Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

The role of light and gravity in the experimental transmission of Echinostoma caproni (Digenea: Echinostomatidae) cercariae to the second intermediate host, Biomphalaria glabrata (Gastropoda: Pulmonata)

Trematode cercariae inhabit predictable environments and respond to trigger cues with genetically fixed releaser responses when foraging for the upstream host. The effect of light and gravity on the transmission of Echinostoma caproni cercariae to Biomphalaria glabrata was investigated experimentally. Transmission chambers were constructed of clear polyvinyl chloride pipe. Snails were constrained within the chamber to prevent movement, while permitting the cercariae to swim freely. A trial consisted of 2 infected B. glabrata shedding E. caproni cercariae placed at the center of the chamber, with 5 uninfected B. glabrata placed 10 cm on either side (or above and below) of the shedding snails as sentinels. There was no significant difference in the prevalence of infection sentinel snails in either experiment (light vs. dark or top vs. bottom); however, mean intensity was significantly higher in sentinel snails in the dark portion of the chamber (42.5 vs. 10.4; P = 0.001) and the top of the transmission chamber (66.1 vs. 38.0; P = 0.0003). There was a high correlation between the number of metacercariae collected from sentinel snails and the total number of infective units (metacercariae + unsuccessful cercariae): r = 0.992 (light vs. dark) and r = 0.957 (top vs. bottom), respectively, at cercariae densities estimated from 22 to 3,304/L. The results suggest that cercariae of E. caproni exhibit negative photo- and geotaxis in searching for a second intermediate host. Stereotypical releaser responses to environmental trigger cues (light and gravity) allow E. caproni cercariae to exploit flexible strategies for completing the life cycle consistent with the broad range second intermediate and definitive hosts used by E. caproni cercariae and adults, respectively.     

Trace metal contamination initiates the apparent auto-aggregation,amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (seeding) and amyloid fibril-free formation of soluble SDS-resistant oligomers (oligomerization) by hydrophobic interaction is an in vitro model thought to propagate -amyloid (A) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimers disease (AD), and other amyloid-associated neurodegenerative diseases. However, A is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical A deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (0.8 µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic A1–42-mediated seeding process and A oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of A1–40 (20 µM) initiated by A1–42 (2 µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble A oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating A amyloidosis and oligomerization.     

Influence of Certain Cations on Activity of Succinyl CoA Synthetase From Tobacco

Succinyl CoA synthetase from Nicotiana tabacum exhibited a requirement for univalent and divalent cations. Mn²⁺ replaced Mg²⁺ in the assay medium and Co²⁺ and Ca²⁺ partially replaced Mg²⁺. Addition of Zn²⁺ resulted in no enzyme activity. The enzyme was activated by univalent cations K⁺, Rb⁺, NH₄⁺, and Na⁺; Li⁺ showed little or no activation. Maximum enzyme activity varied significantly with potassium salts of different anions. Greatest activation was obtained with K₃PO₄ and, respectively, KCl, KNO₃, K₂SO₄ and KF exhibited steadily decreasing enzyme activation.     

YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

Extremophiles - YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular...     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.     

Engineering Triterpene and Methylated Triterpene Production in Plants Provides Biochemical and Physiological Insights into Terpene Metabolism

Linear, branch-chained triterpenes, including squalene (C30), botryococcene (C30), and their methylated derivatives (C31–C37), generated by the green alga Botryococcus braunii race B have received significant attention because of their utility as chemical and biofuel feedstocks. However, the slow growth habit of B. braunii makes it impractical as a production system. In this study, we evaluated the potential of generating high levels of botryococcene in tobacco (Nicotiana tabacum) plants by diverting carbon flux from the cytosolic mevalonate pathway or the plastidic methylerythritol phosphate pathway by the targeted overexpression of an avian farnesyl diphosphate synthase along with two versions of botryococcene synthases. Up to 544 µg g⁻¹ fresh weight of botryococcene was achieved when this metabolism was directed to the chloroplasts, which is approximately 90 times greater than that accumulating in plants engineered for cytosolic production. To test if methylated triterpenes could be produced in tobacco, we also engineered triterpene methyltransferases (TMTs) from B. braunii into wild-type plants and transgenic lines selected for high-level triterpene accumulation. Up to 91% of the total triterpene contents could be converted to methylated forms (C31 and C32) by cotargeting the TMTs and triterpene biosynthesis to the chloroplasts, whereas only 4% to 14% of total triterpenes were methylated when this metabolism was directed to the cytoplasm. When the TMTs were overexpressed in the cytoplasm of wild-type plants, up to 72% of the total squalene was methylated, and total triterpene (C30+C31+C32) content was elevated 7-fold. Altogether, these results point to innate mechanisms controlling metabolite fluxes, including a homeostatic role for squalene.Terpenes and terpenoids represent a distinct class of natural products (Buckingham, 2003) that are derived from two universal five-carbon precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotic fungi and animals, IPP and DMAPP are synthesized via the mevalonate (MVA) pathway, whereas in prokaryotes, they are synthesized via the methylerythritol phosphate (MEP) pathway. In higher plants, the pathways are present in separate compartments and are believed to operate independently. The MVA pathway in the cytoplasm is predominantly responsible for sesquiterpene (C15), triterpene (C30), and polyprenol (greater than C45) biosynthesis and associated with the endoplasmic reticulum (ER) system. The MEP pathway resides in plastids and is dedicated to monoterpenes (C10), diterpenes (C20), carotenoids (C40), and long-chain phytol biosynthesis. All these compounds are usually produced by plants for a variety of physiological (i.e. hormones, aliphatic membrane anchors, and maintaining membrane structure) and ecological (i.e. defense compounds and insect/animal attractants) roles (Kempinski et al., 2015). Terpenes are also important for various industrial applications, ranging from flavors and fragrances (Schwab et al., 2008) to medicines (Dewick, 2009; Niehaus et al., 2011; Shelar, 2011).The utility of terpenes as chemical and biofuel feedstocks has also received considerable attention recently. Isoprenoid-derived biofuels include farnesane (Renninger and McPhee, 2008; Rude and Schirmer, 2009), bisabolene (Peralta-Yahya et al., 2011), pinene dimers (Harvey et al., 2010), isopentenal (Withers et al., 2007), and botryococcene (Moldowan and Seifert, 1980; Hillen et al., 1982; Glikson et al., 1989; Mastalerz and Hower, 1996). The richness of branches within these hydrocarbon scaffolds correlate with their high-energy content, which enables them to serve as suitable alternatives to crude petroleum (Peralta-Yahya and Keasling, 2010). Indeed, some of them are already major contributors to current-day petroleum-based fuels. One of the best examples of this is the triterpene oil accumulating in the green alga Botryococcus braunii race B, which is considered a major progenitor to oil and coal shale deposits (Moldowan and Seifert, 1980). This alga has been well studied, and the major constituents of its prodigious hydrocarbon oil are a group of triterpenes including squalene (C30), organism-specific botryococcene (C30), methylated squalene (C31–C34), and methylated botryococcene (C31–C37; Metzger et al., 1988; Huang and Poulter, 1989; Okada et al., 1995), which can be readily converted into all classes of combustible fuels under hydrocracking conditions (Hillen et al., 1982).The unique biosynthetic mechanism for the triterpenes in B. braunii was recently described by Niehaus et al. (2011), and a series of novel squalene synthase-like genes were identified (Fig. 1). In short, squalene synthase-like enzyme, SSL-1, performs a head-to-head condensation of two farnesyl diphosphate (FPP) molecules into presqualene diphosphate, followed by a reductive rearrangement to yield squalene (C30) by the enzyme SSL-2, or is converted by SSL-3 to form botryococcene through a different reductive rearrangement (Niehaus et al., 2011). Methylated derivatives are the dominant triterpene species generated by B. braunii race B (Metzger, 1985; Metzger et al., 1988), and these derivatives are known to yield higher quality fuels due to their high energy content and the hydrocracking products derived by virtue of having more hydrocarbon branches. Triterpene methyltransferases (TMTs) that can methylate squalene and botryococcene have been successfully characterized by Niehaus et al. (2012). TRITERPENE METHYLTRANSFERASE1 (TMT-1) and TMT-2 prefer squalene C30 as their substrate for the production of monomethylated (C31) or dimethylated (C32) squalene, while TMT-3 prefers botryococcene as its substrate for the biosynthesis of monomethylated (C31) or dimethylated (C32) botryococcene (Fig. 1). These TMTs are believed to be insoluble enzymes; they exhibit large hydrophobic areas, and their activities were only observed in vitro using yeast microsomal preparations (no activity was observed when expressed in bacteria; Niehaus et al., 2012).Open in a separate windowFigure 1.Depiction of the catalytic roles of novel SSL and TMT enzymes in B. braunii race B and their putative contributions to the triterpene constituents (Niehaus et al., 2011; Niehaus et al., 2012). SSL-1 catalyzes the condensation of two farnesyl diphosphate (FPP) molecules to presqualene diphosphate (PSPP), which is converted to either squalene or botryococcene by SSL-2 or SSL-3, respectively. Squalene can also be synthesized directly from the condensation of two FPP molecules catalyzed by squalene synthase (SQS). TMT-1 and TMT-2 transfer the methyl donor group from S-adenosylmethionine (SAM) to squalene to form monomethylated and dimethylated squalene, whereas TMT-3 acts on botryococcene to form monomethylated and dimethylated botryococcene (Niehaus et al., 2012).Like the majority of identified methyltransferases, these TMTs utilize the methyl donor S-adenosyl methionine (SAM), which is ubiquitous in prokaryotes and eukaryotes (Scheer et al., 2011; Liscombe et al., 2012). In plants, SAM is one of the most abundant cofactors (Fontecave et al., 2004; Sauter et al., 2013) and is synthesized exclusively in the cytosol (Wallsgrove et al., 1983; Ravanel et al., 1998, 2004; Bouvier et al., 2006). While it is used predominantly as a methyl donor in the methylation reaction (Ravanel et al., 2004), it also serves as the primary precursor for the biosynthesis of ethylene (Wang et al., 2002b), polyamines (Kusano et al., 2008), and nicotianamine (Takahashi et al., 2003), which play a variety of important roles for plant growth and development (Huang et al., 2012; Sauter et al., 2013). The SAM present in organelles, like the chloroplast, appears to be imported from the cytosol by specific SAM/S-adenosylhomocysteine exchange transporters that reside on the envelope membranes of plastids (Ravanel et al., 2004; Bouvier et al., 2006). The imported SAM is involved in the biogenesis of Asp-derived amino acids (Curien et al., 1998; Jander and Joshi, 2009; Sauter et al., 2013) and serves as the methyl donor for the methylation of macromolecules, such as plastid DNA (Nishiyama et al., 2002; Ahlert et al., 2009) and proteins (Houtz et al., 1989; Niemi et al., 1990; Ying et al., 1999; Trievel et al., 2003; Alban et al., 2014), and small molecule metabolites, such as prenylipids (e.g. plastoquinone, tocopherol, chlorophylls, and phylloquinone; Bouvier et al., 2005, 2006; DellaPenna, 2005).Although plants and microbes are the natural sources for useful terpenes, most of them are produced in very small amounts and often as complex mixtures. In contrast, B. braunii produces large quantities of triterpenes, but its slow growth makes it undesirable as a viable production platform (Niehaus et al., 2011). Nevertheless, metabolic engineering and synthetic biology offer many strategies to manipulate terpene metabolism in various biological systems to achieve high-value terpene production with high yield and high fidelity for particular practical applications (Nielsen and Keasling, 2011). Many successes have been achieved in engineering valuable terpenes in heterotrophic microbes, such as Escherichia coli (Nishiyama et al., 2002; Martin et al., 2003; Ajikumar et al., 2010) and Saccharomyces cerevisiae (Ro et al., 2006; Takahashi et al., 2007; Westfall et al., 2012; Zhuang and Chappell, 2015). The strategies developed in these efforts usually take advantage of specific microbe strains whose innate biosynthetic machinery is genetically modified to accumulate certain prenyldiphosphate precursors (e.g. IPP or FPP), which can be utilized by other introduced terpene synthase(s) for the production of the desired terpene(s). For example, greater than 900 mg L⁻¹ bisabolene was produced when bisabolene synthase genes from plants were introduced into FPP-overproducing E. coli or S. cerevisiae strains (Peralta-Yahya et al., 2011). High levels of farnesane production for diesel fuels were also achieved by reductive hydrogenation of its precursor farnesene, which was generated from a genetically engineered yeast (e.g. Saccharomyces cerevisiae) strain using plant farnesene synthases (Renninger and McPhee, 2008; Ubersax and Platt, 2010). However, terpene production using microbial platforms is still dependent on exogenous feedstocks (i.e. sugars) and elaborate production facilities, both of which add significantly to their production costs.Compared with microbial systems, engineering terpene production in plant systems seems like an attractive target as well. This is because plants can take advantage of photosynthesis by using atmospheric CO₂ as their carbon resource instead of relying on exogenous carbon feedstocks. Moreover, crop plants such as tobacco (Nicotiana tabacum) can generate a large amount of green tissues efficiently when grown for biomass production (Schillberg et al., 2003; Andrianov et al., 2010), making them a robust, sustainable, and scalable platform for large-scale terpene production. Nonetheless, compared with microbial platforms, there are only a few examples of elevating terpene production in bioengineered plants. This is due partly to higher plants being complex multicellular organisms, in which terpene metabolism generally utilizes more complex innate machinery that can be compartmentalized intracellularly and to cell/tissue specificities (Lange and Ahkami, 2013; Kempinski et al., 2015). Significant efforts have been made to overcome these obstacles to improve the production of valuable terpenes in plants, including monoterpenes (Lücker et al., 2004; Ohara et al., 2010; Lange et al., 2011), sesquiterpenes (Aharoni et al., 2003; Kappers et al., 2005; Wu et al., 2006; Davidovich-Rikanati et al., 2008), diterpenes (Besumbes et al., 2004; Anterola et al., 2009), and triterpenes (Inagaki et al., 2011; Wu et al., 2012). Among these, engineering terpene metabolism into a subcellular organelle, where the engineered enzymes/pathways can utilize unlimited/unregulated precursors as substrates, appears most successful. For example, Wu et al. (2006, 2012) expressed an avian farnesyl diphosphate synthase (FPS) with foreign sesquiterpene/triterpene synthases targeted to the plastid to divert the IPP/DMAPP pool from the plastidic MEP pathway to synthesize high levels of the novel sesquiterpenes patchoulol and amorpha-4,11-diene up to 30 µg g⁻¹ fresh weight and the triterpene squalene up to 1,000 µg g⁻¹ fresh weight. This strategy appears to be particularly robust because it avoids possible endogenous regulation of sesquiterpene and triterpene biosynthesis, which occurs normally in the cytoplasm, and relies upon more plastic precursor pools of IPP/DMAPP inherent in the plastid, which are primarily derived from the local CO₂ fixation (Wright et al., 2014).The goal of this study was to evaluate the prospects for engineering advanced features of triterpene metabolism from B. braunii into tobacco and, thus, to probe the innate intricacies of isoprenoid metabolism in plants. In order to achieve this, we first introduced the key steps of botryococcene biosynthesis into specific subcellular compartments of tobacco cells under the direction of constitutive or trichome-specific promoters. The transgenic lines expressing the enzymes in the chloroplast were found to accumulate the highest levels of botryococcene. Triterpene methyltransferases were next introduced into the same intracellular compartments of selected high-triterpene-accumulating lines. A high yield of methylated triterpenes was also achieved in transgenic lines when the TMTs were targeted to the chloroplast. Through careful comparison of the levels of triterpenes and the methylated triterpene products in the various transgenic lines, we have also gained a deeper insight into the subcellular distribution of the triterpene products in these transgenic lines as well as a better understanding of methylation metabolism for specialized metabolites in particular compartments. These findings all contribute to our understanding of the regulatory elements that control carbon flux through the innate terpene biosynthetic pathways operating in plants.