共查询到20条相似文献,搜索用时 0 毫秒
1.
2.
D. Friedecký P. Bednář M. Procházka T. Adam 《Nucleosides, nucleotides & nucleic acids》2013,32(9-11):1233-1236
A pilot study using capillary electrophoresis with mass spectrometry for the analysis of nucleotides in human erythrocytes is presented. Erythrocytes were incubated with 5-amino-4-imidazolecarboxamide riboside in order to mimic situation in defect of purine metabolism—AICA-ribosiduria. Characteristic AICA-ribotides together with normal nucleotides were separated by capillary electrophoresis in acetate buffer (20 mmol/L, pH 4.4) and identified on line by mass spectrometry. 相似文献
3.
《Nucleosides, nucleotides & nucleic acids》2013,32(12):1865-1874
The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were < 1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐7‐iodoisocarbostyril (EN2) showed poor relative‐phosphorylation efficiencies (k cat /K m ) with both TK1 and dGK, but not with TK2. The k cat /K m value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5‐(1′‐(2′‐deoxy‐β‐D‐ribofuranosyl))‐2,4‐difluorotoluene (JW1) and 1‐(1′‐(2′‐deoxy‐β‐D‐ribofuranosyl))‐2,4‐difluoro‐5‐iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with k cat /K m values of 45% relative to that for Thd. 2,5‐Difluoro‐4‐[1‐(2‐deoxy‐β‐L‐ribofuranosyl)]‐aniline (JW5), a L‐nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril‐ and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated. 相似文献
4.
Akira Matsumoto Reiko Matsumoto Kei-ichi Kadoyama Taka-aki Nishimoto Shogo Matsuyama Osamu Midorikawa 《International journal of peptide research and therapeutics》2009,15(3):205-210
Establishment of diagnostic measures for early stage Alzheimer’s disease (AD) and mild cognitive impairment (MCI) is of crucial
importance. Using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS), antibody-assisted
MS of cerebrospinal fluid (CSF) has enabled quantitative analysis of the ratio of β-amyloid (Aβ) peptides, Aβ1-42/Aβ1-40,
which has a diagnostic value for AD/MCI. To apply the MS analysis to a far wider range of CSF samples, we have established
a method to analyze Aβ peptides expressed in 100 μl CSF samples quantitatively. Pretreatment of CSF samples by limit-filtration
to condense peptides, and modified washing procedure using urea as denaturant, Aβ peptides of interest can be assessed with
higher sensitivity by five to tenfolds to the original method. This improvement enables quantitative analysis of Aβ species
from a residual amount of CSF samples, which will be occasionally obtained in case of lumbar anesthesia prior to operation
and spinal tap performed for routine diagnostic purposes. Prevalence of the new procedure as laboratory test, especially among
the elderly consulting for neurological clinic, will enhance the number of subjects diagnosed at early stage of AD/MCI. 相似文献
5.
T. Berthod Y. Pétillot A. Guy J. Cadet E. Forest D. Molkot 《Nucleosides, nucleotides & nucleic acids》2013,32(7-8):1287-1305
Abstract Two oligonucleotides containing FdU (1) have been synthesized. The use of the “Pac-amidites” for the natural nucleosides has allowed the incorporation of the oxidized thymine residue without protection of the aldehydic function. The oligonucleotide composition was confirmed by enzymatic digestion and electrospray mass spectrometry. 相似文献
6.
7.
《Chronobiology international》2013,30(7):868-881
Although daily rhythms regulate multiple aspects of human physiology, rhythmic control of the metabolome remains poorly understood. The primary objective of this proof-of-concept study was identification of metabolites in human plasma that exhibit significant 24-h variation. This was assessed via an untargeted metabolomic approach using liquid chromatography–mass spectrometry (LC-MS). Eight lean, healthy, and unmedicated men, mean age 53.6 (SD?±?6.0) yrs, maintained a fixed sleep/wake schedule and dietary regime for 1 wk at home prior to an adaptation night and followed by a 25-h experimental session in the laboratory where the light/dark cycle, sleep/wake, posture, and calorific intake were strictly controlled. Plasma samples from each individual at selected time points were prepared using liquid-phase extraction followed by reverse-phase LC coupled to quadrupole time-of-flight MS analysis in positive ionization mode. Time-of-day variation in the metabolites was screened for using orthogonal partial least square discrimination between selected time points of 10:00 vs. 22:00?h, 16:00 vs. 04:00?h, and 07:00 (d 1) vs. 16:00?h, as well as repeated-measures analysis of variance with time as an independent variable. Subsequently, cosinor analysis was performed on all the sampled time points across the 24-h day to assess for significant daily variation. In this study, analytical variability, assessed using known internal standards, was low with coefficients of variation <10%. A total of 1069 metabolite features were detected and 203 (19%) showed significant time-of-day variation. Of these, 34 metabolites were identified using a combination of accurate mass, tandem MS, and online database searches. These metabolites include corticosteroids, bilirubin, amino acids, acylcarnitines, and phospholipids; of note, the magnitude of the 24-h variation of these identified metabolites was large, with the mean ratio of oscillation range over MESOR (24-h time series mean) of 65% (95% confidence interval [CI]: 49–81%). Importantly, several of these human plasma metabolites, including specific acylcarnitines and phospholipids, were hitherto not known to be 24-h variant. These findings represent an important baseline and will be useful in guiding the design and interpretation of future metabolite-based studies. (Author correspondence: Jooern. Ang@icr. ac. uk or Florence. Raynaud@icr. ac. uk) 相似文献
8.
《Molecular & cellular proteomics : MCP》2019,18(3):534-545
Highlights
- •Glycosylation of endogenous FcγRIII from neutrophils and matched plasma from more than 40 donors characterized at two sites involved in IgG binding.
- •Glycosylation of soluble FcγRIII glycosylation at N45 can be used to assign FcγRIIIb alleles.
- •FcγRIIIb allele specific differences in glycosylation at N162 may influence differential activity observed for primary cells.
9.
Ma?a Sinreih Sven Zukunft Izidor Sosi? Jo?ko Cesar Stanislav Gobec Jerzy Adamski Tea Lani?nik Ri?ner 《PloS one》2015,10(2)
Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing. 相似文献
10.
Masao Ohnishi Masuo Nakano Yasuhiko Fujino 《Bioscience, biotechnology, and biochemistry》2013,77(3):743-744
Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I–, Br–, semicarbazide, and hydroxylamine. 相似文献
11.
Laurence Conraux Catherine Pech Halim Guerraoui Denis Loyaux Pascual Ferrara Jean-Claude Guillemot Vincent Meininger Pierre-Fran?ois Pradat Fran?ois Salachas Ga?lle Bruneteau Nadine Le Forestier Lucette Lacomblez 《PloS one》2013,8(11)
The diagnostic of Amyotrophic lateral sclerosis (ALS) remains based on clinical and neurophysiological observations. The actual delay between the onset of the symptoms and diagnosis is about 1 year, preventing early inclusion of patients into clinical trials and early care of the disease. Therefore, finding biomarkers with high sensitivity and specificity remains urgent. In our study, we looked for peptide biomarkers in plasma samples using reverse phase magnetic beads (C18 and C8) and MALDI-TOF mass spectrometry analysis. From a set of ALS patients (n=30) and healthy age-matched controls (n=30), C18- or C8-SVM-based models for ALS diagnostic were constructed on the base of the minimum of the most discriminant peaks. These two SVM-based models end up in excellent separations between the 2 groups of patients (recognition capability overall classes > 97%) and classify blinded samples (10 ALS and 10 healthy age-matched controls) with very high sensitivities and specificities (>90%). Some of these discriminant peaks have been identified by Mass Spectrometry (MS) analyses and correspond to (or are fragments of) major plasma proteins, partly linked to the blood coagulation. 相似文献
12.
13.
William M. Hammargren Debra R. Luffer Karl H. Schram Mark L. J. Reimer Katsuyuki Nakano Toshio Yasaka 《Nucleosides, nucleotides & nucleic acids》2013,32(6):1275-1292
Abstract 5′-Deoxy-5′-methylthioguanosine (MTG) has been identified in human urine by combined gas chromatography/mass spectrometry. Preliminary identification of MTG in the urine of a lung cancer patient was based upon mass spectral comparisons of the trimethylsilylated (TMS) urinary component to both MTA-(TMS)3 and guanosine-(TMS)5. Structural confirmation was obtained by comparing the mass spectral and chromatographic characteristics of authentic MTG to those of the urinary component. 相似文献
14.
Ellen Stokvis Susan L. Clugston John F. Honek Albert J. R. Heck 《The protein journal》2000,19(5):389-397
Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-{2-[3-hexyloxybenzoyl]-vinyl}glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme. 相似文献
15.
Wei Huang Krishnakumar?M. Ravikumar Mark?R. Chance Sichun Yang 《Biophysical journal》2015,108(1):107-115
Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest. 相似文献
16.
Biological Trace Element Research - Human blood is a complex sample matrix when elemental analysis is considered. In this study, the effects of Na, a natural component of serum samples, was... 相似文献
17.
Rong Wang Lifang Guo Hua Xie Juanhong Zhang Xiaoyun Li Wenbing Li Jianfeng Wang Xiaoyu Wu Zhengping Jia 《Cell biochemistry and biophysics》2013,67(3):1343-1351
A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG2 cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1 % formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4 mL min?1. The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 → m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL?1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15 %. The mean recovery of (+)-MTX and (?)-MTX in the extracellular fluid of HepG2 cells were 95.30 and 96.53 %, respectively, and the mean recovery of (+)-MTX and (?)-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12 %, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG2 cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of (?)-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and (?)-MTX was (+)-MTX > (?)-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro. 相似文献
18.
Philip L?ssl Knut K?lbel Dirk T?nzler David Nannemann Christian H. Ihling Manuel V. Keller Marian Schneider Frank Zaucke Jens Meiler Andrea Sinz 《PloS one》2014,9(11)
We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2–4, γ1 LEb2–4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2–4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces. 相似文献
19.
Senarath B. P. Athauda Masaaki Nishigai Hideo Arakawa Atsushi Ikai Masanori Ukai Kenji Takahashi 《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):219-224
The inhibitory effects of human α2-macroglobulin (α2-M), a major plasma proteinase inhibitor, on human pepsin and gastricsin were investigated. The activities of pepsin and gastricsin towards a protein substrate (reduced and carboxymethylated ribonuclease A) were significantly inhibited by α2-M at pH 5.5, whereas those towards a peptide substrate (oxidized insulin B-chain) were scarcely inhibited. Under these conditions at pH 5.5, pepsin and gastricsin cleaved α2-M mainly at the His694-Ala695 bond and Leu697-Val698 bond, respectively, in the bait regions sequence of α2-M. The conformation of α2-M was also shown to be markedly altered upon inhibition of these enzymes as examined by native polyacrylamide gel electrophoresis and electron microscopy. These results show the entrapment and concomitant inhibition of those proteinases by α2-M. 相似文献
20.