Connie C. Bonham

Developmental Regulation of Methyl Benzoate Biosynthesis and Emission in Snapdragon Flowers

In snapdragon flowers, the volatile ester methyl benzoate is the most abundant scent compound. It is synthesized by and emitted from only the upper and lower lobes of petals, where pollinators (bumblebees) come in contact with the flower. Emission of methyl benzoate occurs in a rhythmic manner, with maximum emission during the day, which correlates with pollinator activity. A novel S-adenosyl-l-methionine:benzoic acid carboxyl methyl transferase (BAMT), the final enzyme in the biosynthesis of methyl benzoate, and its corresponding cDNA have been isolated and characterized. The complete amino acid sequence of the BAMT protein has only low levels of sequence similarity to other previously characterized proteins, including plant O-methyl transferases. During the life span of the flower, the levels of methyl benzoate emission, BAMT activity, BAMT gene expression, and the amounts of BAMT protein and benzoic acid are developmentally and differentially regulated. Linear regression analysis revealed that production of methyl benzoate is regulated by the amount of benzoic acid and the amount of BAMT protein, which in turn is regulated at the transcriptional level.

Methionine sulfoxide reductase A protects dopaminergic cells from Parkinson’s disease-related insults

Parkinsons disease (PD) is a neurologic disorder characterized by dopaminergic cell death in the substantia nigra. PD pathogenesis involves mitochondrial dysfunction, proteasome impairment, and alpha-synuclein aggregation, insults that may be especially toxic to oxidatively stressed cells including dopaminergic neurons. The enzyme methionine sulfoxide reductase A (MsrA) plays a critical role in the antioxidant response by repairing methionine-oxidized proteins and by participating in cycles of methionine oxidation and reduction that have the net effect of consuming reactive oxygen species. Here, we show that MsrA suppresses dopaminergic cell death and protein aggregation induced by the complex I inhibitor rotenone or mutant alpha-synuclein, but not by the proteasome inhibitor MG132. By comparing the effects of MsrA and the small-molecule antioxidants N-acetylcysteine and vitamin E, we provide evidence that MsrA protects against PD-related stresses primarily via methionine sulfoxide repair rather than by scavenging reactive oxygen species. We also demonstrate that MsrA efficiently reduces oxidized methionine residues in recombinant alpha-synuclein. These findings suggest that enhancing MsrA function may be a reasonable therapeutic strategy in PD.

Near-Isogenic Lines of Maize Differing for Glycinebetaine

A series of near-isogenic glycinebetaine-containing and -deficient F8 pairs of Zea mays L. (maize) lines were developed. The pairs of lines differ for alternative alleles of a single locus; the wild-type allele conferring glycinebetaine accumulation is designated Bet1 and the mutant (recessive) allele is designated bet1. The near-isogenic lines were used to investigate whether glycinebetaine deficiency affects the pool size of the glycinebetaine precursor, choline, using a new method for glycinebetaine and choline determination: stable isotope dilution plasma desorption mass spectrometry. Glycinebetaine deficiency in maize was associated with a significant expansion of the free choline pool, but the difference in choline pool size was not equal to the difference in glycinebetaine pool size, suggesting that choline must down-regulate its own synthesis. Consistent with this, glycinebetaine deficiency was also associated with the accumulation of the choline precursor, serine. A randomly amplified polymorphic DNA marker was identified that detects the bet1 allele. In 62 F8 families tested the 10-mer primer 5[prime]-GTCCTCGTAG produced a 1.2-kb polymerase chain reaction product only when DNA from Bet1/bet1 or bet1/bet1 lines was used as template. All 26 homozygous Bet1/Bet1 F8 families tested were null for this marker.

Characterization of betaines using electrospray MS/MS

Betaines are an important class of naturally occurring compounds that function as compatible solutes or osmoprotectants. Because of the permanent positive charge on the quaternary ammonium moiety, mass spectrometric analysis has been approached by desorption methods, including fast atom bombardment and plasma desorption mass spectrometry. Here we show that electrospray ionization MS gives comparable results to plasma desorption MS for a range of authentic betaine standards and betaines purified from plant extracts by ion exchange chromatography. A distinct advantage of electrospray ionization MS over plasma desorption MS is the capability of obtaining product ion spectra via MS/MS of selected parent ions, and hence structural information to discriminate between ions of identical mass.

Metabolic Fingerprint of Brazilian Maize Landraces Silk (Stigma/Styles) Using NMR Spectroscopy and Chemometric Methods

Aqueous extract from maize silks is used by traditional medicine for the treatment of several ailments, mainly related to the urinary system. This work focuses on the application of NMR spectroscopy and chemometric analysis for the determination of metabolic fingerprint and pattern recognition of silk extracts from seven maize landraces cultivated in southern Brazil. Principal component analysis (PCA) of the (1)H NMR data set showed clear discrimination among the maize varieties by PC1 and PC2, pointing out three distinct metabolic profiles. Target compounds analysis showed significant differences (p < 0.05) in the contents of protocatechuic acid, gallic acid, t-cinnamic acid, and anthocyanins, corroborating the discrimination of the genotypes in this study as revealed by PCA analysis. Thus the combination of (1)H NMR and PCA is a useful tool for the discrimination of maize silks in respect to their chemical composition, including rapid authentication of the raw material of current pharmacological interest.

DISCOVERY OF 3-METHYL-2-BUTEN-1-YL ACETATE, A NEW ALARM COMPONENT IN THE STING APPARATUS OF AFRICANIZED HONEYBEES

We analyzed the alarm pheromone components from five colonies of Africanized honeybees and three colonies of European honeybees collected in Mexico. Analyses revealed a novel alarm pheromone component that was only present in appreciable quantities in the Africanized bee samples. Analysis of the mass spectrum and subsequent synthesis confirmed that this compound is 3-methyl-2-buten-1-yl acetate (3M2BA), an unsaturated derivative of IPA. In Africanized honeybees, sampling from stings of guards showed that 3M2BA was present at levels of 0–38% the amount of isoamyl acetate (IPA). Behavioral assays from three colonies each of Africanized and European bees showed that 3M2BA recruited worker bees from hives of both Africanized bees and European bees at least as efficiently as isopentyl acetate IPA, a compound widely reported to have the highest activity for releasing alarm and stinging behavior in honeybees. However, a mixture of of 3M2BA and IPA (1:2) recruited bees more efficiently than either of the compounds alone. None of the compounds differed in their efficacy for inducing bees to pursue the observers.

Maldi-tof analysis of mixtures of 3-deoxyanthocyanidins and anthocyanins

Abstract The mass spectrometric analysis of 3-deoxyanthocyanidins and anthocyanins present in crude extracts from sorghum plant tissue was carried out by matrix-assisted laser desorption ionization mass spectrometry (MALDI). Sensitivities to as low as 5 pmol\ μ l were easily attained for pure samples of the anthocyanidin, pelargonidin, and the anthocyanin, malvin. Sensitivities to the level of 15 pmol\ μ l were attained for 3-deoxyanthocyanidins present in crude extracts from sorghum plant tissue. Through its capability for analyzing very small quantities of these compounds in unpurified samples, MALDI provided a sensitive means for the detection of these flavonoid pigments in plant tissues.

Trinuclear nickel clusters, [Ni3(μ3-L)(μ3-I)(μ2-dppm)3]n+ (L = I−, CO, CNCH3, CN-2,6-Me2C6H3, CN-i-C3H7, CN-t-C4H9, CN-n-C4H9, NO+) and the “dimer of trimer” hexanuclear nickel clusters {[Ni3(μ3-I)(μ2-dppm)3]2(μ3,μ3′,η1,η1′- CN-R-NC)}2+, (R = −(CH2)6−, p-C6H4): characterization of large clusters by mass spectrometry

Abstract The mass spectra of a series of trinuclear nickel clusters of the general formula [Ni3(μ3-L)(μ3-I)(μ2-dppm)3]n+, where L = I− (1), CO (2), CNCH3 (3), CN-2,6-Me2C6H3 (4, CN-i-C3H7 (5), CN-t-C4H9 (6), CN-n-C4H9 (7), and NO+ (8) and n = 0, 1, 2 were measured by Plasma Desorption (PD) and Fast Atom Bombardment (FAB) mass spectrometry. Strong molecular ion peaks were observed for all of the trinuclear clusters except 8 by both techniques. Ion fragmentation followed similar pathways in both ionization modes. Dimers of trinuclear clusters, {[Ni3(μ3-I)(μ2-dppm)3]2(μ3,μ3′,η1,η1′-CN-R-NC)}2+ where R = −(CH2)6− (9) and 1,4-C6H4 (10) were prepared by the reaction of two equivalents of 1 with one equivalent of the appropriate diisocyanide, CN-R-NC. Clusters 9 and 10 were not distinguishable from simple trimers such as 3–7 by 31P NMR and optical spectroscopies, but were unambiguously identified by PD and FAB mass spectrometry. The parent ions are different for the two techniques: MI+ in the former case and M+ in the latter.

Analysis of anthocyanins and 3-deoxyanthocyanidins by plasma desorption mass spectrometry

Abstract A technique for the mass spectrometric analysis of anthocyanins, 3-hydroxyanthocyanidins and 3-deoxyanthoeyanidins in Sorghum bicolor by plasma desorption was developed. Sensitivities to ng levels with minimum sample preparation and analysis were achieved. The technique provides a sensitive means for the measurement of these flavonoid pigments as well as a means to screen for very small quantities of the compounds within plant tissues.

Hydrolysis of thioesters in an ion trap

The alkenyldiarylmethanes are a class of non-nucleoside reverse transcriptase inhibitors that are currently being developed as potential antivirals for the treatment of HIV infection and AIDS. As part of our continuing investigations on the alkenyldiarylmethanes, a series of thioester analogues were prepared in an effort to improve upon the metabolic stability of the parent lead compound. Hydrolysis of the thioester moieties was consistently observed during ion trap electrospray ionization (ESI) mass spectrometry to the extent that the parent molecular ion was weak in intensity or simply could not be detected. The same hydrolysis observations were also made when the analogues were analyzed by ion trap electron impact (EI) ionization, indicating the hydrolysis event was the result of the ion trap and not ionization technique. Ion-trap-mediated hydrolysis has been observed previously in prior alkenyldiarylmethane studies and prevented characterization of certain intermediates; thus, we wished to investigate whether modifying instrument parameters and protocols affected the instrument-mediated hydrolysis event. Unfortunately, varying the maximum injection time and the number of microscans performed, independent of each other, had little effect on the intensities of the parent ions [MH(+)] or the hydrolysis products] MH(+) -HSCH(3)].