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Stephen Barnes

Publications and source records attributed to Stephen Barnes.

At least 55 records · Page 3Linked to original sources
PubMed2004

Modification of Cytochrome c by 4-hydroxy- 2-nonenal: evidence for histidine, lysine, and arginine-aldehyde adducts.

4-Hydroxy-2-nonenal (4HNE), a major secondary product of lipid peroxidation, has been associated with a number of disease states involving oxidative stress. Despite the recognized importance of post-translational modification of proteins by products such as 4HNE, little is known of the modification of cytochrome c by this reagent and its analysis by mass spectrometry. The purpose of this study was to investigate the chemical interaction of 4HNE and cytochrome c, a protein essential to cellular respiration, under in vitro conditions. Isoelectric focusing of native and 4HNE-modified cytochrome c using immobilized pH gradient (IpG) strips showed a decrease in the pI of the 4HNE-modified protein suggesting modification of charged amino acids. Reaction of 4HNE with cytochrome c resulted in increases in molecular weight consistent with the addition of four 4HNE residues as determined by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS). Samples of both native and 4HNE-modified cytochrome c were enzymatically digested and subjected to peptide mass fingerprinting using MALDI-TOF MS. Analysis of these samples using LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) provided sequence information that was used to determine specific residues to which the aldehyde adducted. Taken together, the data indicated that H33, K87, and R38 were modified by 4HNE. Mapping these results onto the X-ray crystal structure of native cytochrome c suggest that 4HNE adduction to cytochrome c could have significant effects on tertiary structure, electron transport, and ultimately, mitochondrial dysfunction.

Aldehydes↗
PubMed2004

Chemoprevention by grape seed extract and genistein in carcinogen-induced mammary cancer in rats is diet dependent.

Many popular dietary supplements are enriched in polyphenols such as the soy isoflavones, tea catechins, and resveratrol (from grape skins), each of which has been shown to have chemopreventive activity in cellular models of cancer. The proanthocyanidins, which are oligomers of the catechins, are enriched in grape seeds and form the basis of the dietary supplement grape seed extract (GSE). Evidence suggests that the proanthocyanidins may be metabolized to the monomeric catechins. This study was carried out to determine whether GSE added to rodent diets protected against carcinogen-induced mammary tumorigenesis in rats and whether this was affected by the composition of the whole diet. Female rats were begun on 5%, 1.25%, or 0% (control) GSE-supplemented diets at age 35 d. At age 50 d they were administered 7,12-dimethylbenz[a]anthracene (DMBA) in sesame oil at 80 mg/kg body weight. They were weighed and monitored weekly for tumor development until 120 d after DMBA administration. Administration of GSE in AIN-76A diet did not show any protective activity of GSE against DMBA-induced breast cancer. However, administration of GSE in a laboratory dry food diet (Teklad 4% rodent diet) resulted in a 50% reduction in tumor multiplicity. In similar experiments, genistein administered in AIN-76A diet also failed to show chemopreventive activity against the carcinogen N-methyl-N-nitrosourea; however, when administered at the same dose in the Teklad 4% rodent diet, genistein exhibited significant chemopreventive activity (44-61%). These results demonstrate that GSE is chemopreventive in an animal model of breast cancer; moreover, the diet dependency of the chemopreventive activity for both GSE and genistein suggests that whether or not a compound is chemopreventive may depend on the diet in which the agent is administered.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

Dietary phytoestrogens increase metabolic resistance (cold tolerance) in long-chain acyl-CoA dehydrogenase-deficient mice.

We evaluated the role of dietary phytoestrogens (PE) in the disease phenotype of cold intolerance that characterizes long-chain acyl-CoA dehydrogenase-deficient (LCAD-/-) mice, a model of inborn errors of mitochondrial fatty acid beta-oxidation. Male LCAD-/- mice were fed a standard diet containing endogenous PE, a PE-free diet, or a PE-free diet that was supplemented with genistein (250 microg/g diet). The standard diet did not restore complete cold tolerance, but it provided more resistance (P = 0.004) to cold challenge than the PE-free diet. There was a nonsignificant difference (P < 0.07) between LCAD-/- mice fed the genistein-supplemented diet and those fed the PE-free diet. There were no differences in end-point serum glucose concentrations among the 3 groups. Serum FFA were decreased in LCAD-/- mice fed the standard diet compared with those fed the PE-free diet (P = 0.005) and the diet supplemented with genistein (P < 0.001). Serum triglyceride concentrations were greater (P < 0.05) only in LCAD-/- mice fed the genistein-supplemented diet than those fed the standard diet. These results demonstrate the beneficial effects of dietary PE on metabolic tolerance in LCAD-/- mice. Furthermore, they suggest changes that could improve pediatric formula constituents, especially with regard to management of children with inborn errors of fatty acid oxidation.

Acclimatization↗
PubMed2004

Soy isoflavones--phytoestrogens and what else?

The weak estrogen-like properties of isoflavonoids were discovered over 50 y ago. In recent years, the overall effects of the isoflavones in soy on human health have been the subject of lively debate largely based on their presumed estrogenic properties. Missing from our knowledge base is the systematic identification of the cellular and biochemical targets of isoflavones and the mechanisms that they influence. Because of the benign effects of isoflavones on cellular integrity, a concentration of the isoflavones can be reached in cell culture models where almost any process can be modulated. Modern systems biology approaches and high dimensional analysis techniques offer new ways to better understand the function of how cells and integrated biological mechanisms respond to compounds such as isoflavones. Data from experiments using DNA microarray analysis for examining the effects of genistein in the developing rat uterus indicate that genistein alters the expression of 6-8 times as many genes as does a physiological estrogen such as 17 beta-estradiol. Although these new approaches are exciting, their incipient high dimensionality places considerable strain on the quality of experimental design and meaningful statistical interpretation of the resulting data. In another approach using affinity chromatography methods, DING, a novel genistein-binding protein of yet unknown function, was isolated from human breast cancer MCF-7 cells. Its function remains to be established.

Animals↗
PubMed2004

Defining and assessing tolerance in enteral nutrition.

Nutrition support has become widely recognized as an essential component of optimal care for acutely ill patients. Enteral nutrition is preferred over parenteral routes when possible. However, prescribed enteral nutritional regimens are sometimes met with side effects and even complications. These adverse events have been collectively termed "intolerance," and forms of intolerance occur in a spectrum from bothersome at least to life threatening when most severe. Here we discuss nutritional access and its maintenance, introduce and define intolerance, and then review the current literature with regard to principal forms of enteral nutrition intolerance.

Journal Article↗
PubMed2003

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties.

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.

Antioxidants↗
PubMed2003

Rat liver bile acid CoA:amino acid N-acyltransferase: expression, characterization, and peroxisomal localization.

Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver.

Acyltransferases↗
PubMed2003

Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite.

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.

4-Chloro-7-nitrobenzofurazan↗
PubMed2003

Profiling and quantification of isoflavonoids in kudzu dietary supplements by high-performance liquid chromatography and electrospray ionization tandem mass spectrometry.

The kudzu vine (Pueraria sp.) is a rich source of isoflavones. Dietary supplements based on kudzu have become commercially available. In the present study, liquid chromatography coupled with negative and positive electrospray ionization tandem mass spectrometry (MS/MS) and diode array detection (DAD) has been used for the detection and characterization of isoflavonoids in kudzu dietary supplements (KDS). The MS/MS spectrum of the protonated ion of puerarin showed characteristic product ions of the C-glycoside unit itself, whereas daidzin generated an abundant Y(0)(+) aglycon ion in its product ion spectrum. A base peak due to the loss of 120 Da [M + H - 120](+) is the diagnostic ion for C-glycosides. Neutral loss scans allowed for the detection of other C- and O-glycosides in the methanolic extract of KDS, and their structures have been proposed. The concentration of isoflavonoids in the methanolic extract of commercially available KDS was quantified by using DAD-HPLC. Puerarin, rather than daidzin, was the most abundant component (8.44-30.60 mg/capsule) in commercially available KDS.

Chromatography, High Pressure Liquid↗
PubMed2003

Identification of novel interactions in HIV-1 capsid protein assembly by high-resolution mass spectrometry.

The pleomorphic nature of the immature and mature HIV-1 virions has made it difficult to characterize intersubunit interactions using traditional approaches. While the structures of isolated domains are known, the challenge is to identify intersubunit interactions and thereby pack these domains into supramolecular structures. Using high-resolution mass spectrometry, we have measured the amide hydrogen exchange protection factors for the soluble capsid protein (CA) and CA assembled in vitro. Comparison of the protection factors as well as chemical crosslinking experiments has led to a map of the subunit/subunit interfaces in the assembled tubes. This analysis provides direct biochemical evidence for the homotypic N domain and C domain interactions proposed from cryo-electron microscopy image reconstruction of CA tubes. Most significantly, we have identified a previously unrecognized intersubunit N domain-C domain interaction. The detection of this interaction reconciles previously discrepant biophysical and genetic data.

Amides↗
PubMed2003

Mass spectrometric methods for the analysis of chlorinated and nitrated isoflavonoids: a novel class of biological metabolites.

Electrospray ionization combined with tandem mass spectrometry was applied to a study of some representative chlorinated and nitrated isoflavones-potential metabolites of isoflavones in inflammatory cells. Upon collision-induced dissociation of deprotonated [M - H](-) ions of these compounds, a number of structurally characteristic product ions were produced. The product ion analysis of 3'- and 8-chlorodaidzein in the tandom mass spectra led to ready differentiation of these isomers. 3-Nitro derivatives of both genistein and daidzein have product ions due to the losses of HNO(2) and two OH groups. Chlorinated derivatives of isoflavones were detected in cell-based experiments and their structures were proposed by comparing the tandem mass spectra of their product ions with those of standards. This work provides a suitable analytical basis to aid the characterization of chlorinated and nitrated metabolites in studies in vivo and in vitro.

Chlorine↗
PubMed2003

Neutrophil-derived matrix metalloproteinase-9 is increased in severe asthma and poorly inhibited by glucocorticoids.

BACKGROUND: Matrix metalloproteinase (MMP)-9 levels are increased in bronchoalveolar lavage (BAL) fluid from patients with severe asthma on high doses of glucocorticoids (GCs). OBJECTIVE: We sought to identify neutrophils as the source of increased BAL fluid MMP-9 in severe asthma and to evaluate the effects of GCs on this MMP-9. METHODS: MMP-9 protein, activity, and mRNA were measured in BAL fluid and cells at baseline, and after in vitro GCs in patients with severe asthma and controls using enzyme immunoassays, zymography, Western blotting, and real-time PCR. RESULTS: The high molecular weight (HMW) form of MMP-9 was significantly increased in severe asthma (P =.02). Western blotting confirmed a heterodimer of MMP-9 and neutrophil gelatinase-associated lipocalin. The HMW MMP-9 correlated with BAL neutrophils (r =.65, P <.0001). BAL cell supernatant MMP-9 protein levels also tended to be higher in patients with severe asthma (overall, P =.09), whereas the HMW activity form was increased (P =.03). MMP-9 protein (and HMW activity) correlated with neutrophils in the cell pellet (r =.75, P <.0001). In contrast to protein and activity, BAL cell mRNA levels were marginally lower in patients with severe asthma than in control subjects (overall, P =.06). Although GCs decreased BAL cell MMP-9 protein and mRNA in vitro, the effect was significantly smaller in severe asthma (P <.01 for both). GCs decreased the pro-MMP-9 activity in patients with severe asthma and normal control subjects, while having no effects on the HMW form (P =.22). Peripheral blood neutrophil MMP-9 protein was not affected by GCs. CONCLUSIONS: BAL neutrophils contribute to BAL fluid MMP-9 protein and activity and are poorly inhibited by GCs.

Acute-Phase Proteins↗
PubMed2003

Plant genome archaeology: evidence for conserved ancestral chromosome segments in dicotyledonous plant species.

We have developed genetic maps, based on expressed sequence tags (ESTs) that are homologous to Arabidopsis genes, in four dicotyledonous crop plant species from different families. A comparison of these maps with the physical map of Arabidopsis reveals common genome segments that appear to have been conserved throughout the evolution of the dicots. In the four crop species analysed these segments comprise between 16 and 33% of the Arabidopsis genome. Our findings extend the synteny patterns previously observed only within plant families, and indicate that structural and functional information from the model species will be, at least in part, applicable in crop plants with large genomes.

Journal Article↗
PubMed2003

Phyto-oestrogens and osteoporosis: what is a safe dose?

Hormone replacement therapy (HRT) for preventing loss of bone following the menopause is utilised by only 8-10 % of possible users, largely due to a fear of increased risk of breast cancer. Plant oestrogen-like compounds (phyto-oestrogens) have been proposed as an alternative to HRT to prevent osteoporosis. One class of phyto-oestrogens (the isoflavones) is found in soya foods and red clover. The food industry is developing a wide variety of new foods containing soya to substantially increase isoflavone intake, as well as extracting isoflavones from soya and clover to use as additives to non-soya foods. Pharmaceutical companies are also preparing isoflavone extracts to be used in pill form. In each case the targeted delivery is approximately 50 mg of isoflavones/d. Is this dose of isoflavones safe? In this review of the current literature, it is concluded that isoflavones consumed orally and in doses below 2 mg/kg body weight per d should be considered safe for most population groups. Whether these doses are sufficient to prevent osteoporosis is a separate matter.

Breast Neoplasms↗
PubMed2003

Polyphenols, inflammatory response, and cancer prevention: chlorination of isoflavones by human neutrophils.

An important aspect of the risk of cancer is the involvement of the inflammatory response. Currently, antiinflammatory agents are used in chemopreventive strategies. For example, aspirin is recommended for the prevention of colon cancer as well as breast and other cancers. The inflammatory response involves the production of cytokines and proinflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONO2-) produced by neutrophils and macrophages, respectively. These oxidants react with phenolic tyrosine residues on proteins to form chloro- and nitrotyrosine. Diets rich in polyphenols (green tea catechins, soy isoflavones) have also been shown to be chemopreventive. The aromatic nature of polyphenols makes them potential targets of HOCl and ONO2-. These reactions may create novel pharmacophores at the site of inflammation. Previous studies in the neutrophil-like cell line, differentiated HL-60 cells, demonstrated the formation of chlorinated and nitrated isoflavones. In this study we have examined whether similar reactions occur in freshly isolated human neutrophils. After induction of a respiratory burst with a phorbol ester, isoflavones and their metabolites were identified by liquid chromatography-tandem mass spectrometry and then quantitatively measured by LC-mass spectrometry using multiple-reaction ion monitoring. The data obtained indicate that both chlorinated and nitrated genistein are formed by human neutrophils. The extent of chlorination of genistein was markedly increased by the phorbol ester whereas the low level of nitration of genistein was constitutive and unaffected. These data imply a potential role for modified forms of genistein that would be produced in the inflammatory environment in and around a tumor.

Anticarcinogenic Agents↗
PubMed2003

DING, a genistein target in human breast cancer: a protein without a gene.

Because most noncancer cells are tolerant to high micromolar concentrations of genistein (GEN), inhibitory or stimulatory effects of GEN have been claimed for a wide variety of biochemical targets that lead to a plethora of potential mechanisms. However, because GEN is present in tissues in the nanomol-per-liter range, most of these mechanisms are unlikely to be relevant in vivo. To better identify proteins that are targets of GEN, we used a GEN-agarose-affinity phase. Cytosols from human breast cancer MCF-7 cells were fractionated over a Sephadex diethylaminoethyl column, and nonabsorbed proteins in the flow-through were affinity absorbed onto a 2-carboxygenistein-agarose column. After proteins were washed with 100 mmol NaCl/L to remove weakly bound proteins, affinity elution was conducted with 1 mmol 2-carboxygenistein/L. Using this method, a p38 protein was recovered from MCF-7 cells. N-terminal chemical sequencing of the first 30 residues of the protein revealed a peptide sequence similar to those that have been discovered in human tissues (a T-cell attractant protein from synovial fluid from patients with osteoarthritis and an analogous human skin fibroblast protein using a hirudin-affinity column) as well as a cotonine-binding protein from rat brain and related proteins in plants. In each case, the corresponding gene has not been found. In conclusion, although much of the human genome has been sequenced, novel proteins that are not described by genome data remain to be found. The DING protein (N-terminal amino acid sequence Asp-Ile-Asn-Gly) that binds to genistein with high affinity is one of these. Its biological role, however, remains to be defined.

Animals↗