The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.

BACKGROUND: Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera. OBJECTIVES: To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE. METHODS: An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood. RESULTS: Compared with mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (P < 0.05) and a smaller decrease in body temperature (P < 0.01). Results with the basophil histamine release assay corroborated these findings (P < 0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared with placebo (P < 0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges. CONCLUSIONS AND CLINICAL RELEVANCE: Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.

Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants.

RATIONALE: An important property of allergens is their ability to cross-link IgE and activate mast cells and basophils. The effector activity of peanut allergens has not been well characterized. METHODS: Crude extracts of fresh peanut flour were fractionated by gel filtration. Effector function was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from individual sera and from pools of sera of peanut-allergic donors. RESULTS: Following gel filtration, 75 +/- 7% of the applied protein and 76 +/- 16% (n=3) of the applied activity (assayed with a pool of 11 sera) were recovered in the resultant fractions. The majority (85 +/- 2%; n=3) of the recovered activity resided in a fraction with a theoretical average molecular weight of approximately 20 kDa and a range of 13-25 kDa. When all the individual fractions were recombined, the measured activity was similar to that of the original extract [140 +/- 43% when measured with a pool of serum (n=2) and 66 +/- 7% when measured with individual sera (n=4)]; when all individual fractions excluding the 20 kDa fraction were recombined, the measured activity was only 8 +/- 2% (n=2) of the original extract when assayed with the serum pool and 10 +/- 4% (n=3) when assayed with the individual sera. Two-dimensional gel electrophoresis of this biologically active fraction revealed >60 protein spots. Analysis of 50 of the most prominent spots by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and of the full mixture by automated tandem mass spectrometry coupled to online capillary liquid chromatography revealed that >97% of the protein mass consisted of Ara h 2.0101, Ara h 2.0201, Ara h 6 isoforms, and variants of these proteins. CONCLUSIONS: Ara h 2 and Ara h 6 account for the majority of the effector activity found in a crude peanut extract.

Infectious arthritis.

One hundred thirteen patients with 120 episodes of septic arthritis were seen during a 14-year period. The most common bacteria cultured from joint fluid or blood during the acute episodes were gonococci, staphylococci, and streptococci. Seventeen other bacteria were the infecting organisms in one or more cases each. Other infections and medical conditions frequently were present. In some instances the septic arthritis was a complication of another infection. In other patients septic arthritis appeared to occur because of diminished resistance to infection. The majority of patients responded well to medical treatment, but eight died and 26 had persistence of articular pain at follow-up examination.

2-Amino-3-(methylamino)propanoic acid (BMAA) bioavailability in the primate.

2-Amino-3-(methylamino)-propanoic acid (BMAA) is a low potency excitatory amino acid present in the cycad plant that has been proposed as a factor in the high incidence of amyotrophic lateral sclerosis-parkinsonism dementia (ALS-PD) in the western Pacific region. We employed stable isotopic forms of BMAA to assess the oral bioavailability of this compound in cynomolgous monkeys (n = 3). The stable isotope labeled BMAA ([15N]-BMAA) was injected i.v. at the same time that the unlabeled compound was administered orally. Both forms of BMAA were then quantified in a 48h urine sample by gas chromatography-mass spectrometry (GC/MS). Following oral dosing, 80% of the administered BMAA was absorbed into the systemic circulation; thus, oral bioavailability was high and other routes of administration could not result in significantly higher circulating levels of BMAA for a given administered dose.

Measurements of protein carbonyls, ortho- and meta-tyrosine and oxidative phosphorylation complex activity in mitochondria from young and old rats.

Mitochondrial bioenergetic function is often reported to decline with age and the accumulation of oxidative damage is thought to contribute. However, there are considerable uncertainties about the amount and significance of mitochondrial oxidative damage in aging. We hypothesized that, as radical production in mitochondria is greater than the rest of the cell, protein oxidative damage should accumulate more in mitochondria than the cytoplasm, and that this relative accumulation should increase with age. To test these hypotheses we measured the accumulation of three markers of protein oxidative damage in liver, brain, and heart from young and old rats. Ortho- and meta-tyrosine levels in protein hydrolysates were measured by a gas chromatography/mass spectrometry assay, and protein carbonyl content was determined by ELISA. Using these assays we found no evidence for increased protein oxidative damage in mitochondria relative to the cytosol. Most increases found in protein oxidative damage on aging were modest for all three tissues and there was no consistent pattern of increased oxidative damage in mitochondrial proteins on aging. Mitochondrial oxidative phosphorylation complex activities were also assessed revealing 39-42% decreases in F0F1--ATP synthase activity in liver and heart on aging, but not in other oxidative phosphorylation complexes. These findings have implications for the contribution of mitochondrial oxidative damage and dysfunction to aging.

A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: synthesis, characterization, and quantification.

A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine), was synthesized using a simple, single-step condensation between 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access to useful quantities (mg-g) of analytically pure reference material, and with minor modification, to stable isotope-labeled analogues (isotopomers). With reference material and a suitable internal standard available, isotope-dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS/MS) was used to assay the adduct in a model system purged of oxygen, i.e., a gamma-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for quantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the potential for artefactual adduct formation if the appropriate substrates are present under acidic conditions.

2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

We conducted an investigation of the levels of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour. Analysis of 30 flour samples processed from the endosperm of Cycas circinalis seeds collected on Guam indicated that more than 87% of the total BMAA content was removed during processing. Furthermore, in 1/2 the samples almost all (greater than 99%) of the total BMAA was removed. We found no significant regional differences in the BMAA content of flour prepared from cycad seeds collected from several villages on Guam. Testing of different samples prepared by the same Chamorro woman over 2 years suggests that the washing procedure probably varies in thoroughness from preparation to preparation but is routinely efficient in removing at least 85% of the total BMAA from all batches. Analysis of a flour sample that had undergone only 24 hours of soaking indicated that this single wash removed 90% of the total BMAA. We conclude that processed cycad flour as prepared by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which are in the order of only 0.005% by weight (mean values for all samples). Thus, even when cycad flour is a dietary staple and eaten regularly, it seems unlikely that these low levels could cause the delayed and widespread neurofibrillary degeneration of nerve cells observed in amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam (ALS-PD).

Quantification of 3-nitrotyrosine in biological tissues and fluids: generating valid results by eliminating artifactual formation.

Reactive nitrogen species such as peroxynitrite can nitrate specific amino acids, whether free or protein bound, and 3-nitrotyrosine is believed to be one marker of this reaction. To examine the significance of this pathway in biological systems we have developed an accurate, sensitive, and specific assay for 3-nitrotyrosine based on combined liquid chromatography tandem mass spectrometry. Our approach allowed simultaneous analysis of both tyrosine and 3-nitrotyrosine and employs isotopomer standards (i.e., [15N1, 13C9]-tyrosine and [13C6]-3nitrotyrosine). Calibration curves were linear (r2 = 0.999) across the range 0.5-100 pg/microL (i.e., 2.2-442 fmol/microL), and the detection limit for standard samples was 0.5 pg/microL (2.2 fmol/microL, or 10 fmol on column; S/N = 5) or 1 pg/microL (4.4 fmol/microL) for extracted (biological) samples. As a component of this study we have undertaken an extensive investigation of artifactual formation of 3-nitrotyrosine under conditions that exist during sample extraction and derivatization. Our studies show that under appropriate conditions (low pH, elevated temperatures, and in the presence of a vast excess of the two substrates, tyrosine and the nitrate anion), 3-nitrotyrosine can readily be formed as an artifact.

Conserved motifs as the basis for recognition of homologous proteins across species boundaries using peptide-mass fingerprinting.

Two-dimensional gel electrophoresis of any biological system presently resolves a plethora of highly purified proteins for which no function or identity has been determined. Theoretical and experimental data were used to demonstrate that peptide-mass fingerprinting (PMF) could aid in the recognition of conserved motifs across species boundaries, and thereby assist in attributing putative function to some of these molecules. Amino acids residue substitutions produced by biological diversity and phylogenetic distance combine to highlight regions of functional significance within proteins. Using 10 prokaryotic and two eukaryotic elongation factors (EF), up to 25 peptide fragments (> 800 Da) per molecule were compared across species boundaries within a 12 x 12 contingency table (66 cross-species comparisons), based upon the degree of molecular mass and amino acid sequence identity. Total amino acid sequence identity ranged from 29.4-80.9% for these molecules. Peptide fragments with homologous sequence across three or more EF were defined as containing, or being near to, conserved functional motifs. Twelve such fragments (> 800 Da) were found in this group of proteins. In addition, an 808.9 Da peptide of unknown functional significance was seen to occur in three of the 12 molecules studied and in another three EF-Tu molecules. At the 83% (five of six residues) identity level, this fragment was found in a further 35 EF-Tu molecules and in 14 unrelated proteins. Further investigation should reveal a role for this fragment (motif) in structural integrity or protein function. A FASTA search conducted on a peptide fragment containing a conserved GTP-binding motif (GHVDHGK) of EF-Tu from Euglena gracilis was used as an example to putatively attribute partial function to three hypothetical proteins derived from DNA sequencing initiatives.

Dopamine and salsolinol levels in rat hypothalami and striatum after schedule-induced self-injection (SISI) of ethanol and acetaldehyde.

Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS)--selected ion monitoring technique using deuterated internal standards in rats allowed to self-inject either acetaldehyde, ethanol or saline control solution over 20 days for 1 h/day. Significant increases in medial basal hypothalamic (MBH) and striatal salsolinol concentrations were found in animals exposed to acetaldehyde but not ethanol, whereas dopamine concentrations for these animals did not differ significantly from rats exposed to control conditions. The data provides further support for the in vivo formation of salsolinol following acetaldehyde exposure in experimental animals.

Salsolinol and dopamine in rat medial basal hypothalamus after chronic ethanol exposure.

Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS) - selected ion monitoring technique using deuterated internal standards in Long Evans rats chronically exposed to ethanol for ten months. Chronic ethanol exposure produced significant increases of dopamine and salsolinol concentrations in the medial basal hypothalamus but not striatum. The data suggest that the occurrence of salsolinol in rat brain tissue is a consequence of an in vivo Pictet-Spengler cyclization.

Formation of proline thiohydantoin with ammonium thiocyanate: progress towards a viable C-terminal amino-acid-sequencing procedure.

Pure amino acid thiohydantoins are required as reference standards for development of C-terminal-sequencing procedures based on thiohydantoin formation of the C-terminal amino acids of peptides and proteins. Proline thiohydantoin was prepared using a straightforward method involving reaction of acetylproline with ammonium thiocyanate. It was characterized by UV spectrophotometry, mass spectrometry and back-hydrolysis to the free amino acid. These data establish unequivocally that the thiocyanate procedure is applicable to proline as well as to the other common amino acids. This work also validates earlier claims that proline thiohydantoin can be prepared by reaction with thiocyanic acid.

Analysis of the neurotoxic plasticizer n-butylbenzenesulfonamide by gas chromatography combined with accurate mass selected ion monitoring.

The plasticizer, n-butylbenzenesulfonamide (NBBS), is reported to be neurotoxic when inoculated intracisternally or intraperitoneally into rabbits. Because NBBS is commonly used in the production of polyamide (nylon) plastics and is soluble in water, the disposal of NBBS-containing plastics in landfill sites could result in NBBS appearing in the leachate. Further, NBBS could also be leached from packaging into their contents. To allow us to examine the risks posed by NBBS in the environment, we have developed a quantitative assay for this compound. The assay employs a one-step extraction into dichloromethane followed by gas chromatography with accurate mass selected ion recording. The assay incorporates [13C6]NBBS as an internal standard to allow precise quantitation, and four separate ion chromatograms are recorded. NBBS was found in some Australian domestic solidwaste landfill leachate (from less than 0.3 to 94.6 ng/mL), but ground water in the vicinity of a landfill had only trace quantities of NBBS. NBBS was also quantitated in some bottled and cask wines, and levels varied from not detected to 2.17 ng/mL (n = 14). Additional studies are required to assess the public health risks associated with the use of NBBS as a plasticizer.

Quantification of the putative neurotoxin 2-amino-3-(methylamino)propanoic acid (BMAA) in cycadales: analysis of the seeds of some members of the family Cycadaceae.

Over the past 30 years there have been attempts to link the unusually high incidence of amyotrophic lateral sclerosis (ALS) among the Chamorros native to the island of Guam to the consumption of the seeds of Cycas circinalis L., the false sago palm. In support of this relationship it was recently shown that, when given to primates, 2-amino-3-(methylamino)-propanoic acid (BMAA), a minor cycad component, can cause selective degeneration of upper and lower motor neurons in the spinal cord and clinical features similar to those of ALS. In order to test the relationship between ALS and cycads, we have developed a sensitive and precise gas chromatographic/mass spectrometric (GC/MS) assay for BMAA which allows direct assessment of the BMAA content in foods and is directly applicable to the assay of BMAA in biologic tissues and fluids. After the addition of a deuterated isotopomer as an internal standard and transesterication with 2-methyl-1-propanol, BMAA was extracted into dichloromethane and then acylated with pentafluoropropionic anhydride before GC/MS. This method permits precise quantification of BMAA in the low picogram/sample range. Direct quantification of the BMAA content in the female gametophyte tissue (endosperm) of a range of cycad seeds collected from Guam confirmed the presence of BMAA at levels of approximately 1 g/g (dry weight). The presence of BMAA in the seed extract was confirmed after derivatization of an aliquot of the extract and GC/MS analysis in the scanning mode. BMAA was found to be present, albeit at lower levels, in the endosperm of the seeds of C. revoluta (0.32 mg/g) and C. media (0.29 mg/g).

Contribution of Ara h 2 to peanut-specific, immunoglobulin E-mediated, cell activation.

BACKGROUND: Ara h 2 is a potent peanut allergen but its contribution to the ability of a crude peanut extract (CPE) to cross-link IgE and activate mast cells has not been rigorously evaluated. OBJECTIVE: To measure the contribution that Ara h 2 makes to the effector function of a CPE. METHODS: Ara h 2 was specifically removed from a CPE as demonstrated by immunoblots, 2D gels, and an inhibitory ELISA. Functional assays of sham-treated and Ara h 2-depleted CPEs were performed with RBL SX-38 cells sensitized with IgE from highly peanut-allergic subjects and with naturally sensitized basophils. RESULTS: Depletion of approximately 99% of the Ara h 2 from the CPE led to an increase in the concentration of the CPE necessary to give 50% of maximal degranulation (EC50) of the SX-38 cells following sensitization with sera that contain anti-Ara h 2 IgE. Assays with a pool of 10 sera showed a small but significant increase in the EC50 following depletion of Ara h 2 (1.65+/-0.15-fold; P<0.05) and assays of seven individual sera showed a similar increase in the average EC50 (1.7+/-0.2-fold; P<0.02). The percent of the anti-peanut IgE that binds Ara h 2 correlated with an increase in the EC50 of the CPE following depletion of Ara h 2 (r=0.83; P<0.02). On the other hand, data from three of these patients studied with a basophil histamine release assay did not show a significant effect of depletion of Ara h 2. CONCLUSION: Based on its ability to cross-link IgE effectively, Ara h 2 is clearly an important peanut allergen. Its ability to cross-link IgE effectively from a specific serum is related to the proportion of anti-Ara h 2 in that serum but Ara h 2 does not account for a majority of the effector activity of the CPE for any of the sera studied.

A review of approaches to the analysis of 3-nitrotyrosine.

Our understanding of in vivo tyrosine nitration has been confounded by problems associated with the analytical approaches that have been employed to quantify 3-nitroyrosine (3-NT). Trace analysis is a demanding task under the best of circumstances, but 3-NT offers some special concerns. This review examines some of these concerns and discusses approaches to ensuring that carefully validated analytical data are generated.