RESUMEN
Biomimetic carbonation carried out with carbonic anhydrase (CA) in CO2-absorbing solutions, such as methyldiethanolamine (MDEA), is one approach that has been developed to accelerate the capture of CO2. However, there are several practical issues, such as high cost and limited enzyme stability, that need to be overcome. In this study, the capacity of CA immobilization on a porous solid support was studied to improve the instability in the tertiary amine solvent. We have shown that a 63% porosity macroporous carbon foam support makes separation and reuse facile and allows for an efficient supply and presentation of CO2 to an aqueous solvent and the enzyme catalytic center. These enzymatic supports conserved 40% of their initial activity after 42 days at 70 °C in an amine solvent, whereas the free enzyme shows no activity after 1 h in the same conditions. In this work, we have overcome the technical barrier associated with the recovery of the biocatalyst after operation, and most of all, these electropolymerized enzymatic supports have shown a remarkable increase of thermal stability in an amine-based CO2 sequestration solvent.
Asunto(s)
Dióxido de Carbono/química , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Enzimas Inmovilizadas/química , Etanolaminas/químicaRESUMEN
In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 µM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.
Asunto(s)
Glutamato-ARNt Ligasa/metabolismo , Helicobacter pylori/enzimología , Transferasas de Grupos Nitrogenados/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia de Glutamina/metabolismo , Helicobacter pylori/genética , Hidrólisis , Interferometría , Cinética , Modelos Biológicos , Estabilidad del ARNRESUMEN
The electrolysis of carbon capture solutions bypasses energy-intensive CO2 recovery steps that are often required to convert CO2 into value-added products. We report herein an electrochemical flow reactor that converts carbon capture solutions containing carbonic anhydrase enzymes into carbon-based products. Carbonic anhydrase enzymes benefit CO2 capture by increasing the rate of reaction between CO2 and weakly alkaline solutions by 20-fold. In this study, we reduced CO2-enriched bicarbonate solutions containing carbonic anhydrase ("enzymatic CO2 capture solutions") into CO at current densities of 100 mA cm-2. This result demonstrated how to electrolyse enzymatic CO2 capture solutions, but the selectivity for CO production was two-thirds less than bicarbonate solutions without carbonic anhydrase. This reduction in performance occurred because carbonic anhydrase deactivated the catalyst surface. A carbon microporous layer was found to suppress this deactivation.
Asunto(s)
Anhidrasas Carbónicas , Bicarbonatos , Carbono , Dióxido de Carbono , ElectrólisisRESUMEN
SFKs (Src family kinases) are central regulators of many signalling pathways. Their functions are tightly regulated through SH (Src homology) domain-mediated protein-protein interactions. A yeast two-hybrid screen using SH3 domains as bait identified Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X] as a novel Hck (haemopoietic cell kinase) SH3 domain interactor. The Alix-Hck-SH3 interaction was confirmed in vitro by a GST (glutathione transferase) pull-down assay and in intact cells by a mammalian two-hybrid assay. Furthermore, the interaction was demonstrated to be biologically relevant in cells. Through biophysical experiments, we then identified the PRR (proline-rich region) motif of Alix that binds Hck-SH3 and determined a dissociation constant of 34.5 µM. Heteronuclear NMR spectroscopy experiments were used to map the Hck-SH3 residues that interact with an ALIX construct containing the V and PRR domains or with the minimum identified interacting motif. Finally, SAXS (small-angle X-ray scattering) analysis showed that the N-terminal PRR of Alix is unfolded, at least before Hck-SH3 recognition. These results indicate that residues outside the canonical PxxP motif of Alix enhance its affinity and selectivity towards Hck-SH3. The structural framework of the Hck-Alix interaction will help to clarify how Hck and Alix assist during virus budding and cell-surface receptor regulation.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Ciclo Celular/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Proteínas Proto-Oncogénicas c-hck/química , Dominios Homologos src , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas Proto-Oncogénicas c-hck/metabolismo , Dispersión del Ángulo Pequeño , Técnicas del Sistema de Dos Híbridos , Liberación del VirusRESUMEN
Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania.
Asunto(s)
Aneuploidia , Antimonio/farmacología , Expresión Génica , Leishmania infantum/efectos de los fármacos , Leishmania infantum/genética , Tripanocidas/farmacología , Animales , Resistencia a Medicamentos/genética , Amplificación de Genes , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Leishmania infantum/metabolismo , Monosomía , MutaciónRESUMEN
Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.
Asunto(s)
Antiportadores/genética , Proteínas de Unión al ADN/genética , Endometrio/metabolismo , Ciclo Menstrual/metabolismo , Antiportadores/análisis , Antiportadores/fisiología , Células Cultivadas , Ciclooxigenasa 2 , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/fisiología , Endometrio/química , Femenino , Humanos , Inmunohistoquímica , Interleucina-1/farmacología , Proteínas de la Membrana , Transportadores de Anión Orgánico , Prostaglandina-Endoperóxido Sintasas/análisis , Prostaglandinas/metabolismo , ARN Mensajero/análisisRESUMEN
CONTEXT: Prostaglandins (PGs) E2 and PGF2α are produced in the endometrium and are important for menstruation and fertility. Dysmenorrhea is associated with increased production of PGF2α relative to PGE2, and the opposite is true for menorrhagia. The pathways leading to PGE2 biosynthesis are well described, but little is known for PGF2α. Aldoketoreductase (AKR)-1C3, the only PGF synthase identified in the human, cannot explain the production of PGF2α by endometrial cells. AKR1B1 appears to be an alternate candidate with promising therapeutic value. OBJECTIVE: The objective of the study was to address whether AKR1B1 (gene ID 231) is a functional PGF2α synthase in the human endometrium and a valid therapeutic target for menstrual pain. DESIGN: The design of the study was basic laboratory analyses to identify gene expression and protein levels associated with PGF2α production in endometrial tissues and endometrial cells from cycling women aged between 23 and 52 yr undergoing biopsies or hysterectomy for diverse gynecological disorders. RESULTS: AKR1B1 is expressed at a high level during the menstrual cycle during the secretory phase and in both epithelial and stromal cells, whereas AKR1C3 was found only in epithelial cells. Purified recombinant AKR1B1 protein, gene silencing, and transient transfection experiments all concur to demonstrate that this enzyme is a functional PGF synthase. Ponalrestat, a specific inhibitor developed to block AKR1B1 activity, reduced PGF2α production in response to IL-1ß in both cultured endometrial cells and endometrial explants. CONCLUSIONS: The human aldose reductase AKR1B1 currently associated with diabetes complications is also a highly functional PGF synthase responsible for PGF2α production in the human endometrium and a potential target for treatment of menstrual disorders.
Asunto(s)
Aldehído Reductasa/metabolismo , Endometrio/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Ciclo Menstrual/metabolismo , Adulto , Aldehído Reductasa/genética , Análisis de Varianza , Western Blotting , Línea Celular , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Humanos , Hidroxiprostaglandina Deshidrogenasas/genética , Inmunohistoquímica , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismoRESUMEN
Prostaglandins (PG) are primary regulators of reproductive function. In ruminants, the relative production of PGE2 and PGF2alpha determines the return to a new oestrous cycle or to the establishment of pregnancy in response to a viable embryo. PG action depends on biosynthesis, transport and interaction with their receptors, which are all expressed differentially during the oestrous cycle. PGs are, however, local mediators and thus the onsite degradation by enzymes such as 15-hydroxyprostaglandin dehydrogenase (HPGD), also known as 15-PGDH, is another factor to consider in the regulation of physiological action. Little information is available on PG catabolism in the endometrium during the oestrous cycle or early pregnancy. The purpose of this study was to clone the bovine 15-PGDH, produce the recombinant protein and generate a specific antibody to study its activity and its expression in the endometrium during the oestrous cycle. We have found that the bovine 15-PGDH is highly homologous to the rat and human isoforms. 15-PGDH is localized principally in the glandular epithelium and to a lesser extent in stromal and luminal epithelial cells. The enzyme expression is regulated during the oestrous cycle and it reaches its maximal level on days 16-18. Transient expression is observed in luminal epithelial and trophoblast cells on day 21 of pregnancy. The mRNA is expressed at a constant high level throughout the cycle. The activity of the recombinant 15-PGDH was also tested and was found comparable for PGF2alpha and PGE2. These data suggest that 15-PGDH contributes to the tight regulation of PG action in the endometrium especially at the critical period of recognition of pregnancy.
Asunto(s)
Bovinos/metabolismo , Endometrio/química , Estro/metabolismo , Hidroxiprostaglandina Deshidrogenasas/análisis , Preñez/metabolismo , Animales , Northern Blotting/métodos , Western Blotting/métodos , Femenino , Hidroxiprostaglandina Deshidrogenasas/genética , Inmunohistoquímica/métodos , Embarazo , ARN Mensajero/análisisRESUMEN
Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.
Asunto(s)
Leishmania infantum/crecimiento & desarrollo , Leishmania infantum/genética , Estadios del Ciclo de Vida , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Transcripción Genética , Animales , Electroforesis en Gel Bidimensional , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Genes Protozoarios , Leishmania infantum/química , Leishmania infantum/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/química , Mapeo Peptídico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/análisis , Proteínas Protozoarias/genética , ARN Mensajero/metabolismoRESUMEN
During the epididymal transit, mammalian spermatozoa acquire new surface proteins necessary for male gamete function. We have previously shown that membranous vesicles, called epididymosomes, interact with spermatozoa allowing the transfer of some proteins to sperm surface within the epididymal lumen. The protein composition of those vesicles has been investigated to document the mechanisms of protein transfer from epididymosomes to spermatozoa. Electrophoretic analysis revealed that protein composition is different from the epididymal soluble compartment as well as from similar vesicles present in the semen. Protein association with epididymosome is very strong as revealed by resistance to extraction with detergent. Matrix-assisted laser desorption ionization time-of-flight as well as immunodetection techniques have been used to identify some proteins associated to epididymosomes and spermatozoa. An aldose reductase known for its 20alpha-hydroxysteroid dehydrogenase activity and the cytokine (macrophage migration inhibitory factor) have been identified. These two proteins have been immunolocalized in principal cells of the epididymal epithelium, a more intense signal being detected in the distal epididymal segment as well as in the vas deferens. Database search revealed that these two proteins are characterized by the lack of a signal peptide. These results are discussed with regard to a possible apocrine mode of secretion of these proteins acquired by spermatozoa during the epididymal transit.
Asunto(s)
Aldehído Reductasa/fisiología , Epidídimo/fisiología , Factores Inhibidores de la Migración de Macrófagos/fisiología , Espermatozoides/fisiología , Animales , Western Blotting , Bovinos , Centrifugación por Gradiente de Densidad , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Epidídimo/citología , Inmunohistoquímica , Técnicas In Vitro , Masculino , Péptidos/química , Proteínas/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Conducto Deferente/metabolismoRESUMEN
Prostaglandins are important regulators of reproductive function. In particular, prostaglandin F2 alpha (PGF(2 alpha)) is involved in labor and is the functional mediator of luteolysis to initiate a new estrous cycle in many species. These actions have been extensively studied in ruminants, but the enzymes involved are not clearly identified. Our objective was to identify which prostaglandin F synthase is involved and to study its regulation in the endometrium and in endometrial primary cell cultures. The expression of all previously known prostaglandin F synthases (PGFSs), two newly discovered PGFS-like genes, and a 20 alpha-hydroxysteroid dehydrogenase was studied by Northern blot and reverse transcription PCR. These analyses revealed that none of the known PGFS or the PGFS-like genes were significantly expressed in the endometrium. On the other hand, the 20 alpha-hydroxysteroid dehydrogenase gene was strongly expressed in the endometrium at the time of luteolysis. The corresponding recombinant enzyme has a K(m) of 7 microM for PGH(2) and a PGFS activity higher than the lung PGFS. This enzyme has two different activities with the ability to terminate the estrous cycle; it metabolizes progesterone and synthesizes PGF(2 alpha). Taken together, these data point to this newly identified enzyme as the functional endometrial PGFS.