ABSTRACT
Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (Mentha x piperita) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in Escherichia coli. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.
Subject(s)
Expressed Sequence Tags , Plant Extracts/genetics , Plant Oils/metabolism , DNA, Complementary/metabolism , Databases, Factual , Gene Library , Mentha piperita , Models, Chemical , Molecular Sequence Data , Plant Cells , Plant Oils/chemistry , Sequence Analysis, DNA , Terpenes/chemistry , Terpenes/metabolismABSTRACT
We synthesized two sulfhydryl-reactive fluorescent probes, Br-ANT (2-amino-benzoic acid, 2-(bromoacetyl)hydrazide) and Br-MANT (N-[2-[(bromoacetyl)amino]ethyl]-2-(methylamino)benzamide). Br-ANT and Br-MANT contain an anthraniloyl and N-methyl anthraniloyl group, respectively, linked to the sulfhydryl-reactive bromoacetyl moiety. The cysteine adducts have absorption maxima at 323 and 326 nm, with molar extinction coefficients of 2100 and 2900 M-1 cm-1, for Br-ANT and Br-MANT, respectively, making these probes excellent acceptors for tryptophan. The absorption spectra and quantum yields were constant at pH levels useful for protein studies (pH 5-8). Quantum yields of Br-ANT and Br-MANT were 0.16 and 0.42, emission maxima were 432 and 440 nm, and fluorescence lifetimes were 1.3 and 7.8 ns, respectively. The emission of Br-ANT-Cys and Br-MANT-Cys shifted to shorter wavelengths with decreasing solvent polarity. Polarization values were maximal between 330 and 375 nm. Both probes reacted selectively and stoichiometrically with the single cysteine residue of a model protein. The labeled protein exhibited relatively long lifetimes (9-10 ns), suggesting that these probes will be generally useful for rotational studies.
Subject(s)
Benzamides/chemical synthesis , Fluorescent Dyes/chemical synthesis , Sulfhydryl Reagents/chemistry , ortho-Aminobenzoates/chemical synthesis , Benzamides/chemistry , Fluorescent Dyes/chemistry , Magnetic Resonance Spectroscopy , Mass Spectrometry , Spectrometry, Fluorescence , ortho-Aminobenzoates/chemistryABSTRACT
Coordination complexes of chromium (Cr) and two pyridine carboxylate isomers, nicotinate (nic) and picolinate (pic) were synthesized and analyzed. Cr mono and dinicotinate complexes were formed with 1:1 and 1:2 ratios of Cr3+ and nic at pH 7.5. Cr dinicotinate was the only complex formed from a 1:3 ratio of Cr3+ and nic. Mono, di, and tri picolinate complexes were formed with 1:1, 1:2, and 1:3 ratios of Cr3+ and pic at pH 7.5. Cr is coordinated with nic through the carboxyl carbon while Cr is coordinated with pic through both the pyridine nitrogen and the carboxyl carbon. Cr dinicotinate enhanced insulin activity in isolated adipose tissue. None of the other complexes were active in this assay system. In contrast, Cr tripicolinate, which is lipophilic, increased glucose uptake by skeletal muscle cultures but none of the other complexes were effective. In addition, dietary supplements of Cr tripicolinate increased rate of lean body mass development in humans and decreased hemoglobin glycation in aging rats. None of the other complexes was effective in these in vivo assays. The results of this investigation prove that the chemical properties of Cr nic and Cr pic complexes differ markedly. The chemical differences result in a vast difference in the biological action of the complexes.
