RESUMEN
Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.
Asunto(s)
Elastina , Lectinas , Péptidos , Solubilidad , TemperaturaRESUMEN
We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water, where the temperature of these transitions varied with ELP concentration, nature of the methionine alkylating group, and nature of the sulfonium counterions. These studies show that introduction and controlled spacing of methionine sulfonium residues into ELPs can be used as a means both to tune their solubility transition temperatures in water using a variety of different parameters and to introduce new side-chain functionality.
Asunto(s)
Cationes/química , Elastina/química , Metionina/química , Péptidos/química , Agua/química , Solubilidad , TemperaturaRESUMEN
We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were found to be a useful means to precisely adjust the temperature responsive behavior of ELPs in aqueous solutions. In particular, lower critical solution temperatures were found to be elevated in oxidized sample solutions, but were not eliminated. These transition temperatures were found to be further tunable by the use of solvents containing different Hofmeister salts. Overall, the ability to selectively and fully oxidize methionine residues in ELPs proved to be a convenient postmodification strategy for tuning their transition temperatures in aqueous media.
Asunto(s)
Elastina/química , Metionina/química , Péptidos/química , Agua/química , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Oxidación-Reducción , Temperatura de TransiciónRESUMEN
Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose-binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene.
Asunto(s)
Escherichia coli/metabolismo , Vectores Genéticos , Hepcidinas/genética , Periplasma/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Hepcidinas/química , HumanosRESUMEN
Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. We report herein the recombinant expression of three hydrophobic ELPs (VPGIG)n with variable lengths (n = 20, 40, 60) and sub-ambient transition temperatures. These ELPs were purified from the cytoplasmic soluble fraction of Escherichia coli by inverse transition cycling, and their exact molecular weight was confirmed by various mass spectrometry techniques. Transition temperatures of ELP20, ELP40, and ELP60 were measured at 18.6 °C, 12.4 °C and 11.7 °C, respectively.
Asunto(s)
Elastina/biosíntesis , Péptidos/genética , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos/genética , Elastina/genética , Escherichia coli/genética , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/genética , Temperatura de TransiciónRESUMEN
Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass.
Asunto(s)
Elastina/biosíntesis , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Proteínas de Unión a Maltosa/genética , Péptidos/metabolismo , Plásmidos/química , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Secuencia de Bases , Materiales Biomiméticos , Elastina/química , Elastina/aislamiento & purificación , Enteropeptidasa/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Datos de Secuencia Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Plásmidos/metabolismo , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Temperatura de TransiciónRESUMEN
BACKGROUND: Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. Although relevant, such systems permit analysis of various aspects of the biofilm state but often after several hours of bacterial growth. RESULTS: Here we describe a simple and easy-to-use system for growing P. aeruginosa biofilm based on the medium adsorption onto glass wool fibers. This approach which promotes bacterial contact onto the support, makes it possible to obtain in a few minutes a large population of sessile bacteria. Using this growth system, we demonstrated the feasibility of exploring the early stages of biofilm formation by separating by electrophoresis proteins extracted directly from immobilized cells. Moreover, the involvement of protein synthesis in P. aeruginosa attachment is demonstrated. CONCLUSIONS: Our system provides sufficient sessile biomass to perform biochemical and proteomic analyses from the early incubation period, thus paving the way for the molecular analysis of the early stages of colonization that were inaccessible to date.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Pseudomonas aeruginosa/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Vidrio , Proteómica/métodos , Pseudomonas aeruginosa/metabolismo , Propiedades de SuperficieRESUMEN
The prerequisite to monitor gene expression is the selection of reference genes for normalization of RT-qPCR results. Using 13 sputum samples collected from 9 CF patients, we demonstrated that PA2875 and PA3340 are better reference genes than the previously used clpX and oprL genes.
Asunto(s)
Fibrosis Quística/microbiología , Genes Bacterianos , Pseudomonas aeruginosa/genética , Esputo/microbiología , Perfilación de la Expresión Génica/métodos , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix is developed, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation leads to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, longitudinal micro-CT analysis is performed, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration.
Asunto(s)
Regeneración Ósea , Elastina , Ingeniería de Tejidos , Animales , Elastina/química , Ratas , Ingeniería de Tejidos/métodos , Regeneración Ósea/efectos de los fármacos , Humanos , Andamios del Tejido/química , Durapatita/química , Durapatita/farmacología , Huesos , Ratas Sprague-Dawley , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Microtomografía por Rayos XRESUMEN
Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery. Elastin-like polypeptides (ELPs) are good candidates for these applications because they are biosourced, biocompatible and biodegradable. With the aim of developing ELP-based micelles for drug delivery applications we have synthesized 15 acyl-ELP compounds by conjugating myristic, palmitic, stearic, oleic or linoleic acid to the N-terminus of three ELPs differing in molar mass. The ELP-fatty acid conjugates have interesting solution behavior. They form micelles at low temperatures and aggregate above the cloud point temperature (Tcp). The critical micelle concentration depends on the fatty acid nature while the micelle size is mainly determined by the ELP block length. We were able to show that ELPs were better hydrated in the micelles than in their individual state in solution. The micelles are stable in phosphate-buffered saline at temperatures below the Tcp, which can vary between 20 °C and 38 °C depending on the length or hydrophilicity of the ELP. Acyl-ELP micelles were loaded with the small hydrophobic molecule Nile red. The encapsulation efficiency and release kinetics showed that the best loading conditions were achieved with the largest ELP conjugated to stearic acid.
RESUMEN
Lack of compensatory or even reduced food intake is frequently observed in weight-losing cancer patients and contributes to increased morbidity and mortality. Our previous work has shown increased transcription factor expression in the hypothalamus and ventral striatum of anorectic rats bearing small tumors. mRNA expression of molecules known to be involved in pathways regulating appetite in these structures was therefore assessed in this study. Given that pain, pro-inflammatory cytokines and metabolic hormones can modify food intake, spinal cord cellular activation patterns and plasma concentrations of cytokines and hormones were also studied. Morris hepatoma 7777 cells injected subcutaneously in Buffalo rats provoked a 10% lower body weight and 15% reduction in food intake compared to free-feeding tumor-free animals 4 weeks later when the tumor represented 1-2% of body mass. No differences in spinal cord activation patterns or plasma concentration of pro-inflammatory cytokines were observed between groups. However, the changes in plasma ghrelin and leptin concentrations found in food-restricted weight-matched rats in comparison to ad libitum-fed animals did not occur in anorectic tumor-bearing animals. Real-time PCR showed that tumor-bearing rats did not display the increase in hypothalamic agouti-related peptide mRNA observed in food-restricted weight-matched animals. In addition, microarray analysis and real-time PCR revealed increased ventral striatal prostaglandin D synthase expression in food-restricted animals compared to anorectic tumor-bearing rats. These findings indicate that blunted hypothalamic AgRP mRNA expression, probably as a consequence of relatively high leptin and low ghrelin concentrations, and reduced ventral striatal prostaglandin D synthesis play a role in maintaining cancer-associated anorexia.
Asunto(s)
Regulación del Apetito/fisiología , Ganglios Basales/metabolismo , Caquexia/metabolismo , Carcinoma Hepatocelular/metabolismo , Hipotálamo/metabolismo , Neoplasias Hepáticas/metabolismo , Adaptación Fisiológica , Proteína Relacionada con Agouti/genética , Proteína Relacionada con Agouti/metabolismo , Análisis de Varianza , Animales , Peso Corporal/fisiología , Caquexia/etiología , Caquexia/fisiopatología , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/fisiopatología , Citocinas/sangre , Modelos Animales de Enfermedad , Ingestión de Alimentos/fisiología , Regulación de la Expresión Génica , Ghrelina/genética , Ghrelina/metabolismo , Inmunohistoquímica , Oxidorreductasas Intramoleculares/metabolismo , Leptina/genética , Leptina/metabolismo , Lipocalinas/metabolismo , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/fisiopatología , Masculino , Análisis por Apareamiento , Neoplasias Experimentales/complicaciones , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/fisiopatología , Percepción del Dolor/fisiología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas BUF , Médula Espinal/metabolismo , Pérdida de Peso/fisiologíaRESUMEN
Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.
RESUMEN
Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Escherichia coli/metabolismo , Magnetismo , Modelos BiológicosRESUMEN
Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional tissue, taking into account that the nerve systems closely interact with blood vessels in the bone tissue. The aim of this work is to develop a cell-free and growth factor-free hydrogel capable to promote angiogenesis and innervation. To this end, we have used elastin-like polypeptides (ELPs), poly(ethylene glycol) (PEG) and increasing concentrations of the adhesion peptide IKVAV (25% (w/w) representing 1.7â¯mM and 50% (w/w) representing 4.1â¯mM) to formulate and produce hydrogels. When characterized in vitro, hydrogels have fine-tunable rheological properties, microporous structure and are biocompatible. At the biological level, 50% IKVAV composition up-regulated Runx2, Osx, Spp1, Vegfa and Bmp2 in mesenchymal stromal cells and Tek in endothelial cells, and sustained the formation of long neurites in sensory neurons. When implanted subcutaneously in mice, hydrogels induced no signals of major inflammation and the 50% IKVAV composition induced higher vessel density and formation of nervous terminations in the peripheral tissue. This novel composite has important features for tissue engineering, showing higher osteogenic, angiogenic and innervation potential in vitro, being not inflammatory in vivo, and inducing angiogenesis and innervation subcutaneously. STATEMENT OF SIGNIFICANCE: One of the main limitations in the field of tissue engineering remains the sufficient vascularization and innervation during tissue repair. In this scope, the development of advanced biomaterials that can support these processes is of crucial importance. Here, we formulated different compositions of Elastin-like polypeptide-based hydrogels bearing the IKVAV adhesion sequence. These compositions showed controlled mechanical properties, and were degradable in vitro. Additionally, we could identify in vitro a composition capable to promote neurite formation and to modulate endothelial and mesenchymal stromal cells gene expression, in view of angiogenesis and osteogenesis, respectively. When tested in vivo, it showed no signs of major inflammation and induced the formation of a highly vascularized and innervated neotissue. In this sense, our approach represents a potential advance in the development of new strategies to promote tissue regeneration, taking into account both angiogenesis and innervation.
Asunto(s)
Inductores de la Angiogénesis/química , Materiales Biocompatibles/química , Hidrogeles/química , Andamios del Tejido/química , Inductores de la Angiogénesis/metabolismo , Animales , Materiales Biocompatibles/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Proliferación Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Elastina/química , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Hidrogeles/metabolismo , Laminina/química , Células Madre Mesenquimatosas/metabolismo , Ratones , Neuronas/metabolismo , Osteogénesis/efectos de los fármacos , Osteopontina/genética , Osteopontina/metabolismo , Fragmentos de Péptidos/química , Péptidos/química , Polietilenglicoles/química , Porosidad , Implantación de Prótesis , Ratas Wistar , Reología , Factor de Transcripción Sp7/genética , Factor de Transcripción Sp7/metabolismo , Propiedades de Superficie , Ingeniería de Tejidos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.
Asunto(s)
Elastina/química , Hidrogeles/química , Péptidos/química , Ingeniería de Tejidos , Secuencia de Aminoácidos/genética , Animales , Elastina/genética , Elastina/aislamiento & purificación , Elastina/farmacología , Hidrogeles/farmacología , Laminina/química , Laminina/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Péptidos/genética , Péptidos/aislamiento & purificación , Péptidos/farmacología , Ratas , Reología , Células Receptoras Sensoriales/química , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living cells. Using a simple protocol based on the use of a standard real-time thermocycler, we confirmed that the actions of the antimicrobial peptides LL-37 and magainin 2 on bacterial cells are different.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Péptidos Catiónicos Antimicrobianos/metabolismo , Catelicidinas , Colorantes Fluorescentes/química , Magaininas , Compuestos Orgánicos/química , Proteínas de Xenopus/farmacologíaRESUMEN
Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Pseudomonas aeruginosa/metabolismo , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/genética , Biopelículas , Regulación Bacteriana de la Expresión Génica , Vidrio/química , Proteoma/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Reproducibilidad de los Resultados , Transducción de Señal/genética , Transducción de Señal/fisiología , Propiedades de Superficie , Factores de TiempoRESUMEN
Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Genes de Plantas , Proteínas de Plantas/genética , Almidón Sintasa/genética , Aciltransferasas/biosíntesis , Aciltransferasas/genética , Aldehído Oxidorreductasas/biosíntesis , Aldehído Oxidorreductasas/genética , Arabidopsis/enzimología , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Liasas de Carbono-Carbono , Perfilación de la Expresión Génica , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/metabolismo , Plantones/metabolismo , Almidón Sintasa/biosíntesis , Almidón Sintasa/metabolismo , Factores de TiempoRESUMEN
Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to its mammalian orthologues at both structural and sequence levels. The gene is expressed in the embryo mostly in the endoderm-derived tissues. Accordingly it is induced in pluripotent animal cap cells by FGF, activin or a combination of vegT/ß-catenin. Modulating leap2 expression level by gain-of-function strategy impaired normal embryonic development. When overexpressed in pluripotent embryonic cells derived from blastula animal cap explant, leap2 stimulated FGF while it reduced the activin response. Finally, we demonstrate that LEAP2 blocks FGF-induced migration of HUman Vascular Endothelial Cells (HUVEC). Altogether these findings suggest a model in which LEAP2 could act at the extracellular level as a modulator of FGF and activin signals, thus opening new avenues to explore it in relation with cellular processes such as cell differentiation and migration.
Asunto(s)
Activinas/genética , Péptidos Catiónicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Desarrollo Embrionario/genética , beta Catenina/genética , Activinas/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inmunidad Innata/genética , Transducción de Señal , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo , beta Catenina/metabolismoRESUMEN
The expression of acetyl-CoA carboxylase (ACC) in mouse peripheral nervous system (PNS) was investigated. Both ACC 265 and ACC 280 isoforms were expressed in the sciatic nerve, although ACC 265 was predominant. ACC 265 transcripts originating from promoters P1 and P2 could be detected in the developing nerve, as well as the two splice products, which are characterized by the presence or the absence of a 24-base sequence before the codon serine-1200. The mRNA levels for ACC 265 parallel those of other lipogenic genes whose expression is linked to the myelination process. In addition, ACC 265 mRNA and protein levels in the nerves of the trembler mutant, which is a mouse model of PNS dysmyelination, represented around 30% of the normal values. The expression of the sterol regulatory element-binding proteins (SREBPs) was also studied. SREBP 1 mRNAs were expressed at a constant level during nerve development, and their quantities were normal in trembler. On the contrary, SREBP 2 mRNA quantities varied during the myelination period similarly to the lipogenic gene mRNAs, and the levels measured in trembler represented only 10% of the normal values. Taken together, these results suggest that the coordinate expression of several lipogenic genes, which occurs during PNS myelination, could possibly be regulated by SREBP 2.