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The liver manifestations of Alagille syndrome (ALGS) are highly variable, and factors affecting its prognosis are poorly understood. We asked whether or not the composition of bile acids in ALGS patients with good clinical outcomes differs from those with poor outcomes, and whether bile acids could be used as prognostic biomarkers. Blood for bile acid profiling were collected from genetically confirmed JAG1-associated ALGS patients before one year of age. Good prognosis was defined as survival with native liver and total bilirubin (TB) discovery cohort. The same trend was also observed in the molar ratios of GHCA to GCDCA and THCA to TCDCA (both AUC = 0.836). A validation cohort confirmed these findings. Notably, tauro‐2β,3α,7α,12α-THBA achieved the highest prediction accuracy of 88.00% (92.31% sensitivity and 83.33% specificity); GHCA at > 607.69 nmol/L was associated with native liver survival (hazard ratio: 6.456, 95% CI: 1.173–35.545, P = 0.032). We identified two poly-hydroxylated bile acids as the liver prognostic biomarkers of ALGS patients. Enhanced hydroxylation of bile acids may result in better clinical outcomes.

Hypereosinophilic syndrome (HES) is a rare but life-threatening multi-organ disease which can be complicated by stroke, with devastating outcomes. Eosinophils can accumulate in multiple organs, most commonly involving the heart, skin, lungs, spleen, and liver. Neurological end-organ complications in hypereosinophilic syndrome are unusual and have been established to be of three types: brain infarction, encephalopathy and sensory polyneuropathy. We present a case where acute ischaemic stroke and encephalopathy are early manifestations of Idiopathic HES. It is important to consider HES as an aetiology for stroke and a high eosinophil count is an initial diagnostic clue. Early initiation of steroid therapy can potentially prevent disease progression.

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3), severe liver diseases associated with genetic defects in the biliary bile acid transporter bile salt export pump (BSEP; ABCB11) or phosphatidylcholine transporter multidrug resistance protein 3 (MDR3; ABCB4), respectively. Mdr2−/− mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the nonmicelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep−/− mice could protect Mdr2−/− mice from progressive liver damage. We generated double-KO (DKO: Bsep−/− and Mdr2−/−) mice. Their bile acid composition resembles that of Bsep−/− mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBAs), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2−/− littermates. The livers of DKO mice have gene expression profiles very similar to Bsep−/− mice, with 4,410 of 6,134 gene expression changes associated with the Mdr2−/− mutation being suppressed. Feeding with THBAs partially alleviates liver damage in the Mdr2−/− mice. Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2−/− (PFIC3) mutation.

The aim was to determine if liver biochemistry indices can be used as biomarkers to help differentiate patients with neonatal Dubin-Johnson syndrome (nDJS) from those with biliary atresia (BA).Patients with genetically-confirmed nDJS or cholangiographically confirmed BA were retrospectively enrolled and randomly assigned to discovery or verification cohorts. Their liver chemistries, measured during the neonatal period, were compared. Predictive values were calculated by receiver operating characteristic curve analysis.A cohort of 53 nDJS patients was recruited, of whom 13 presented with acholic stools, and 14 underwent diagnostic cholangiography or needle liver biopsy to differentiate from BA. Thirty-five patients in the cohort, with complete biochemical information measured during the neonatal period, were compared with 133 infants with cholangiographically confirmed BA. Total and direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bile acids, alkaline phosphatase, and gamma-glutamyl transferase were significantly lower in nDJS than in BA. In the discovery cohort, the areas under the curve for ALT and AST were 0.908 and 0.943, respectively. In the validation cohort, 13/15 patients in the nDJS group were classified as nDJS, and 10/53 in the BA control group were positive (Having assembled and investigated the largest cohort of nDJS patients reported to date, we found that nDJS patients could be distinguished from BA patients using the serum AST level as a biomarker. The finding may be clinically useful to spare cholestatic nDJS patients unnecessary invasive procedures.

Introduction: In acute lymphoblastic leukemia (ALL) adolescents and young adults (AYA), there are benefits when using pediatric over adult treatment regimes. There is improved overall survival (OS), progression free survival (PFS) and reduced need for hematopoietic stem cell transplantation; all while having an acceptable toxicity profile. Yet, studies supporting this were largely performed in a western population (e.g., United Kingdom and United States) and thus may have had limited representation of the Asian population. Whether these results are generalizable and applicable to an Asian population is in question. MASPORE, a locally designed pediatric regiment based on BFM regimen, which has been used successfully in Singapore and Malaysia's AYA cohort since 2007. MASPORE protocol utilized PCR-based minimal residual disease (MRD) marker to risk stratify patients according to disease severity and used a 3- or 4-drug induction regimen depending on intermediate or high-risk stratification (L-asparaginase, Vincristine, Dexamethasone +/- daunorubicin). In contrast HyperCVAD does not use MRD to stratify or guide management. In this retrospective study performed in Singapore, we aim to demonstrate that MASPORE is an effective treatment option for AYA ALL in Asian population. Methods: Patient registries (IRB approved) spanning from January 2005 to June 2021 from three tertiary hospitals in Singapore were reviewed. ALL patients who were AYA, defined as less than 40 years of age, treated during this time were included and analyzed. Patients were treated with either the pediatric protocol MASPORE or with the adult protocol HyperCVAD. Patients who are Philadelphia positive were excluded from the MASPORE arm up to 2020. Patients' demographics, functional status, risk profile, adverse events, as well as the hematological response and transplant status were collected and analyzed with Pearson chi-square test. Kaplan Meier curve analysis were performed for OS and PFS with SPSS software. Results: In this retrospective study, 116 patients were analyzed. Among these patients 29 (25%) received MASPORE and 87 (75%) received HyperCVAD. The median age was 23 (range 18-40) for MASPORE arm vs 28 years (range 15-40) for the HCVAD arm. median follow-up time was 4.5 vs 10.8 years. Both groups were similar in gender, race, ECOG status, and ALL risk status at diagnosis. Likewise, both arms did not have differences in starting hematological parameters: hemoglobin, total white blood cell count, platelet count, absolute neutrophil count, and bone marrow blast percentages. Median time from diagnosis to treatment was 3 and 4 days for MASPORE and HyperCVAD arms respectively. There were increased adverse rates for patients in the MASPORE arm. They had more (13.8 vs 0.0%, p < 0.001) of which 75% of the pancreatitis were CTCAE grade 3/4, avascular necrosis (13.8 vs 2.2%, p = 0.016), cerebral venous thrombosis (13.8 vs 1.1%, p = 0.004) and other thrombosis (27.6 vs 5.7%, p = 0.004). For the MASPORE and HyperCVAD arms there was one induction death, one from sepsis and the other from pneumonia . Both arms managed to achieve complete response (CR) with equivalent rates (82.5 vs 80.5%, p = 0.752). However, there was less relapsed or refractory disease in those treated with MASPORE (10.3 vs 44.8%, p < 0.001). The 5- OS was better in the MASPORE versus the HyperCVAD arm (4.4 vs 3.7 years; p = 0.049). Likewise, the 5-year PFS was superior in the MASPORE arm (4.2 vs 3.2 years, p = 0.034). Furthermore, a smaller proportion of patients required consolidative stem cell transplant (13.8 vs 49.4%, p < 0.001). Conclusion: The pediatric-inspired protocol MASPORE achieved similar CR rates with OS and PFS benefit, reduced relapse rates, and less need for consolidative stem cell transplant. MASPORE serves as a viable treatment option when treating AYA patients with ALL. Larger prospective studies are needed to further explore its use. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

We use topographic data to show that impact craters with pitted floor deposits are among the deepest on Mars. This is consistent with the interpretation of pitted materials as primary crater-fill impactite deposits emplaced during crater formation. Our database consists of 224 pitted material craters ranging in size from ∼1 to 150 km in diameter. Our measurements are based on topographic data from the Mars Orbiter Laser Altimeter (MOLA) and the High-Resolution Stereo Camera (HRSC). We have used these craters to measure the relationship between crater diameter and the initial post-formation depth. Depth was measured as maximum rim-to-floor depth, ( d r ), but we also report the depth measured using other definitions. The database was down-selected by refining or removing elevation measurements from “problematic” craters affected by processes and conditions that influenced their d r /D, such as pre-impact slopes/topography and later overprinting craters. We report a maximum (deepest) and mean scaling relationship of d r = ( 0.347 ± 0.021 ) D 0.537 ± 0.017 and d r = ( 0.323 ± 0.017 ) D 0.538 ± 0.016 , respectively. Our results suggest that significant variations between previously-reported MOLA-based d r vs. D relationships may result from the inclusion of craters that: 1) are influenced by atypical processes ( e.g. , highly oblique impact), 2) are significantly degraded, 3) reside within high-strength regions, and 4) are transitional (partially collapsed). By taking such issues into consideration and only measuring craters with primary floor materials, we present the best estimate to date of a MOLA-based relationship of d r vs. D for the least-degraded complex craters on Mars. This can be applied to crater degradation studies and provides a useful constraint for models of complex crater formation.

Bile acids are key components of bile required for human health. In humans and mice, conditions of reduced bile flow, cholestasis, induce bile acid detoxification by producing tetrahydroxylated bile acids (THBA), more hydrophilic and less cytotoxic than the usual bile acids, which are typically di- or tri-hydroxylated. Mice deficient in the Bile Salt Export Pump (Bsep, or Abcb11), the primary bile acid transporter in liver cells, produce high levels of THBA, and avoid the severe liver damage typically seen in humans with BSEP deficiencies. THBA can suppress bile acid-induced liver damage in Mdr2-deficient mice, caused by their lack of phospholipids in bile exposing their biliary tracts to unbound bile acids. Here we review THBA-related works in both animals and humans, and discuss their potential relevance and applications as a class of functional bile acids.

Bile acid imbalance causes progressive familial intrahepatic cholestasis type 2 (PFIC2) or type 3 (PFIC3), severe liver diseases associated with genetic defects in the biliary bile acid transporter bile salt export pump (BSEP; ABCB11) or phosphatidylcholine transporter multidrug resistance protein 3 (MDR3; ABCB4), respectively. Mdr2(−/−) mice (a PFIC3 model) develop progressive cholangitis, ductular proliferation, periportal fibrosis, and hepatocellular carcinoma (HCC) because the nonmicelle-bound bile acids in the bile of these mice are toxic. We asked whether the highly hydrophilic bile acids generated by Bsep(−/−) mice could protect Mdr2(−/−) mice from progressive liver damage. We generated double-KO (DKO: Bsep(−/−) and Mdr2(−/−)) mice. Their bile acid composition resembles that of Bsep(−/−) mice, with increased hydrophilic muricholic acids, tetrahydroxylated bile acids (THBAs), and reduced hydrophobic cholic acid. These mice lack the liver pathology of their Mdr2(−/−) littermates. The livers of DKO mice have gene expression profiles very similar to Bsep(−/−) mice, with 4,410 of 6,134 gene expression changes associated with the Mdr2(−/−) mutation being suppressed. Feeding with THBAs partially alleviates liver damage in the Mdr2(−/−) mice. Hydrophilic changes to biliary bile acid composition, including introduction of THBA, can prevent the progressive liver pathology associated with the Mdr2(−/−) (PFIC3) mutation.

Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (

BACKGROUND & AIMS Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. METHODS We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. RESULTS The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Hereditary cholestasis in childhood and infancy with normal serum gamma‐glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low‐GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow‐up. Liver biopsy specimens revealed giant‐cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP‐deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated‐cholestasis patients (11 of 38 vs. 0 of 13). Conclusion: MYO5B deficiency may underlie 20% of previously undiagnosed low‐GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655‐1669).

We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.

Tryptophan (Trp) oxidation leading to atypical degradation of a protein (Fab) formulated with polysorbate 20 (PS20) was investigated. Such atypical Trp oxidation was discussed in relation to a kinetic model that involves initiation of oxidizing free radical through an autocatalytic reaction.Ion-exchange chromatography and peptide mapping were used to determine Trp oxidation. Peroxides in PS20 and free radicals in Fab samples were detected by fluorometric assay and electron paramagnetic resonance (EPR), respectively.PS20 with increased peroxides level led to degradation of Fab stored at 30°C. Degradation was characterized as Trp50 oxidation, which was not observed in a Fab variant where His31 was replaced. EPR peaks related to known spin adducts of 5,5 dimethylpyrroline N-oxide were detected in Fab exhibiting Trp oxidation, indicating free radicals were present. Trp oxidation of Fab observed in several drug product lots with different degradation rates fits an autocatalytic reaction model that involves free radicals. EDTA, catalase, and free tryptophan prevented oxidation.A metal-binding amino acid, His31, was responsible for Trp50 oxidation of Fab induced by peroxides in PS20 present in the protein formulation. Oxidation was induced by autocatalytic degradation of PS20 and could be inhibited by antioxidants.

The biliary secretion of bile acids is critical for multiple liver functions including digesting fatty nutrients and driving bile flow. When this process is impaired, the accumulating bile acids cause inflammatory liver injury. Multiple ABC transporters in the liver are key players to safeguard the hepatocyte and avoid toxicity due to bile acid over-accumulation. BSEP provides for efficient secretion of bile acids across the canalicular membrane against a steep concentration gradient. MDR3/Mdr2 and ABCG5/G8 secrete phosphatidylcholine and cholesterol, respectively, in coordination with BSEP-mediated bile acid secretion to mask the detergent/toxic effects of bile acids in the bile ductular space. Several lines of evidence indicate that when these critical steps are compromised, bile acid toxicity in vivo leads to inflammatory liver injury and liver cancer. In bsep-/- mice, liver cancer is rare. These mice display greatly increased expression of alternative bile acid transporters, such as Mdr1a/1b, Mrp3 and Mrp4. We believe these alternative transport systems provide an additional safeguard to avoid bile acid overload in liver. Such backup systems appear to be under-utilized in humans, as defects in BSEP and MDR3 lead to severe, often fatal childhood diseases. It is possible, therefore, that targeting ABC transporters and modulating the toxicity of the bile acid pool could be vital interventions to alleviate chronic inflammation and reduce the incidence of liver cancer in high-risk populations. The combination of an alternative ABC transporter with a novel substrate may prove an effective chemo-preventive or therapeutic strategy.

Pancreatic cancer is an aggressive, drug-resistant disease, its first-line chemotherapeutic, gemcitabine, is only marginally effective. Intracellular depletion of glutathione, a major free-radical scavenger, has been associated with growth arrest and reduced drug resistance (chemosensitization) of cancer cells. In search of a new therapeutic approach for pancreatic cancer, we sought to determine whether specific inhibition of the plasma membrane xc&minus, cystine transporter could lead to reduced uptake of cysteine, a key precursor of glutathione, and subsequent glutathione depletion. Sulfasalazine (approximately 0.2 mmol/L), an anti-inflammatory drug with potent xc&minus, inhibitory properties, markedly reduced l-[14C]-cystine uptake, glutathione levels, and growth and viability of human MIA PaCa-2 and PANC-1 pancreatic cancer cells in vitro. These effects were shown to result primarily from inhibition of cystine uptake mediated by the xc&minus, cystine transporter and not from inhibition of nuclear factor &kappa, B activation, another property of sulfasalazine. The efficacy of gemcitabine could be markedly enhanced by combination therapy with sulfasalazine both in vitro and in immunodeficient mice carrying xenografts of the same cell lines. No major side effects were observed in vivo.

Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals.

Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.

The x(c)(-) cystine transporter enhances biosynthesis of glutathione, a tripeptide thiol important in drug resistance and cellular defense against oxidative stress, by enabling cellular uptake of cystine, a rate-limiting precursor. Because it is known to regulate glutathione levels and growth of various cancer cell types, and is expressed in the pancreas, we postulate that it is involved in growth and drug resistance of pancreatic cancer. To examine this, we characterised expression of the x(c)(-) transporter in pancreatic cancer cell lines, MIA PaCa-2, PANC-1 and BxPC-3, as subjected to cystine-depletion and oxidative stress. The results indicate that these cell lines depend on x(c)(-)-mediated cystine uptake for growth, as well as survival in oxidative stress conditions, and can modulate x(c)(-) expression to accommodate growth needs. Immunohistochemical analysis showed that the transporter was differentially expressed in normal pancreatic tissues and overexpressed in pancreatic cancer tissues from two patients. Furthermore, gemcitabine resistance of cells was associated with elevated x(c)(-) expression and specific x(c)(-) inhibition by monosodium glutamate led to growth arrest. The results suggest that the x(c)(-) transporter by enhancing glutathione biosynthesis plays a major role in pancreatic cancer growth, therapy resistance and represents a potential therapeutic target for the disease.

Metastatic prostate cancer is a terminal disease, and the development of reliable prognostic tools and more effective therapy is critically important for improved disease survival and management. This study was aimed at identifying genes that are differentially expressed in metastatic and nonmetastatic prostate cancer cells and, as such, could be critical in the development of metastasis. Long-SAGE analysis was used to compare a transplantable human metastatic prostate cancer subline, PCa1-met, with a nonmetastatic counterpart, PCa2. Both sublines were developed from a patient's prostate cancer specimen via subrenal capsule grafting and subsequent orthotopic implantation into SCID mice. Among various differentially expressed genes identified, ASAP1, an 8q24 gene encoding an ADP-ribosylation factor GTPase-activating protein not previously associated with prostate cancer, was up-regulated in the metastatic subline as confirmed by quantitative real-time PCR. Immunohistochemistry of xenograft sections showed that cytoplasmic ASAP1 protein staining was absent or weak in benign tissue, significantly stronger in nonmetastatic PCa2 tissue, and strongest in PCa1-met tissue. In clinical specimens, ASAP1 protein staining was elevated in 80% of primary prostate cancers and substantially higher in metastatic lesions compared with benign prostate tissue. Moreover, additional ASAP1 gene copies were detected in 58% of the primary prostate cancer specimens. Small interfering RNA–induced reduction of ASAP1 protein expression markedly suppressed in vitro PC-3 cell migration (∼50%) and Matrigel invasion (∼67%). This study suggests that the ASAP1 gene plays a role in prostate cancer metastasis and may represent a therapeutic target and/or biomarker for metastatic disease. [Cancer Res 2008;68(11):4352–9]

Cell transplantation is a potential therapy for acquired or inherited liver diseases. Donor-derived hepatocytes (DDH) have been found in humans and mice after bone marrow transplantation (BMT) but with highly variable frequencies in different disease models. To test the effect of liver repopulation after BMT in inherited cholestatic liver diseases, spgp (sister of P-glycoprotein, or bile salt export pump, abcb11) knockout mice, a model for human progressive intrahepatic cholestasis type 2 with defects in excreting bile salts across the hepatocyte canalicular membrane, were transplanted with bone marrow cells from enhanced green fluorescent protein (EGFP) transgenic donor mice after lethal irradiation. One to 6 months later, scattered EGFP-positive DDHs with positive spgp staining were observed in the liver. These hepatocytes had been incorporated into hepatic plates and stained positively with hepatocyte-specific marker albumin. RT-PCR for the spgp gene revealed positive expression in the liver of sgsp knockout mice that had received the transplant. Bile acid analysis of bile samples showed that these mice also had higher levels of total biliary bile acid and taurocholic acid concentration than knockout mice without transplantation, indicating that BMT partially improved biliary bile acid secretion. Our results indicate that bone marrow cells could serve as a potential source for restoration of hepatic functions in chronic metabolic liver disease.

Objective Overexpression of members of the adenosine triphosphate binding cassette (ABC) transporter superfamily has been implicated in multidrug resistance in cancer, but results in acute myeloid leukemia (AML) have been inconsistent. We investigated the expression and activity of ABC transporters in patient total blasts and subpopulations along the leukemic stem cell hierarchy. Materials and Methods Using quantitative reverse transcriptase polymerase chain reaction, we measured expression of the ABC transporter superfamily in the blast cells from AML patients prior to chemotherapy. In addition, we measured ex vivo daunorubicin resistance of subpopulations with or without ABC inhibitors. Results In the total blasts, no consistent difference was observed in 18 patients achieving complete remission (CR) and 13 patients who were refractory to induction chemotherapy (NR). However, among the subpopulation of CD34 + CD38 − AML cells (candidate "leukemic stem cells"), elevated expression of MDR1 and/or BCRP1 , two ABC transporters associated with drug resistance, was found in 8 of 10 NR patients as compared to 0 of 7 CR patients. No such association was observed in the more differentiated CD34 + CD38 + or CD34 − subpopulations. There was no significant difference in MRP1 expression between CR and NR patient samples in any of the subpopulations examined. The increased expression of MDR1 and BCRP1 in leukemic cells correlated with increased cellular daunorubicin resistance, which could be reversed by the ABC transporter inhibitors verapamil and PSC-833. Conclusion Expression of MDR1 and BCRP1 in leukemic stem cells correlates with chemotherapy response both at the cellular level and in AML patients.

The AU-rich element (ARE) is a stability determinant found in the 3' UTR of a number of short-lived mRNAs. The best characterized ARE is the Shaw-Kamen (SK) box or AUUUA motif. Previously, a fungal metabolite, radicicol analog A (RAA), was shown to destabilize SK box-containing mRNAs based on 16 mRNAs examined [T. Kastelic et al., Cytokine 8 (1996) 751-761]. Using serial analysis of gene expression (SAGE) to examine the global effect of RAA on mRNA expression in interferon-gamma/lipopolysaccharide-stimulated THP-1 human monocytes, we observed that the expression level of greater than 99% of the SAGE tags was unchanged by RAA treatment and only 34 of the 17,608 unique tags annotated were reduced (p< or =0.0001). RAA destabilized approximately half of the down-regulated transcripts. Whereas all the destabilized mRNAs possessed at least one SK box, for transcripts not destabilized but nonetheless down-regulated, RAA appears to function by a SK box-independent mechanism not currently understood.

Human bile salt export pump ( BSEP) mutations underlie progressive familial intrahepatic cholestasis type 2 (PFIC2). In the PFIC2 animal model, Bsep−/−mice, biliary secretion of bile salts (BS) is decreased, but that of phospholipids (PL) and cholesterol (CH) is increased. Under physiological conditions, the biliary secretion of PL and CH is positively related (“coupled”) to that of BS. We aimed to elucidate the mechanism of increased biliary lipid secretion in Bsep−/−mice. The secretion of the BS tauro-β-muricholic acid (TβMCA) is relatively preserved in Bsep−/−mice. We infused Bsep−/−and Bsep+/+(control) mice with TβMCA in stepwise increasing dosages (150–600 nmol/min) and determined biliary bile flow, BS, PL, and CH secretion. mRNA and protein expression of relevant canalicular transporters was analyzed in livers from noninfused Bsep−/−and control mice. TβMCA infusion increased BS secretion in both Bsep−/−and control mice. The secreted PL or CH amount per BS, i.e., the “coupling,” was continuously two- to threefold higher in Bsep−/−mice ( P < 0.05). Hepatic mRNA expression of canalicular lipid transporters Mdr2, Abcg5, and Abcg8 was 45–55% higher in Bsep−/−mice (Abcg5; P < 0.05), as was canalicular Mdr2 and Abcg5 protein expression. Potential other explanations for the increased coupling of the biliary secretion of PL and CH to that of BS in Bsep−/−mice could be excluded. We conclude that the mechanism of increased biliary lipid secretion in Bsep−/−mice is based on increased expression of the responsible canalicular transporter proteins.

The problem of multidrug resistance (MDR) in human cancers led to the discovery 30 years ago of a single protein P-glycoprotein (P-gp), capable of mediating resistance to multiple structurally diverse drugs. P-gp became the archetypal eukaryotic ABC transporter gene, and studies of P-gp and related ABC transporters in both eukaryotes and bacteria have led to a basic mechanistic understanding of the molecular basis of MDR. Particular milestones along the way have been the identification of the homology between P-gp and bacterial transport proteins, the purification and functional reconstitution of P-gp into synthetic lipid systems, and the development of targeted therapies that attempt to overcome MDR by inhibiting P-gp. This preface places into this context some of the less well-explored themes developed in the MDR field, particularly various alternative models of P-gp action, evidence for parallel physiological roles for P-gp, and the unusual relationship between the substrate recognition capabilities of ABC transporters and their evolutionary history.

Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.

The transporter associated with antigen presentation (TAP) is an ATP-binding cassette (ABC) protein which transports peptides for presentation to the immune system. TAP is composed of two half transporters, TAP1 (ABCB2) and TAP2 (ABCB3), which heterodimerize to function. In humans, the TAP family consists of TAP1, TAP2, and TAPL (ABCB9). While the TAP1-TAP2 complex is well characterized, TAPL's dimerization state and function are unknown. To identify interactions within the human TAP family, we adapted the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to half ABC transporters. This assay has been shown to be suitable for the study of membrane-bound proteins in vivo [Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Science 283, 990-993]. With this method, in vivo TAP1-TAP2 heterodimerization was confirmed, no homodimerizations were detected with TAP1 or TAP2, and TAPL did not show any interaction with TAP1 or TAP2. However, we found strong evidence that TAPL forms homodimers. These results provide evidence of a novel homomeric TAPL interaction and demonstrate that the DHFR PCA will be of general utility in studies of half ABC transporter interactions in vivo.

Studies in our laboratory on the role of P-glycoprotein (Pgp, coded by mdr1 gene) have led to the hypothesis that over-expression of Pgp is closely associated with the development of cancer. It was conceived therefore that inhibitors of Pgp should inhibit the development of cancer. We have reported that PSC833 (PSC), a potent inhibitor of Pgp, inhibits the development of liver cancer in rats. Similarly, based on the intrinsic over-expression of Pgp in experimental mammary carcinogenesis, we studied the effect of PSC on N-methyl-N-nitrosourea induced mammary cancer in female Sprague-Dawley rats. The study indicates that PSC at daily dietary doses of 15 (PSC15) and 30 mg/kg (PSC30) body wt resulted in dose-dependent inhibition of the incidence as well as the growth of mammary tumors. Compared with controls, PSC15 and PSC30 inhibited: (i) mean tumor multiplicity by 32 and 67%, (ii) median tumor burden by 46 and 93% and (iii) incidence of ulcerated tumors by 40 and 82%, respectively. Most remarkably, PSC delayed median tumor incidence by 8 weeks, and exerted a 100% inhibitory effect on the incidence of large tumors, 4 cm(3) and greater. In all the cases, although the inhibitory effect of PSC was evident at both doses, only PSC30 exhibited statistical significance. A possible compounding effect that was also observed in PSC30-treated rats was a decrease in body weight gain not attributed to diminished food consumption. All in all, consistent with recent reports, which have demonstrated inhibition of cancer development by compromising Pgp function, this study introduces a novel role for Pgp in breast cancer and potentially an unexplored therapeutic approach in treating the disease.

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5β-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7α-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3α,6β,7β,12α-tetrahydroxy-5β-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.

P-Glycoprotein (P-gp) is comprised of a small family of plasma membrane proteins, and its presence in high amounts often correlates with multidrug resistance in cultured cells. Dramatically increased levels of a single member of P-gp mRNA (pgp2) have been observed in experimental liver carcinogenesis models, during liver regeneration, upon culturing of hepatocytes and in the uterus of pregnant animals. In all cases, the increase in mRNA level appears to be the result of an increase in mRNA half-life (stability). Previously, we have used transcriptional inhibitors alpha-amanitin and actinomycin D to measure P-gp mRNA half-life in normal liver and in liver tumors. We showed that the level of all three P-gp mRNAs decreased with time in the presence of transcriptional inhibitors, yielding measured half-lives of less than 2 h in liver but greater than 12 h in liver tumors. This observation raised the possibility that regulation of P-gp mRNA stability plays a role in liver carcinogenesis. In the present study, we measured P-gp mRNA half-life in other normal tissues to determine if a short P-gp mRNA half-life is unique to the liver. Our study reveals that in contrast to liver, measured P-gp mRNA half-lives in most tissues examined are greater than 12 h. Moreover, we observed an unexpected, marked increase in the level of pgp2 mRNA with time after injection of transcriptional inhibitors. This can only be explained if the transcriptional inhibitors directly or indirectly inhibit the normally high degradation rate of pgp2 mRNA, resulting in the superinduction of this mRNA. These findings have implications for our understanding of the regulation of P-gp gene expression and drug resistance in vivo.

Lysosome-associated protein transmembrane 4 alpha (LAPTM4 alpha) and homologues comprise a family of conserved proteins, which are found in mammals, insects and nematodes. LAPTM4 alpha functions to regulate the intracellular compartmentalization of amphipathic solutes and possibly the sensitivity of cells toward anthracyclines, antibiotics, ionophores, nucleobases and organic cations. This is similar to the multidrug-resistance phenotype exhibited by cells synthesizing high levels of P-glycoprotein. Accordingly, it is possible that LAPTM4 alpha may be a suitable target for development of novel chemotherapeutic agents. LAPTM4 alpha contains four putative membrane-spanning domains and a 55 amino acid C-terminal region that faces the cytoplasm. Localization of LAPTM4 alpha to endosomes and lysosomes appears to be tightly controlled as transient high-level expression of LAPTM4 alpha in cultured cells resulted in no detectable protein on the cell surface. Mutagenic analysis of the C-terminus of LAPTM4 alpha indicated that two tandomly arranged tyrosine-containing motifs in the cytoplasmic domain are required for efficient localization of LAPTM4 alpha to vesicles containing the lysosomal marker lysosomal glycoprotein 120. Although a number of membrane proteins that localize to endosomes/lysosomes contain more than one independently functioning sorting signal, to our knowledge, LAPTM4 alpha is the first example of a membrane protein that requires two tandemly arranged tyrosine-based sorting signals for efficient localization in these compartments.

Secretion of hemolysin is directed by a signal sequence located within its C-terminal 60 amino acids. Deletion analyses have indicated that the extreme end of this C-terminus is critical for transport; however, it is not known if this region contains structural features necessary for function. In this study, we have used a combinatorial approach to generate two contiguous 8-residue random libraries (Cterm1 and Cterm2) in the signal sequence to investigate the functional specificity of the last 16 residues. The large number of variants generated had provided us with a rich data set to determine if a restricted subset of sequences was actually required for function in the extreme C-terminus. We observed that over 90% of the random sequences in the Cterm1 region were secreted at close to wild-type level, while the Cterm2 region was more restricted with only 50% of the random sequences supporting wild-type-like transport. It appeared that, in the Cterm2 region, the relative lack of positive charge is favored for function. These findings, along with previous results, allow us to propose a model for recognition and transport of hemolysin that emphasizes secondary structure and general biophysical properties over primary sequence. This model may have implications for understanding the broad substrate specificity common among ATP-binding cassette transporters.

To obtain a more comprehensive profile of bile acids (BAs) in blood, we developed an ultrahigh performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC-MRM-MS) method for the separation and detection of 50 known BAs. This method utilizes phospholipid-depletion solid-phase extraction as a new high-efficiency sample preparation procedure for BA assay. UPLC/scheduled MRM-MS with negative ion electrospray ionization enabled targeted quantitation of 43 and 44 BAs, respectively, in serum samples from seven individuals with and without fasting, as well as in plasma samples from six cholestatic gene knockout mice and six age- and gender-matched wild-type (FVB/NJ) animals. Many minor BAs were identified and quantitated in the blood for the first time. Method validation indicated good quantitation precision with intraday and interday relative standard deviations of ≤9.3% and ≤10.8%, respectively. Using a pooled human serum sample and a pooled mouse plasma sample as the two representative test samples, the quantitation accuracy was measured to be 80% to 120% for most of the BAs, using two standard-substance spiking approaches. To profile other potential BAs not included in the 50 known targets from the knockout versus wild-type mouse plasma, class-specific precursor/fragment ion transitions were used to perform UPLC-MRM-MS for untargeted detection of the structural isomers of glycine- and taurine-conjugated BAs and unconjugated tetra-hydroxy BAs. As a result, as many as 36 such compounds were detected. In summary, this UPLC-MRM-MS method has enabled the quantitation of the largest number of BAs in the blood thus far, and the results presented have revealed an unexpectedly complex BA profile in mouse plasma.