Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.     

Till Death Do Us Part: Stable Sponge-Bacteria Associations under Thermal and Food Shortage Stresses

Sporadic mass mortality events of Mediterranean sponges following periods of anomalously high temperatures or longer than usual stratification of the seawater column (i.e. low food availability) suggest that these animals are sensitive to environmental stresses. The Mediterranean sponges Ircinia fasciculata and I. oros harbor distinct, species-specific bacterial communities that are highly stable over time and space but little is known about how anomalous environmental conditions affect the structure of the resident bacterial communities. Here, we monitored the bacterial communities in I. fasciculata (largely affected by mass mortalities) and I. oros (overall unaffected) maintained in aquaria during 3 weeks under 4 treatments that mimicked realistic stress pressures: control conditions (13°C, unfiltered seawater), low food availability (13°C, 0.1 µm-filtered seawater), elevated temperatures (25°C, unfiltered seawater), and a combination of the 2 stressors (25°C, 0.1 µm-filtered seawater). Bacterial community structure was assessed using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences and transmission electron microscopy (TEM). As I. fasciculata harbors cyanobacteria, we also measured chlorophyll a (chl a) levels in this species. Multivariate analysis revealed no significant differences in bacterial T-RFLP profiles among treatments for either host sponge species, indicating no effect of high temperatures and food shortage on symbiont community structure. In I. fasciculata, chl a content did not significantly differ among treatments although TEM micrographs revealed some cyanobacteria cells undergoing degradation when exposed to both elevated temperature and food shortage conditions. Arguably, longer-term treatments (months) could have eventually affected bacterial community structure. However, we evidenced no appreciable decay of the symbiotic community in response to medium-term (3 weeks) environmental anomalies purported to cause the recurrent sponge mortality episodes. Thus, changes in symbiont structure are not likely the proximate cause for these reported mortality events.     

Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism

It is now well established that passive exposure to inhaled OVA leads to a state of immunological tolerance. Therefore, to elicit allergic sensitization, researchers have been compelled to devise alternative strategies, such as the systemic delivery of OVA in the context of powerful adjuvants, which are alien to the way humans are exposed and sensitized to allergens. The objectives of these studies were to investigate immune-inflammatory responses to intranasal delivery of a purified house dust mite (HDM) extract and to evaluate the role of GM-CSF in this process. HDM was delivered to BALB/c mice daily for 10 days. After the last exposure, mice were killed, bronchoalveolar lavage was performed, and samples were obtained. Expression/production of Th2-associated molecules in the lymph nodes, lung, and spleen were evaluated by real-time quantitative PCR and ELISA, respectively. Using this exposure protocol, exposure to HDM alone generated Th2 sensitization based on the expression/production of Th2 effector molecules and airway eosinophilic inflammation. Flow cytometric analysis demonstrated expansion and activation of APCs in the lung and an influx of activated Th2 effector cells. Moreover, this inflammation was accompanied by airways hyper-responsiveness and a robust memory-driven immune response. Finally, administration of anti-GM-CSF-neutralizing Abs markedly reduced immune-inflammatory responses in both lung and spleen. Thus, intranasal delivery of HDM results in Th2 sensitization and airway eosinophilic inflammation that appear to be mediated, at least in part, by endogenous GM-CSF production.     

Mechanistic studies of the oxidation of oxyhemoglobin by peroxynitrite

The strong oxidizing and nitrating agent peroxynitrite has been shown to diffuse into erythrocytes and oxidize oxyhemoglobin (oxyHb) to metHb. Because the value of the second-order rate constant for this reaction is on the order of 10(4) M(-)(1) s(-)(1) and the oxyHb concentration is about 20 mM (expressed per heme), this process is rather fast and oxyHb is considered a sink for peroxynitrite. In this work, we showed that the reaction of oxyHb with peroxynitrite, both in the presence and absence of CO(2), proceeds via the formation of oxoiron(iv)hemoglobin (ferrylHb), which in a second step is reduced to metHb and nitrate by its reaction with NO(2)(*). In the presence of physiological relevant amounts of CO(2), ferrylHb is generated by the reaction of NO(2)(*) with the coordinated superoxide of oxyHb (HbFe(III)O(2)(*)(-)). This reaction proceeds via formation of a peroxynitrato-metHb complex (HbFe(III)OONO(2)), which decomposes to generate the one-electron oxidized form of ferrylHb, the oxoiron(iv) form of hemoglobin with a radical localized on the globin. CO(3)(*)(-), the second radical formed from the reaction of peroxynitrite with CO(2), is also scavenged efficiently by oxyHb, in a reaction that finally leads to metHb production. Taken together, our results indicate that oxyHb not only scavenges peroxynitrite but also the radicals produced by its decomposition.     

The plant landscape as inferred from a basket of the Roman town of Privernum (Latium,central Italy)

Abstract

Privernum was a rich Roman colony located 70 km southwest of Rome (southern Latium, central Italy). The archaeobotanical investigations focused on the garden and related structures of the luxury domus della Soglia nilotica. They are archaeologically and radiocarbon dated to the second half of the 1st century AD. The remains of a charred basket were found in the filling of the euripus, an ornamental water basin of the garden. The weaving was made with twisted strands of the leaves of Ampelodesmos mauritanicus (Poir.) T. Durand and Schinz; for the bottom and the handle/s of the basket, wood of evergreen oaks and ash and/or elm, respectively were probably used. The basket contained Pinus pinea seeds and cone scales, and Prunus persica endocarps, which were probably burnt in summer. The sediment in the drainage system and in the kitchen was processed for macro- and microremains. The results indicate the presence of spontaneous ruderal and weed flora elements, typical of human settlement areas, and crops.     

The murine gammaherpesvirus-68 gp150 acts as an immunogenic decoy to limit virion neutralization

Herpesviruses maintain long-term infectivity without marked antigenic variation. They must therefore evade neutralization by other means. Immune sera block murine gammaherpesvirus-68 (MHV-68) infection of fibroblasts, but fail to block and even enhance its infection of IgG Fc receptor-bearing cells, suggesting that the antibody response to infection is actually poor at ablating virion infectivity completely. Here we analyzed this effect further by quantitating the glycoprotein-specific antibody response of MHV-68 carrier mice. Gp150 was much the commonest glycoprotein target and played a predominant role in driving Fc receptor-dependent infection: when gp150-specific antibodies were boosted, Fc receptor-dependent infection increased; and when gp150-specific antibodies were removed, Fc receptor-dependent infection was largely lost. Neither gp150-specific monoclonal antibodies nor gp150-specific polyclonal sera gave significant virion neutralization. Gp150 therefore acts as an immunogenic decoy, distorting the MHV-68-specific antibody response to promote Fc receptor-dependent infection and so compromise virion neutralization. This immune evasion mechanism may be common to many non-essential herpesvirus glycoproteins.