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Results: 1 to 10 of 33

Publication Record


Simplified LC/MS assay for the measurement of isolevuglandin protein adducts in plasma and tissue samples.
Yermalitsky VN, Matafonova E, Tallman K, Li Z, Zackert W, Roberts LJ, Amarnath V, Davies SS
(2019) Anal Biochem 566: 89-101
MeSH Terms: Aldehydes, Animals, Chromatography, Liquid, Ketones, Lipids, Mice, Mice, Inbred C57BL, Protein Processing, Post-Translational, Proteins, Tandem Mass Spectrometry
Show Abstract · Added July 17, 2019
Isolevuglandins (IsoLGs) are a family of highly reactive 4-ketoaldehydes formed by lipid peroxidation that modify the lysyl residues of cellular proteins. Modification of proteins by IsoLGs have been shown to contribute to disease processes such as the development of hypertension. Accurate quantitation of the extent of protein modification by IsoLGs is essential for understanding the mechanisms whereby these modifications contribute to disease and the efficacy of interventions designed to prevent this modification. The previously described LC/MS assay to quantitate IsoLG protein adducts was extremely labor-intensive and time consuming, and while it offered reasonably low intra-day variation for replicate samples, variation when replicate samples were processed on separate days was significant. These limitations significantly restricted utilization of this approach. We therefore performed a series of studies to optimize the assay. We now report a significantly simplified LC/MS assay for measurement of IsoLG protein adducts with increased sensitivity and lower intra-day and inter-day variability.
Copyright © 2018. Published by Elsevier Inc.
1 Communities
1 Members
0 Resources
10 MeSH Terms
Modification by isolevuglandins, highly reactive γ-ketoaldehydes, deleteriously alters high-density lipoprotein structure and function.
May-Zhang LS, Yermalitsky V, Huang J, Pleasent T, Borja MS, Oda MN, Jerome WG, Yancey PG, Linton MF, Davies SS
(2018) J Biol Chem 293: 9176-9187
MeSH Terms: Aldehydes, Animals, Apolipoprotein A-I, Apolipoprotein A-II, Cells, Cultured, Cholesterol, Female, Humans, Hyperlipoproteinemia Type II, Ketones, Lipid Metabolism, Lipids, Lipoproteins, HDL, Macrophages, Male, Mice, Mice, Inbred C57BL, Phosphatidylethanolamines
Show Abstract · Added August 3, 2018
Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia ( = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls ( = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
4 Members
0 Resources
18 MeSH Terms
To Avoid Chasing Incorrect Chemical Structures of Chiral Compounds: Raman Optical Activity and Vibrational Circular Dichroism Spectroscopies.
Polavarapu PL, Covington CL, Raghavan V
(2017) Chemphyschem 18: 2459-2465
MeSH Terms: Alcohols, Alkynes, Circular Dichroism, Furans, Ketones, Lactones, Molecular Structure, Quantum Theory, Sesquiterpenes, Spectrum Analysis, Raman
Show Abstract · Added April 10, 2018
A chemical structure (CS) identifies the connectivities between atoms, and the nature of those connections, for a given elemental composition. For chiral molecules, in addition to the identification of CS, the identification of the correct absolute configuration (AC) is also needed. Several chiral natural products are known whose CSs were initially misidentified and later corrected, and these errors were often discovered during the total synthesis of natural products. In this work, we present a new and convenient approach that can be used with Raman optical activity (ROA) and vibrational circular dichroism (VCD) spectroscopies, to distinguish between the correct and incorrect CSs of chiral compounds. This approach involves analyzing the spectral similarity overlap between experimental spectra and those predicted with advanced quantum chemical theories. Significant labor needed for establishing the correct CSs via chemical syntheses of chiral natural products can thus be avoided.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
0 Communities
1 Members
0 Resources
MeSH Terms
Electrophilic Modification of PKM2 by 4-Hydroxynonenal and 4-Oxononenal Results in Protein Cross-Linking and Kinase Inhibition.
Camarillo JM, Ullery JC, Rose KL, Marnett LJ
(2017) Chem Res Toxicol 30: 635-641
MeSH Terms: Aldehydes, Cell Line, Tumor, Chromatography, Liquid, Click Chemistry, Enzyme Inhibitors, Humans, Ketones, Pyruvate Kinase, Tandem Mass Spectrometry
Show Abstract · Added April 22, 2018
Rapidly proliferating cells require an increased rate of metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway, and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is strongly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly termed the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 in vitro, including Cys424 and His439, which play a role in protein-protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of the electrophile, most notably in the case of ONE, result in substantial protein-protein cross-linking in vitro and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation.
0 Communities
1 Members
0 Resources
MeSH Terms
Discovery and SAR of a novel series of potent, CNS penetrant M4 PAMs based on a non-enolizable ketone core: Challenges in disposition.
Wood MR, Noetzel MJ, Tarr JC, Rodriguez AL, Lamsal A, Chang S, Foster JJ, Smith E, Chase P, Hodder PS, Engers DW, Niswender CM, Brandon NJ, Wood MW, Duggan ME, Conn PJ, Bridges TM, Lindsley CW
(2016) Bioorg Med Chem Lett 26: 4282-6
MeSH Terms: Allosteric Regulation, Animals, Central Nervous System, Drug Discovery, Humans, Ketones, Molecular Structure, Receptor, Muscarinic M1, Structure-Activity Relationship
Show Abstract · Added April 6, 2017
This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.
Copyright © 2016 Elsevier Ltd. All rights reserved.
0 Communities
2 Members
0 Resources
9 MeSH Terms
A peptide-based positron emission tomography probe for in vivo detection of caspase activity in apoptotic cells.
Hight MR, Cheung YY, Nickels ML, Dawson ES, Zhao P, Saleh S, Buck JR, Tang D, Washington MK, Coffey RJ, Manning HC
(2014) Clin Cancer Res 20: 2126-35
MeSH Terms: Amino Acid Chloromethyl Ketones, Animals, Apoptosis, Caspase 3, Caspase Inhibitors, Cell Line, Tumor, Colonic Neoplasms, Colorectal Neoplasms, Female, Fluorine Radioisotopes, Fluorobenzenes, Humans, Imidazoles, Immunoblotting, Immunohistochemistry, Indoles, Mice, Inbred C57BL, Mice, Nude, Organophosphates, Peptides, Positron-Emission Tomography, Protein Kinase Inhibitors, Quinazolines, Quinolines, Radiopharmaceuticals, Sulfonamides, Tissue Distribution, Xenograft Model Antitumor Assays
Show Abstract · Added March 20, 2014
PURPOSE - Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET).
EXPERIMENTAL DESIGN - Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer.
RESULTS - Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size.
CONCLUSIONS - These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.
©2014 AACR.
1 Communities
3 Members
0 Resources
28 MeSH Terms
Preconditioning mesenchymal stem cells with caspase inhibition and hyperoxia prior to hypoxia exposure increases cell proliferation.
Saini U, Gumina RJ, Wolfe B, Kuppusamy ML, Kuppusamy P, Boudoulas KD
(2013) J Cell Biochem 114: 2612-23
MeSH Terms: Amino Acid Chloromethyl Ketones, Animals, Caspase 1, Caspase 3, Caspase 6, Caspase 7, Caspase 9, Cell Hypoxia, Cell Proliferation, Cells, Cultured, Male, Mesenchymal Stem Cells, Rats
Show Abstract · Added February 21, 2015
Myocardial infarction is a leading cause of mortality and morbidity worldwide. Occlusion of a coronary artery produces ischemia and myocardial necrosis that leads to left ventricular (LV) remodeling, dysfunction, and heart failure. Stem cell therapy may decrease infarct size and improve LV function; the hypoxic environment, however, following a myocardial infarction may result in apoptosis, which in turn decreases survival of transplanted stem cells. Therefore, the effects of preconditioned mesenchymal stem cells (MSC) with hyperoxia (100% oxygen), Z-VAD-FMK pan-caspase inhibitor (CI), or both in a hypoxic environment in order to mimic conditions seen in cardiac tissue post-myocardial infarction were studied in vitro. MSCs preconditioned with hyperoxia or CI significantly decreased apoptosis as suggested by TUNEL assay and Annexin V analysis using fluorescence assisted cell sorting. These effects were more profound when both, hyperoxia and CI, were used. Additionally, gene and protein expression of caspases 1, 3, 6, 7, and 9 were down-regulated significantly in MSCs preconditioned with hyperoxia, CI, or both, while the survival markers Akt1, NF-κB, and Bcl-2 were significantly increased in preconditioned MSCs. These changes ultimately resulted in a significant increase in MSC proliferation in hypoxic environment as determined by BrdU assays compared to MSCs without preconditioning. These effects may prove to be of great clinical significance when transplanting stem cells into the hypoxic myocardium of post-myocardial infarction patients in order to attenuate LV remodeling and improve LV function.
© 2013 Wiley Periodicals, Inc.
0 Communities
1 Members
0 Resources
13 MeSH Terms
Site-specific AGE modifications in the extracellular matrix: a role for glyoxal in protein damage in diabetes.
Voziyan P, Brown KL, Chetyrkin S, Hudson B
(2014) Clin Chem Lab Med 52: 39-45
MeSH Terms: Aldehydes, Diabetes Mellitus, Extracellular Matrix, Extracellular Matrix Proteins, Glycation End Products, Advanced, Glyoxal, Humans, Ketones
Show Abstract · Added February 25, 2014
Non-enzymatic modification of proteins in hyperglycemia is a major proposed mechanism of diabetic complications. Specifically, advanced glycation end products (AGEs) derived from hyperglycemia-induced reactive carbonyl species (RCS) can have pathogenic consequences when they target functionally critical protein residues. Modification of a small number of these critical residues, often undetectable by the methodologies relying on measurements of total AGE levels, can cause significant functional damage. Therefore, detection of specific sites of protein damage in diabetes is central to understanding the molecular basis of diabetic complications and for identification of biomarkers which are mechanistically linked to the disease. The current paradigm of RCS-derived protein damage places a major focus on methylglyoxal (MGO), an intermediate of cellular glycolysis. We propose that glyoxal (GO) is a major contributor to extracellular matrix (ECM) damage in diabetes. Here, we review the current knowledge and provide new data about GO-derived site-specific ECM modification in experimental diabetes.
1 Communities
3 Members
0 Resources
8 MeSH Terms
High-throughput quantification of bioactive lipids by MALDI mass spectrometry: application to prostaglandins.
Manna JD, Reyzer ML, Latham JC, Weaver CD, Marnett LJ, Caprioli RM
(2011) Anal Chem 83: 6683-8
MeSH Terms: Animals, Cell Line, Tumor, Cyclooxygenase 2, High-Throughput Screening Assays, Ketones, Lipids, Metabolome, Mice, Prostaglandins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added March 7, 2014
Analysis and quantification of analytes in biological systems is a critical component of metabolomic investigations of cell function. The most widely used methods employ chromatographic separation followed by mass spectrometric analysis, which requires significant time for sample preparation and sequential chromatography. We introduce a novel high-throughput, separation-free methodology based on MALDI mass spectrometry that allows for the parallel analysis of targeted metabolomes. Proof-of-concept is demonstrated by analysis of prostaglandins and glyceryl prostaglandins. Derivatization to incorporate a charged moiety into ketone-containing prostaglandins dramatically increases the signal-to-noise ratio relative to underivatized samples. This resulted in an increased dynamic range (15-2000 fmol on plate) and improved linearity (r(2) = 0.99). The method was adapted for high-throughput screening methods for enzymology and drug discovery. Application to cellular metabolomics was also demonstrated.
1 Communities
3 Members
0 Resources
10 MeSH Terms
Targeting colon cancer stem cells using a new curcumin analogue, GO-Y030.
Lin L, Liu Y, Li H, Li PK, Fuchs J, Shibata H, Iwabuchi Y, Lin J
(2011) Br J Cancer 105: 212-20
MeSH Terms: Animals, Antineoplastic Agents, Benzene Derivatives, Carcinoma, Cell Line, Tumor, Colonic Neoplasms, Curcumin, Drug Delivery Systems, Female, HCT116 Cells, HT29 Cells, Humans, Ketones, Mice, Mice, Inbred NOD, Mice, SCID, Neoplastic Stem Cells, Xenograft Model Antitumor Assays
Show Abstract · Added June 14, 2013
BACKGROUND - Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer, including colon cancer. To date, whether STAT3 is activated and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, in colon cancer stem cells are still unknown.
METHODS - Flow cytometry was used to isolate colon cancer stem cells, which are characterised by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulations (ALDH(+)/CD133(+)). The levels of STAT3 phosphorylation and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, that targets STAT3 in colon cancer stem cells were examined.
RESULTS - Our results observed that ALDH(+)/CD133(+) colon cancer cells expressed higher levels of phosphorylated STAT3 than ALDH-negative/CD133-negative colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. GO-Y030 and curcumin inhibited STAT3 phosphorylation, cell viability, tumoursphere formation in colon cancer stem cells. GO-Y030 also reduced STAT3 downstream target gene expression and induced apoptosis in colon cancer stem cells. Furthermore, GO-Y030 suppressed tumour growth of cancer stem cells from both SW480 and HCT-116 colon cancer cell lines in the mouse model.
CONCLUSION - Our results indicate that STAT3 is a novel therapeutic target in colon cancer stem cells, and inhibition of activated STAT3 in cancer stem cells by GO-Y030 may offer an effective treatment for colorectal cancer.
0 Communities
1 Members
0 Resources
18 MeSH Terms