Microgram Of Total Protein Research Articles

Selecting patients with stage II/III colorectal cancer for 5-fluorouracil-based adjuvant chemotherapy using proteomic analysis.

708 Background: 5-fluorouracil (5-FU) is a common adjuvant treatment for stage III and high-risk stage II colorectal cancer (CRC). However, about 20% of patients relapse within 48 months of treatment with 5-FU, even when combined with oxaliplatin. To improve patient selection, tumor biomarkers that predict sensitivity to 5-FU have been proposed. These include proteins involved in 5-FU activation or metabolism such as uridine-cytidine kinase 2 (UCK2) and thymidylate synthase (TYMS). We used multiplexed mass spectrometry to evaluate the utility these biomarkers in the archived tumor samples of patients with stage II/III CRC. Methods: Tumor samples were from 143 patients with stage II/III CRC who received adjuvant 5-FU, folinic acid, and oxaliplatin during 2000-2014; 83% of patients received 12 cycles and the others received ≤ 11 cycles. Tumor cells were microdissected and solubilized, and 67 candidate biomarkers were quantitated using mass spectrometry. Overall survival (OS) and relapse-free survival (RFS) were assessed using the Kaplan-Meier method and log-rank test. Protein expression by tumor stage, lymph node (LN) status, tumor sidedness was compared using the Student’s t-test. Results: Of 143 patients, 45 had recurrence and 98 patients did not. UCK2 was detected in all samples, ranging from 187 to 1606 attomoles per microgram of total protein (amol/µg). Patients with UCK2 expression above 335 amol/μg (n = 109) had significantly longer OS than patients with lower expression (n = 34; HR: 0.42; p= 0.009). There was no significant difference in RFS (HR: 0.6; p= 0.088). UCK2 expression did not differ by disease stage, LN metastasis status, or tumor sidedness. TYMS expression was not associated with survival in this cohort. Analysis of other biomarkers associated with response to 5-FU and platinum is in progress. Conclusions: In stage II/III CRC, UCK2 expression above 335 amol/μg identifies a subgroup of 5-FU-treated CRC patients with longer survival, suggesting that quantitated UCK2 has potential for use in selecting patients for treatment. These findings warrant validation in larger cohorts.

Abstract 3871: Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics

Abstract The dog serves as a useful model of spontaneously occurring cancer by virtue of similar biology, therapies and mechansims of chemotherapeutic resistance. The ABC transporters are a superfamily of membrane bound proteins that are widely distributed across mammalian tissues and constitute a substantial component of variability in anticancer drug pharmakokinetics and tumor cell resistance. Thus a quantitative atlas of ATP-binding cassette (ABC) transporters across canine tissues is crucial for improving our ability to extrapolate data from dog to human. We have employed a quantitative proteomic approach based on multiple reaction monitoring in tandem mass spectrometry that relies on detection of novel peptide sequences following trypsin digestion of biological samples and separation of peptides utilizing high-pressure liquid chromatography. The 14 proteins evaluated include P-gp, Bcrp, Bsep, Mrp1-6, Mrp7, Mrp9, and the products of canine Abcg5 and Abcg8. Peptide sequences were identified via an in-silico trypsin digestion of proteins and signature peptides were generated for identification and quantitation using stable isotope-labeled signature peptides as an internal reference. Signature peptides were chosen based on lack of posttranslational modification sites and known polymorphisms. Isolated brain microvessels, liver, kidney, duodenum, jejunum, ileum and lung were collected at necropsy from ten normal dogs and membrane fractions of each tissue were prepared. Additional samples were collected for isolation of mRNA. Membrane protein fractions were digested with trypsin and total protein concentrations determined by BCA assay. Calibration curves for signature peptides were prepared in a bovine serum albumin digestate and ranged from 0.25 fmol to 200 fmol on column. Quantitation of peptides in tissue samples was performed via linear regression analysis of calibration curves with extrapolation using at least two transitions for each peptide. Results were obtained as femtomole of peptide per microgram of total protein in the sample. Differential protein levels across tissues were compared to quantitative real time RT-PCR results for the corresponding genes and differences between tissues with each method were concordant. Our results provide the first quantitative atlas of ABC transporters across normal dog tissues and provide a framework for improved understanding of pharmacokinetic and pharmacodynamic variability among dogs receiving anticancer therapies. In addition, this panel will allow evaluation of the potential role that each of these transporters plays in resistance to chemotherapy in vitro and in vivo in dogs, a relevant preclinical model of naturally occurring cancers. Citation Format: Luke A. Wittenburg, Holly Conger, Dominique Ramirez, Daniel Gustafson. Simultaneous absolute quantitation of ATP-binding cassette transporters in canine tissues via targeted proteomics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3871.

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Limited packaging capacity has hampered adeno-associated virus (AAV)-mediated gene therapy for many common genetic diseases such as cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD). Trans-splicing AAV (tsAAV) vectors double AAV packaging capacity but their transduction efficiency has been too low to be useful. We have recently overcome this hurdle by rational vector design. We have shown that a pair of optimized mini-dystrophin tsAAV vectors can reach the same transduction efficiency as that of a single AAV vector after local injection in dystrophic muscle. However, global gene transfer is required to treat diseases like DMD. To test whether systemic delivery can be achieved with tsAAV vectors, we generated a set of optimized alkaline phosphatase (AP) tsAAV vectors. We delivered AAV serotype 9 pseudotyped AP tsAAV intravenously to newborn mice. Six weeks later, we observed high-level transduction in all body skeletal muscle and the heart, the tissues that are affected in DMD. We also detected efficient transduction in the lung, the primary organ affected in CF. Our results provide the first evidence of whole-body transduction with tsAAV vectors and further raise the hope of tsAAV gene therapy for DMD and CF.

Increased Expression of Mcl-1 Is Required for Protection against Serum Starvation in Phosphatase and Tensin Homologue on Chromosome 10 Null Mouse Embryonic Fibroblasts, but Repression of Bim Is Favored in Human Glioblastomas

Inactivating mutations in the tumor suppressor gene phosphatase and tensin homologue on chromosome 10 (PTEN) result in elevated levels of phosphatidylinositol (3,4,5)-trisphosphate, activation of protein kinase B (PKB), and protection against apoptotic insults such as withdrawal of survival factors. Protection may arise through the inhibition of the pro-apoptotic protein Bim, which is normally repressed by a PKB-dependent mechanism. Here we show that PTEN-/- immortalized mouse embryonic fibroblasts (MEFs) exhibit elevated PKB phosphorylation and are resistant to serum withdrawal-induced death, but exhibit normal Bim expression following withdrawal of serum. In contrast, expression of Mcl-1, a prosurvival member of the Bcl-2 family, was elevated in PTEN-/- MEFs. Transient or stable overexpression of Mcl-1 in PTEN+/- MEFs conferred resistance to serum withdrawal, whereas ablating expression of Mcl-1 in PTEN-/- MEFs, using RNA interference, abolished their resistance to serum withdrawal-induced apoptosis. To determine if Mcl-1 is selected for overexpression in human tumors we examined human glioblastoma cell lines but found that loss of PTEN had no effect on Mcl-1 expression. In contrast, two of three PTEN-/- glioblastoma cell lines exhibited low expression of Bim, which was refractory to serum withdrawal. These results indicate that the resistance of PTEN-/- MEFs to serum withdrawal is largely due to the up-regulation of Mcl-1 but that loss of PTEN in tumor cell lines is more complex and may favor de-regulation of different apoptotic regulators such as Bim.

Trout ovulatory proteins are partially responsible for the anti-proteolytic activity found in trout coelomic fluid.

After ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Using a chromogenic peptide substrate, the anti-protease activity of brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activities were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 microgram of total protein, respectively. Using subtractive cDNA cloning, we have previously characterized a set of ovarian proteins called TOPs (trout ovulatory proteins) that are secreted into the coelomic fluid after ovulation. TOPs are most homologous to mammalian antileukoprotease, a heat- and acid-stable serine protease inhibitor. On the basis of this homology, we hypothesized that the anti-trypsin activity observed in the coelomic fluid was related to the presence of TOPs. In the present study, this hypothesis was supported by the acid- and heat-stability of the anti-trypsin activity present in coelomic fluid. Coelomic fluid could be heated to 50 degrees C or treated at a pH less than 5.2 without a significant decrease in the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls. We propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to act as protease inhibitors following ovulation.

Growth phase variation of integration host factor level in Escherichia coli.

We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12. Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures. IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity. Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase. We attribute this reduction to the loss of stabilizing interactions between subunits. A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated. We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites. Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites.

Ontogeny of a Human Polychlorinated Biphenyl-Binding Protein: Level of Expression in Tracheal Aspirates in Bronchopulmonary Dysplasia

A human lung polychlorinated biphenyl binding protein (PCB-BP, M_r 13 kd) has recently been purified from lavage fluid. Polyclonal monospecific antibodies against PCB-BP were produced and used for immunohistochemical staining in sections of human lung tissue. PCB-BP was found to localize preferentially to the nonciliated (Clara) cells of the lung, whereas the alveolar cells and ciliated cells of the larger airways were devoid of staining. Tracheal aspirates from infants receiving mechanical ventilation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and Western immunoblotting. The antibodies to human PCB-BP detected a single band of the expected molecular weight, and a quantitative analysis of the ontogeny of PCB-BP in tracheal aspirates was performed by construction of Western immunblot standard curves. A significant increase in the levels of PCB-BP in late gestation (gestational weeks 39 to 41) was demonstrated. In similar experiments, levels of PCB-BP in tracheal aspirates obtained from infants with bronchopulmonary dysplasia (BPD) at the postconceptional age of less than 38 weeks were found to be significantly elevated as compared with a normal study group of similar gestational age (21.8 ± 4.8 vs 3.1 ± 0.8 ng of PCB-BP per microgram of total protein, p<0.005). It is suggested that the high levels of PCB-BP at the postconceptional age of less than 38 weeks observed in infants with BPD may reflect inflammatory injury and regeneration of airway epithelium, associated with proliferation of Clara cells.

Deficiency of Alveolar Fluid Glutathione in Patients with Sepsis and the Adult Respiratory Distress Syndrome

The adult respiratory distress syndrome (ARDS) is a devastating clinical illness characterized by refractory hypoxemia and high-permeability pulmonary edema. Reactive oxygen species such as hydrogen peroxide and hypochlorous acid may play a key role in the pathogenesis of the acute lung injury. Glutathione (GSH) is a tripeptide that is able to react with and effectively neutralize oxidants such as hydrogen peroxide and hypochlorous acid. The present study found that the alveolar epithelial lining fluid of patients with ARDS was deficient in total GSH compared to normal subjects (21.7 mumols +/- 7.8 mumols vs 91.8 mumols +/- 14.5 mumols; p = 0.002). In addition, if GSH was measured in unconcentrated bronchoalveolar lavage (BAL) fluid and indexed to total BAL protein, there was also a deficiency in patients with ARDS compared to normal subjects (0.004 +/- 0.003 nmol of GSH per microgram of total protein vs 0.026 +/- 0.005 nmol of GSH per microgram of total protein; p = 0.002). Since patients with ARDS are subjected to an increased burden of oxidants in the alveolar fluid, principally released by recruited neutrophils, this deficiency of GSH may predispose these patients to enhanced lung cell injury.

Rapid clonal growth and serial passage of human diploid fibroblasts in a lipid-enriched synthetic medium supplemented with epidermal growth factor, insulin, and dexamethasone.

A serum-free, hormone-supplemented medium, enriched with a mixture of lipids, has been developed that supports rapid clonal growth of human diploid fetal lung fibroblasts (Flow 2000, WI-38, MRC-5, and IMR-90) and of low-passage human foreskin fibroblasts. The medium, which contains less than 1 microgram of total protein per ml, also supports serial passage of Flow 2000 cells under totally serum-free conditions. It provides lipid at a total of 10 micrograms/ml as a liposome prepared from a mixture of soybean lecithin, cholesterol, sphingomyelin, vitamin E, and vitamin E acetate. The soybean lecithin, which contains a variety of naturally occurring phospholipids, can be replaced with a mixture of highly purified phospholipids. Except for possible contaminants in the substances used in its preparation, the serum-free medium is fully defined chemically. It consists of an optimized basal nutrient medium, MCDB 110, supplemented with insulin, epidermal growth factor, dexamethasone, prostaglandins E1 and F2 alpha, phosphoenolpyruvate, dithiothreitol, glutathione, and the lipids listed above.

Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein.

A method is described for radioiodination to high specific activity of fixed and stained proteins within sodium dodecyl sulfate-polyacrylamide gels, without elution of the proteins from the gel. Following radioiodination, the proteins can be removed from the gel by trypsin treatment and the peptides analyzed. This procedure offers a means to structurally compare the proteins of multicomponent systems when purification of each component to homogeneity is unfeasible. Using this technique, we have compared the tryptic peptides of all the major protein components of Moloney and Rauscher leukemia viruses using only 50 to 100 microgram of total protein from each virus. Additionally, we have analyzed the membrane proteins of Dictyostelium discoideum at various stages in development. The validity of the technique and its value as a tool for comparative studies and identification of precursor-product relationships is discussed.