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1.  Measuring Vincristine-Induced Peripheral Neuropathy in Children with Acute Lymphoblastic Leukemia 
Cancer nursing  2013;36(5):E49-E60.
Vincristine-induced peripheral neuropathy (VIPN) is difficult to quantify in children.
The study objective was to examine the reliability, validity, and clinical feasibility of several VIPN measures for use in children with acute lymphoblastic leukemia.
Children (N = 65) aged 1–18 years receiving vincristine at four academic centers participated in the study. Baseline and pre-vincristine VIPN assessments were obtained using the Total Neuropathy Score-Pediatric Vincristine (TNS-PV), the National Cancer Institute Common Terminology Criteria for Adverse Events, the Balis grading scale, and the FACES pain scale. TNS-PV scores (n = 806) were obtained over 15 weeks. Blood was obtained at several time-points to quantify pharmacokinetic parameters.
Cronbach’s alpha for a reduced TNS-PV scale was 0.84. TNS-PV scores correlated with cumulative vincristine dosage (r = 0.53, p = 0.01), pharmacokinetic parameters (r = 0.41, p = 0.05), and grading scale scores (r = 0.46 – 0.52; p = 0.01). FACES scores correlated with the TNS-PV neuropathic pain item (r = 0.48; p = 0.01), and were attainable in all ages. A 2-item V-Rex score (vibration and reflex items) was the most responsive to change (es 0.65, p < 0.001). TNS-PV scores were attainable in 95% of children ≥ 6 years.
The TNS-PV is reliable and valid for measuring VIPN. It is sensitive to change over time (15 weeks) and feasible for use in children ≥ 6 years of age.
Implications for Practice
The TNS-PV may be a useful tool for assessing vincristine toxicity in children with acute lymphoblastic leukemia.
PMCID: PMC3951303  PMID: 23842524
2.  Warfarin Pharmacogenomics in Children 
Pediatric blood & cancer  2013;60(9):1402-1407.
Warfarin is the most commonly used oral anticoagulant worldwide. Warfarin has a narrow therapeutic index, requiring frequent monitoring of the INR to achieve therapeutic anticoagulation. The role of pharmacogenomics in warfarin disposition and response has been well-established in adults, but remains unclear for pediatric patients. In this review, we focus on the important CYP2C9 and VKORC1 variants involved in warfarin response, our current understanding of warfarin disposition and pharmacogenomics, and recent warfarin pharmacogenetic studies in pediatric patients. Finally, we discuss the need for future prospective pediatric studies and the clinical implications of developing pharmacogenetic-based dosing algorithms in children.
PMCID: PMC3786327  PMID: 23682017
warfarin; pharmacogenomics; CYP2C9; VKORC1
3.  Effect of drug transporter genotypes on pravastatin disposition in European- and African-American participants 
Pharmacogenetics and genomics  2007;17(8):647-656.
Our aims were to evaluate the effects of polymorphisms in the hepatic drug uptake transporter organic anion transporting polypeptide 1B1 (OATP1B1, SLCO1B1) and efflux transporters multidrug resistance-associated protein 2 (MRP2, ABCC2), bile salt export pump (BSEP, ABCB11), and breast cancer-related protein (BCRP, ABCG2) on single-dose pravastatin pharmacokinetics in healthy European- and African-American participants.
The pharmacokinetics of a single oral 40mg dose of pravastatin was determined in 107 participants (69 European-Americans and 38 African-Americans). Participants were genotyped for known OATP1B1, MRP2, BSEP, and BCRP polymorphisms. Baseline serum total and unconjugated plasma bilirubin concentrations were also determined.
OATP1B1 genotypes were ethnicity-dependent with a 521C allele frequency of ~15% in European-Americans and ~1% in African-Americans. SLCO1B1 521TC genotype was associated with significantly higher pravastatin area under the curve [AUC(0–5)] (P =0.01) and Cmax values (P< 0.05). When analyzed by diplotype, SLCO1B1*1a/*15 (N =8) participants exhibited 45 and 80% higher AUC values than SLCO1B1*1a/*1a (N=29) (P=0.013) and SLCO1B1*1b/*1b (N=34) (P=0.001) carriers, respectively. SLCO1B1*15/*15 (N=2) participants exhibited 92 and 149% higher AUC values than SLCO1B1*1a/*1a (P=0.017) and SLCO1B1*1b/*1b (P= 0.011) carriers, respectively. European-Americans had significantly higher plasma pravastatin AUC(0–5) (P =0.01) and Cmax values (P=0.009) than African-Americans. Neither ABCC2, ABCB11, nor ABCG2 genotypes were associated with differences in pravastatin pharmacokinetics. We did not observe an effect of SLCO1B1 genotype on baseline total or unconjugated bilirubin levels.
SLCO1B1 genotype, in particular the 521C allele, had a significant effect on the pharmacokinetics of pravastatin. Even when adjusted for the presence of the SLCO1B1 521C or 388G variant allele, European-Americans demonstrated significantly higher pravastatin AUC and Cmax values than African-Americans.
PMCID: PMC4063287  PMID: 17622941
ABCB11; ABCC2; ABCG2; BCRP; bilirubin; BSEP; MRP2; OATP1B1; pharmacokinetics; pravastatin; SLCO1B1; transporter
4.  Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe 
Pharmacogenetics and genomics  2008;18(5):439-448.
Breast cancer resistance protein (BCRP) is an efflux transporter expressed in tissues that act as barriers to drug entry. Given that single nucleotide polymorphisms (SNPs) in the ABCG2 gene encoding BCRP are common, the possibility exists that these genetic variants may be a determinant of interindividual variability in drug response. The objective of this study is to confirm the human BCRP-mediated transport of sulfasalazine in vitro, evaluate interindividual variation in BCRP expression in human intestine and to determine the role of ABCG2 SNPs to drug disposition in healthy patients using sulfasalazine as a novel in vivo probe. To evaluate these objectives, pinch biopsies were obtained from 18 patients undergoing esophagogastro–duodenoscopy or colonoscopy for determination of BCRP expression in relation to genotype. Wild-type and variant BCRP were expressed in a heterologous expression system to evaluate the effect of SNPs on cell-surface trafficking. A total of 17 healthy individuals participated in a clinical investigation to determine the effect of BCRP SNPs on sulfasalazine pharmacokinetics. In vitro, the cell surface protein expression of the common BCRP 421 C>A variant was reduced in comparison with the wild-type control. Intestinal biopsy samples revealed that BCRP protein and mRNA expression did not significantly differ between patients with 34GG/421CC versus patients with 34GG/421CA genotypes. Remarkably, in subjects with 34GG/421CA genotype, sulfasalazine area under the concentration-time curve was 2.4-fold greater compared with 34GG/421CC subjects (P<0.05). This study links commonly occurring SNPs in BCRP with significantly increased oral sulfasalazine plasma exposure in humans. Accordingly, sulfasalazine may prove to have utility as in vivo probe for assessing the clinical impact of BCRP for the disposition and efficacy of drugs.
PMCID: PMC4043148  PMID: 18408567
breast cancer resistance protein/ATP binding cassette; drug transport; member 2; pharmacogenetics; pharmacokinetics; subfamily G (WHITE); sulfasalazine
Pediatric Transplantation  2012;16(5):438-442.
Engraftment and overall survival after umbilical cord blood transplant is highly dependent on the total nucleated cell count (TNC). The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant is not well described. To investigate the amount of cell loss after washing and its impact on major outcomes compared to pre-cryopreserved TNC, we analyzed data from patients prospectively enrolled on a National Heart, Lung and Blood Institute sponsored cord blood transplant study between 1999 and 2003. There were 310 patients ≤18 years of age with malignant (N=218) or non-malignant (N=92) disease enrolled on this trial. Only single cord blood units were used. All cord blood units were thawed and washed using an identical process. The median TNC after thawing and washing (PTW) was 5.43 × 107/kg (79% recovery of cells). The cumulative incidence of neutrophil engraftment was significantly higher in patients receiving a PTW TNC ≥ 2.5 × 107/kg (p=0.01). The cumulative incidence of transplant related mortality was higher among patients receiving post thaw and wash TNC < 2.5 × 107/kg (p=0.039). In conclusion, receiving a PTW TNC of < 2.5 × 107/kg resulted in worse neutrophil engraftment and increased transplant related mortality compared to a PTW TNC of ≥ 2.5 × 107/kg
PMCID: PMC3391317  PMID: 22533817
Cord blood transplant; Pediatrics; Cell dose; Washing and Thawing
7.  Interaction of three regio-specific amino acid residues is required for OATP1B1 gain of OATP1B3 substrate specificity 
Molecular Pharmaceutics  2012;9(4):986-995.
The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds including bile acids, steroid conjugates, hormones, and drugs including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.
PMCID: PMC3319192  PMID: 22352740
hepatic uptake transport; organic anion transporting polypeptide; site-directed mutagenesis
8.  CCDC-55 is required for larval development and distal tip cell migration in C. elegans 
Mechanisms of Development  2012;128(11-12):548-559.
The C. elegans distal tip cells (DTCs) are an in vivo model for the study of developmentally regulated cell migration. In this study we characterize a novel role for CCDC-55, a conserved coiled-coil domain containing protein, in DTC migration and larval development in C. elegans. Although animals homozygous for a probable null allele, ccdc-55(ok2851), display an early larval arrest, RNAi depletion experiments allow the analysis of later phenotypes and suggest that CCDC-55 is needed within the DTC for migration to cease at the end of larval morphogenesis. The ccdc-55 gene is found in an operon with rnf-121 and rnf-5, E3 ubiquitin ligases that target cell migration genes such as the β-integrin PAT-3. Genetic interaction studies using RNAi depletion and the deletion alleles rnf-121(ok848) and rnf-5(tm794) indicate that CCDC-55 and the RNF genes act at least partially in parallel to promote termination of cell migration in the adult DTC.
PMCID: PMC3295921  PMID: 22285439
coiled-coil domain containing proteins; E3 ubiquitin ligases; C. elegans; cell migration
9.  t(17;19) in Children with Acute Lymphocytic Leukemia: A Report of 3 Cases and a Review of the Literature 
Case Reports in Hematology  2013;2013:563291.
Several cytogenetic abnormalities identified in patients with childhood acute lymphocytic leukemia (ALL) have been associated with a poor prognosis. There are several case reports in the literature describing t(17;19) in children with ALL. This translocation has been associated with hypercalcemia, coagulopathy, and poor outcome. We present three cases of ALL with t(17;19) treated at our institution and review the outcome of children reported in the medical literature.
PMCID: PMC3549381  PMID: 23346431
10.  Functional Characterization of Genetic Variants in the Apical Sodium-Dependent Bile Acid Transporter (ASBT; SLC10A2) 
Background and Aims
The major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity.
SNPs in SLC10A2 from genomic DNA of ethnically-defined healthy individuals were identified using a PCR-based temperature gradient capillary electrophoresis (TGCE) system. A heterologous gene expression system was used to assess transport activity of SLC10A2 nonsynonymous variants and missense mutations. Total and cell surface protein expression of wild-type and variant ASBT was assessed by western blot analysis and immunofluorescence confocal microscopy. Expression of ASBT mRNA and protein was also measured in human intestinal samples.
The studies revealed two nonsynonymous SNPs, 292G>A and 431G>A, with partially impaired in vitro taurocholate transport. A novel variant, 790A>G, was also shown to exhibit near complete loss of taurocholate transport, similar to the previously identified ASBT missense mutations. Examination of ASBT protein expression revealed no significant differences in expression or trafficking to the cell surface amongst variants versus wild-type ASBT. Analysis of ASBT mRNA and protein expression in human intestinal samples revealed modest intersubject variability.
Genome sequencing and in vitro studies reveal the presence of multiple functionally relevant variants in SLC10A2 that may influence bile acid homeostasis and physiology.
PMCID: PMC3170668  PMID: 21649730
Biliary physiology; Bile acid metabolism; Intestinal lipid and bile acid transport
11.  Dapsone-Induced Methemoglobinemia: A Dose Related Occurrence? 
Cancer  2011;117(15):3485-3492.
Dapsone, used for Pneumocystis jiroveci (PCP) prophylaxis, is associated with increased risk of methemoglobinemia. Absence of cytochrome b5 reductase enzyme activity (CYB5RA) causes congenital methemoglobinemia, but its role in dapsone-associated methemoglobinemia is unknown. We sought to elucidate drug-related risk factors for dapsone-associated methemoglobinemia in pediatric oncology patients, including contribution of CYB5RA.
Patients and Methods
Among 167 pediatric patients treated for hematologic malignancies or aplastic anemia who received dapsone for PCP prophylaxis, demographic and dapsone treatment data were retrospectively collected. Drug-related risk factors were evaluated by Cox proportional hazards, and in a cross-sectional subgroup of 40 patients, CYB5RA was assessed.
Methemoglobinemia (median methemoglobin level = 9.0% [3.5–22.4]) was documented in 32 patients (19.8%). There was a 73% risk reduction in methemoglobinemia with dosing ≥20% below the target dose 2mg/kg/day (HR = 0.27; 95% confidence interval (CI) 0.09, 0.78; p=0.016), while methemoglobinemia risk was increased with dosing ≥20% above the target dose (HR = 6.25; 95% CI 2.45, 15.93; p<0.001). Sex, body mass index, and age were not associated with increased risk. CYB5RA did not differ by methemoglobinemia status (median 8.6 IU/g Hb; [5.5 – 12.1] vs. 9.1 IU/g Hb; [6.7 – 12.7]). No patient developed PCP on dapsone.
Methemoglobinemia occurred in almost 20% of pediatric oncology patients receiving dapsone for PCP prophylaxis. Higher dapsone dosing is associated with increased risk. A cross-sectionally acquired CYB5RA level was not associated with methemoglobinemia risk. Studies are needed to define biologic correlates of methemoglobinemia and evaluate lower dapsone doses for PCP prophylaxis.
PMCID: PMC3138875  PMID: 21246536
pediatric oncology; HIV; dapsone; methemoglobinemia; Drug-related complications; cytochrome b5 reductase
12.  Identification of novel functional Organic Anion-transporting Polypeptide 1B3 (OATP1B3) polymorphisms and assessment of substrate specificity 
Pharmacogenetics and genomics  2011;21(3):103-114.
The uptake carrier Organic Anion-transporting Polypeptide 1B3 (OATP1B3, gene SLCO1B3) is involved in the hepatic clearance of xenobiotics including statins, taxanes and mycophenolic acid. We thought to assess the SLCO1B3 coding region for yet unidentified polymorphisms, and to analyze their functional relevance.
We used DNA of ethnically diverse subjects for polymerase chain reaction (PCR), and determined polymorphisms by sequencing or temperature-dependent capillary electrophoresis. We then created variant OATP1B3 expression plasmids by site-directed mutagenesis, which were transiently expressed and functionally characterized in HeLa cells using radiolabeled substrates.
We identified six non-synonymous polymorphisms including novel variants 439A>G (Thr147Ala), 767G>C (Gly256Ala), 1559A>C (His520Pro) and 1679T>C (Val560Ala). Allelic frequencies occurred ethnicity-dependent, with the latter observed only in African Americans (3.6%). After expression in HeLa cells, variants His520Pro, Val560Ala, and Met233Ile or Met233Ile_Ser112Ala haplotype demonstrated decreased uptake activity compared to wildtype for cholecystokinin-8 (CCK8) and rosuvastatin, but not atorvastatin. Kinetic CCK8 analysis revealed reduced Vmax without altering Km. His520Pro and Val560Ala exhibited decreased total and plasma membrane protein expression. Val560 mapped onto a structural model of OATP1B3 revealed this is a key region for substrate–transporter interaction. His520 resides in a predicted extracellular region thought to be critical to the pH-dependent component of OATP1B3 activity. Loss of activity at pH 7.4 and 8.0 relative to pH 6.5 was significantly greater for the Pro520 variant.
OATP1B3 polymorphisms that result in altered expression, substrate specificity, and pH-dependent activity may be of potential relevance to hepatic clearance of substrate drugs in vivo.
PMCID: PMC3044558  PMID: 21278621
OATP1B3 polymorphisms; drug transporter; drug disposition
13.  High Risk Patients with Hematuria Are Not Evaluated According to Guideline Recommendations 
Cancer  2010;116(12):2954-2959.
To determine whether high risk patients with hematuria receive evaluation according to guideline recommendations.
Materials and Methods
We recently performed a screening study for bladder cancer using a urine-based tumor marker in 1502 subjects at high risk based on age over 50, ≥10-year smoking history, and/or a 15 year or more environmental exposure. We evaluated use of urinalysis (UA) within 3 years preceding the screening study. Chart review was performed to determine if this subset with microhematuria received any additional evaluation.
Of 1502 study participants, routine urinalysis was performed in 73.2% and 164 (14.9%) subjects had documented hematuria (>3 RBCs/HPF) prior to inclusion. Of these, 42.1% had no further evaluation. Additional testing included repeat urinalysis (36%), urine culture (15.2%), cytology (10.4%), imaging (22.6% overall: 15.9% CT, 4.3% IVP; 2.4% MRI) and cystoscopy (12.8%).
Three subjects with microscopic hematuria (2%) were subsequently found to have bladder cancer during the screening study but were not referred for evaluation based on their hematuria. The source of hematuria was unknown in 65%, infection in 22%, benign prostatic enlargement in 10% and renal stone disease in 4% but these results are based on incomplete evaluation since only 12.8% underwent cystoscopy.
Subjects at high risk for bladder cancer based on ≥10 years of smoking or environmental exposure with microscopic hematuria are rarely evaluated thoroughly and only 12.8% were referred for urologic evaluation. Further studies are needed to evaluate both the utilization and effectiveness of guidelines for hematuria.
PMCID: PMC2940122  PMID: 20564400
Hematuria; Guidelines Recommendations; bladder cancer
14.  Polymorphic variants of the human bile salt export pump (ABCB11): functional characterization and interindividual variability 
Pharmacogenetics and genomics  2010;20(1):45-57.
Our aims were to identify and functionally characterize coding region nonsynonymous single nucleotide polymorphisms in the hepatic efflux transporter, bile salt export pump (BSEP; ABCB11) and to assess interindividual variability in BSEP expression.
We identified 24 single nucleotide polymorphisms, including nine nonsynonymous variants, in ABCB11 from genomic DNA of approximately 250 ethnically diverse healthy individuals using denaturing high-performance liquid chromatography analysis and DNA sequencing. Wild type and variant BSEP were generated and functionally characterized for taurocholate transport activity in vitro in HeLa cells using a recombinant vaccinia-based method. BSEP expression was assessed by real-time mRNA analysis, western blot analysis, and immunofluorescence confocal microscopy.
For the most part, polymorphisms were rare and ethnicity dependent. In-vitro functional studies revealed several rare variants, including 616A>G, 1674G>C, 1772A>G, and 3556G>A, to be associated with significantly impaired taurocholate transport activity while the 890A>G variant trended towards impaired function but was not statistically significant. The 3556G>A variant was associated with reduced cell surface – total protein expression compared with wild-type BSEP. Expression of BSEP by mRNA and protein analysis was determined from a bank of human liver samples. Wide interindividual variability was noted in both mRNA (19-fold) and protein (31-fold) expression levels. The common variant 1331T>C was associated with significantly reduced hepatic BSEP mRNA levels.
Accordingly, our study indicates that there are functionally relevant polymorphisms in ABCB11, which may be of potential relevance in the predisposition to acquired liver disorders such as drug-induced cholestasis.
PMCID: PMC2883163  PMID: 20010382
ABCB11; bile acid; bile salt export pump; cholestasis; pharmacogenetics; polymorphism; transporter
15.  Interplay between the nuclear receptor PXR and the uptake transporter OATP1A2 selectively enhances estrogen effects in breast cancer 
Cancer research  2008;68(22):9338-9347.
The ligand-activated nuclear receptor PXR is known to play a role in the regulated expression of drug metabolizing enzymes and transporters. Recent studies suggest a potential clinically relevant role of PXR in breast cancer. However, the relevant pathway or target genes of PXR in breast cancer biology and progression have not yet been fully clarified.
In this study, we show that mRNA expression of OATP1A2, a transporter capable of mediating the cellular uptake of estrogen metabolites, is nearly 10-fold greater in breast cancer compared to adjacent healthy breast tissues. Immunohistochemistry revealed exclusive expression of OATP1A2 in breast cancer tissue. Interestingly, treatment of breast cancer cells in vitro with the PXR agonist rifampin induced OATP1A2 expression in a time- and concentration-dependent manner. Consistent with a role as a hormone uptake transporter, induction of OATP1A2 was associated with increased uptake of estrone 3-sulfate. The rifampin response was abrogated after si-RNA targeting of PXR. We then identified a PXR response element in the human OATP1A2 promoter, located approximately 5.7 kb upstream of the transcription initiation site. The specificity of PXR-OATP1A2 promoter interaction was confirmed using chromatin immunoprecipitation. Importantly we utilized a novel potent and specific antagonist of PXR (A-792611) to demonstrate the reversal of the rifampin effect on the cellular uptake of E1S.
These data provide important new insights into the interplay between a xenobiotic nuclear receptor PXR and OATP1A2 that could contribute to the pathogenesis of breast cancer and may also prove to be heretofore unrecognized targets for breast cancer treatment.
PMCID: PMC2597047  PMID: 19010908
OATP1A2; Estrone 3-Sulfate uptake; breast cancer; PXR; A-792611
16.  Ablation of AMP-Activated Protein Kinase α2 Activity Exacerbates Insulin Resistance Induced by High-Fat Feeding of Mice 
Diabetes  2008;57(11):2958-2966.
OBJECTIVE—We determined whether muscle AMP-activated protein kinase (AMPK) has a role in the development of insulin resistance.
RESEARCH DESIGN AND METHODS—Muscle-specific transgenic mice expressing an inactive form of the AMPK α2 catalytic subunit (α2i TG) and their wild-type littermates were fed either a high-fat (60% kcal fat) or a control (10% kcal fat) diet for 30 weeks.
RESULTS—Compared with wild-type mice, glucose tolerance in α2i TG mice was slightly impaired on the control diet and significantly impaired on the high-fat diet. To determine whether the whole-body glucose intolerance was associated with impaired insulin sensitivity in skeletal muscle, glucose transport in response to submaximal insulin (450 μU/ml) was measured in isolated soleus muscles. On the control diet, insulin-stimulated glucose transport was reduced by ∼50% in α2i TG mice compared with wild-type mice. High-fat feeding partially decreased insulin-stimulated glucose transport in wild-type mice, while high-fat feeding resulted in a full blunting of insulin-stimulated glucose transport in the α2i TG mice. High-fat feeding in α2i TG mice was accompanied by decreased expression of insulin signaling proteins in gastrocnemius muscle.
CONCLUSIONS—The lack of skeletal muscle AMPK α2 activity exacerbates the development of glucose intolerance and insulin resistance caused by high-fat feeding and supports the thesis that AMPK α2 is an important target for the prevention/amelioration of skeletal muscle insulin resistance through lifestyle (exercise) and pharmacologic (e.g., metformin) treatments.
PMCID: PMC2570392  PMID: 18728234
17.  Dissociation of AMP-activated Protein Kinase and p38 Mitogen-activated Protein Kinase Signaling in Skeletal Muscle 
AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle. The p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. Here we used several different models of altered AMPK activity to determine whether p38 MAPK is a downstream intermediate of AMPK-mediated signaling in skeletal muscle. First, L6 myoblasts and myotubes were treated with AICAR, an AMPK stimulator. AMPK phosphorylation was significantly increased, but there was no change in p38 MAPK phosphorylation. Similarly, AICAR incubation of isolated rat extensor digitorum longus (EDL) muscles did not increase p38 phosphorylation. Next, we used transgenic mice expressing an inactive form of the AMPKα2 catalytic subunit in skeletal muscle (AMPKα2i TG mice). AMPKα2i TG mice did not exhibit any defect in basal or contraction-induced p38 MAPK phosphorylation. We also used transgenic mice expressing an activating mutation in the AMPKγ1 subunit (γ1R70Q TG mice). Despite activated AMPK, basal p38 MAPK phosphorylation was not different between wild type and γ1R70Q TG mice. In addition, muscle contraction-induced p38 MAPK phosphorylation was significantly blunted in the γ1R70Q TG mice. In conclusion, increasing AMPK activity by AICAR and AMPKγ1 mutation does not increase p38 MAPK phosphorylation in skeletal muscle. Furthermore, AMPKα2i TG mice lacking contraction-stimulated AMPK activity have normal p38 MAPK phosphorylation. These results suggest that p38 MAPK is not a downstream component of AMPK-mediated signaling in skeletal muscle.
PMCID: PMC2040310  PMID: 17709097
18.  Skeletal Muscle-Selective Knockout of LKB1 Increases Insulin Sensitivity, Improves Glucose Homeostasis, and Decreases TRB3▿ †  
Molecular and Cellular Biology  2006;26(22):8217-8227.
LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a >80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.
PMCID: PMC1636784  PMID: 16966378
19.  Structure-Activity of Sulfones and Sulfonamides on Dihydropteroate Synthetase from Neisseria meningitidis 
The molecular interaction of various sulfones and sulfonamides with partially purified dihydropteroate synthetase from Neisseria meningitidis M-166 has been examined. The mode of action of the sulfones was similar to that of the sulfonamides. Both groups of drugs were competitive inhibitors of dihydropteroate synthetase with respect to p-aminobenzoate in a partially purified enzyme preparation. 4,4′-Diaminodiphenylsulfone was three times more effective than sulfadiazine and nine times more effective than sulfanilamide as a competitive inhibitor of dihydropteroate synthetase. The inhibitory activity of 4-amino-4′-acetamidodiphenylsulfone and 4-amino-4′-formamidodiphenylsulfone in this system eliminated their prior conversion to 4,4′-diaminodiphenylsulfone as a requirement for activity.
PMCID: PMC429223  PMID: 808157
20.  Alterations in Dihydropteroate Synthetase in Cell-Free Extracts of Sulfanilamide-Resistant Neisseria meningitidis and Neisseria gonorrhoeae 
Extracts from Neisseria meningitidis and N. gonorrhoeae with varying susceptibility to sulfanilamide have been investigated for dihydropteroate synthetase activity. Sulfanilamide was a competitive inhibitor of dihydropteroate synthetase with respect to p-aminobenzoate in extracts from both species. Though the Km for p-aminobenzoate was unaffected, the Ki for sulfanilamide increased and the Vmax decreased as the strains' resistance to sulfanilamide increased. Temperature studies have revealed differences in the dihydropteroate synthetase from N. meningitidis and N. gonorrhoeae. A direct relationship was observed between the minimal inhibitory concentration of sulfanilamide determined in vitro and the ratio of Ki/Km. This ratio may be a molecular explanation of sulfanilamide resistance for both N. meningitidis and N. gonorrhoeae.
PMCID: PMC428980  PMID: 15825393
21.  Long-Term Use of the Thrombopoietin-Mimetic Romiplostim in Children With Severe Chronic Immune Thrombocytopenia (ITP) 
Pediatric Blood & Cancer  2014;62(2):208-213.
Treatment of chronic severe pediatric ITP is not well studied. In a phase 1/2 12–16-week study, 15/17 romiplostim-treated patients achieved platelet counts ≥50 × 109/L, and romiplostim treatment was well tolerated. In a subsequent open-label extension (≤109 weeks), 20/22 patients received romiplostim; all achieved platelet counts >50 × 109/L. Twelve patients continued in a second extension (≤127 weeks). Longitudinal data from start of romiplostim treatment through the two extensions were evaluated to investigate the safety and efficacy of long-term romiplostim treatment in chronic severe pediatric ITP.
Patients received weekly subcutaneous romiplostim, adjusted by 1 µg/kg/week to maintain platelet counts (50–200 × 109/L, maximum dose 10 µg/kg). Bone marrow examinations were not required.
At baseline, patients were median age 10.0 years; median ITP duration 2.4 years; median platelet count 13 × 109/L; 73% were male; and 36% had prior splenectomy. Median romiplostim treatment duration was 167 weeks (Q1, Q3: 78,227 weeks), and median average weekly dose was 5.4 µg/kg (Q1, Q3: 4.3, 8.0 µg/kg). Seven patients discontinued treatment: four withdrew consent, two were noncompliant, and one received alternative therapy. None withdrew because of adverse events (AEs). After the first 12 weeks, median platelet counts remained >50 × 109/L. Eight (36.4%) patients received rescue medication, and 14 (63.6%) used concurrent ITP therapy. Seven patients (31.8%) reported serious AEs, and two (9.1%) reported life-threatening AEs (both thrombocytopenia); there were no serious AEs attributed to treatment and no fatalities.
Long-term romiplostim treatment in this small cohort increased and maintained platelet counts for over 4 years in children with ITP with good tolerability and without significant toxicity. Pediatr Blood Cancer 2015;62:208–213. © 2014. The Authors. Pediatr Blood & Cancer published by Wiley Periodicals, Inc.
PMCID: PMC4309514  PMID: 25345874
autoimmunity; bleeding; platelets; thrombopoietin

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