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In the past decade, there has been an increase in allergic reactions to peanut proteins, sometimes resulting in fatal anaphylaxis. The development of improved methods for diagnosis and treatment of peanut allergies requires a better understanding of the structure of the allergens. Ara h 1, a major peanut allergen belonging to the vicilin family of seed storage proteins, is recognized by serum IgE from >90% of peanut-allergic patients. In this communication, Ara h 1 was shown to form a highly stable homotrimer. Hydrophobic interactions were determined to be the main molecular force holding monomers together. A molecular model of the Ara h 1 trimer was constructed to view the stabilizing hydrophobic residues in the three dimensional structure. Hydrophobic amino acids that contribute to trimer formation are at the distal ends of the three dimensional structure where monomer-monomer contacts occur. Coincidentally, the majority of the IgE-binding epitopes are also located in this region, suggesting that they may be protected from digestion by the monomer-monomer contacts. On incubation of Ara h 1 with digestive enzymes, various protease-resistant fragments containing IgE-binding sites were identified. The highly stable nature of the Ara h 1 trimer, the presence of digestion resistant fragments, and the strategic location of the IgE-binding epitopes indicate that the quaternary structure of a protein may play a significant role in overall allergenicity.

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Cetylpyridinium chloride (CPC), a water-soluble, neutral pH, colorless compound, is widely used in oral hygiene products to inhibit bacteria responsible for plaque. Previously, researchers have demonstrated that CPC not only reduces Salmonella typhimurium on poultry but also prevents cross-contamination. To determine the effectiveness of CPC against pathogens associated with lean and adipose beef surfaces, several spray-washing experiments (862 kPa, 15 s, 35 degrees C) with 1% (wt/vol) CPC were conducted. On lean beef surfaces, CPC immediately reduced 5 to 6 log10 CFU/cm2 of Escherichia coli O157:H7 and Salmonella typhimurium to virtually undetectable levels (0 log10 CFU/cm2), as well as after 35 days of refrigerated (4 degrees C), vacuum-packaged storage. On adipose beef surfaces, 5 log10 CFU/cm2 Salmonella typhimurium and E. coli O157:H7 were reduced immediately (>2.5 log10 CFU/cm2) with 1% CPC; by day 35 the reduction was <1.3 log10 CFU/cm2. Further plate overlay analyses indicated that the effectiveness of CPC against pathogens on adipose surfaces was not hampered by the presence of meat components or fatty acids. Additional chemical and microbiological analyses of 1% CPC-treated beef surfaces subjected to a secondary water wash (following contact times of 0, 5, 10, 15, or 30 min) or grinding did reduce pathogenic bacteria and CPC levels. However, residual CPC levels following any of the treatments were considered excessive for human consumption. Despite the residual levels, this study is the first to demonstrate the effect of CPC on pathogenic bacteria associated with beef surfaces immediately after treatment and also after long-term, refrigerated, vacuum-packaged storage.

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Cetylpyridinium chloride (CPC) has been found to be effective in reducing contamination of chicken carcasses from a variety of microorganisms, including Escherichia coli O157:H7, Salmonella typhimurium, Campylobacter jejuni, Aeromonas hydrophila, Listeria monocytogenes, and Staphylococcus aureus. A procedure has been developed to determine residue levels on chicken carcasses after CPC treatment. For the analysis, chicken carcasses were extracted with 95% ethanol. The CPC concentration in the extract was measured by high-performance liquid chromatography (HPLC) with ultraviolet detection using dodecylpyridinium chloride (DPC) as an internal standard. The method was validated in the concentration range of 3-200 microg/ml CPC in ethanolic extract. This assay is rapid, precise, and accurate.

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The pharmacokinetics of inhaled (R,S)-albuterol following pulmonary absorption were studied in healthy human subjects. Ten subjects (5 females and 5 males) inhaled two puffs (180 microg) of albuterol via a metered-dose inhaler and spacer device. All subjects were nonsmoking and had normal pulmonary function. Charcoal slurries were ingested to block gastrointestinal absorption of drug. Venous samples were obtained from each subject at thirteen time points from 0 through 12 h post dose. (R,S)-Albuterol concentration in plasma was measured using a gas chromatography-mass spectrometry (GC-MS) assay. The plasma concentration-time profiles conformed to a two-compartment extravascular model with first-order absorption kinetics. The drug levels reached maximum in 12.6 +/- 2.2 (SD) minutes, which is in contrast with previous reports that maximum plasma concentrations occur within 2 to 4 h. The mean peak plasma level was 1469 +/- 410 pg/mL. The mean half-life of distribution was 17.9 +/- 8.2 min. The mean half-life of elimination was 4.4 +/- 1.5 h. Female subjects achieved peak concentration more rapidly than male subjects (10.4 vs 14.8 min, p = 0.01) and had a higher mean peak concentration (1778 vs 1159 pg/mL, p = 0.04).

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Allergy to peanut is a significant IgE-mediated health problem because of the high prevalence, potential severity, and chronicity of the reaction. Ara h1, an abundant peanut protein, is recognized by serum IgE from >90% of peanut-sensitive individuals. It has been shown to belong to the vicilin family of seed storage proteins and to contain 23 linear IgE binding epitopes. In this communication, we have determined the critical amino acids within each of the IgE binding epitopes of Ara h1 that are important for immunoglobulin binding. Surprisingly, substitution of a single amino acid within each of the epitopes led to loss of IgE binding. In addition, hydrophobic residues appeared to be most critical for IgE binding. The position of each of the IgE binding epitopes on a homology-based molecular model of Ara h1 showed that they were clustered into two main regions, despite their more even distribution in the primary sequence. Finally, we have shown that Ara h1 forms a stable trimer by the use of a reproducible fluorescence assay. This information will be important in studies designed to reduce the risk of peanut-induced anaphylaxis by lowering the IgE binding capacity of the allergen.

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Comparative Molecular Field Analysis (CoMFA) was applied to a comprehensive data set of heterogeneous nitroaromatics tested in Salmonella typhimurium TA98 and TA100 with and without S9 microsomal activation. The four CoMFA models developed agree with postulated mechanisms of mutagenicity, and explain over 70% of the corresponding mutagenic variance The standard deviation coefficient contours common in the four models included high electronic density regions equivalent to C4-C5 in the pyrene ring, and an electron deficient site equivalent to C6. These areas are associated with high mutagenicity. Electron deficient areas may be related with the nitroreductive bioactivation of nitroaromatics. Electron rich sites may be involved with oxidative mechanisms analogous to the bioactivation pathway of polycyclic aromatic hydrocarbons. The contribution of steric factors to mutagenicity follows the order TA98 + S9 > TA98 > TA100 + S9 > TA100. The models indicated that increasing bulk perpendicular to the aromatic plane would decrease mutagenicity, but increasing the aromatic ring system along a region corresponding to C6-C7 in 1-nitropyrene would increase mutagenicity.

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Albuterol is a beta2-adrenergic agonist commonly used as a bronchodilator for the treatment of patients with asthma. We have developed an assay to determine plasma levels as low as 50 pg/ml of albuterol by gas chromatography-mass spectrometry (GC-MS). This assay utilizes isotopically labeled albuterol ([13C]albuterol) as an internal standard. In this assay albuterol and the internal standard are recovered from 1 ml of plasma using solid-phase extraction. The samples are then derivatized to trimethylsilyl ethers using N,O-bis(trimethylsilyl)trifluoro-acetamide with 1% trimethylchlorosilane. The samples are then analyzed by GC-MS with selected-ion monitoring (SIM) for the ions m/z 369.15 and 370.15. The method has been validated for a concentration range of 50-10000 pg/ml in plasma.

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The photoactivated antiviral and cytotoxic activities of the naturally occurring thiophene, alpha-terthienyl (1), and 15 synthetic analogues were evaluated against murine cytomegalovirus and Sindbis virus, and murine mastocytoma cells. After irradiation with near UV light, alpha-terthienyl and most of its analogues had significant toxicity, with minimum inhibitory concentrations in the range of 0.02-40 microM. In the absence of near UV irradiation, only one analogue had antiviral activity and five were cytotoxic. The most active analogues were those containing carboxylic acid, hydroxyl, or cyano substituents. Quantitative structure-activity relationship analysis of thiophene phototoxicity suggested that the rate of singlet oxygen production is the primary determinant of antiviral and cytotoxic activities. For phototoxicity against murine cytomegalovirus, a significant role for hydrophobicity was also demonstrated. Tricyclic thiophenes show significant potential for photochemotherapy of viral infections and cancer, and further evaluation in animal models is recommended.

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The role of hydrophobic and electronic effects on the kinetic constants kcat and Km for the papain hydrolysis of a series of 22 substituted N-benzoylglycine pyridyl esters was investigated. The series studied comprises a wide variety of substituents on the N-benzoyl ring, with about a 300,000-fold range in their hydrophobicities, and 2.1-fold range in their electronic Hammet constants (sigma). It was found that the variation in the log kcat and log 1/Km constants could be explained by the following quantitative-structure activity relationships (QSAR): log 1/Km = 0.40 pi 4 + 4.40 and log 1/kcat = 0.45 sigma + 0.18. The substituent constant, pi 4, is the hydrophobic parameter for the 4-N-benzoyl substituents. QSAR analysis of two smaller sets of glycine phenyl and methyl esters produced similar results. A clear separation of the substituent effects indicates that in the case of these particular esters, acylation appears to be the rate limiting catalytic step.

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The relationship between structure and the Michaelis-Menten constants (Km) for the papain hydrolysis of a series of 37 N-benzoylglycine esters was investigated. The series studied comprises a wide range of aromatic and aliphatic esters with a 5000-fold variation in their Km constants and essentially constant kcat values. It was found that the variation in the Km constants could be rationalized by the following quantitative structure-activity relationship (QSAR): log 1/Km = 8.13F + 0.33Z + 1.27II3' + 1.95. In this equation F is the field inductive parameter, II3' is the hydrophobic constant for the more lipophilic of the two possible meta substituents and Z is the Van der Waals distance from oxygen through the end of the molecule, in the direction of the 4 position of the aromatic ester moiety.

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The relationship between sweetness and structure was studied for several analogues of the intensely sweet sesquiterpene, hernandulcin. These derivatives were prepared synthetically, and were spectroscopic and conformational analysis. With the exception of the parent substance, none of the derivatives tested proved to be sweet. Evidence gathered in this study suggests that hernandulcin binds to its putative receptor through a three-point interaction, involving the C-1 carbonyl and C-1' hydroxyl groups, and the double bond between C-4' and C-5'. In the course of a preliminary safety assessment, the 3-desmethyl derivative of hernandulcin was found to be mutagenic toward Salmonella typhimurium strain TM677.

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Steviol (ent-13-hydroxykaur-16-en-19-oic acid), the aglycone of various plant-derived glycoside sweeteners consumed by human populations, is known to be mutagenic toward Salmonella tymphimurium strain TM677 when metabolically activated using a 9000 x g supernatant fraction derived from the liver of Aroclor 1254-pretreated rats. Mass spectral analysis of this diterpenoid and some analogs revealed characteristic patterns reflecting differential stereochemistry at the C/D rings and variations in the nature of the substituents present. Such information has been used to help identify several in vitro metabolites of steviol in conditions known to produce a mutagenic response, when analyzed by gas chromatography/mass spectrometry. The major pathways of such steviol mammalian metabolism proved to be allylic oxidation and epoxidation. 15-Oxosteviol, a product of oxidation of the major steviol metabolite, 15alpha-hydroxysteviol, was found to be a direct-acting mutagen [corrected].

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The constituents of the volatile oils of MONTANOA TOMENTOSA and LIPPIA GRAVEOLENS, were investigated using capillary GC/MS. Borneol acetate, beta-cubebene, and beta-caryophyllene were found to be the major constituents of the volatile oil of M. TOMENTOSA, while P-cymene, 1,8-cineol, thymol, and carvacrol were the major volatile components of L. GRAVEOLENS. The possible correlation between the high concentration of monoterpenes and the alleged antifertility effect of the title plants is discussed.

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The intensely sweet compounds, stevioside [1] and rebaudioside A [2], and 15 of their derivatives, were tested for feeding deterrent activity against the aphid, Schizaphis graminum. Included with these compounds was steviol [4], the aglycone of stevioside and rebaudioside A that was one of the most active compounds in this investigation. Loss of feeding deterrent activity of steviol was observed on acetylation or glycosylation of the C-13 tertiary hydroxy group or on methylation of the C-19 carboxylic acid substituent. In contrast, the antifeedant activity of steviol was not greatly affected by modification of either the C-16 exomethylene group or the C/D-ring junction stereochemistry. 13C-nmr data have been obtained for 12 of the test compounds investigated.

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Stevioside is a sweet-tasting diterpene glycoside that is derived from Stevia rebaudiana (Bertoni) Bertoni (Compositae). It is used commercially in Japan and other parts of the world as a sucrose substitute. Whereas stevioside demonstrates no mutagenic activity in a variety of test systems, the aglycone, steviol (13-hydroxy-ent-kaurenoic acid), is mutagenic toward Salmonella typhimurium strain TM677 in the presence of a metabolic activating system derived from the liver of Aroclor 1254-pretreated rats. The required activating component is localized in the microsomal fraction of rat liver, suggestive of a cytochrome P-450-mediated reaction. Partially purified epoxide hydrolase does not inhibit steviol-induced mutagenicity, indicating that an active metabolite is not an epoxide that serves as a substrate for this enzyme preparation. The 13-hydroxy group of steviol is required for the expression of mutagenicity since ent-kaurenoic acid is nonmutagenic, and acetylation of steviol at this position negates mutagenicity. Similarly, diterpenes bearing a strong structural resemblance to steviol, cafestol and kahweol, were found to demonstrate no mutagenic activity toward Salmonella typhimurium TM677, as were their respective acetates and palmitic acid esters. Conversely, 19-O-beta-D-glucopyranosyl steviol, a potential hydrolysis product of stevioside, is mutagenic and bactericidal in the presence of a metabolic activating system. Additionally, in contrast to the nonmutagenic diterpenes cafestol and kahweol that are effective as inducers of glutathione S-transferase activity, evaluation by administration to mice proved steviol, isosteviol and various steviol glycosides to be inactive in this process. Thus, structural differences among these naturally occurring and semi-synthetic diterpenes appear to impart major differences in biological activity that may relate to human health upon dietary ingestion.

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Lippia dulcis Trev. (Verbenaceae) is the source of hernandulcin, the first known intensely sweet sesquiterpenoid, a compound which is a volatile oil constituent. The literature on the uses of this species, dating back to early colonial times in Mexico, has been examined. This plant began to be used as an official drug in the late 19th century for the treatment of coughs and bronchitis, and at that time preliminary phytochemical investigations were undertaken. Field work carried out in Mexico in 1981 and 1982 has indicated that there is still an active trade involving L. dulcis, which is sold primarily in market places for its alleged abortifacient activity. We have obtained no evidence, either from the literature or from field inquiries, that L. dulcis has ever been used for sweetening foods or beverages. Fourteen L. dulcis volatile oil constituents, mainly mono- and sesquiterpenoids, were identified by gas chromatography/mass spectrometry. The toxic compound, camphor, was found to constitute 53% w/w of the volatile oil of this species. The potential use of L. dulcis for the extraction of hernandulcin is discussed.

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Stevioside, a constituent of Stevia rebaudiana, is commonly used as a noncaloric sugar substitute in Japan. Consistent with reports in the literature, we have found that stevioside is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system. Similar negative results were obtained with several structurally related sweet-tasting glycosides. However, steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of a 9000 X g supernatant fraction derived from the livers of Aroclor 1254-pretreated rats. Expression of mutagenic activity was dependent on both pretreatment of the rats with Aroclor 1254 and addition of NADPH; unmetabolized steviol was not active. The structurally related species, isosteviol, was not active regardless of metabolic activation. Similarly, chemical reduction of the unsaturated bond linking the carbon-16 and -17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B, that were not mutagenic. In addition, ent-kaurenoic acid was found to be inactive. It is therefore clear that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxy group at position 13 and an unsaturated bond joining the carbon atoms at positions 16 and 17. A potential metabolite of steviol, steviol-16 alpha,17-epoxide, was synthesized chemically and found to be ineffective as a direct-acting mutagen. Thus, although stevioside itself appears innocuous, it would seem prudent to expeditiously and unequivocally establish the human metabolic disposition of this substance.

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Ancient Mexican botanical literature was systematically searched for new plant sources of intensely sweet substances. Lippia dulcis Trev., a sweet plant, emerged as a candidate for fractionation studies, and hernandulcin, a sesquiterpene, was isolated and judged by a human taste panel as more than 1000 times sweeter than sucrose. The structure of the sesquiterpene was determined spectroscopically and confirmed by chemical synthesis. Hernandulcin was nontoxic when administered orally to mice, and it did not induce bacterial mutation.

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Altogether, 110 species of the genus Stevia, comprising both herbarium and fresh leaf samples, were screened for the presence of sweet ent-kaurene glycosides, using a combination of tlc and hplc, followed by gc/ms. Stevioside and rebaudiosides A and C were detected in a Stevia rebaudiana herbarium specimen collected in Paraguay in 1919, and stevioside was observed as a constituent of a Stevia phlebophylla herbarium specimen collected in Mexico in 1889. Steviol glycosides were not detected in any of the other 108 Stevia species studied. The phytochemical results obtained in this study are correlated with those of preliminary organoleptic tests on the sweetness of these Stevia samples, and the chemotaxonomic implications of the present findings are discussed.

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