional MDR-activity on almost 60 Rheumatoid Arthritis patients, which can be interpreted as a sign of the immune-suppressive effect of the MTX-treatment.

Sulfonation of raloxifene in HEK293 cells overexpressing SULT1A3: Involvement of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in excretion of sulfate metabolites.

Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 µM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 µM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular excretion of the raloxifene sulfates was mainly mediated by BCRP and MRP4.

Combined circulating epigenetic markers to improve mesothelin performance in the diagnosis of malignant mesothelioma.

OBJECTIVES: Malignant mesothelioma (MM) is a highly aggressive tumor with poor prognosis. A major challenge is the development and application of early and highly reliable diagnostic marker(s). Serum biomarkers, such as 'soluble mesothelin-related proteins' (SMRPs), is the most studied and frequently used in MM. However, the low sensitivity of SMRPs for early MM limits its value; therefore, additional biomarkers are required. In this study, two epigenetically regulated markers in MM (microRNA-126, miR-126, and methylated thrombomodulin promoter, Met-TM) were combined with SMRPs and evaluated as a potential strategy to detect MM at an early stage. MATERIALS AND METHODS: A total of 188 subjects, including 45 MM patients, 99 asbestos-exposed subjects, and 44 healthy controls were prospectively enrolled, serum samples collected, and serum levels of SMRPs, miR-126 and Met-TM evaluated. Logistic regression analysis was performed to evaluate the diagnostic value of the three biomarkers. Using this approach, the performance of the '3-biomarker classifier' was tested by calculating the overall probability score of the MM and control samples, respectively, and the ROC curve was generated. RESULTS AND CONCLUSION: The combination of the three biomarkers was the best predictor to differentiate MM patients from asbestos-exposed subjects and healthy controls. The accuracy and cancer specificity was confirmed in a second validation cohort and lung cancer population. We propose that the combination of the two epigenetic biomarkers with SMRPs as a diagnosis for early MM overcomes the limitations of using SMRPs alone.

Lenalidomide treatment induced the normalization of marker protein levels in blood plasma of patients with 5q-myelodysplastic syndrome.

A specific type of myelodysplastic syndrome (MDS) is associated with isolated deletion on the long arm of chromosome 5, i.e., 5q-syndrome (del(5q)). The treatment approaches for MDS del(5q) include the immunomodulating drug lenalidomide (LEN). Thirteen MDS del(5q) patients were included in this study. We found elevated activities of lactate dehydrogenase (LDH) and matrix metalloproteinase 9 (MMP-9) in the blood plasma of MDS del(5q) patients as compared with healthy controls. This was stabilized to control values after LEN treatment. Similar behavior we registered also for the thioredoxin and calnexin contents in BP. Peripheral blood mononuclear cells (PBMC) from patients with MDS del(5q) prior to and after treatment with LEN did not exhibit any detectable amount of P-glycoprotein (P-gp) gene transcript. However, we detected a measurable amount of multidrug resistance associated protein 1 (MRP1) mRNA in PBMCs from three patients prior to LEN treatment and in one patient during LEN treatment but it was not present prior to treatment. These data indicated on usefulness of applied protein markers estimation for monitoring of MDS del(5q) patient treatment effectiveness by LEN. Expression of MRP1 seems to be independent on LEN treatment and reflects probably the molecular variability in the ethiopathogenesis of MDS del(5q).

Impact of parenteral nutrition versus fasting on hepatic bile acid production and transport in a rabbit model of prolonged critical illness.

BACKGROUND: Cholestatic liver dysfunction frequently occurs during critical illness. Administration of parenteral nutrition (PN) is thought to aggravate this. Underlying mechanisms are not clear. METHODS: In a burn model of prolonged critical illness, rabbits were randomized to a nutritional strategy either accepting caloric deficits (fasted, n = 11) or covering caloric needs by PN (fed, n = 10). At baseline and after 7 days of critical illness, markers of hepatotoxicity, circulating bile acids, and the hepatobiliary transport system were studied. RESULTS: Fasted animals had lower circulating alanine aminotransferase/aspartate aminotransferase levels than did the fed animals at day 7. Compared with baseline values, fed animals displayed lower serum unconjugated cholic acid (CA) and deoxycholic acid (DCA) levels. Unconjugated DCA remained unaltered in fasted animals. Unconjugated lithocholic acid was increased comparably in all animals, whereas hyodeoxycholic acid was not altered. In contrast, fasting induced a shift from unconjugated CA and DCA to glyco-CA and glyco-DCA. Total bile acids did not correlate with the bile acid-producing enzyme CYP7A1, but with the basolateral efflux transporter MRP3. Fasting increased protein expression of the basolateral (MRP3) and the canalicular (BSEP) transporter, whereas the canalicular efflux pump MRP2 was suppressed. Gene expression levels of the nuclear receptor farnesoid X receptor were lower with fasting and correlated inversely with MRP3. The heterodimer partner of farnesoid X receptor, retinoid X receptor α, was increased with fasting and correlated positively with MRP3. CONCLUSIONS: During prolonged critical illness, withholding PN improved markers for hepatocyte injury and accentuated bile acid transport toward the blood. This suggests that the latter is an adaptive rather than a dysfunctional feedback to illness.

A predictive equation to adjust for clinical variables in soluble mesothelin-related protein (SMRP) levels.

BACKGROUND: Malignant mesothelioma (MM) is an aggressive pleural tumor which is difficult to diagnose in its early stages. Thus, biomarkers for MM including soluble mesothelin-related protein (SMRP) are currently an area of intense research interest. However, SMRP is affected by several factors other than malignancy which need to be taken into account in the individual patient. This study aimed to evaluate factors required to adjust SMRP levels for such variables and produce a useful prediction equation for clinical application. METHODS: Serum SRMP levels were measured in 535 subjects formerly exposed to asbestos and silica, including many with asbestos-related disorders (ARDs). Linear regression analyses were used to quantify the strength and " direction " of the relationship between SMRP and several independent variables,and 2 × 2 tables were used to determine the diagnostic accuracy of SMRP levels, taking into account clinical variables. RESULTS: SMRP levels were affected by age and glomerular filtration rate (GFR), which were strong confounders in this study. Body mass index (BMI) was also an initial confounder but lost significance after other factors were taken into account.SMRP was also affected by smoking. Poor sensitivity (15.1 % )for SMRP values among subjects with non-malignant asbestos-related disorders was found when compared to currently healthy subjects with a history of asbestos exposure. CONCLUSIONS: The present study proposes an equation based on age and GFR to improve diagnostic accuracy of SMRP.The poor sensitivity of SMRP found in this study suggests that further work is needed to fi nd new candidate biomarkers for diagnosing early stage MM.

Reduction of cAMP and cGMP inhibitory effects in human platelets by MRP4-mediated transport.

Cyclic nucleotide-dependent inhibition of platelets represents the most important physiological way to limit thrombus formation. cAMP and cGMP increase in platelets as a consequence of prostacyclin and nitric oxide production by endothelial cells and act through PKA and PKG, respectively. The cytosolic concentration of cyclic nucleotides in platelets is regulated by AC- and GC-dependent synthesis and PDE-dependent degradation. In some cells cyclic nucleotides are eliminated also through MRP4/5/8-dependent efflux. As only MRP4 is expressed in platelets, at high levels in dense granules, we determined its role in the elimination of cyclic nucleotides from platelet cytosol. We studied the effects of MRP4 inhibition on cAMP/cGMP effects in platelets. Cyclic nucleotide inhibitory effects triggered by cAMP and cGMP-elevating agents on platelet aggregation are strongly enhanced by MRP4 inhibition and so is cyclic nucleotide-dependent phosphorylation of the common substrate VASP. MRP4 inhibition decreases cAMP concentration in platelet granules and both cAMP and cGMP compete with an established substrate of MRP4 (fluo-cAMP) for entrance in granules. Here we provide the first evidence of the transport of cyclic nucleotides mediated by MRP4 as part of their physiological mechanism of elimination in human platelets, which might represent a novel target to increase cyclic nucleotide-dependent inhibition.

Multiplex analysis of tumor multidrug-resistance genes expression with photonic suspension array.

An easy-operated suspension array based on silica colloidal crystal beads is developed for multiplex analysis of tumor multidrug-resistance genes expression, such as multidrug resistance 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1), and potentially single nucleotide polymorphism. In order to obtain high fluorescence intensity, controlled PCR was used to amplify targets at the samples pretreatment stage. By optimizing the conditions a hybridization procedure, which is similar to nucleic acids analysis with binary probes, was established. Small amounts of analytes 10(-19) M could be detected by the method. The K562 cell, human myeloma cell, and its multidrug-resistance string, adriamycin-selected P-glycoprotein-overexpressed K562/A02, were analyzed by using an established procedure to validate feasibility. Clinical blood samples were detected by our method and real-time PCR simultaneously to validate accuracy. Moreover, when combined with multiplex controlled PCR, the method successfully meets the requirements of multiplex analysis. Hence, the method presented is a good method for multiplex analysis of tumor multidrug-resistance genes expression.

Association of multidrug resistance-associated protein 2 single nucleotide polymorphism rs12762549 with the basal plasma levels of phase II metabolites of isoflavonoids in healthy Japanese individuals.

OBJECTIVES: Multidrug resistance-associated protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter that mediates the efflux of anionic drugs and phase II metabolites. Our aim was to elucidate the impact of a single nucleotide polymorphism, rs12762549 (G>C), on the in-vivo activity of MRP2. METHODS: Plasma specimens collected from 18 healthy volunteers were subjected to an untargeted metabolomic analysis using liquid chromatography-mass spectrometry. The role of MRP2 in the disposition of substances of interest was then examined in vivo in Mrp2-deficient mutant rats (Eisai hyperbilirubinemic rats; EHBRs) and in vitro in human MRP2-expressing membrane vesicles. RESULTS: A multivariate analysis model using liquid chromatography-mass spectrometry data sets successfully differentiated the GG and the CC genotypes of rs12762549. The C allele is associated with the basal plasma levels of the five phase II metabolites of genistein and dihydrogenistein. Such phase II metabolites were also accumulated in EHBR, compared with normal rats, partly because of the reduced biliary excretion. Following oral administration of genistein and daidzein, the plasma concentrations of total genistein and total daidzein, which were mainly accounted for by sulfoglucuronide conjugates, were markedly higher in EHBR than in normal rats. ATP-dependent uptake of sulfoglucuronides and glucuronides of the isoflavonoids was observed only in MRP2-expressing membrane vesicles. CONCLUSION: MRP2 plays a significant role in preventing the accumulation of phase II metabolites of isoflavonoids. The rs12762549 is associated with an interindividual difference in the plasma levels of MRP2 substrates, phase II metabolites of isoflavonoids, suggesting that this single nucleotide polymorphism is associated with variations in in-vivo MRP2 activity.

[Expression of P-glycoprotein and multidrug resistance-associated protein in peripheral blood mononuclear cells from multidrug resistant tuberculosis patients].

OBJECTIVE: To evaluate the expression of P-glycoprotein (P-pg) and multidrug resistance-associated protein (MRP) mRNA levels in peripheral blood mononuclear cells from multidrug resistant tuberculosis (MDR-TB) patients. METHODS: The subjects of this study included 3 groups: a non-TB control group, a TB control group and a MDR-TB group. The 31 subjects in the non-TB control group were staff from Wuhan Tuberculosis Prevention and Treatment Institute. The 55 cases in the TB control group were in-patients during September 2004 to December 2007 who were diagnosed as having pulmonary tuberculosis. The 89 cases in the MDR-TB group were in-patients during the same period, but who were diagnosed as having MDR-TB. Peripheral mononuclear cells were isolated and mRNA levels of P-pg and MRP were measured by real-time PCR. Comparisons of the data between different groups were performed using one-way ANOVA, and SNK q was used for comparison between 2 groups. RESULTS: There was no significant difference in the relative P-pg mRNA levels among the MDR-TB group (1.34 ± 0.32), the non-TB control group (1.05 ± 0.16) and the TB control group (1.12 ± 0.23), F = 0.536, P > 0.05. The relative MRP mRNA level (3.45 ± 0.43) was the highest in the MDR-TB group (3.45 ± 0.43), as compared to the TB control group (1.23 ± 0.34) and the non-TB control group (1.04 ± 0.12), F = 24.241, P < 0.05. CONCLUSION: Higher expression of MRP in peripheral mononuclear cells might be related to multidrug resistance in MDR-TB patients.