Biomedical subjects
Publications and source records attributed to A Daniel Jones.
Methods for network-wide analysis are increasingly showing that the textbook view of the regulation of plant metabolism is often incomplete and misleading. Recent innovations in small-molecule analysis have created the ability to rapidly identify and quantify numerous compounds, and these data are creating new opportunities for understanding plant metabolism and for plant metabolic engineering.
The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of alpha subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease.
Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (1) to 6-hydroxymethyl-7,8-dihydropterin (4) in the folate biosynthetic pathway. Substitution of a conserved tyrosine residue at the active site of DHNA by phenylalanine converts the enzyme to a cofactor-independent oxygenase, which generates mainly 7,8-dihydroxanthopterin (6) rather than 4. 6 is generated via the same enol intermediate as in the wild-type enzyme-catalyzed reaction, but this species undergoes an oxygenation reaction to form 6. The conserved tyrosine residue plays only a minor role in the formation of the enol reaction intermediate but a critical role in the protonation of the enol intermediate to form 4.
The phytohormone jasmonic acid (JA) regulates the synthesis of secondary metabolites in a wide range of plant species. Here, we show that exogenous methyl-JA (MeJA) elicits massive accumulation of caffeoylputrescine (CP) in tomato leaves. A mutant (jai1) that is defective in jasmonate perception failed to accumulate CP in flowers and MeJA-treated leaves. Conversely, a transgenic tomato line (called 35S::PS) that exhibits constitutive JA signaling accumulated high levels of leaf CP in the absence of jasmonate treatment. RNA blot analysis showed that genes encoding enzymes in the phenylpropanoid and polyamine pathways for CP biosynthesis are upregulated in MeJA-treated wild-type plants and in untreated 35S::PS plants. These results indicate that CP accumulation in tomato is tightly controlled by the jasmonate signaling pathway, and provide proof-of-concept that the production of some plant secondary metabolites can be enhanced by transgenic manipulation of endogenous JA levels.
Fluorinated, silica-based stationary phases are becoming increasingly popular alternatives to traditional alkyl phases owing to their differential selectivity and retention for a variety of analyte classes. In this report, the ion-exchange mechanisms characteristic of a fluorinated phase are exploited to rapidly develop separation conditions for ephedrine alkaloids and synephrine using a mobile phase compatible with mass spectrometry. A linear relationship of basic analyte retention with the reciprocal of ammonium acetate concentration is first established. This linear relationship can then be used to optimize retention and selectivity in just two experiments. The relationship of retention with temperature is also explored. Greater retention with increasing temperature is demonstrated on the fluorinated phase at high percentages of organic modifier, which is in contrast to behavior observed in typical reversed-phase separations. The unexpected observation is explicated based on the reduction in solvent solvating power with increasing temperature. As solvation power of the mobile phase decreases, decreased solvation of both mobile phase and ionized surface groups of the stationary phase leads to stronger interactions between analyte and stationary phase. Both mobile phase ion concentration and temperature are shown to be powerful tools for the manipulation of analyte retention and selectivity.
The structural attributes and molecular interactions contributing to "U-shape" retention on pentafluorophenylpropyl (PFPP) HPLC stationary phases are systematically investigated. Only basic analytes exhibit retention that increases with the acetonitrile content in mixtures of acetonitrile and aqueous ammonium acetate, with some basic analytes not eluting at all from PFPP columns using 100% acetonitrile. U-shaped retention as a function of mobile phase acetonitrile content was more dramatic on a PFPP column relative to C18. Retention of the quaternary ammonium salt bretylium on these stationary phases and on the same bare silica support showed minimal influence of ion-exchange mechanisms on the C18 phase, however, a significant influence of ion-exchange mechanisms was observed for both PFPP and bare silica. The retention of bretylium on PFPP was only slightly less than on bare silica. These findings suggest ion-exchange mechanisms dominate retention of basic analytes in the high acetonitrile realm on PFPP. The PFPP stationary phase exhibits a substantial increase in effects of ionized surface silanol groups compared to the alkyl phase despite similar surface coverage. Retention of some basic analytes on a PFPP phase was enhanced relative to retention on silica alone, and implicates other dispersive interactions that might be exploited for selectivity different from either alkyl phases or silica alone.
A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [(14)C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A(3) (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase beta-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nucleophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria.
Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soil-bound trifluralin residues, 14C-labeled herbicide was incubated for 7 weeks with four soils under anoxic conditions. As determined by radiocounting, trifluralin binding ranged between 10 and 53% of the initial 14C depending on the soil tested. 19F NMR analyses of the methanol extracts and different fractions of the extracted soil suggested that bound residue formation largely involved reduced metabolites of the herbicide. A 2,6-diamino product of trifluralin reduction with zero-valent iron (Fe-TR), and the standard of a 1,2-diaminotrifluralin derivative (TR6) formed covalent bonds with fulvic acid (FA), as indicated by the 19F NMR spectra taken periodically over a 3-week contact time. At short contact times, TR6 and Fe-TR formed weak physical bonds with FA, as the respective spin-lattice relaxation times (T1) decreased from the range 1300-1831 ms for TR6 or Fe-TR analyzed in the absence of FA to the range 150-410 ms for TR6/FA or Fe-TR/FA mixtures. In general, the results indicated that trifluralin immobilization involved a variety of mechanisms (covalent binding, adsorption, sequestration), and with time it became increasingly stable.
Spermidine/spermine-N1-acetyltransferase (SSAT1) is a short-lived polyamine catabolic enzyme inducible by polyamines and polyamine analogues. Induction of SSAT1 plays an important role in polyamine homoeostasis, since the N1-acetylated polyamines can be excreted or oxidized by acetylpolyamine oxidase. We have purified a recombinant human acetyltransferase (SSAT2) that shares 45% identity and 61% homology with human SSAT1, but is only distally related to other known members of the GNAT (GCN5-related N-acetyltransferase) family. Like SSAT1, SSAT2 is widely expressed, but did not turn over rapidly, and levels were unaffected by treatments with polyamine analogues. Despite similarity in sequence to SSAT1, polyamines were found to be poor substrates of purified SSAT2, having K(m) values in the low millimolar range and kcat values of <0.01 s(-1). The kcat/K(m) values for spermine and spermidine for SSAT2 were <0.0003% those of SSAT1. Expression of SSAT2 in NIH-3T3 cells was not detrimental to growth, and did not reduce polyamine content or increase acetylpolyamines. These results indicate that SSAT2 is not a polyamine catabolic enzyme, and that polyamines are unlikely to be its natural intracellular substrates. A promising candidate for the physiological substrate of SSAT2 is thialysine [S-(2-aminoethyl)-L-cysteine], which is acetylated predominantly at the epsilon-amino group with K(m) and kcat values of 290 muM and 5.2 s(-1). Thialysine is a naturally occurring modified amino acid that can undergo metabolism to form cyclic ketimine derivatives found in the brain and as urinary metabolites, which can undergo further reaction to form antioxidants. SSAT2 should be renamed 'thialysine N(epsilon)-acetyltransferase', and may regulate this pathway.
Chromophoric or colored dissolved organic matter (CDOM) is one of the principal light adsorbing components of seawater, particularly in the ultraviolet, where it attenuates over 90% of downwelling ultraviolet radiation. In highly productive coastal regions and throughout most of the global ocean, in situ biological production is the major source of CDOM. However, little is known about CDOM composition on the molecular level, and there are only a few reports that link CDOM composition to autochthonous biological sources. Here we report the isolation and characterization of CDOM components from one coastal and two open-ocean sites. Each sample contains a complex mixture of light absorbing (300-400 nm) components, including 2,4-dichlorobenzoic acid and a suite of novel, polychlorinated biphenyl carboxylic acids that closely resemble polychlorinated biphenyls (PCBs) of anthropogenic origin. However, the global inventory and isomer distribution of dissolved chlorinated aromatic acids suggest they are derived from in situ biological production rather than anthropogenic contaminants. These novel chlorinated aromatic acids account for a significant amount of CDOM adsorption in the ultraviolet.
Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.
The green sulfur bacterium Chlorobium tepidum is a strict anaerobe and an obligate photoautotroph. On the basis of sequence similarity with known enzymes or sequence motifs, nine open reading frames encoding putative enzymes of carotenoid biosynthesis were identified in the genome sequence of C. tepidum, and all nine genes were inactivated. Analysis of the carotenoid composition in the resulting mutants allowed the genes encoding the following six enzymes to be identified: phytoene synthase (crtB/CT1386), phytoene desaturase (crtP/CT0807), zeta-carotene desaturase (crtQ/CT1414), gamma-carotene desaturase (crtU/CT0323), carotenoid 1',2'-hydratase (crtC/CT0301), and carotenoid cis-trans isomerase (crtH/CT0649). Three mutants (CT0180, CT1357, and CT1416 mutants) did not exhibit a discernible phenotype. The carotenoid biosynthetic pathway in C. tepidum is similar to that in cyanobacteria and plants by converting phytoene into lycopene using two plant-like desaturases (CrtP and CrtQ) and a plant-like cis-trans isomerase (CrtH) and thus differs from the pathway known in all other bacteria. In contrast to the situation in cyanobacteria and plants, the construction of a crtB mutant completely lacking carotenoids demonstrates that carotenoids are not essential for photosynthetic growth of green sulfur bacteria. However, the bacteriochlorophyll a contents of mutants lacking colored carotenoids (crtB, crtP, and crtQ mutants) were decreased from that of the wild type, and these mutants exhibited a significant growth rate defect under all light intensities tested. Therefore, colored carotenoids may have both structural and photoprotection roles in green sulfur bacteria. The ability to manipulate the carotenoid composition so dramatically in C. tepidum offers excellent possibilities for studying the roles of carotenoids in the light-harvesting chlorosome antenna and iron-sulfur-type (photosystem I-like) reaction center. The phylogeny of carotenogenic enzymes in green sulfur bacteria and green filamentous bacteria is also discussed.
The stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) was investigated using a combination of high-performance liquid chromatography, solid-phase extraction, photodiode array spectrophotometric detection, and mass spectrometric (MS) characterization. The degradation of amino acid derivatives, generated using beta-mercaptoethanol as a nucleophile, was characterized under a variety of environmental influences, with a focus on understanding the degradation kinetics and identifying the degradation products. The predominant degradation product observed under most reaction conditions was the nonfluorescent lactam form of the originally fluorescent isoindole derivative. First, the time-dependent degradation of the isoindole derivative L-serine-NDA-beta-mercaptoethanol was found to follow pseudo-first order kinetics with a half-life of 2.0 min at pH 9.2 and room temperature. The isoindole derivative was observed to react further with methanol to form a more stable fluorescent methoxy-isoindole, shedding new light on the basis for enhanced stability of these derivatives in methanol. Tandem mass spectrometry (MS/MS) experiments were used to demonstrate unimolecular degradation of the protonated isoindole in the absence of solvent or atmosphere, suggesting an intramolecular reaction mechanism involving the hydroxyethylthio group. Finally, in photobleaching studies, NDA derivatives rapidly degraded into a variety of products within the first 2 min of photobleaching versus timed controls, with the predominant product being the lactam. These results suggest that the degradation pathway for NDA derivatives is similar to the previously reported pathway for o-phthalaldehyde derivatives and clearly identifies the reaction and degradation products under a variety of conditions.
Nonenzymatic glycosylation (glycation) has been recognized as an important posttranslational modification underlying alterations of structure and function of extracellular proteins during aging and diabetes. Intracellular proteins may also be affected by this modification, and glycation has been suggested to contribute to aging-related impairment in skeletal muscle function. Glycation is the chemical reaction of reducing sugars with primary amino groups resulting in the formation of irreversible advanced glycation end products. Glutathione is an abundant tripeptide in skeletal muscle. To understand the effect of glutathione on glycated myosin function, we used a single-fiber in vitro motility assay in which myosin is extracted from a single muscle fiber segment to propel fluorescent-labeled actin filaments. Myosin function responded to glucose exposure in a dose-dependent manner, i.e., motility speeds were reduced by 10, 34, and 90% of preincubation values after 30-min exposure to 1, 3, and 6 mM glucose, respectively. The 30-min 6 mM glucose incubation was followed by a 20-min 10 mM glutathione incubation. Glutathione treatment restored motility (0.98 +/- 0.06 microm/s, n = 3; P < 0.001) after glucose exposure (0.10 +/- 0.07 microm/s, n = 3), close to preincubation levels (1.12 +/- 0.06 microm/s, n = 3). It is concluded that glucose modifies myosin function in a dose-dependent manner and that glutathione reverses the effect of glucose on myosin function.
The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains genes identified as menD and menE, homologs of Escherichia coli genes that code for 2-succinyl-6-hydroxyl-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase and O-succinylbenzoic acid-CoA ligase in the menaquinone biosynthetic pathway. In cyanobacteria, the product of this pathway is 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone), a molecule used exclusively as an electron transfer cofactor in Photosystem (PS) I. The menD(-) and menE(-) strains were generated, and both were found to lack phylloquinone. Hence, no alternative pathways exist in cyanobacteria to produce O-succinylbenzoyl-CoA. Q-band EPR studies of photoaccumulated quinone anion radical and optical kinetic studies of the P700(+) [F(A)/F(B)](-) backreaction indicate that in the mutant strains, plastoquinone-9 functions as the electron transfer cofactor in the A(1) site of PS I. At a light intensity of 40 microE m(-2) s(-1), the menD(-) and menE(-) mutant strains grew photoautotrophically and photoheterotrophically, but with doubling times slower than the wild type. Both of which are sensitive to high light intensities. Low-temperature fluorescence studies show that in the menD(-) and menE(-) mutants, the ratio of PS I to PS II is reduced relative to the wild type. Whole-chain electron transfer rates in the menD(-) and menE(-) mutant cells are correspondingly higher on a chlorophyll basis. The slower growth rate and high-light sensitivity of the menD(-) and menE(-) mutants are therefore attributed to a lower content of PS I per cell.
Many aspects of the toxicity of xenobiotic compounds have been attributed to the consequences of covalent modification of specific proteins, but the nature and specificity of protein targets for classes of electrophilic toxins remain largely uncharacterized. For inhaled toxicants, the point of exposure or absorption lies with epithelial cells lining the pulmonary tree. In this study, abundant proteins in human bronchial epithelial cells that are arylated in vitro by two quinonoid compounds, 1,4-benzoquinone (BQ) and 1,4-naphthoquinone (NQ) have been detected using (14)C-labeled quinones and two-dimensional gel electrophoresis. These proteins were identified using matrix assisted laser desorption/ionization mass spectrometry for tryptic mass mapping followed by sequence database searching. Corroborative identification of protein targets was obtained from the apparent isoelectric points, molecular weights, and the use of antibody probes. There were subtle differences in the protein targets of BQ and NQ, but both associated with the following abundant proteins, nucleophosmin, galectin-1, probable protein disulfide isomerase, protein disulfide isomerase, 60 kDa heat shock protein, mitochondrial stress-70 protein, epithelial cell marker protein, and S100-type calcium binding protein A14. We further delineate the properties of these proteins that make them preferred targets and the evidence these adducts present for delivery of these quinones to subcellular compartments.
Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to well-characterized allergens such as Lol p 1 and Phl p 1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 micro g mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function.
CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA'-'lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5- to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by Esigma(70). We investigated the influence of sigma(S) on cstA expression and found that a sigma(S) deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA Shine-Dalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.