Isoprenoid biosynthesis in the erythrocytic stages of Plasmodium falciparum

The development of new drugs is one strategy for malaria control. Biochemical pathways localised in the apicoplast of the parasite, such as the synthesis of isoprenic precursors, are excellent targets because they are different or absent in the human host. Isoprenoids are a large and highly diverse group of natural products with many functions and their synthesis is essential for the parasite's survival. During the last few years, the genes, enzymes, intermediates and mechanisms of this biosynthetic route have been elucidated. In this review, we comment on some aspects of the methylerythritol phosphate pathway and discuss the presence of diverse isoprenic products such as dolichol, ubiquinone, carotenoids, menaquinone and isoprenylated proteins, which are biosynthesised during the intraerythrocytic stages of Plasmodium falciparum.

Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1.

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.

Catabolite repression control of pyocyanin biosynthesis at an intersection of primary and secondary metabolism in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the catabolite repression control (Crc) protein repressed the formation of the blue pigment pyocyanin in response to a preferred carbon source (succinate) by interacting with phzM mRNA, which encodes a key enzyme in pyocyanin biosynthesis. Crc bound to an extended imperfect recognition sequence that was interrupted by the AUG translation initiation codon.

Study of chemical modification of alkaline lignin by the glyoxalation reaction

In this study, glyoxalated alkaline lignins with a non-volatile and non-toxic aldehyde, which can be obtained from several natural resources, namely glyoxal, were prepared and characterized for its use in wood adhesives. The preparation method consisted of the reaction of lignin with glyoxal under an alkaline medium. The influence of reaction conditions such as the molar ratio of sodium hydroxide-to-lignin and reaction time were studied relative to the properties of the prepared adducts. The analytical techniques used were FTIR and 1H-NMR spectroscopies, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results from both the FTIR and 1H-NMR spectroscopies showed that the amount of introduced aliphatic hydroxyl groups onto the lignin molecule increased with increasing reaction time and reached a maximum value at 10 h, and after they began to decrease. The molecular weights remained unchanged until 10 h of reaction time, and then started to increase, possibly due to the repolymerization reactions. DSC analysis showed that the glass transition temperature (Tg) decreased with the introduction of glyoxal onto the lignin molecule due to the increase in free volume of the lignin molecules. TGA analysis showed that the thermal stability of glyoxalated lignin is not influenced and remained suitable for wood adhesives. Compared to the original lignin, the improved lignin is reactive and a suitable raw material for adhesive formula

The suitability of steam exploded vitis vinifera and alkaline lignin for the manufacture of fiberboard

The main objective of this study was to explore the suitability of Vitis vinifera as a raw material and alkaline lignin as a natural binder for fiberboard manufacturing. In the first step, Vitis vinifera was steam- exploded through a thermo-mechanical vapor process in a batch reactor, and the obtained pulp was dried, ground, and pressed to produce the boards. The effects of pretreatment factors and pressing conditions on the chemical composition of the fibers and the physico-mechanical properties of binderless fiberboards were evaluated, and the conditions that optimize these properties were found. A response surface method based on a central composite design and multiple-response optimization was used. The variables studied and their respective variation ranges were: pretreatment temperature (Tr: 190-210ºC), pretreatment time (tr: 5-10 min), pressing temperature (Tp: 190-210ºC), pressing pressure (Pp: 8-16MPa), and pressing time (tp: 3-7min). The results of the optimization step show that binderless fiberboards have good water resistance and weaker mechanical properties. In the second step, fiberboards based on alkaline lignin and Vitis vinifera pulp produced at the optimal conditions determined for binderless fiberboards were prepared and their physico-mechanical properties were tested. Our results show that the addition of about 15% alkaline lignin leads to the production of fiberboards that fully meet the requirements of the relevant standard specifications

Synthesis and characterization of kraft lignin-based epoxy resins

Epoxidization is an interesting way to develop a new application of lignin and therefore to improve its application potential. In this work, kraft lignin-based epoxy resins were obtained by the epoxidization reaction, using the kraft lignin recovered directly from pulping liquor and modified by a methylolation reaction. The methylolated lignins were obtained by the reaction of original kraft lignin with formaldehyde and glyoxal, which is a less volatile and less toxic aldehyde. 1H-NMR spectroscopy showed that methylolated kraft lignin has more hydroxymethyl groups than glyoxalated kraft lignin. For the epoxidization reaction we studied the influence of the lignin:NaOH (w/w) ratio, temperature, and time of the reaction on the properties of the prepared epoxidized lignins. The structures of lignin-based epoxy resins were followed by epoxy index test and FTIR spectroscopy. Optimal conditions were obtained for lignin-based epoxy resin produced at lignin/NaOH = 1/3 at 70 ºC for 3h. Thermogravimetry analysis (TGA) revealed that the epoxidization enhances the thermal stability of lignins and may allow a wider temperature range for applications with lignin epoxy-PF blends

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function.

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K(+) increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin.

The antimicrobial metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) contributes to the capacity of Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soilborne pathogens. A 2, 4-DAPG-negative Tn5 insertion mutant of strain CHA0 was isolated, and the nucleotide sequence of the 4-kb genomic DNA region adjacent to the Tn5 insertion site was determined. Four open reading frames were identified, two of which were homologous to phlA, the first gene of the 2,4-DAPG biosynthetic operon, and to the phlF gene encoding a pathway-specific transcriptional repressor. The Tn5 insertion was located in an open reading frame, tentatively named phlH, which is not related to known phl genes. In wild-type CHA0, 2, 4-DAPG production paralleled expression of a phlA'-'lacZ translational fusion, reaching a maximum in the late exponential growth phase. Thereafter, the compound appeared to be degraded to monoacetylphloroglucinol by the bacterium. 2,4-DAPG was identified as the active compound in extracts from culture supernatants of strain CHA0 specifically inducing phlA'-'lacZ expression about sixfold during exponential growth. Induction by exogenous 2,4-DAPG was most conspicuous in a phlA mutant, which was unable to produce 2, 4-DAPG. In a phlF mutant, 2,4-DAPG production was enhanced severalfold and phlA'-'lacZ was expressed at a level corresponding to that in the wild type with 2,4-DAPG added. The phlF mutant was insensitive to 2,4-DAPG addition. A transcriptional phlA-lacZ fusion was used to demonstrate that the repressor PhlF acts at the level of transcription. Expression of phlA'-'lacZ and 2,4-DAPG synthesis in strain CHA0 was strongly repressed by the bacterial extracellular metabolites salicylate and pyoluteorin as well as by fusaric acid, a toxin produced by the pythopathogenic fungus Fusarium. In the phlF mutant, these compounds did not affect phlA'-'lacZ expression and 2, 4-DAPG production. PhlF-mediated induction by 2,4-DAPG and repression by salicylate of phlA'-'lacZ expression was confirmed by using Escherichia coli as a heterologous host. In conclusion, our results show that autoinduction of 2,4-DAPG biosynthesis can be countered by certain bacterial (and fungal) metabolites. This mechanism, which depends on phlF function, may help P. fluorescens to produce homeostatically balanced amounts of extracellular metabolites.

Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.

Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

Control of jasmonate biosynthesis and senescence by miR319 targets.

Considerable progress has been made in identifying the targets of plant microRNAs, many of which regulate the stability or translation of mRNAs that encode transcription factors involved in development. In most cases, it is unknown, however, which immediate transcriptional targets mediate downstream effects of the microRNA-regulated transcription factors. We identified a new process controlled by the miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes. In contrast to other miRNA targets, several of which modulate hormone responses, TCPs control biosynthesis of the hormone jasmonic acid. Furthermore, we demonstrate a previously unrecognized effect of TCPs on leaf senescence, a process in which jasmonic acid has been proposed to be a critical regulator. We propose that miR319-controlled TCP transcription factors coordinate two sequential processes in leaf development: leaf growth, which they negatively regulate, and leaf senescence, which they positively regulate.

Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis.

A few bacterial species are known to produce and excrete hydrogen cyanide (HCN), a potent inhibitor of cytochrome c oxidase and several other metalloenzymes. In the producer strains, HCN does not appear to have a role in primary metabolism and is generally considered a secondary metabolite. HCN synthase of proteobacteria (especially fluorescent pseudomonads) is a membrane-bound flavoenzyme that oxidizes glycine, producing HCN and CO2. The hcnABC structural genes of Pseudomonas fluorescens and P. aeruginosa have sequence similarities with genes encoding various amino acid dehydrogenases/oxidases, in particular with nopaline oxidase of Agrobacterium tumefaciens. Induction of the hcn genes of P. fluorescens by oxygen limitation requires the FNR-like transcriptional regulator ANR, an ANR recognition sequence in the -40 region of the hcn promoter, and nonlimiting amounts of iron. In addition, expression of the hcn genes depends on a regulatory cascade initiated by the GacS/GacA (global control) two-component system. This regulation, which is typical of secondary metabolism, manifests itself during the transition from exponential to stationary growth phase. Cyanide produced by P. fluorescens strain CHA0 has an ecological role in that this metabolite accounts for part of the biocontrol capacity of strain CHA0, which suppresses fungal diseases on plant roots. Cyanide can also be a ligand of hydrogenases in some anaerobic bacteria that have not been described as cyanogenic. However, in this case, as well as in other situations, the physiological function of cyanide is unknown.