A. Daniel Jones

Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils

Nitric oxide (˙NO) plays a central role in the pathogenesis of diverse inflammatory and infectious disorders,. The toxicity of ˙NO is thought to be engendered, in part, by its reaction with superoxide (O˙−2), yielding the potent oxidant peroxynitrite (ONOO−). However, evidence for a role of ONOO− in vivo is based largely upon detection of 3-nitrotyrosine in injured tissues. We have recently demonstrated that nitrite (NO2−), a major end-product of ˙NO metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (˙NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase,. We now show that activated human polymorphonuclear neutrophils convert NO2− into NO2Cl and ˙NO2 through myeloperoxidase-dependent pathways. Polymorphonuclear neutrophil-mediated nitration and chlorination of tyrosine residues or 4-hydroxyphenylacetic acid is enhanced by addition of NO2− or by fluxes of ˙NO. Addition of 15NO2− led to 15N enrichment of nitrated phenolic substrates, confirming its role in polymorphonuclear neutrophil-mediated nitration reactions. Polymorphonuclear neutrophil-mediated inactivation of endothelial cell angiotensin-converting enzyme was exacerbated by NO2−, illustrating the physiological significance of these reaction pathways to cellular dysfunction. Our data reveal that NO2− may regulate inflammatory processes through oxidative mechanisms, perhaps by contributing to the tyrosine nitration and chlorination observed in vivo.

Steroid induced exocytosis: The human sperm acrosome reaction

By a combination of organic precipitation and high pressure liquid chromatography, human sperm acrosome reaction inducing activity has been purified from the fluid aspirated from preovulatory human ovarian follicles and identified as 4-pregnen-3,20-dione (progesterone) and 4-pregnen-17 alpha-ol-3,20-dione (17 alpha-hydroxyprogesterone). It is argued that progesterone is present at the site of fertilization of placental mammals in concentrations sufficient for activity, and hence provides a mechanism of inducing the acrosome reaction, an exocytotic event, in vivo.

Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid A NOVEL MECHANISM FOR NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO−). We show here that reaction of nitrite (NO−2), the autoxidation product of nitric oxide (·NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO−2 with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO−2/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO−2/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO−2/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO−2/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (·NO2) and tyrosyl radical. We propose that NO−2 reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl· and ·NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO−.

Regulation and Function of Arabidopsis JASMONATE ZIM-Domain Genes in Response to Wounding and Herbivory

Jasmonate (JA) and its amino acid conjugate, jasmonoyl-isoleucine (JA-Ile), play important roles in regulating plant defense responses to insect herbivores. Recent studies indicate that JA-Ile promotes the degradation of JASMONATE ZIM-domain (JAZ) transcriptional repressors through the activity of the E3 ubiquitin-ligase SCFCOI1. Here, we investigated the regulation and function of JAZ genes during the interaction of Arabidopsis (Arabidopsis thaliana) with the generalist herbivore Spodoptera exigua. Most members of the JAZ gene family were highly expressed in response to S. exigua feeding and mechanical wounding. JAZ transcript levels increased within 5 min of mechanical tissue damage, coincident with a large (approximately 25-fold) rise in JA and JA-Ile levels. Wound-induced expression of JAZ and other CORONATINE-INSENSITIVE1 (COI1)-dependent genes was not impaired in the jar1-1 mutant that is partially deficient in the conversion of JA to JA-Ile. Experiments performed with the protein synthesis inhibitor cycloheximide provided evidence that JAZs, MYC2, and genes encoding several JA biosynthetic enzymes are primary response genes whose expression is derepressed upon COI1-dependent turnover of a labile repressor protein(s). We also show that overexpression of a modified form of JAZ1 (JAZ1Δ3A) that is stable in the presence of JA compromises host resistance to feeding by S. exigua larvae. These findings establish a role for JAZ proteins in the regulation of plant anti-insect defense, and support the hypothesis that JA-Ile and perhaps other JA derivatives activate COI1-dependent wound responses in Arabidopsis. Our results also indicate that the timing of JA-induced transcription in response to wounding is more rapid than previously realized.

A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis

Jasmonic acid (JA) and its biologically active derivatives (bioactive JAs) perform a critical role in regulating plant responses to wound stress. The perception of bioactive JAs by the F-box protein COI1 triggers the SCF(COI1)/ubiquitin-dependent degradation of JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the expression of JA-response genes. JA is required for many wound-inducible systemic defense responses, but little is known about the role of the hormone in long-distance signal relay between damaged and undamaged leaves. Here, we show that the wounding of Arabidopsis thaliana leaves results in the rapid (<5 min) accumulation of jasmonoyl-l-isoleucine (JA-Ile), the bioactive form of JA, in leaves distal to the wound site. The rapid systemic increase in JA-Ile preceded the onset of early transcriptional responses, and was associated with JAZ degradation. Wound-induced systemic production of JA-Ile required the JA biosynthetic enzyme 12-oxo-phytodienoic acid (OPDA) reductase 3 (OPR3) in undamaged responding leaves, but not in wounded leaves, and was largely dependent on the JA-conjugating enzyme JAR1. Interestingly, the wound-induced synthesis of JA/JA-Ile in systemic leaves was correlated with a rapid decline in OPDA levels. These results are consistent with a model in which a rapidly transmitted wound signal triggers the systemic synthesis of JA, which, upon conversion to JA-Ile, activates the expression of early response genes by the SCF(COI1)/JAZ pathway.

Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments.

Decomposition products formed/released during ammonia fiber expansion (AFEX) and dilute acid (DA) pretreatment of corn stover (CS) were quantified using robust mass spectrometry based analytical platforms. Ammonolytic cleavage of cell wall ester linkages during AFEX resulted in the formation of acetamide (25mg/g AFEX CS) and various phenolic amides (15mg/g AFEX CS) that are effective nutrients for downstream fermentation. After ammonolysis, Maillard reactions with carbonyl-containing intermediates represent the second largest sink for ammonia during AFEX. On the other hand, several carboxylic acids were formed (e.g. 35mg acetic acid/g DA CS) during DA pretreatment. Formation of furans was 36-fold lower for AFEX compared to DA treatment; while carboxylic acids (e.g. lactic and succinic acids) yield was 100-1000-fold lower during AFEX compared to previous reports using sodium hydroxide as pretreatment reagent.

Particle concentrations, gas-particle partitioning, and species intercorrelations for polycyclic aromatic hydrocarbons (PAH) emitted during biomass burning

Abstract Eight types of agricultural and forest fuels including 4 cereal crop residues and 4 wood fuels were burned in a combustion wind tunnel to simulate the open burning of biomass. Concentrations for 19 PAH species in particulate matter were found to range between 120 and 4000 mg kg−1, representing between 1 and 70% of total PAH emission. Weakly flaming spreading fires in the cereals were observed to produce higher levels of heavier PAH than more robust fires, with greater partitioning of PAH to the particle phase. Individual species concentrations appeared well correlated within groups based primarily on molecular weight, but no single species was observed to correlate with all others to serve as an indicator of PAH emission strength. Equilibrium gas-particle partitioning did not appear to be achieved within the 3–5 s residence time prior to sampling for sampling temperatures between 32 and 87°C, and in particular for the heavier species emitted from wood fuel pile fires with higher stack gas temperatures and shorter residence times. Total PAH emission, particle-phase concentrations, and fraction of PAH on particles were more strongly influenced by burning conditions than by fuel type.

Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato

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.

Changes in myosin structure and function in response to glycation

Nonenzymatic glycosylation (glycation) is recognized as an important post‐translational modification underlying alterations of structure and function of extracellular proteins. The effect of glycation on intracellular proteins is, on the other hand, less well known despite the vital importance of intracellular proteins for cell, tissue, and organ function. The aim of this study was to explore the effects of glycation on the structure and function of skeletal muscle myosin. My‐osin was incubated for up to 30 min with glucose and subsequently tested for structural and functional modifications by matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry and a single‐fiber in vitro motility assay, respectively. MALDI spectra revealed glycation‐related structural alterations as evidenced by the disappearance of specific Lys‐C proteolysis products and the appearance of higher mass peaks that are attributed to cross‐linking by glucose. This change was paralleled by a significant reduction in the in vitro motility speed, suggesting a structure‐related decline in myosin mechanics in response to glucose exposure. Further evidence that early glycation products form in the regulatory regions of the myosin molecule is derived from the fact that there is complete reversal of motility speed after reaction with the Schiff base‐cleaving agent hydroxylamine hydrochloride. Thus, gly‐cation of skeletal muscle myosin has a significant effect on both the structural and functional properties of the protein, a finding that is important in understanding the mechanisms underlying the impairment in muscle function associated with aging and diabetes.—Ramamurthy, B., Höök, P., Jones, A. D., Larsson, L. Changes in myosin structure and function in response to glycation. FASEB J. 15, 2415–2422 (2001)

Recruitment of a Foreign Quinone into the A1 Site of Photosystem I I. GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF PHYLLOQUINONE BIOSYNTHETIC PATHWAY MUTANTS IN SYNECHOCYSTIS SP. PCC 6803

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified inSynechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway inEscherichia coli. Targeted inactivation of themenA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 μE m−2 s−1), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 μE m−2 s−1). The menA andmenB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menAand menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50–60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from themenA and menB mutant strains contain the full complement of polypeptides, show photoreduction of FA and FB at 15 K, and support 82–84% of the wild type rate of electron transfer from cytochrome c 6 to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A1site, where it functions as an efficient cofactor in electron transfer from A0 to the iron-sulfur clusters.