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1.
Stingl C van Vilsteren FG Guzel C Ten Kate FJ Visser M Krishnadath KK Bergman JJ Luider TM 《Journal of proteome research》2011,10(1):288-298
Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis. 相似文献
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Saadet Gümüşlü Süreyya Bi˙lmen Dijle Ki˙pmen Korgun Piraye Yargiçoğlu Aysel Ağar 《Free radical research》2013,47(6):621-627
Antioxidant defenses within the lung are pivotal in preventing damage from oxidative toxicants. There have also been several reports with conflicting results on the antioxidant system during aging. In this study, we attempted to investigate age-related alterations in both antioxidant enzyme activities and thiobarbituric acid-reactive substances (TBARS), a product of lipid peroxidation, in the whole lung of control and sulfur dioxide (SO2) exposed rats of different age groups (3-, 12-, and 24-months-old). Swiss-Albino Male rats were exposed to 10 ppm SO2 1 hr/day, 7 days/week for 6 weeks. The antioxidant enzymes examined include Cu,Zn-superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST). A mixed pattern of age-associated alterations in antioxidant activities was observed. SOD, GSH-Px and GST activities were increased with age, but CAT activity was decreased. Lung SOD, GSH-Px and GST activities were also increased in response to SO2. The level of TBARS was increased with age. SO2 exposure stimulated lipid peroxide formation in the lung as indicated by an increase in the level of TBARS. These findings suggest that both aging and SO2 exposure may impose an oxidative stress to the body. We conclude that the increase in the activities of the antioxidant enzymes of the lung during aging, could be interpreted as a positive feedback mechanism in response to rising lipid peroxidation. 相似文献
4.
Alexei Kharitonenkov John M. Beals Radmila Micanovic Beth A. Strifler Radhakrishnan Rathnachalam Victor J. Wroblewski Shun Li Anja Koester Amy M. Ford Tamer Coskun James D. Dunbar Christine C. Cheng Christopher C. Frye Thomas F. Bumol David E. Moller 《PloS one》2013,8(3)
Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7–14 days resulted in a 25–50% lowering of plasma glucose coupled with a 10–30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans. 相似文献
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Devrim Coskun Dev T. Britto Yuel-Kai Jean Imtiaz Kabir Inci Tolay Ayfer A. Torun Herbert J. Kronzucker 《PloS one》2013,8(2)
Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K+) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with 42K, coupled with growth and tissue K+ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K+ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K+ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K+ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na+ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K+ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K+ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na+ accumulation is the key factor in performance decline on NaCl stress. 相似文献
6.
Abdulkadir Kucukbayrak Saadet Cakmak Ismail Necati Hakyemez Tekin Tas Hayrettin Akdeniz 《Folia microbiologica》2013,58(4):343-347
Since the 1990s, blood donors have been scanned for anti-hepatitis C virus (anti-HCV) antibodies, which can be defined by enzyme immunoassay as a screening test. In this population, false-reactive ratios have been high. Recently, some authors have aimed to find a cutoff value for anti-HCV different from those established by test manufacturers to predict HCV infection. In this study, 321 patients, after two repeating tests, had reactive results in s/co <10 titers on anti-HCV test. The patients were 29.6 % (n?=?95) in women and 70.4 % (n?=?226) in men. The patients were classified into three groups by Western blot (WB) results (PS, positive; NG, negative; and ID, indeterminate). The average anti-HCV titer of the whole group was 2.61?±?1.96. Anti-HCV titers of subgroups were 2.43?±?1.95 in NG, 4.93?±?2.53 in PS, and 2.50?±?1.65 in ID (p?<?0.001). There was a significant difference between NG and PS and between PS and ID subgroups (p?<?0.001). There was a positive correlation between WB and anti-HCV titers in all patients (r?=?0.298, p?<?0.001), in women (r?=?0.282, p?<?0.001), and in men (r?=?0.337, p?=?0.002). According to receiver operator characteristic curve analysis, the cutoff value of anti-HCV titer to predict hepatitis C infection was >2.61 s/co, with 74.1 % sensitivity and 71.6 % specificity (area under the curve, 0.820; 95 % confidence interval, 0.753 to 0.887). We suggest that an effective cutoff value for anti-HCV other than that established by the manufacturer cannot be assigned to predict hepatitis C infection for blood donors in low-prevalence areas. 相似文献
7.
Martin Neumann Ebru Coskun Lars Fransecky Liliana H. Mochmann Isabelle Bartram Nasrin Farhadi Sartangi Sandra Heesch Nicola G?kbuget Stefan Schwartz Christian Brandts Cornelia Schlee Rainer Haas Ulrich Dührsen Martin Griesshammer Hartmut D?hner Gerhard Ehninger Thomas Burmeister Olga Blau Eckhard Thiel Dieter Hoelzer Wolf-Karsten Hofmann Claudia D. Baldus 《PloS one》2013,8(1)
Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup. 相似文献
8.
Hacer Esen Saadet Alpdağtaş Mehmet Mervan Çakar 《Preparative biochemistry & biotechnology》2019,49(5):529-534
AbstractSeveral protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD+ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD+ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH. 相似文献
9.
The response of V(1) ATPase of the tobacco hornworm Manduca sexta to Mg(2+) and nucleotide binding in the presence of the enhancer methanol has been studied by CuCl(2)-induced disulfide formation, fluorescence spectroscopy, and small-angle X-ray scattering. When the V(1) complex was supplemented with CuCl(2) nucleotide-dependence of A-B-E and A-B-E-D cross-linking products was observed in absence of nucleotides and presence of MgADP+Pi but not when MgAMP.PNP or MgADP were added. A zero-length cross-linking product of subunits D and E was formed, supporting their close proximity in the V(1) complex. The catalytic subunit A was reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP.PNP, -ATP, -ADP+Pi, or -ADP. Differences in the fluorescence emission of these nucleotide-binding states were monitored using the intrinsic tryptophan fluorescence. The structural composition of the V(1) ATPase from M. sexta and conformational alterations in this enzyme due to Mg(2+) and nucleotide binding are discussed on the basis of these and previous observations. 相似文献
10.
Reeve JR Liddle RA McVey DC Vigna SR Solomon TE Keire DA Rosenquist G Shively JE Lee TD Chew P Green GM Coskun T 《American journal of physiology. Gastrointestinal and liver physiology》2004,287(2):G326-G333
Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent. 相似文献