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.

Biosynthesis of panaxynol and panaxydol in Panax ginseng

The natural formation of the bioactive C17-polyacetylenes (−)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13CO2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-13C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots.

Effectiveness of antioxidants in preventing oxidation of palm oil enriched with heme iron: A model for iron fortification in baked products

Bakery products such as biscuits, cookies, and pastries represent a good medium for iron fortification in food products, since they are consumed by a large proportion of the population at risk of developing iron deficiency anemia, mainly children. The drawback, however, is that iron fortification can promote oxidation. To assess the extent of this, palm oil added with heme iron and different antioxidants was used as a model for evaluating the oxidative stability of some bakery products, such as baked goods containing chocolate. The palm oil samples were heated at 220°C for 10 min to mimic the conditions found during a typical baking processing. The selected antioxidants were a free radical scavenger (tocopherol extract (TE), 0 and 500 mg/kg), an oxygen scavenger (ascorbyl palmitate (AP), 0 and 500 mg/kg), and a chelating agent (citric acid (CA), 0 and 300 mg/kg). These antioxidants were combined using a factorial design and were compared to a control sample, which was not supplemented with antioxidants. Primary (peroxide value and lipid hydroperoxide content) and secondary oxidation parameters (p-anisidine value, p-AnV) were monitored over a period of 200 days in storage at room temperature. The combination of AP and CA was the most effective treatment in delaying the onset of oxidation. TE was not effective in preventing oxidation. The p-AnV did not increase during the storage period, indicating that this oxidation marker was not suitable for monitoring oxidation in this model.

Effectiveness of antioxidants in preventing oxidation of palm oil enriched with heme iron: A model for iron fortification in baked products

Bakery products such as biscuits, cookies, and pastries represent a good medium for iron fortification in food products, since they are consumed by a large proportion of the population at risk of developing iron deficiency anemia, mainly children. The drawback, however, is that iron fortification can promote oxidation. To assess the extent of this, palm oil added with heme iron and different antioxidants was used as a model for evaluating the oxidative stability of some bakery products, such as baked goods containing chocolate. The palm oil samples were heated at 220°C for 10 min to mimic the conditions found during a typical baking processing. The selected antioxidants were a free radical scavenger (tocopherol extract (TE), 0 and 500 mg/kg), an oxygen scavenger (ascorbyl palmitate (AP), 0 and 500 mg/kg), and a chelating agent (citric acid (CA), 0 and 300 mg/kg). These antioxidants were combined using a factorial design and were compared to a control sample, which was not supplemented with antioxidants. Primary (peroxide value and lipid hydroperoxide content) and secondary oxidation parameters (p-anisidine value, p-AnV) were monitored over a period of 200 days in storage at room temperature. The combination of AP and CA was the most effective treatment in delaying the onset of oxidation. TE was not effective in preventing oxidation. The p-AnV did not increase during the storage period, indicating that this oxidation marker was not suitable for monitoring oxidation in this model.

Zim17/Tim15 links mitochondrial iron–sulfur cluster biosynthesis to nuclear genome stability

Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron–sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron–sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron–sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.

Biosynthesis of panaxynol and panaxydol in Panax ginseng

The natural formation of the bioactive C17-polyacetylenes (−)-(R)-panaxynol and panaxydol was analyzed by 13C-labeling experiments. For this purpose, plants of Panax ginseng were supplied with 13CO2 under field conditions or, alternatively, sterile root cultures of P. ginseng were supplemented with [U-13C6]glucose. The polyynes were isolated from the labeled roots or hairy root cultures, respectively, and analyzed by quantitative NMR spectroscopy. The same mixtures of eight doubly 13C-labeled isotopologues and one single labeled isotopologue were observed in the C17-polyacetylenes obtained from the two experiments. The polyketide-type labeling pattern is in line with the biosynthetic origin of the compounds via decarboxylation of fatty acids, probably of crepenynic acid. The 13C-study now provides experimental evidence for the biosynthesis of panaxynol and related polyacetylenes in P. ginseng under in planta conditions as well as in root cultures. The data also show that 13CO2 experiments under field conditions are useful to elucidate the biosynthetic pathways of metabolites, including those from roots.

Transferència d’electrò i protó als intermediaris de reacció de les hemo-catalases

Projecte de recerca elaborat a partir d’una estada a l’University of Pennsylvania, EUA, entre els mesos d’agosta a desembre del 2006. Les hemo-catalases són enzims que protegeixen les cèl•lules dels efectes tòxics del peròxid d'hidrogen. Aquesta reacció té lloc en dues etapes, via l'intermediari Compost I (Cpd I). Tanmateix, el Compost I pot seguir una reacció secundària, a través de l'intermediari Compost II. Hi ha dos tipus d'hemo-catalases: les hemo-b (com la d'Helicobacter pylori, HPC) i les hemo-d catalases (com la de Penicillium vitale, PVC). Experimentalment s'observa que les hemo-b catalases formen Cpd II més fàcilment que les hemo-d. La formació del Cpd II consta de dos processos: la reducció del catió radical porfirínic i la protonació del grup oxoferril. Durant l'estada, es va estudiar el procès de transferència electrònica a la porfirina utilitzant una metodologia desenvolupada recentment. Els resultats mostren que per PVC la reducció és més fàcil que per HPC. Posteriorment hem realitzat una sèrie de optimitzacions de geometria CPMD QM/MM al llarg del camí per la transferència de protó (PT) de la histidina distal a l'oxoferril. Mentre que per HPC aquesta PT és espontània, per PVC l'isòmer hidroxoferrílic és menys estable que el catió imidazoli.

Aspectos metabólicos y multivitamínicos del maíz

La deficiencia de vitamina A causa 2.2 millones de muertes al año. Por tanto, investigadores intentan elevar el contenido de β-caroteno y otros carotenoides en cereales mediante la ingeniería genética. Mi trabajo consistió en analizar carotenoides en maíz y callos de arroz transgénico con el fin de indentificar y cuantificar estos pigmentos y además analizar la ruta metabólica de los mismos. Por tal motivo, desarrollaré una técnica analítica por HPLC y UHPLC que me permitió separar la mezcla de carotenoides. Estas moléculas se detectaton utilizando un detector de arreglo de diodos y masas. Con éste último se probaron distintas técnicas de ionización (ESI, APCI y APPI) para encontrar la mejor técnica que ionizara carotenos y xantófilas. Además se hallaron transiciones para identificar a cada uno de los carotenoides. Para mostrar la confiabilidad del método analítico, realicé la validación del mismo y determiné factores importantes que influyen en el análisis de carotenoides, como por ejemplo, su estabilidad química. Para analizar la ruta metabólica de los carotenoides, se realizaron distintos experimentos, entre ellos, introducir distintas combinaciones de genes en el maíz y arroz que permitieran entender cuáles eran las enzimas importantes que permitían la acumulación de carotenoides. También se analizó la biosíntesis de carotenoides a distintas etapas del desarrollo de la semilla de maíz, desde los 15 días después de la polinización (DAP) hasta los 60 DAP y su estabilidad química a lo largo de todo este período. Finalmente, se profundizó en la identificación de nuevos carotenoides encontrados en las muestras, como los oxo-carotenoides (utilizados en la industria como colorantes). Para llevar acabo su identificación, se realizaron pruebas químicas, se determinaron sus espectros visibles y sus fragmentos por masas.   

Poliamines en estrès abiòtic i mecanismes moleculars

Les poliamines (PAs) putrescina (Put), espermidina (Spd) i espermina (Spm) són mol•lècules policatiòniques de baix pes molecular, presents en els microorganismes, animals i plantes. Les PAs han estat implicades en diversos processos cel•lulars importants, incloent la resposta de les plantes a l'estrès. No obstant això, el seu mode d'acció està es desconeix. En les plantes, es van acumulant evidències de que les PAs interactuen amb macromolècules i estructures cel•lulars, com ara proteïnes de membrana, i la seva possible participació en transducció de senyals s'ha convertit en una creixent i interessant àrea d'estudi. En aquesta tesi, la possible interacció entre les poliamines i les vies de senyalització de fosfolípids és investigada. Resultats previs, han posat de manifest que alteracions en els nivells endògens de poliamines (PAs), per sobreexpressió gènica o pèrdua de funció de gens de biosíntesi, redueixen o milloren, respectivament, la capacitat de les plantes d'Arabidopsis per tolerar agressions per estrès abiòtic, produint en alguns casos notables alteracions en el desenvolupament. En aquestes plantes amb nivells alterats de PAs s'han detectat canvis importants en l'expressió gènica i s'ha trobat una connexió entre el contingut de PAs ii la biosíntesi / senyalització d'àcid abscísic (ABA). La hipòtesi actual de treball és que aquestes alteracions en l'expressió gènica poden estar mediades, si més no en part, pel catabolisme de PAs, i l’acció directa o indirecta de les espècies reactives d'oxigen (ROS) que se’n deriven.

Relaxation of selective constraints causes independent selenoprotein extinction in insect genomes

BACKGROUND: Selenoproteins are a diverse family of proteins notable for the presence of the 21st amino acid, selenocysteine. Until very recently, all metazoan genomes investigated encoded selenoproteins, and these proteins had therefore been believed to be essential for animal life. Challenging this assumption, recent comparative analyses of insect genomes have revealed that some insect genomes appear to have lost selenoprotein genes. METHODOLOGY/PRINCIPAL FINDINGS: In this paper we investigate in detail the fate of selenoproteins, and that of selenoprotein factors, in all available arthropod genomes. We use a variety of in silico comparative genomics approaches to look for known selenoprotein genes and factors involved in selenoprotein biosynthesis. We have found that five insect species have completely lost the ability to encode selenoproteins and that selenoprotein loss in these species, although so far confined to the Endopterygota infraclass, cannot be attributed to a single evolutionary event, but rather to multiple, independent events. Loss of selenoproteins and selenoprotein factors is usually coupled to the deletion of the entire no-longer functional genomic region, rather than to sequence degradation and consequent pseudogenisation. Such dynamics of gene extinction are consistent with the high rate of genome rearrangements observed in Drosophila. We have also found that, while many selenoprotein factors are concomitantly lost with the selenoproteins, others are present and conserved in all investigated genomes, irrespective of whether they code for selenoproteins or not, suggesting that they are involved in additional, non-selenoprotein related functions. CONCLUSIONS/SIGNIFICANCE: Selenoproteins have been independently lost in several insect species, possibly as a consequence of the relaxation in insects of the selective constraints acting across metazoans to maintain selenoproteins. The dispensability of selenoproteins in insects may be related to the fundamental differences in antioxidant defense between these animals and other metazoans.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200,000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A SNP located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (OR = 2.31, permutation p-value = 0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

Evolutionary analysis of genes of two pathways involved in placental malaria infection

Placental malaria is a special form of malaria that causes up to 200,000 maternal and infant deaths every year. Previous studies show that two receptor molecules, hyaluronic acid and chondroitin sulphate A, are mediating the adhesion of parasite-infected erythrocytes in the placenta of patients, which is believed to be a key step in the pathogenesis of the disease. In this study, we aimed at identifying sites of malaria-induced adaptation by scanning for signatures of natural selection in 24 genes in the complete biosynthesis pathway of these two receptor molecules. We analyzed a total of 24 Mb of publicly available polymorphism data from the International HapMap project for three human populations with European, Asian and African ancestry, with the African population from a region of presently and historically high malaria prevalence. Using the methods based on allele frequency distributions, genetic differentiation between populations, and on long-range haplotype structure, we found only limited evidence for malaria-induced genetic adaptation in this set of genes in the African population; however, we identified one candidate gene with clear evidence of selection in the Asian population. Although historical exposure to malaria in this population cannot be ruled out, we speculate that it might be caused by other pathogens, as there is growing evidence that these molecules are important receptors in a variety of host-pathogen interactions. We propose to use the present methods in a systematic way to help identify candidate regions under positive selection as a consequence of malaria.