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Stephen Barnes

Publications and source records attributed to Stephen Barnes.

68 records · Page 4Linked to original sources
PubMed2002

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization.

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.

Actins↗
PubMed2002

Review of the methods used in the determination of phytoestrogens.

Interest in analytical methods for plant estrogens (phytoestrogens) has risen sharply in the past 10 years. In this review, we examine the existing analytical methods based on separations by gas-liquid chromatography, high-performance liquid chromatography and capillary electrophoresis in addition to methods of detection by ultraviolet absorption, fluorescence, electrochemical oxidation/reduction and mass spectrometry. These methods are compared with other methods of phytoestrogen analysis utilizing immunoassay approaches. The advantages and disadvantages of each of these methods are highlighted and potential areas for further development identified.

Chromatography, High Pressure Liquid↗
PubMed2002

Conserved residues in the putative catalytic triad of human bile acid Coenzyme A:amino acid N-acyltransferase.

Human bile acid-CoA:amino acid N-acyltransferase (hBAT), an enzyme catalyzing the conjugation of bile acids with the amino acids glycine or taurine has significant sequence homology with dienelactone hydrolases and other alpha/beta hydrolases. These enzymes have a conserved catalytic triad that maps onto the mammalian BATs at residues Cys-235, Asp-328, and His-362 of the human sequence, albeit that the hydrolases contain a serine instead of a cysteine. In the present study, the function of the putative catalytic triad of hBAT was examined by chemical modification with the cysteine alkylating reagent N-ethylmaleimide (NEM) and by site-directed mutagenesis of the triad residues followed by enzymology studies of mutant and wild-type hBATs. Treatment with NEM caused inactivation of wild-type hBAT. However, preincubation of wild-type hBAT with the substrate cholyl-CoA before NEM treatment prevented loss of N-acyltransferase activity. Substitution of His-362 or Asp-328 with alanine results in inactivation of hBAT. Although substitution of Cys-235 with serine generated an hBAT mutant with lower N-acyltransferase activity, it substantially increased the bile acid-CoA thioesterase activity compared with wild type. In summary, data from this study support the existence of an essential catalytic triad within hBAT consisting of Cys-235, His-362, and Asp-328 with Cys-235 serving as the probable nucleophile and thus the site of covalent attachment of the bile acid molecule.

Acyl Coenzyme A↗
PubMed2002

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes.

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.

Animals↗
PubMed2002

Comparing Arabidopsis to other flowering plants.

Comparisons of the Arabidopsis genomic sequence with sequences from other flowering plants have revealed that substantial colinearity exists between species in the arrangement of genes within chromosomal blocks. Although seen most clearly in short sequences (at the Megabase scale), this colinearity can also be found using dense genetic maps that are based on expressed sequence tags. The genomes of most diploid Angiosperms show evidence of polyploid ancestry, and the resulting duplicated blocks, which have been subject to deletion and rearrangements during evolution, form complex networks of homology both within and between species. These homologies should prove to be of value in exploiting the Arabidopsis sequence to identify candidate genes in defined chromosomal regions within genomes that are less well characterised.

Arabidopsis↗
PubMed2002

Effect of soy protein on endogenous hormones in postmenopausal women.

BACKGROUND: The long-term clinical effects of soy protein containing various concentrations of isoflavones on endogenous hormones are unknown. OBJECTIVE: We examined the effects of ingestion of soy protein containing various concentrations of isoflavones on hormone values in postmenopausal women. DESIGN: Seventy-three hypercholesterolemic, free-living, postmenopausal women participated in a 6-mo double-blind trial in which 40 g protein as part of a National Cholesterol Education Program Step I diet was provided as casein from nonfat dry milk (control), isolated soy protein (ISP) containing 56 mg isoflavones (ISP56), or ISP containing 90 mg isoflavones (ISP90). Endogenous hormone concentrations were measured at baseline and at 3 and 6 mo. RESULTS: The concentration of thyroxine and the free thyroxine index were higher in the ISP56 group, and the concentration of thyroid-stimulating hormone was higher in the ISP90 group than in the control group at 3 and 6 mo (P < 0.05). Triiodothyronine was significantly higher in the ISP90 group only at 6 mo. Thyroxine, free thyroxine index, and thyroid-stimulating hormone at 6 mo were inversely associated with measures of baseline estrogenicity. No significant differences were found for endogenous estrogens, cortisol, dehydroepiandrosterone sulfate, insulin, glucagon, or follicle-stimulating hormone after baseline hormone values were controlled for. CONCLUSIONS: This study does not provide evidence that long-term ingestion of soy protein alters steroid hormone values, but it suggests that soy protein may have small effects on thyroid hormone values that are unlikely to be clinically important. The thyroid effects are, however, consistent with previous findings in animals and highlight the need for future research investigating possible mechanisms of action.

Bone Density↗
PubMed2002

Gaining insight into the health effects of soy but a long way still to go: commentary on the fourth International Symposium on the Role of Soy in Preventing and Treating Chronic Disease.

Research into the health effects of soyfoods and soybean constituents has increased at a phenomenal pace over the past decade. This research includes a wide range of areas, such as cancer, coronary heart disease, osteoporosis, cognitive function, menopausal symptoms and renal function. Importantly, there are an increasing number of clinical studies being conducted in this field, which was quite evident from the findings presented at the Fourth International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, November 4-7, 2001, in San Diego, California. There is no doubt that progress in understanding the health effects of soy is being made, but much of the data are frustratingly inconsistent. For example, there were conflicting results presented at the symposium on the role of isoflavones in bone health. Similarly, presentations painted an unclear picture of the role of isoflavones in cholesterol reduction. The relatively short duration and small sample size of many of the human studies in this field likely contribute to the inconsistent results. Although there are some controversies regarding the safety of soy for certain subsets of the population, special sessions at the symposium on breast cancer and cognitive function did much to alleviate concerns that soy could have detrimental effects in these areas. Furthermore, published data and new research presented at this meeting suggest that the consumption of even 10 g (typical of Asian intake) of isoflavone-rich soy protein per day may be associated with health benefits. If this modest amount of soy protein were to be incorporated in the American diet, it would represent only approximately 15% of total U. S. protein intake.

Animals↗
PubMed2002

Molecular cloning and expression of rat liver bile acid CoA ligase.

Bile acid CoA ligase (BAL) is responsible for catalyzing the first step in the conjugation of bile acids with amino acids. Sequencing of putative rat liver BAL cDNAs identified a cDNA (rBAL-1) possessing a 51 nucleotide 5'-untranslated region, an open reading frame of 2,070 bases encoding a 690 aa protein with a molecular mass of 75,960 Da, and a 138 nucleotide 3'-nontranslated region followed by a poly(A) tail. Identity of the cDNA was established by: 1) the rBAL-1 open reading frame encoded peptides obtained by chemical sequencing of the purified rBAL protein; 2) expressed rBAL-1 protein comigrated with purified rBAL during SDS-polyacrylamide gel electrophoresis; and 3) rBAL-1 expressed in insect Sf9 cells had enzymatic properties that were comparable to the enzyme isolated from rat liver. Evidence for a relationship between fatty acid and bile acid metabolism is suggested by specific inhibition of rBAL-1 by cis-unsaturated fatty acids and its high homology to a human very long chain fatty acid CoA ligase. In summary, these results indicate that the cDNA for rat liver BAL has been isolated and expression of the rBAL cDNA in insect Sf9 cells results in a catalytically active enzyme capable of utilizing several different bile acids as substrates.

Amino Acid Sequence↗
PubMed2002

Epiploic appendagitis: an emerging diagnosis for general surgeons.

The increased use of sonography and computed tomography in the assessment of lower abdominal pain has led to the greater recognition of epiploic appendagitis. Although epiploic appendagitis is increasingly described and diagnosed by radiologists it is rarely discussed in the surgical literature. We describe five recent cases of epiploic appendagitis managed at our center and review the literature regarding this clinical entity. All cases were diagnosed by computed tomography and none underwent surgery. General surgeons need to include epiploic appendagitis in their differential diagnosis for abdominal pain and be aware of the natural history of this condition when considering therapy.

Abdominal Pain↗
PubMedDate not supplied

Prospective highlights of functional skin proteomics.

Although a wide variety of protein profiles have been extensively constructed via proteomic analysis, the comprehensive proteomic profiling of the skin, which is considered to be the largest organ of the human body, is still far from complete. Our efforts to establish the functional skin proteome, a protein database describing the protein networks that underlie biological processes, has set in motion the identification and characterization of proteins expressed in the epidermis and dermis of the BALB/c mice. In this review, we will highlight various cutaneous proteins we have characterized and discuss their biological functions associated with skin distress, immunity, and cancer. This type of research into functional skin proteomics will provide a critical step toward understanding disease and developing successful therapeutic strategies.

Animals↗
PubMedDate not supplied

A design and statistical perspective on microarray gene expression studies in nutrition: the need for playful creativity and scientific hard-mindedness.

OBJECTIVES: Our purpose is to highlight some of the past and potential future uses of microarray in nutrition research, while also commenting on some aspects of the design conduct and analysis of microarray data that will leave to improved data quality. METHODS: In this review article we outline some of the aspects of microarray experimentation that must be considered before and during these experiments. These topics include: identification of the experiment's objective (hypothesis), the experimental design, sample size, statistical analysis, data verification, data handling, and experimental interpretation. RESULTS: In order to illustrate the principles we outline in this article we use the methods to layout the design of a microarray experiment to study one aspect of the observation that a diet high in soy is associated with lower rates of breast cancer. CONCLUSIONS: Microarrays are a very powerful tool for studying virtually every nutrition-related disease and trait and can provide valuable insights that are not obtainable with other techniques. However, unless nutrition researchers conduct their studies with scientific hard-mindedness, the studies will be of lower power at least if not completely misleading.

Animals↗