Histone acetyltransferase p300 acetylates Pax5 and strongly enhances Pax5-mediated transcriptional activity.

Pax5/B cell lineage specific activator protein (BSAP) is a B lineage-specific regulator that controls the B lineage-specific gene expression program and immunoglobulin gene V(H) to DJ(H) recombination. Despite extensive studies on its multiple functions, little is known about how the activity of Pax5 is regulated. Here, we show that co-expression of histone acetyltransferase E1A binding protein p300 dramatically enhances Pax5-mediated transcriptional activation. The p300-mediated enhancement is dependent on its intrinsic histone acetyltransferase activity. Moreover, p300 interacts with the C terminus of Pax5 and acetylates multiple lysine residues within the paired box DNA binding domain of Pax5. Mutations of lysine residues 67 and 87/89 to alanine within Pax5 abolish p300-mediated enhancement of Pax5-induced Luc-CD19 reporter expression in HEK293 cells and prevent Pax5 to activate endogenous Cd19 and Blnk expression in Pax5(-/-) murine pro B cells. These results uncover a novel level of regulation of Pax5 function by p300-mediated acetylation.

Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo.

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.

Occidiofungin, a unique antifungal glycopeptide produced by a strain of Burkholderia contaminans.

Bacterial strain Burkholderia contaminans MS14 was isolated from soil that suppressed brown patch disease of lawn grass. An antifungal compound was purified from the liquid culture of this bacterium. In this study, complete covalent structures of two purified closely related antifungal compounds were determined by the experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR, and ESI-MS and GC. The analysis of monoisotopic masses of the purified preparation indicated the presence of two related compounds with masses determined to be 1199.543 and 1215.518 Da; the difference corresponds to the mass of an oxygen atom. GC analysis identified a xylose sugar attached to the antifungal compound. NMR experiments revealed that the compound is cyclic and composed of eight amino acids, two of which are beta-hydroxy derivatives of Tyr and Asn, and one being a novel amino acid. The novel amino acid serves as the scaffold for the attachment of the xylose and a short acyl chain. The spectrum and concentration of antifungal activity were determined using a microtiter plate assay. The antifungal compound demonstrated potent antifungal activities against a broad panel of fungal plant and animal pathogens, as well as two Pythium spp. Microscopic observations showed that the antifungal compound disrupts normal membrane morphology. The cells fill with large inclusion bodies and the membrane becomes irregularly shaped and swollen following the exposure to subinhibitory concentrations of the antifungal compound. Our data support the identification of a novel fungicide and the compound has been named occidiofungin, meaning fungal killer.

UV-A-induced structural and functional changes in human lens deamidated alphaB-crystallin.

PURPOSE: To determine comparative effects of ultraviolet (UV)-A irradiation on structural and functional properties of wild type (WT) alphaB-crystallin and its three deamidated mutant proteins (alphaB-Asn78Asp, alphaB-Asn146Asp, and alphaB-Asn78/146Asp). METHODS: Three deamidated mutants previously generated from recombinant WT alphaB-crystallin, using a site-specific mutagenesis procedure as previously described [32], were used. The WT alphaB-crystallin and its three deamidated species were exposed to UV-A light (320-400 nm) at intensities of 20 or 50 J/cm(2). The UV-A-unexposed and UV-A-exposed preparations were examined for their chaperone activity, and their activities were correlated with the UV-A-induced structural changes. The structural properties studied included dimerization and degradation, intrinsic tryptophan (Trp) fluorescence, ANS (8-anilino-1-naphthalenesulfate)-binding, far ultraviolet circular dichroism (UV-CD) spectral analysis, molecular sizes by dynamic light scattering, and oxidation of Trp and methionine (Met) residues. RESULTS: The WT alphaB-crystallin and its three deamidated mutant proteins showed enhanced dimerization to 40 kDa species and partial degradation with increasing doses during UV-A-exposure. Compared to the deamidation of asparagines (Asn) 78 residue to aspartic acid (Asp) or both Asn78 and Asn146 residues to Asp, the deamidation of Asn146 residue to Asp resulted in a greater loss of chaperone activity. The UV-A-induced loss of chaperone activity due to structural changes was studied. The ANS-binding data suggested that the alphaB-Asn146Asp mutant protein had a relatively compact structure and an increase in surface hydrophobic patches compared to WT and two other deamidated proteins. Similarly, UV-A-exposure altered the Trp microenvironment in the deamidated mutant proteins compared to the WT alphaB-crystallin. Far-UV CD spectral analyses showed almost no changes among WT and deamidated species on UV-A-exposure except that the alphaB-Asn146Asp mutant protein showed maximum changes in the random coil structure relative to WT alphaB-crystallin and two other deamidated proteins. The UV-A-exposure also resulted in the aggregation of WT and the three deamidated mutant proteins with species of greater mass compared to the non-UV-A exposed species. Among the four spots recovered after two-dimensional (2D)-gel electrophoresis from WT and the three deamidated species, the Met and Trp residues of alphaB-Asn146Asp mutant showed maximum oxidation after UV-A exposure, which might account for its greater loss in chaperone activity compared to WT alphaB-crystallin and two other deamidated species. CONCLUSIONS: After UV-A-exposure, the deamidated alphaB-Asn146Asp mutant protein showed a complete loss of chaperone activity compared to WT alphaB and alphaB-Asn78Asp and alphaB-Asn78/146Asp deamidated species. Apparently, this loss of chaperone activity was due to oxidative changes leading to its greater structural alteration compared to other alphaB-species.

2D gel proteomics: an approach to study age-related differences in protein abundance or isoform complexity in biological samples.

This chapter describes protocols for two-dimensional (2D) gel electrophoresis (isoelectric focusing [IEF] followed by sodium-dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis [PAGE]), staining of gels with the fluorescent dye Sypro Ruby, 2D gel image analysis, peptide mass fingerprint (PMF) analysis using matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS), liquid chromatography (LC)-tandem mass spectrometry (MS/MS), Western blot analysis of protein oxidations, and mass spectrometric mapping of sites of protein oxidations. Many of these methods were used to identify proteins affected in rat brain following ingestion of grape seed extract (GSE), a dietary supplement touted for anti-oxidant activity. Although beneficial actions in cell and animal models of chronic disease have been described for GSE, it has not been shown whether specific proteins were affected, or the nature of the effects. Applying 2D gel proteomics technology allowed discovery of proteins targeted by GSE without a priori knowledge of which one(s) might be affected. The newer 2D blue native (BN) electrophoresis methodology, which resolves protein complexes in a nondenaturing first dimension and then the components of these complexes in a denaturing second dimension, is discussed as a complementary approach. Analysis of protein oxidations and protein-protein interactions have special relevance to aging-related research, since oxidative stress and altered protein interactions may be at the heart of aging-related diseases. Finally, quality control issues related to implementation of high throughput technologies are addressed, to underscore the importance of minimizing bias and randomizing human and technical error in generating large datasets that are expensive and time-consuming to repeat.

A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress.

The combination of proteomics with highly specific and sensitive affinity techniques is important for the identification of posttranslational modifications by reactive oxygen and nitrogen species (ROS/RNS). One of the most pressing problems with this approach is to determine accurately the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample. A number of techniques relevant to free radical biology use biotin tagging as a method to follow protein modification with high sensitivity and specificity. To realize the potential of this approach to provide quantitative data, we have prepared a series of biotinylated proteins through the modification of lysine residues. These proteins were then used as quantitative standards in electrophoretic separation of protein samples labeled with biotin-conjugated iodoacetamide. The utility of the approach was assessed by measuring modification of thiols in response to exposure to thiol oxidants, as well as the amount of protein adduct formation with a biotin-tagged electrophilic lipid. Furthermore, using a combination of native and biotin-tagged cytochrome c, this method was used to quantitate the amount of thiol relative to the amount of protein in a given spot on a two-dimensional gel. Thus, we have developed a versatile, cost-effective standard that can be used in proteomic methods to quantitate biotin tags in response to oxidative stress.

6-Benzylthioinosine analogues as subversive substrate of Toxoplasma gondii adenosine kinase: activities and selective toxicities.

Toxoplasma gondii adenosine kinase (EC.2.7.1.20) is the major route of adenosine metabolism in this parasite. The enzyme is significantly more active than any other enzyme of the purine salvage in T. gondii and has been established as a potential chemotherapeutic target for the treatment of toxoplasmosis. Certain 6-substituted purine nucleosides act as subversive substrates of T. gondii, but not the human, adenosine kinase. Therefore, these compounds are preferentially metabolized to their respective nucleotides and become selectively toxic against the parasites but not their host. Herein, we report the testing of newly synthesized 6-benzylthioinosine analogues with various substituents on the phenyl ring of their benzyl group as subversive substrates of T. gondii adenosine kinases. The binding affinity of these compounds to T. gondii adenosine kinase and their efficacy as antitoxoplasmic agents varied depending on the nature and position of the various substituents on the phenyl ring of their benzyl group. p-Cyano-6-benzylthioinosine and 2,4-dichloro-6-benzylthioinosine were the best ligands. In general, analogues with substitution at the para position of the phenyl ring were better ligands than those with the same substitutions at the meta or ortho position. The better binding of the para-substituted analogues is attributed to the combined effect of hydrophobic as well as van der Waals interactions. The 6-benzylthioinosine analogues were devoid of host-toxicity but all showed selective anti-toxoplasmic effect in cell culture and animal models. These results further confirm that toxoplasma adenosine kinase is an excellent target for chemotherapy and that 6-substituted purine nucleosides are potential selective antitoxoplasmic agents.

Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells.

We examined the effects of reactive oxygen-nitrogen intermediates on chloride (Cl-) currents across murine tracheal epithelial (MTE) cells isolated from CD-1 mice. MTE cells were cultured on permeable supports until they formed water-tight monolayers with transepithelial resistances (Rt)>500 Omega/cm2 and then were mounted in Ussing chambers. Baseline short-circuit current (ISC) values, prior to and following the addition of 10 microM amiloride (an inhibitor of sodium-transport pathways) into the apical side, were 65 +/- 1.9 microA/cm2 and 7.6 +/- 0.51 microA/cm2, respectively (X +/- 1 SE, n=32). The addition of 3-morpholinosydnominine (SIN-1, 1 mM), which generates both superoxide and nitric oxide anions, to amiloride-treated monolayers resulted in a transient increase of ISC to a peak value of 35 +/- 1.3 microA/cm2 (X +/- SE, n=14) within the next 30-60 min. After this, the ISC decreased gradually and returned to its pre-SIN-1 value. These changes were blocked by glibenclamide (200 microM), an inhibitor of cystic fibrosis transmembrane regulator, or reduced by glutathione (GSH, 5 mM), a scavenger of peroxynitrite. Forskolin (10 microM) augmented the SIN-1 effect when added at the peak of the SIN-1 response but not when ISC had returned to its baseline value. Perfusion of MTE cells with SIN-1 also increased whole cell Cl- currents 4-fold and the open probability of CFTR-type single-channel currents from 0.041 to 0.92 in a transient fashion. Decomposed SIN-1, but not pure SIN-1c (the stable decomposition product of SIN-1), also increased ISC with an EC50 of 5 microM. Electrospray mass spectroscopy revealed several unique and uncharacterized compounds formed during the decomposition of SIN-1 as well as the reaction of SIN-1c with peroxynitrite. Formation of these compounds was inhibited by GSH. We conclude that compounds formed by the reaction of peroxynitrite with by-products of SIN-1, rather than reactive oxygen-nitrogen species per se, were responsible for the modulation of Cl- secretion across primary cultures of MTE cells.

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.

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.

Production of androgens by microbial transformation of progesterone in vitro: a model for androgen production in rivers.

We have previously documented the presence of progesterone and androstenedione in the water column and bottom sediments of the Fenholloway River, Taylor County, Florida. This river receives paper mill effluent and contains masculinized female mosquitofish. We hypothesized that plant sterols (e.g., ss-sitosterol) derived from the pulping of pine trees are transformed by bacteria into progesterone and subsequently into 17alpha-hydroxyprogesterone, androstenedione, and other androgens. In this study, we demonstrate that these same androgens can be produced in vitro from the bacterium Mycobacterium smegmatis. In a second part to this study, we reextracted and reanalyzed the sediment from the Fenholloway River and verified the presence of androstadienedione, a delta1 steroid with androgen activity.

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.

Androstenedione and progesterone in the sediment of a river receiving paper mill effluent.

The Fenholloway River near Perry, Florida, receives effluent from a paper mill and contains populations of masculinized female eastern mosquitofish, Gambusia holbrooki. A previous study identified the androgen precursor androstenedione at a low concentration (0.14 nM) in water samples from the river. The present study makes use of a toxicity identification and evaluation approach that includes solid phase extraction and high pressure liquid chromatography purification, androgen receptor transcription assays, and liquid chromatography mass spectroscopy to identify and characterize steroids in the Fenholloway River sediment. Androstenedione (2.4 nM) and progesterone (155 nM) were identified in the river sediment at concentrations greater than in the river water column (0.14 nM androstenedione, and 6.5 nM progesterone). Spring Creek, a comparison stream that does not receive mill effluent, contained low levels of progesterone (0.3 nM) but no androstenedione in the sediment. The data are consistent with the hypothesis that pine pulp-derived phytosteroids in the paper mill effluent accumulate in river sediment where they are converted by microbes into progesterone and this into androstenedione and other bioactive steroids. Equally important is that normal streams with much less organic matter still contain progesterone, but at dramatically lower levels. The presence of androgens and androgen precursors in the river water and sediment likely contributes to the masculinized phenotype of the female Gambusia holbrooki in the Fenholloway River.

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.

Proteomics analysis of rat brain protein modulations by grape seed extract.

Dietary supplements such as grape seed extract (GSE) enriched in proanthocyanidins (PA) (oligomeric polyphenols) have been suggested to have multiple health benefits, due to antioxidant and other beneficial activities of the PA. However, a systematic analysis of the molecular basis of these benefits has not been demonstrated. Because the brain is vulnerable to age-related oxidative damage and other insults including inflammation, it was hypothesized that rats ingesting GSE would experience changes in expression or modifications of specific brain proteins that might protect against pathologic events. Normal adult female rats were fed diets supplemented with 5% GSE for 6 weeks. Proteomics analysis (2D electrophoresis and mass spectrometry) of brain homogenates from these animals identified 13 proteins that were altered in amount and/or charge. Because many of these changes were quantitatively in the opposite direction from previous findings for the same proteins in either Alzheimer disease or mouse models of neurodegeneration, the data suggest that these identified proteins may mediate the neuroprotective actions of GSE. This is the first identification and quantitation of specific proteins in mammalian tissues modulated by a dietary supplement, as well as the first to demonstrate links of such proteins with any disease.

LambdaSa1 and LambdaSa2 prophage lysins of Streptococcus agalactiae.

Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity.

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

Differential biliary excretion of genistein metabolites following intraduodenal and intravenous infusion of genistin in female rats.

The purpose of this study was to determine whether bioflavonoid glucoside O-conjugates are absorbed from the intestine in the intact form or as their aglycones following hydrolysis by intestinal beta-glucosidases. In this study, the intestinal absorption of genistin, the beta-glucoside of the isoflavone genistein, was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. To first establish whether genistein, once absorbed, was converted into unique metabolites, genistin was infused into the femoral or portal veins and bile samples quantitatively collected. Analysis of bile samples by HPLC-mass spectrometry revealed that almost full recovery of the genistein component occurred in the form of unreacted genistin ( approximately 20%) and genistein 7beta-O-glucuronide ( approximately 80%). However, when genistin was infused into the upper small intestine, only genistein 7beta-O-glucuronide and the aglycone genistein appeared in the bile. There was no evidence for any biliary secretion of the unreacted genistin, thereby excluding its uptake in the intact form from the small intestine in this animal model.

Characterization of covalent multimers of crystallins in aging human lenses.

The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the water-insoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25- and 41-year-old subjects showed distinct 5- and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52- and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25- and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. alphaA, alphaB, betaA3, betaA4, betaB1, betaB2, gammaS, and gammaD), and the second type of alpha-, beta-, and gamma-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: alphaA-fragment (C-terminally truncated, residues 1-157), alphaB-fragment (residues 83-90), betaB1-crystallin (residues 60-71), betaA3 (residues 33-44), betaA4 (residues 106-117), filensin (residues 78-90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in alphaA-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.

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.