Characterization of the acyl-ACP thioesterases from Koelreuteria paniculata reveals a new type of FatB thioesterase.

Koelreuteria paniculata is a deciduous tree, popular in temperate regions for its ornamental value, which accumulates unusual cyanolipids in its seeds. The seed oil of this plant is rich in the unusual cis-11-eicosenoic fatty acid (20:1, or gondoic acid), a monounsaturated oil of interest to the oleochemical industry. In higher plants, de novo fatty acid biosynthesis takes place in the plastids, a process that is terminated by hydrolysis of the thioester bond between the acyl moiety and the ACP by acyl-ACP thioesterases. The specificity of acyl-ACP thioesterases is fundamental in controlling the fatty acid composition of seed oil. To determine the mechanisms involved in fatty acid biosynthesis in K. paniculata seeds, we isolated, cloned and sequenced two cDNAs encoding acyl-ACP thioesterases in this plant, KpFatA and KpFatB. Both of them were expressed heterologously in Escherichia coli and characterized with different acyl-ACP substrates. The K. paniculata FatB2 displayed unusual substrate specificity, so that unlike most FatB2 type enzymes, it displayed preference for oleoyl-ACP instead of palmitoyl-ACP. This specificity was consistent with the changes in E. coli and N. benthamiana fatty acid composition following heterologous expression of this enzyme. KpFatB also showed certain genetic divergence relative to other FatB-type thioesterases and when modelled, its structure revealed differences at the active site. Together, these results suggest that this thioesterase could be a new class of FatB not described previously.

Molecular and biochemical characterization of the sunflower (Helianthus annuus L.) cytosolic and plastidial enolases in relation to seed development.

In the present study, we describe the molecular and biochemical characterization of sunflower (Helianthus annuus L.) enolase (ENO, EC 4.2.1.11) proteins, which catalyze the formation of phosphoenolpyruvate, the penultimate intermediate in the glycolytic pathway. We cloned and characterized three cDNAs encoding different ENO isoforms from developing sunflower seeds. Studies using fluorescently tagged ENOs confirmed the predicted subcellular localization of ENO isoforms: HaENO1 in the plastid while HaENO2 and HaENO3 were found in the cytosol. The cDNAs were used to express the corresponding 6(His)-tagged proteins in Escherichia coli. The proteins were purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Recombinant HaENO1 and HaENO2, but not HaENO3 were shown to have enolase activity, in agreement with data obtained with the Arabidopsis homolog proteins. Site directed mutagenesis of several critical amino acids was used to attempt to recover enolase activity in recombinant HaENO3, resulting in very small increases that were not additive. A kinetic characterization of the two active isoforms showed that pH had similar effect on their velocity, that they had similar affinity for 2-phosphoglycerate, but that the kcat/Km of the plastidial enzyme was higher than that of the cytosolic isoform. Even though HaENO2 was always the most highly expressed transcript, the levels of expression of the three ENO genes were remarkably distinct in all the vegetative and reproductive tissues studied. This indicates that in seeds the conversion of 2-phosphoglycerate to phosphoenolpyruvate takes place through the cytosolic and the plastidial pathways therefore both routes could contribute to the supply of carbon for lipid synthesis. The identity of the main source of carbon during the period of stored products synthesis is discussed.

Hemiparesia súbita secundaria a cisticercosis cerebral: presentación de un caso clínico / Sudden hemiparesis secondary to cerebral cysticercosis: presentation of a clinical case

Introducción: La neurocisticercosis es una infección parasitaria del sistema nervioso central, ocasionada por la etapa larval del parásito Taenia solium. Su prevalencia mundial es de 9,1%. Se presenta a continuación un caso clínico en el que destaca una clínica infrecuente y tamaño de la lesión poco común; caso que fue resuelto con éxito mediante neurocirugía. Presentación del caso: Hombre de 48 años, residente en localidad rural, consultó por cuadro súbito de hemiparesia derecha de predominio crural, posteriormente con aumento de paresia braquial ipsilateral. Al examen físico destacó síndrome piramidal derecho. Se estudió con Tomografía Computarizada que evidenció quiste frontal izquierdo con compresión del área motora, sugerente de lesión parasitaria. Resonancia Nuclear Magnética de Cerebro (RNMC) informó proceso expansivo quístico fronto-parietal parasagital izquierdo de 5 x 5,5 cm, con efecto de masa. Por tamaño, tipo de lesión y clínica se decidió realizar cirugía abierta con extirpación total y biopsia que informó cisticercosis cerebral. El paciente evolucionó con recuperación total de su déficit motor posterior a la cirugía. Discusión: La neurocisticercosis se encuentra dentro de los diagnósticos diferenciales de lesiones quísticas encefálicas. Su clínica es inespecífica, manifestándose comúnmente con convulsiones y cefalea. La RNMC permite el diagnóstico y localización de las lesiones. El tratamiento es médico, quirúrgico o combinado. Generalmente, el manejo se basa en antiepilépticos y antiparasitarios sistémicos, sin embargo en este caso, se decidió el manejo quirúrgico que llevó a una mejoría total del paciente, lo que avala la cirugía precoz como principal medida en lesiones de este tipo

Introduction: Neurocysticercosis is a parasitic infection of the central nervous system, caused by the larval stage of the parasite Taenia solium. Its worldwide prevalence is 9.1%. We present below a clinical case with an infrequent clinical and uncommon lesion size, with surgical resolved. Case report: A 48 year old man, who was a resident of a rural locality, consulted for sudden hemiparesis on the right side of the crural predominance, subsequently with increased ipsilateral brachial paresis. Physical examination highlighted right pyramidal syndrome. It was studied with Computed Axial Tomography that showed left frontal cyst with compression of the motor area, suggestive of parasite lesion. Brain Nuclear Magnetic Resonance (BNMR) reported left parasagittal fronto-parietal cystic expansive process of 5 x 5.5 cm, with mass effect. By size, type of lesion and clinic it was decided to perform open surgery with total extirpation and biopsy that reported cerebral cysticercosis. The patient evolved with complete recovery of motor deficit after surgery. Discussion: Neurocysticercosis is a differential diagnosis of brain cystic lesions. Its clinic is non-specific, commonly manifesting with seizures and headache. The RNMC allows the diagnosis and location of the lesions. The treatment is medical, surgical or combined. Usually, the management is based on antiepileptics and systemic antiparasitic, however in this case, it was decided the surgical management that led to the total improvement of the patient, which guarantees the early surgery as the main measure in lesions of this type

Antipolar and Anticlinic Mesophase Order in Chromatin Induced by Nucleosome Polarity and Chirality Correlations.

Contrary to the usual "rigid supermolecular assembly" paradigm of chromatin structure, we propose to analyze its eventual ordered state in terms of symmetry properties of individual nucleosomes that give rise to mesophase order parameters, like in many other soft-matter systems. Basing our approach on the Landau-de Gennes phenomenology, we describe the mesoscale order in chromatin by antipolar and anticlinic correlations of chiral individual nucleosomes. This approach leads to a unifying physical picture of a whole series of soft locally ordered states with different apparent structures, including the recently observed heteromorphic chromatin, stemming from the antipolar arrangement of nucleosomes complemented by their chiral twisting. Properties of these states under an external force field can reconcile apparently contradictory results of single-molecule experiments.

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.

Diseminación leptomeníngea en un carcinoma nasosinusal de tipo intestinal: una forma de presentación inusual / Leptomeningeal dissemination in an intestinal-type sinonasal adenocarcinoma: an uncommon presentation

Presentamos el caso de un paciente con antecedentes laborales de exposición al polvo de madera durante 30 años, con el diagnóstico anatomopatológico de adenocarcinoma tipo intestinal mucinoso nasosinusal. El cuadro clínico por el que consultó el paciente era neurológico, objetivándose en el momento del diagnóstico una diseminación tumoral leptomeníngea intra y extracraneal, forma de presentación excepcional en este tipo de tumores(AU)

We present the case of a patient with a 30-year history of exposure to sawdust who was diagnosed with mucinous intestinal-type sinonasal adenocarcinoma after histological examination. The patient presented with neurological symptoms; moreover, intra- and extra-cranial leptomeningeal involvement, which is exceedingly rare in this type of tumors, was observed at the time of diagnosis(AU)

[Leptomeningeal dissemination in an intestinal-type sinonasal adenocarcinoma: an uncommon presentation]. / Diseminación leptomeníngea en un carcinoma nasosinusal de tipo intestinal: una forma de presentación inusual.

We present the case of a patient with a 30-year history of exposure to sawdust who was diagnosed with mucinous intestinal-type sinonasal adenocarcinoma after histological examination. The patient presented with neurological symptoms; moreover, intra- and extra-cranial leptomeningeal involvement, which is exceedingly rare in this type of tumors, was observed at the time of diagnosis.

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation.

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 µmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds.

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.

Phospholipase Dalpha from sunflower (Helianthus annuus): cloning and functional characterization.

D type phospholipases (PLD) are enzymes that hydrolyze the head group of phospholipids to produce phosphatidic acid. This activity is ubiquitous in plant tissues, and has been isolated and characterized from different species and organs. Several families of these proteins have been described in plants on the basis of their gene sequences (PLD alpha, beta, gamma, delta, zeta and epsilon). They have been shown to be involved in many metabolic events, such as response to abiotic stress, signal transduction, and membrane lipid turnover and degradation. In the present study, PLD activity was measured in the soluble fractions isolated from different organs of this plant. A PLD of alpha type was cloned from leaf cDNA that was responsible for most of this activity. The gene encoding this 810 aa protein was heterologously expressed in E. coli. This protein was not lethal for the eukaryotic host, although it altered its phospholipid profile. PLDalpha was purified to almost homogeneity by His-tag affinity chromatography, displaying an optimum pH of 6.5 and strong dependence on the presence of Ca(2+) and SDS in the assay medium. The enzyme was active towards phosphatidylcholine, Phosphatidylethanolamine and phosphatidylglycerol. Furthermore, the HaPLDalpha gene was found to be expressed at high levels in leaf and stem tissues.

Degeneración walleriana bilateral de las fibras pontocerebelosas secundaria a ictus pontinos: presentación de 4 casos / Bilateral wallerian degeneration of the pontocerebellar fibers secondary to pontine stroke: a report of 4 cases

La degeneración walleriana es una desmielinización de los axones neuronales distales por una daño proximal de cualquier etiología.Se presentan los hallazgos de resonancia magnética en 4 pacientes con ictus de tronco y signos de degeneración walleriana de los haces pontocerebelosos. Para ello se revisan las resonancias magnéticas de 4 pacientes con lesiones pontinas en fase subaguda o crónica, y las alteraciones de señal en los pedúnculos cerebelosos medios. Los hallazgos en las secuencias potenciadas en T2 y en difusión se relacionan con el tiempo de evolución y la etiología del ictus (AU)

Wallerian degeneration occurs after demyelination of the distal neuronal axons due to proximal damage of any type.AbstractWe present the magnetic resonance findings in four patients with brainstem stroke and signs of Wallerian degeneration in the pontocerebellar tracts.AbstractWe reviewed the magnetic resonance studies in four patients with subacute or chronic stage pontine lesions and the signal alterations at the level of the medial cerebellar peduncles. We correlated the findings in T2-weighted sequences and diffusion-weighted sequences with the time of evolution and etiology of the stroke (AU)

[Bilateral wallerian degeneration of the pontocerebellar fibers secondary to pontine stroke: a report of 4 cases]. / Degeneración walleriana bilateral de las fibras pontocerebelosas secundaria a ictus pontinos: presentación de 4 casos.

Wallerian degeneration occurs after demyelination of the distal neuronal axons due to proximal damage of any type. We present the magnetic resonance findings in four patients with brainstem stroke and signs of Wallerian degeneration in the pontocerebellar tracts. We reviewed the magnetic resonance studies in four patients with subacute or chronic stage pontine lesions and the signal alterations at the level of the medial cerebellar peduncles. We correlated the findings in T2-weighted sequences and diffusion-weighted sequences with the time of evolution and etiology of the stroke.

Effect of the ferredoxin electron donor on sunflower (Helianthus annuus) desaturases.

Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants: light-regulated photosynthetic ferredoxins and heterotrophic ferredoxins whose activity is not influenced by light. Genes encoding the two forms of ferredoxin have been cloned and characterized in developing sunflower cotyledons. Here, these genes were overexpressed in Escherichia coli and they were purified by ion exchange and size exclusion chromatography to study their capacity to supply electrons to two different sunflower desaturases: soluble stearoyl-ACP desaturase from sunflower cotyledons, and membrane bound desaturase FAD7 expressed in yeast. In both cases photosynthetic ferredoxin was the form that promoted the strongest desaturase activity.

cDNA cloning, expression levels and gene mapping of photosynthetic and non-photosynthetic ferredoxin genes in sunflower (Helianthus annuus L.).

Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5'-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.

Functional characterization of a plastidial omega-3 desaturase from sunflower (Helianthus annuus) in cyanobacteria.

Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane and, as a consequence, they are fundamental for the proper growth and development of all living organisms. We have used sequences from the conserved regions of known omega-3-desaturases to design degenerated oligonucleotides and clone a cDNA encoding a plastidial omega-3 desaturase from sunflower (HaFAD7). From its presumed full-length sequence, we predict that Hafad7 encodes a protein of 443 amino acids with a molecular mass of 50.8 kDa, and that it contains a putative chloroplast transit peptide of 51 amino acids. The predicted hydrophobicity of the protein identifies four potential membrane-spanning regions and, according to the TargetP algorithm, the protein should be targeted to the plastid/chloroplast membrane. RT-PCR analysis of its expression shows the transcript is preferentially expressed in photosynthetically active tissues. Heterologous expression of this protein in the unicellular cyanobacterium Synechocystis sp. PCC 6803 confirmed that the protein produced from this cDNA has omega-3 desaturase activity.

Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.).

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K(m)) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the delta-subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.