Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana. Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.

Single cell tuning of Myc expression by antigen receptor signal strength and interleukin-2 in T lymphocytes.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses.

GROWTH, YIELD AND POSTHARVEST QUALITY OF WAX APPLE AS AFFECTED BY NAPHTHALENE ACETIC ACID APPLICATION

Results of this study represent the first report of the effect of Naphthalene Acetic Acid (NAA) on the pre and post harvest quality of wax apple fruit. The wax apple trees were spray treated with 0, 5, 10 and 20 mg L-1 NAA under field conditions during 2008 to 2011. The experiments were carried out in Completely Randomized Design (CRD) with six replications. Leaf chlorophyll content, chlorophyll fluorescence, photosynthetic yield, net photosynthetic rate, drymatter content of leaves and total soluble solids and K+content of wax apple fruits were significantly increased after treatments with 10 mg L-1. Polygalacturonase activity significantly decreased with NAA treatments. The application of 5 mg L-1 NAA increased 27% more bud and reduced 42% less fruit drop compared to the control. In addition, higher protein and phosphate synthase activity of leaves, fruit set, fruit growth, larger fruit size and yield were recorded in NAA treated plants. In storage, treated fruits exhibited higher TSS and firmness and less weight loss, browning, titratable acidity, respiration and ethylene production than the control. It is concluded that spraying with 5 and 10 mg L-1 NAA once a week under field conditions produced better fruit growth and yield of the wax apple and maintained better fruit quality in postharvest storage.

STUDY ON THE SUGAR-ACID RATIO AND RELEVANT METABOLIZING ENZYME ACTIVITIES IN NAVEL ORANGE FRUITS FROM DIFFERENT ECO-REGIONS

ABSTRACT The flavor quality of citrus fruits is largely determined by the sugar-acid ratio, but it remains uncertain how sugar- and/or acid-metabolizing enzymes regulate the sugar-acid ratio of navel oranges and further affect the fruit quality. In the present study, Robertson navel oranges (Citrus sinesis Osb.) were collected from six representative habitats in three eco-regions of Sichuan, China. The changes in the sugar-acid ratio and the activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), cytosolic cio-aconitase (ACO), and isocitrate dehydrogenase (IDH) were examined in navel oranges during fruit development. The results indicated that the sugar-acid ratio of fruits in different eco-regions changed significantly from 150 days after full bloom. The SPS and cytosolic ACO fruit activities had minor changes among different ecoregions throughout the experimental periods, whereas the activities of SS and IDH changed significantly in fruits among three eco-regions. Furthermore, the sugar-acid ratio and the activities of SS in the synthetic direction and IDH were the highest in south subtropics and the lowest in north mid-subtropics, probably due to the effects of climate conditions and/or other relevant eco-factors. It demonstrated that SS in the synthetic direction and IDH were of greater importance in regulating the sugar-acid ratio of navel oranges in different eco-regions, which provided new insights into the factors that determine the flavor quality of navel oranges and valuable data for guiding relevant agricultural practices.

Soybean (Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

Antifungal activities of SCY-078 (MK-3118) and standard antifungal agents against clinical non-Aspergillus mold isolates.

The limited armamentarium of active and oral antifungal drugs against emerging non-Aspergillus molds is of particular concern. Current antifungal agents and the new orally available beta-1,3-d-glucan synthase inhibitor SCY-078 were tested in vitro against 135 clinical non-Aspergillus mold isolates. Akin to echinocandins, SCY-078 showed no or poor activity against Mucoromycotina and Fusarium spp. However, SCY-078 was highly active against Paecilomyces variotii and was the only compound displaying some activity against notoriously panresistant Scedosporium prolificans.

Inflammation in schizophrenia: A question of balance.

In the past decade, there has been renewed interest in immune/inflammatory changes and their associated oxidative/nitrosative consequences as key pathophysiological mechanisms in schizophrenia and related disorders. Both brain cell components (microglia, astrocytes, and neurons) and peripheral immune cells have been implicated in inflammation and the resulting oxidative/nitrosative stress (O&NS) in schizophrenia. Furthermore, down-regulation of endogenous antioxidant and anti-inflammatory mechanisms has been identified in biological samples from patients, although the degree and progression of the inflammatory process and the nature of its self-regulatory mechanisms vary from early onset to full-blown disease. This review focuses on the interactions between inflammation and O&NS, their damaging consequences for brain cells in schizophrenia, the possible origins of inflammation and increased O&NS in the disorder, and current pharmacological strategies to deal with these processes (mainly treatments with anti-inflammatory or antioxidant drugs as add-ons to antipsychotics).

The Aldosterone/Renin Ratio as a Diagnostic Tool for the Diagnosis of Primary Hypoaldosteronism in Newborns and Infants.

BACKGROUND/AIMS: Primary hypoaldosteronism is a rare inborn disorder with life-threatening symptoms in newborns and infants due to an aldosterone synthase defect. Diagnosis is often difficult as the plasma aldosterone concentration (PAC) can remain within the normal range and thus lead to misinterpretation and delayed initiation of life-saving therapy. We aimed to test the eligibility of the PAC/plasma renin concentration (PRC) ratio as a tool for the diagnosis of primary hypoaldosteronism in newborns and infants. Meth ods: Data of 9 patients aged 15 days to 12 months at the time of diagnosis were collected. The diagnosis of primary hypoaldosteronism was based on clinical and laboratory findings over a period of 12 years in 3 different centers in Switzerland. To enable a valid comparison, the values of PAC and PRC were correlated to reference methods. RESULTS: In 6 patients, the PAC/PRC ratio could be determined and showed constantly decreased values <1 (pmol/l)/(mU/l). In 2 patients, renin was noted as plasma renin activity (PRA). PAC/PRA ratios were also clearly decreased. The diagnosis was subsequently genetically confirmed in 8 patients. CONCLUSION: A PAC/PRC ratio <1 pmol/mU and a PAC/PRA ratio <28 (pmol/l)/(ng/ml × h) are reliable tools to identify primary hypoaldosteronism in newborns and infants and help to diagnose this life-threatening disease faster. © 2015 S. Karger AG, Basel.

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09.

OBJECTIVE: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced non-small-cell lung cancer are promising for further investigation. METHODS: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. RESULTS: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16 cycles. PFS at 6 months was 70%, median PFS was 14 months, and ORR was 70%. Biopsy at progression was safe and successful in 71% of the cases. CONCLUSIONS: Both combination therapies were promising for further studies. Biopsy at progression was feasible and will be part of future SAKK studies to investigate molecular mechanisms of resistance.

Germination, senescence and pathogenic attack in soybean (Glycine max. L.): Identification of the cytosolic aconitase participating in the glyoxylate cycle

During our study of the glyoxylate cycle in soybean (Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between P-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the CI aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release.

BACKGROUND: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown. RESULTS: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype. CONCLUSIONS: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.

Role of Inflammatory Monocytes in Vaccine-Induced Reduction of Helicobacter felis Infection.

Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. In this study, we evaluated the role of inflammatory monocytes in the vaccine-induced reduction of Helicobacter felis infection. We first showed by using flow cytometric analysis that Ly6C(low) major histocompatibility complex class II-positive chemokine receptor type 2 (CCR2)-positive CD64(+) inflammatory monocytes accumulate in the stomach mucosa during the vaccine-induced reduction of H. felis infection. To determine whether inflammatory monocytes played a role in the protection, these cells were depleted with anti-CCR2 depleting antibodies. Indeed, depletion of inflammatory monocytes was associated with an impaired vaccine-induced reduction of H. felis infection on day 5 postinfection. To determine whether inflammatory monocytes had a direct or indirect role, we studied their antimicrobial activities. We observed that inflammatory monocytes produced tumor necrosis factor alpha and inducible nitric oxide synthase (iNOS), two major antimicrobial factors. Lastly, by using a Helicobacter in vitro killing assay, we showed that mouse inflammatory monocytes and activated human monocytes killed H. pylori in an iNOS-dependent manner. Collectively, these data show that inflammatory monocytes play a direct role in the immunization-induced reduction of H. felis infection from the gastric mucosa.

Heme oxygenase-1 induction by endogenous nitric oxide: influence of intracellular glutathione.

To investigate the influence of glutathione (GSH) on cellular effects of nitric oxide (NO) formation, human colon adenocarcinoma cells were transfected with a vector allowing controlled expression of inducible nitric oxide synthase (iNOS). Protein levels of oxidative stress-sensitive heme oxygenase-1 (HO-1) were analyzed in the presence or absence of GSH depletion using L-buthionine-[S,R]-sulfoximine and iNOS induction. While no effect was observed in the presence of iNOS activity alone, a synergistic effect on HO-1 expression was observed in the presence of iNOS expression and GSH depletion. This effect was prevented by addition of N-methyl-L-arginine. Therefore, targeting of endogenous NO may be modulated by intracellular GSH.

The nitric oxide pathway in pig isolated calyceal smooth muscle.

In pig and humans, whose kidneys have a multi-calyceal collecting system, the initiation of ureteral peristalsis takes place in the renal calyces. In the pig and human ureter, recent evidence suggests that nitric oxide (NO) is an inhibitory mediator that may be involved in the regulation of peristalsis. This study was designed to assess whether the NO synthase/NO/cyclic GMP pathway modulates the motility of pig isolated calyceal smooth muscle. Immunohistochemistry revealed a moderate overall innervation of the smooth muscle layer, and no neuronal or inducible NO synthase (NOS) immunoreactivities. Endothelial NOS immunoreactivities were observed in the urothelium and vascular endothelium, and numerous cyclic GMP-immunoreactive (-IR) calyceal smooth muscle cells were found. As measured by monitoring the conversion of L-arginine to L-citrulline, Ca(2+)-dependent NOS activity was moderate. Assessment of functional effects was performed in tissue baths and showed that NO and SIN-1 decreased spontaneous and induced contractions of isolated preparations in a concentration-dependent manner. In strips exposed to NO, there was a 10-fold increase of the cyclic GMP levels compared with control preparations (P < 0.01). It is concluded that a non-neuronal NOS/NO/cyclic GMP pathway is present in pig calyces, where it may influence motility. The demonstration of cyclic GMP-IR smooth muscle cells suggests that NO acts directly on these cells. This NOS/NO/cyclic GMP pathway may be a target for drugs inhibiting peristalsis of mammalian upper urinary tract. Neurourol. Urodynam. 18:673-685, 1999.