Limited research with rodents and humans suggests that oral ingestion of pinitol (3-0-methyl-D-chiro-inositol) might positively influence glucose tolerance. This double-blinded, placebo-controlled, and cross-over study assessed the effects of acute pinitol supplementation on plasma pinitol concentration, glucose tolerance, insulin sensitivity, and activation of the skeletal muscle insulin receptor. Fifteen older, nondiabetic subjects (62 +/- 1 years, mean +/- SEM) completed four, 1-day trials. Subjects consumed a non-nutritive beverage with nothing (placebo) or 1000 mg pinitol. Sixty minutes later, the Subjects consumed beverages that were either energy- and carbohydrate-free (Sham) or contained 75g glucose (OGTT). Blood samples were collected frequently over the 240-min testing period. For the OGTT trials only, vastus lateralis samples were obtained before the placebo and pinitol supplementation and 60 min after consuming the 75g glucose beverage. Plasma pinitol concentration increased and was maintained for 240 min. Pinitol did not influence the fasting state and 180 min area under the curves for plasma glucose and insulin during the Sham and OGTT trials or hepatic (placebo 0.83 +/- 0.08; pinitol 0.80 +/- 0.08) and whole-body (placebo 6.10 +/- 0.54; pinitol 6.22 +/- 0.52) insulin sensitivities. Activation of the muscle insulin receptor was increased by 140% with glucose ingestion (Pre 0.62 +/- 0.12; Post 1.49 +/- 0.35), but pinitol did not influence this response. These results show that the pinitol supplement was quickly absorbed, but did not acutely influence indices of whole-body glucose tolerance and insulin sensitivity, or the activation of the skeletal muscle insulin receptor in older, nondiabetic humans.

ABSTRACT Flavonoids are valuable natural products derived from the phenylpropanoid pathway. The objective of this study was to create a host for the biosynthesis of naringenin, the central precursor of many flavonoids. This was accomplished by introducing the phenylpropanoid pathway with the genes for phenylalanine ammonia lyase ( PAL ) from Rhodosporidium toruloides , 4-coumarate:coenzyme A (CoA) ligase ( 4CL ) from Arabidopsis thaliana , and chalcone synthase ( CHS ) from Hypericum androsaemum into two Saccharomyces cerevisiae strains, namely, AH22 and a pad1 knockout mutant. Each gene was cloned and inserted into an expression vector under the control of a separate individual GAL10 promoter. Besides its PAL activity, the recombinant PAL enzyme showed tyrosine ammonia lyase activity, which enabled the biosynthesis of naringenin without introducing cinnamate 4-hydroxylase (C4H). 4CL catalyzed the conversion of both trans -cinnamic acid and p -coumaric acid to their corresponding CoA products, which were further converted to pinocembrin chalcone and naringenin chalcone by CHS. These chalcones were cyclized to pinocembrin and naringenin. The yeast AH22 strain coexpressing PAL, 4CL, and CHS produced approximately 7 mg liter −1 of naringenin and 0.8 mg liter −1 of pinocembrin. Several by-products, such as 2′,4′,6′-trihydroxydihydrochalcone and phloretin, were also identified. Precursor feeding studies indicated that metabolic flux to the engineered flavonoid pathway was limited by the flux to the precursor l -tyrosine.

In this study, we have begun to analyze phosphotyrosyl and associated proteins present in a DT40 chicken B cell line overexpressing the nonreceptor protein-tyrosine kinase, Syk. An anti-phosphotyrosine antibody was used to select tyrosine-phosphorylated proteins. After tryptic digestion, peptides were subjected to a beta-elimination reaction and phosphotyrosine-containing peptides were enriched via immobilized metal affinity chromatography. Several known substrates and candidate substrates for Syk and the location of 22 tyrosine phosphorylation sites were identified.

Conidia of Cochliobolus carbonum secrete a toxin (HC-toxin) during appressorium formation on maize leaves. Plasma desorption mass spectrometry revealed that approximately 70ng of toxin per 106 conidia were secreted during the first 16h of morphogenesis. Growth of the fungus was monitored microscopically. Extensive fungal growth occurred in the susceptible interaction by 24h. In spite of a substantial amount of HC-toxin, the fungus failed to become established in the resistant host even after 36h. Results suggest that the resistance conditioned by Hm1, which encodes a toxin reductase, causes inactivation of the toxin early in the interaction.

Proteomic analysis is becoming a popular field in science. Analysis of protein modifications is useful in deciphering cellular functions and errors in pathways that can result in disease. There has been increased interest in the phosphotyrosine proteome. Due to the difficulty in finding the location of the tyrosine phosphorylation site in the tyrosine phosphorylated peptide or even to verify that the parent protein is a phosphotyrosyl-protein, new analytical tools are being developed. The phosphotyrosine immonium ion can be produced via skimmer CID for detection via ion trap mass spectrometry and is a useful marker for the indication of the presence of a phosphotyrosine residue. Skimmer CID analysis can also be used to differentiate phosphotyrosine-containing peptides from other phosphorylated peptides. In this study, phosphotyrosine-containing peptides were analyzed by skimmer CID in an ion trap mass spectrometer. The factors affecting the signal abundance of the phosphotyrosine immonium ion were investigated.

Ocotea odorifera , a tree native to the Atlantic rainforest in south Brazil, has been used as a source of sassafras oil rich in safrole [5-(2-propenyl)-1,3-benzodioxole], an aromatic ether used as a flavoring agent and also in the manufacture of insecticides. The intensive deforestation process of the Brazilian Atlantic rainforest has threatened with extinction some species including O. odorifera . In this context, O. odorifera cell cultures might be an interesting alternative for the production of secondary metabolites of value (i.e., safrole), without risk of damage to the native germplasm. Insights into the secondary metabolites of organosolvent extracts of prototrophic cell cultures were performed by GC-MS and MALDI-TOF MS. GC-MS analysis revealed the occurrence of safrole in concentration of ca. 62.6 µg ml −1 and allowed the identification of other compounds besides safrole in the crude extract of these cultures. Thus, alkyl phenol (C 8 ) [ m / z = 206], C 14 myristic acid [ m / z = 228], long chain olefin or alcohol and essential oil were detected. However, safrole [ m / z = 162] was not detected by MALDI-TOF MS, indicating that it is not easily protonated (M + H) + .

Glycinebetaine (GB) accumulation has been suggested to be an adaptive response to several abiotic environmental stresses. Genetic and metabolic studies of GB accumulation in maize (Zea mays L.) indicate that recessive alleles of a single locus are responsible for the phenotype of GB nonaccumulation. The present study was undertaken to determine whether a similar genetically determined range of GB levels exists in the related C4 species Sorghum bicolor (L.) Moench. In a preliminary analysis of 240 sorghum genotypes, sampled at the postflowering stage, total quaternary ammonium compound (QAC) levels in the betaine fraction of the flag leaves were found to range from as low as 0.1 μmol g−1 FW to as much as 33 μmol g−1 FW. Stable isotope dilution desorption chemical ionization mass spectrometry of six genotypes with high QAC levels and five genotypes with low QAC levels confirmed that this variation could be attributed almost exclusively to genetic variability for GB level. GB‐nonaccumulating sorghum genotypes were confirmed to be GB‐non‐accumulating in a second year of field‐testing, and in greenhouse studies under salinized and non‐salinized conditions. GB levels increased with seedling age and/or salinization in GB‐accumulating genotypes. Also, GB levels were highest in the youngest leaves of GB‐accumulating sorghum genotypes. This work shows that GB is the major QAC in sorghum, that genetic differences in GB accumulation exist in sorghum as they do in maize, and that the level of GB in GB‐accumulating lines is developmentally and environmentally regulated. A list of GB levels of publicly available lines of sorghum is also provided.

Electrospray ionization (ESI) mass spectrometry was employed to obtain both molecular weight confirmation and structural information for a series of novel alkenyldiarylmethane (ADAM) analogs. The mass spectral data were intended for use during the structure elucidation of ester hydrolysis products formed during an in vitro metabolism study of a series of novel ADAM analogs. The data on the precursor molecules show the presence of the molecular ion peak, [M+H](+), as well as a peak consistent with the hydrolysis product of the original ester ([MH-ROH+H2O](+)). However, chemical ionization mass spectrometry, elemental analysis and H-1 NMR data indicated the presence of only the intact diester compounds, suggesting that the formation of the hydrolysis product was an instrumental artifact, i.e., in-beam hydrolysis during ESI or a result of longer ion residence times of the ion trap mass analyzer. Copyright (C) 2003 John Wiley Sons, Ltd.

Floral volatiles, which are small and generally water-insoluble, must move from their intracellular sites of synthesis through the outermost cuticle membrane before release from the flower surface. To determine whether petal cuticle might influence volatile emissions, we performed the first analysis of petal cuticle development and its association with the emission of flower volatiles using Antirrhinum majus L. (snapdragon) as a model system. Petal cuticular wax amount and composition, cuticle thickness and ultrastructure, and the amounts of internal and emitted methylbenzoate (the major snapdragon floral scent compound) were examined during 12 days, from flower opening to senescence. Normal (n-) alkanes were found to be the major wax class of snapdragon petals (29.0% to 34.3%) throughout the 12 days examined. Besides n-alkanes, snapdragon petals possessed significant amounts of methyl branched alkanes (23.6-27.8%) and hydroxy esters (12.0-14.0%). Hydroxy esters have not been previously reported in plants. Changes in amount of methylbenzoate inside the petals followed closely with levels of methylbenzoate emission, suggesting that snapdragon petal cuticle may provide little diffusive resistance to volatile emissions. Moreover, clear associations did not exist between methylbenzoate emission and the cuticle properties examined during development. Nevertheless, the unique wax composition of snapdragon petal cuticles shows similarities with those of other highly permeable cuticles, suggesting an adaptation that could permit rapid volatile emission by scented flowers.

Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

The 3-deoxyanthocyanidin phytoalexins produced in sorghum leaves in response to Colletotrichum sublineolum have chemical structure similarities to the 3-deoxy flavonoids that are precursors of phlobaphene pigments. Phlobaphenes are commonly observed in the pericarp of mature sorghum grains, while synthesis of 3-deoxyanthocyanidin phytoalexins is a site-specific response to infection with C. sublineolum. We have taken a genetic approach to investigate the possible overlap between the two sub-branches of flavonoid biosynthesis in sorghum that lead to phlobaphenes and 3-deoxyanthocyanidin phytoalexins. A sorghum line with a functional y1 gene synthesizes 3-deoxyanthocyanidins as well as phlobaphenes. However, a progenitor line with the mutable Y1-candystripe (Y1-cs) allele shows variable levels of biosynthesis of these compounds. The Y1-cs allele carries a copy of the Candystripe1 (Cs1) transposable element in the y1 gene. We demonstrate here that the variability in the expression of 3-deoxyanthocyanidins produced in individual mesocotyls of hyper-mutable Y1-cs plants is a function of the activity of the y1 gene. TheCs1 insertion in the Y1-cs allele blocks y1 function, while excision of Cs1 out of they1 locus restores the gene to a functional state. The combined molecular and biochemical characterization of sibling plants confirms that the allelic state of the y1 gene is completely correlated with the production of phytoalexins in response to fungal infection. These results provide support for the idea that the y1 gene regulates the biosynthesis of both 3-deoxyanthocyanidin phytoalexins and phlobaphene pigments in sorghum.

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.

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

MALDI-TOF MS analysis of supercritical CO 2 extracted samples obtained from Mandevilla velutinacell cultures allowed the detection of the anti-bradykinin pregnanic steroid, velutinol A, using low amount of sample (1 g lyophilized cells), with minimum analyte isolation.

Gas chromatography/mass spectrometry (GC/MS), with an ion trap mass analyzer, was used to examine the very-long-chain cuticular acid and certain non-acid wax constituents on the leaf sheath surface of Sorghum bicolor before and during 36 hours of light exposure. The mass spectra of the trimethylsilylated acids and alcohols did not match any of those published in searchable mass spectral libraries. The observed differences can be related to the interaction between water and the trimethylsilylated acids and alcohols. Understanding the observed mass spectra of the very-long-chain plant waxes is critical for studies that employ GC/MS with the ion trap mass analyzer to elucidate cuticular wax compositions on plants.

The rapid evolution of mass spectrometry in the past 15 years has moved mass spectrometry facilities from the traditional model in which instruments were located in and used for a single department's samples to a distributed model servicing entire universities. In this paper we describe two such shared instrument facilities that have evolved from a base in a single department to facilities that service a broad clientele. The Purdue University Campus-wide Mass Spectrometry Center (CWMSC) is a decentralized facility with multiple sites on campus. The CWMSC is a limited-access facility in which samples are run by service facility personnel in close cooperation with investigators. The Vanderbilt University Mass Spectrometry Research Center (VU-MSRC) is a centralized facility in the medical school that provides services to the university at large. The VU-MSRC is an open-access facility in which users are expected to prepare and analyze their own samples under the guidance of a trained operator. Perhaps the most significant benefit achieved by these models has been the minimization of academic barriers and the resultant intellectual cross-fertilization that has greatly enriched research at institutions where this approach has been adopted. The advantages and limitations of both models are discussed in terms of the traditional academic paradigm of service, research and education.

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\μl were easily attained for pure samples of the anthocyanidin, pelargonidin, and the anthocyanin, malvin. Sensitivities to the level of 15 pmol\μl 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.