Plants and tortoises: mutations in the Arabidopsis jasmonate pathway increase feeding in a vertebrate herbivore.

Photosynthetic tissues, the major food source of many invertebrates and vertebrates, are well defended. Many defence traits in leaves are controlled via the jasmonate signalling pathway in which jasmonate acts as a hormone by binding to a receptor to activate responses that lead to increased resistance to invertebrate folivores. We predicted that mutations in jasmonate synthesis might also increase the vulnerability of leaves to vertebrate folivores and tested this hypothesis using the Eastern Hermann's tortoise (Eurotestudo boettgeri) and an Arabidopsis thaliana (Brassicaceae) allene oxide synthase (aos) mutant unable to synthesize jasmonate. Tortoises preferred the aos mutant over the wild type (WT). Based on these results, we then investigated the effect of mutating jasmonate perception using a segregating population of the recessive A. thaliana jasmonate receptor mutant coronatine insensitive1-1 (coi1-1). Genotyping of these plants after tortoise feeding revealed that the homozygous coi1-1 receptor mutant was consumed more readily than the heterozygous mutant or the WT. Therefore, the plant's ability to synthesize or perceive jasmonate reduces feeding by a vertebrate herbivore. We also tested whether or not tortoise feeding behaviour was influenced by glucosinolates, the principal defence chemicals in Arabidopsis leaves with known roles in defence against many generalist insects. However, in contrast to what has been observed with such insects, leaves in which the levels of these compounds were reduced genetically were consumed at a similar rate to those of the WT.

Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa.

Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa.

FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development.

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.

Combination of lentivector immunization and low-dose chemotherapy or PD-1/PD-L1 blocking primes self-reactive T cells and induces anti-tumor immunity.

In the last two decades, anti-cancer vaccines have yielded disappointing clinical results despite the fact that high numbers of self/tumor-specific T cells can be elicited in immunized patients. Understanding the reasons behind this lack of efficacy is critical in order to design better treatment regimes. Recombinant lentivectors (rLVs) have been successfully used to induce antigen-specific T cells to foreign or mutated tumor antigens. Here, we show that rLV expressing a murine nonmutated self/tumor antigen efficiently primes large numbers of self/tumor-specific CD8(+) T cells. In spite of the large number of tumor-specific T cells, however, no anti-tumor activity could be measured in a therapeutic setting, in mice vaccinated with rLV. Accumulating evidence shows that, in the presence of malignancies, inhibition of T-cell activity may predominate overstimulation. Analysis of tumor-infiltrating lymphocytes revealed that specific anti-tumor CD8(+) T cells fail to produce cytokines and express high levels of inhibitory receptors such as programmed death (PD)-1. Association of active immunization with chemotherapy or antibodies that block inhibitory pathways often leads to better anti-tumor effects. We show here that combining rLV vaccination with either cyclophosphamide or PD-1 and PD-L1 blocking antibodies enhances rLV vaccination efficacy and improves anti-tumor immunity.

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

The role of macrophage migration inhibitory factor in mouse islet transplantation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. METHODS: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. RESULTS: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. CONCLUSION: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.

Predicting the physicochemical profile of diastereoisomeric histidine-containing dipeptides by property space analysis.

OBJECTIVES: This study aimed at measuring the lipophilicity and ionization constants of diastereoisomeric dipeptides, interpreting them in terms of conformational behavior, and developing statistical models to predict them. METHODS: A series of 20 dipeptides of general structure NH(2) -L-X-(L or D)-His-OMe was designed and synthetized. Their experimental ionization constants (pK(1) , pK(2) and pK(3) ) and lipophilicity parameters (log P(N) and log D(7.4) ) were measured by potentiometry. Molecular modeling in three media (vacuum, water, and chloroform) was used to explore and sample their conformational space, and for each stored conformer to calculate their radius of gyration, virtual log P (preferably written as log P(MLP) , meaning obtained by the molecular lipophilicity potential (MLP) method) and polar surface area (PSA). Means and ranges were calculated for these properties, as was their sensitivity (i.e., the ratio between property range and number of rotatable bonds). RESULTS: Marked differences between diastereoisomers were seen in their experimental ionization constants and lipophilicity parameters. These differences are explained by molecular flexibility, configuration-dependent differences in intramolecular interactions, and accessibility of functional groups. Multiple linear equations correlated experimental lipophilicity parameters and ionization constants with PSA range and other calculated parameters. CONCLUSION: This study documents the differences in lipophilicity and ionization constants between diastereoisomeric dipeptides. Such configuration-dependent differences are shown to depend markedly on differences in conformational behavior and to be amenable to multiple linear regression. Chirality 24:566-576, 2012. © 2012 Wiley Periodicals, Inc.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.

Stereoselective Acetate Aldol Reactions From Metal Enolates

This review deals with metal enolate-mediated stereoselective acetate aldol reactions. It summarizes recent advances on aldol additions of unsubstituted metal enolates from chiral auxiliaries, stoichiometric and catalytic Lewis acids, or acting in substrate- controlled reactions, which provide stereocontrolled aldol transformations that allow the efficient synthesis of structurally complex natural products.

Full establishment of arbuscular mycorrhizal symbiosis in rice occurs independently of enzymatic jasmonate biosynthesis.

Development of the mutualistic arbuscular mycorrhiza (AM) symbiosis between most land plants and fungi of the Glomeromycota is regulated by phytohormones. The role of jasmonate (JA) in AM colonization has been investigated in the dicotyledons Medicago truncatula, tomato and Nicotiana attenuata and contradicting results have been obtained with respect to a neutral, promotive or inhibitory effect of JA on AM colonization. Furthermore, it is currently unknown whether JA plays a role in AM colonization of monocotyledonous roots. Therefore we examined whether JA biosynthesis is required for AM colonization of the monocot rice. To this end we employed the rice mutant constitutive photomorphogenesis 2 (cpm2), which is deficient in JA biosynthesis. Through a time course experiment the amount and morphology of fungal colonization did not differ between wild-type and cpm2 roots. Furthermore, no significant difference in the expression of AM marker genes was detected between wild type and cpm2. However, treatment of wild-type roots with 50 μM JA lead to a decrease of AM colonization and this was correlated with induction of the defense gene PR4. These results indicate that JA is not required for AM colonization of rice but high levels of JA in the roots suppress AM development likely through the induction of defense.

Surface passivation of crystalline silicon by Cat-CVD amorphous and nanocrystalline thin silicon films

In this work, we study the electronic surface passivation of crystalline silicon with intrinsic thin silicon films deposited by Catalytic CVD. The contactless method used to determine the effective surface recombination velocity was the quasi-steady-state photoconductance technique. Hydrogenated amorphous and nanocrystalline silicon films were evaluated as passivating layers on n- and p-type float zone silicon wafers. The best results were obtained with amorphous silicon films, which allowed effective surface recombination velocities as low as 60 and 130 cms -1 on p- and n-type silicon, respectively. To our knowledge, these are the best results ever reported with intrinsic amorphous silicon films deposited by Catalytic CVD. The passivating properties of nanocrystalline silicon films strongly depended on the deposition conditions, especially on the filament temperature. Samples grown at lower filament temperatures (1600 °C) allowed effective surface recombination velocities of 450 and 600 cms -1 on n- and p-type silicon.

Stability of hydrogenated nanocrystalline silicon thin-film transistors

Hydrogenated nanocrystalline silicon thin-films were obtained by catalytic chemical vapour deposition at low substrate temperatures (150°C) and high deposition rates (10 Å/s). These films, with crystalline fractions over 90%, were incorporated as the active layers of bottom-gate thin-film transistors. The initial field-effect mobilities of these devices were over 0.5 cm 2/V s and the threshold voltages lower than 4 V. In this work, we report on the enhanced stability of these devices under prolonged times of gate bias stress compared to amorphous silicon thin-film transistors. Hence, they are promising candidates to be considered in the future for applications such as flat-panel displays.

Regio- and stereoselective microwave-assisted synthesis of 5-alkyl-4-alkenyl-4-phenyl-1,3-oxazolidin-2-ones

Chiral symmetrical alk-2-yne-1,4-diols have been stereoselectively transformed into 5-alkyl-4-alkenyl-4-phenyl-1,3-oxazolidin- 2-ones, which are precursors of quaternary α-amino β-hydroxy acids. The key step was the cyclization of the bis(tosylcarbamates) of 2- phenylalk-2-yne-1,4-diols, easily obtained from the starting chiral diols. These cyclizations were accomplished with complete regioselectivity and up to 92:8 dr in the presence of catalytic amounts of Ni(0) or Pd (II) derivatives under microwave heating.

Total synthesis of entecavir

Entecavir (BMS-200475) was synthesized from 4-trimethylsilyl-3-butyn-2-one and acrolein. The key features of its preparation are: (i) a stereoselective boron-aldol reaction to afford the acyclic carbon skeleton of the methylenecylopentane moiety; (ii) its cyclization by a Cp2TiCl-catalyzed intramolecular radical addition of an epoxide to an alkyne; and (iii) the coupling with a purine derivative by a Mitsunobu reaction.