RESUMEN
BACKGROUND: Optimal consolidation for young patilents with relapsed/refractory (R/R) follicular lymphoma (FL) remains uncertain in the rituximab era, with an unclear benefit of autologous stem cell transplantation (ASCT). The multicenter, randomized, phase III FLAZ12 (NCT01827605) trial compared anti-CD20 radioimmunotherapy (RIT) with ASCT as consolidation after chemoimmunotherapy, both followed by rituximab maintenance. PATIENTS AND METHODS: Patients (age 18-65 years) with R/R FL and without significant comorbidities were enrolled and treated with three courses of conventional, investigator-chosen chemoimmunotherapies. Those experiencing at least a partial response were randomized 1 : 1 to ASCT or RIT before CD34+ collection, and all received postconsolidation rituximab maintenance. Progression-free survival (PFS) was the primary endpoint. The target sample size was 210 (105/group). RESULTS: Between August 2012 and September 2019, of 164 screened patients, 159 were enrolled [median age 57 (interquartile range 49-62) years, 55% male, 57% stage IV, 20% bulky disease]. The study was closed prematurely because of low accrual. Data were analyzed on 8 June 2023, on an intention-to-treat basis, with a 77-month median follow-up from enrollment. Of the 141 patients (89%), 70 were randomized to ASCT and 71 to RIT. The estimated 3-year PFS in both groups was 62% (hazard ratio 1.11, 95% confidence interval 0.69-1.80, P = 0.6662). The 3-year overall survival also was similar between the two groups. Rates of grade ≥3 hematological toxicity were 94% with ASCT versus 46% with RIT (P < 0.001), and grade ≥3 neutropenia occurred in 94% versus 41%, respectively (P < 0.001). Second cancers occurred in nine patients after ASCT and three after radioimmunotherapy (P = 0.189). CONCLUSIONS: Even if prematurely discontinued, our study did not demonstrate the superiority of ASCT versus RIT. ASCT was more toxic and demanding for patients and health services. Both strategies yielded similar, favorable long-term outcomes, suggesting that consolidation programs milder than ASCT require further investigation in R/R FL.
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Trasplante de Células Madre Hematopoyéticas , Linfoma Folicular , Humanos , Masculino , Persona de Mediana Edad , Adolescente , Adulto Joven , Adulto , Anciano , Femenino , Linfoma Folicular/radioterapia , Radioinmunoterapia , Rituximab , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Supervivencia sin Enfermedad , Trasplante Autólogo , Trasplante de Células MadreAsunto(s)
Sistemas de Transporte de Aminoácidos Neutros/química , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Treonina/fisiología , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Transporte Biológico/genética , Femenino , Proteínas de Insectos/genética , Activación del Canal Iónico/genética , Manduca , Potenciales de la Membrana/genética , Oocitos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Treonina/química , Treonina/genética , Xenopus laevisRESUMEN
K+-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAATI shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na+, K+, Li+) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(Il)(l,10-phenanthroline)3, showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na+ protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Glicina/genética , Proteínas de Insectos/genética , Mutación , Alanina/genética , Alanina/metabolismo , Alanina/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/fisiología , Aminoácidos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Secuencia Conservada/genética , Relación Dosis-Respuesta a Droga , Glicina/metabolismo , Glicina/fisiología , Proteínas de Insectos/metabolismo , Proteínas de Insectos/fisiología , Litio/metabolismo , Litio/farmacología , Manduca/genética , Manduca/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Potasio/metabolismo , Potasio/farmacología , Homología de Secuencia de Aminoácido , Sodio/metabolismo , Sodio/farmacología , Xenopus laevisRESUMEN
K-activated amino acid transporter 1 (KAAT1) and cation-anion-activated amino acid transporter/channel 1 (CAATCH1) are amino acid cotransporters, belonging to the Na/Cl-dependent neurotransmitter transporter family (also called SLC6/NSS), that have been cloned from Manduca sexta midgut. They have been thoroughly studied by expression in Xenopus laevis oocytes, and structure/function analyses have made it possible to identify the structural determinants of their cation and amino acid selectivity. About 40 mutants of these proteins have been studied by measuring amino acid uptake and current/voltage relationships. The results obtained since the cloning of KAAT1 and CAATCH1 are here discussed in the light of the 3D model of the first crystallized member of the family, the leucine transporter LeuT.
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Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Insectos/metabolismo , Manduca/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/química , Animales , Proteínas Portadoras/química , Proteínas de Insectos/química , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Xenopus laevisRESUMEN
PURPOSE: We evaluated the impact of premature extrauterine life on brain maturation. PATIENTS AND METHODS: Twelve neonates underwent MR imaging at 40 (39.64 +/- 0.98) weeks (full term). Fifteen premature infants underwent 2 MR imaging examinations, after birth (preterm at birth) and at 40 weeks (41.03 +/- 1.33) (preterm at term). A 3D MR imaging technique was used to measure brain volumes compared with intracranial volume: total brain volume, cortical gray matter, myelinated white matter, unmyelinated white matter, basal ganglia (BG), and CSF. RESULTS: The average absolute volume of intracranial volume (269.8 mL +/- 36.5), total brain volume (246.5 +/- 32.3), cortical gray matter (85.53 mL +/- 22.23), unmyelinated white matter (142.4 mL +/-14.98), and myelinated white matter (6.099 mL +/-1.82) for preterm at birth was significantly lower compared with that for the preterm at term: the average global volume of intracranial volume (431.7 +/- 69.98), total brain volume (391 +/- 66,1), cortical gray matter (179 mL +/- 41.54), unmyelinated white matter (185.3 mL +/- 30.8), and myelinated white matter (10.66 mL +/- 3.05). It was also lower compared with that of full-term infants: intracranial volume (427.4 mL +/- 53.84), total brain volume (394 +/- 49.22), cortical gray matter (181.4 +/- 29.27), unmyelinated white matter (183.4 +/- 27.37), and myelinated white matter (10.72 +/- 4.63). The relative volume of cortical gray matter (30.62 +/- 5.13) and of unmyelinated white matter (53.15 +/- 4.8) for preterm at birth was significantly different compared with the relative volume of cortical gray matter (41.05 +/- 5.44) and of unmyelinated white matter (43.22 +/- 5.11) for the preterm at term. Premature infants had similar brain tissue volumes at 40 weeks to full-term infants. CONCLUSION: MR segmentation techniques demonstrate that cortical neonatal maturation in moderately premature infants at term and term-born infants was similar.
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Encéfalo/anatomía & histología , Encéfalo/crecimiento & desarrollo , Recien Nacido Prematuro/crecimiento & desarrollo , Imagen por Resonancia Magnética , Humanos , Recién Nacido , Estudios ProspectivosRESUMEN
We investigated the role of the Q291 glutamine residue in the functioning of the rat gamma-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter.
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Secuencia Conservada , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Glutamina/metabolismo , Ácido gamma-Aminobutírico/farmacología , Secuencia de Aminoácidos , Animales , Cisteína/genética , Cisteína/metabolismo , Electrofisiología , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Regulación de la Expresión Génica , Glutamina/genética , Humanos , Litio/farmacología , Datos de Secuencia Molecular , Mutación/genética , Técnicas de Placa-Clamp , Ratas , Alineación de SecuenciaRESUMEN
We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/fisiología , Animales , Línea Celular , Electrofisiología/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Técnicas de Transferencia de Gen , Oocitos/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/química , Subunidades de Proteína/fisiología , Ratas , Xenopus laevisRESUMEN
PURPOSE: To validate the use of water as contrast agent for the delineation of the small intestine on the planning CT of external beam in patients treated with conformal radiotherapy for gynaecological tumours. PATIENTS AND METHODS: From March to September 2003, 20 patients received an external irradiation for a gynaecological carcinoma (13 with cervix carcinoma, seven with endometrial carcinoma) in the radiotherapy department of the Centre G.F. Leclerc of Dijon. The protocol of opacification of the small intestine consisted in administration of a "negative" contrast agent: water. The protocol commonly used for the bladder filling, i.e. absorption of 500 cm(3) of water from 60 to 30 min before the CT-scan, was applied for the evaluation of the visualisation of the small intestine in the 12 first patients (group I). For the last eight patients (group II), the absorption of the same amount of water was fractionated, every 10 min within half an hour before the start of the examination. RESULTS: The small bowel identification was possible in 100% of cases without any need of administration of a "positive" contrast agent. In overall, the identification of the small intestine was considered as easy in 14 patients (70%) and as difficult in two patients (10%). In group I, the delineation was considered as easy in 50% of cases, moderately easy in 33% of cases and none easy in 17% of cases. Conversely, no difficulty was encountered for the definition of the small bowel in all patients of group II. CONCLUSIONS: Water is an efficient "negative" contrast agent for the differentiation of the small bowel from the colon on the planning abdomino-pelvic CT. Nevertheless, the delineation was really made easier only when the fractionated protocol of water absorption within half an hour before CT was used.
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Neoplasias de los Genitales Femeninos/radioterapia , Intestino Delgado/anatomía & histología , Traumatismos por Radiación/prevención & control , Agua , Adulto , Colon/anatomía & histología , Medios de Contraste/administración & dosificación , Femenino , Humanos , Planificación de Atención al Paciente , Tomografía Computarizada por Rayos X , Agua/administración & dosificaciónRESUMEN
To investigate the peculiar ionic specificity of KAAT1, an Na+- and K+-coupled amino acid cotransporter from Lepidoptera, a detailed analysis of membrane topology predictions was performed, together with sequence comparison with strictly Na+-dependent mammalian cotransporters from the same family. The analysis identified aspartate 338, a residue present also in the other cotransporter accepting K+ (CAATCH1), but absent in most mammalian transporters that have, instead, an asparagine in the corresponding position. Mutation of D338 in KAAT1 led either to non-functional transporters (D338G, D338C), or to an altered ionic selectivity (D338E, D338N), observable in uptake experiments and in electrophysiological properties. In particular, in D338E, the transport activity, while persisting in the presence of Na+, appeared to be completely abolished in the presence of K+. D338E also showed uncoupling between transport-associated current and uptake. The opposite mutation in the gamma-aminobutyric acid transporter rGAT-1 (N327D) resulted in complete loss of function. In conclusion, aspartate 338 in KAAT1 appears to be important in allowing K+, in addition to Na+, to drive the transport mechanism, although other residues in different parts of the protein may also play a role in the complete determination of ionic selectivity.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Ácido Aspártico/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Insectos , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Secuencia de Aminoácidos , Animales , Ácido Aspártico/genética , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática , Leucina/metabolismo , Manduca/genética , Manduca/metabolismo , Glicoproteínas de Membrana/genética , Potenciales de la Membrana , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Técnicas de Placa-ClampRESUMEN
The effects of reducing external Cl- on the electrophysiological properties of the Na+/Cl(-)-dependent GABA transporter rGAT1 expressed in Xenopus oocytes were investigated. In agreement with a recently proposed kinetic scheme, the effects of Cl- are complex but preserve the mutual relationship that links the transport-associated current, I(tr), measured in saturating GABA concentration, and the transient current, I(pre), recorded in the absence of GABA following a voltage step from the holding potential Vh to V. In particular, I(tr) (V) - I(tr) (Vh) = r integral I(pre) (V) dt, where r is the relaxation rate of I(pre) at the same membrane potential and Cl- concentration. The model also predicts a relationship between charge relaxation rate and apparent affinity for GABA, which is also verified in the presence of lowered Na+ or Cl- concentrations. In these conditions, the binding rate of GABA to the transporter is increased. All these effects are consistent with the hypothesis that interaction of the organic substrate with rGAT1 induces a conversion from a capacitive to a conductive mode of operation without strongly altering either the amount or the rate of charge movement.
Asunto(s)
Proteínas Portadoras/metabolismo , Cloro/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Ácido gamma-Aminobutírico/metabolismo , Animales , Femenino , Proteínas Transportadoras de GABA en la Membrana Plasmática , Oocitos/metabolismo , Sodio/metabolismo , Xenopus laevisRESUMEN
Biochemical maturation of the brain can be studied noninvasively by (1)H magnetic resonance spectroscopy (MRS) in human infants. Detailed time courses of cerebral tissue contents are known for the most abundant metabolites only, and whether or not premature birth affects biochemical maturation of the brain is disputed. Hence, the last trimester of gestation was observed in infants born prematurely, and their cerebral metabolite contents at birth and at expected term were compared with those of fullterm infants. Successful quantitative short-TE (1)H MRS was performed in three cerebral locations in 21 infants in 28 sessions (gestational age 32-43 weeks). The spectra were analyzed with linear combination model fitting, considerably extending the range of observable metabolites to include acetate, alanine, aspartate, cholines, creatines, gamma-aminobutyrate, glucose, glutamine, glutamate, glutathione, glycine, lactate, myo-inositol, macromolecular contributions, N-acetylaspartate, N-acetylaspartylglutamate, o-phosphoethanolamine, scyllo-inositol, taurine, and threonine. Significant effects of age and location were found for many metabolites, including the previously observed neuronal maturation reflected by an increase in N-acetylaspartate. Absolute brain metabolite content in premature infants at term was not considerably different from that in fullterm infants, indicating that prematurity did not affect biochemical brain maturation substantially in the studied population, which did not include infants of extremely low birthweight.
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Química Encefálica , Encéfalo/embriología , Encéfalo/metabolismo , Recien Nacido Prematuro/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Ácido Aspártico/metabolismo , Colina/metabolismo , Desarrollo Embrionario y Fetal , Femenino , Edad Gestacional , Ácido Glutámico/metabolismo , Glutatión/metabolismo , Humanos , Lactante , Recién Nacido , Inositol/metabolismo , Masculino , Embarazo , Tercer Trimestre del Embarazo , Valores de Referencia , Sensibilidad y EspecificidadRESUMEN
1. The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2. At neutral pH, the transport-associated current vs. voltage (I-V) relationship of the mutated transporter was different from wild-type, and the pre-steady-state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mM at -60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 microM at -60 mV). 3. In both isoforms changes in [Na+]o shifted the voltage dependence of the pre-steady-state and of the transport-associated currents by similar amounts. 4. In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport-associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I-V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5. These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6. A possible explanation for the complex effect of pH on the transport-associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Transportadores de Anión Orgánico , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Transportadoras de GABA en la Membrana Plasmática , Expresión Génica/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida/fisiología , Oocitos/fisiología , Técnicas de Placa-Clamp , Mutación Puntual , Ratas , Sodio/farmacología , Relación Estructura-Actividad , Xenopus laevis , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The cloned canine betaine-GABA cotransporter BGT-1 has been heterologously expressed in Xenopus laevis oocytes in order to characterize its electrophysiological properties. Voltage-clamp experiments on transfected oocytes reveal the presence of three types of membrane current which are absent in non-injected oocytes: (i) an organic substrate-independent current (uncoupled current); (ii) a transport-associated current, seen upon addition of betaine or GABA; (iii) presteady-state currents induced by voltage changes. The three kinds of current are analogous to those reported in structurally similar cotransporters. The transport-associated current is strictly dependent on the presence of Na(+). The good correlation between the amount of charge underlying the presteady-state currents and the transport-associated current indicates that both processes are due to the activity of the transporter.
Asunto(s)
Betaína/metabolismo , Proteínas Portadoras/química , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Cationes Monovalentes , Perros , Proteínas Transportadoras de GABA en la Membrana Plasmática , Potenciales de la Membrana , Oocitos/metabolismo , Técnicas de Placa-Clamp , Transfección , Xenopus laevis , Ácido gamma-Aminobutírico/metabolismoRESUMEN
We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC). The procedure makes use of iterative rounds of homologous recombination in Escherichia coli. Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required. They are transferred first into a temperature-sensitive replicon and then into the BAC of choice. We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli-STREPTOMYCES: artificial chromosome (ESAC). Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids.
Asunto(s)
Cromosomas Artificiales Bacterianos/genética , ADN Recombinante/genética , Escherichia coli/genética , Recombinación Genética , Clonación Molecular/métodos , Cósmidos/genética , Electroforesis en Gel de Campo Pulsado , Plásmidos/genética , Rec A Recombinasas/genética , Streptomyces/genéticaRESUMEN
Two oligonucleotide probes derived from conserved motifs in peptide synthetases were hybridized with a cosmid library of Planobispora rosea genomic DNA. Detailed characterization of the physical organization of the positive cosmids indicated the existence of at least eight unlinked contigs containing multiple fragments that hybridized to both probes. Partial sequences of PCR products from the positive cosmids confirmed the existence of peptide synthetase genes. The combined results of hybridizations and physical mapping indicate that, in all likelihood, the isolated P. rosea contigs encode over 40 putative peptide synthetase modules. Similar results were obtained on screening a cosmid library of Actinoplanes teichomyceticus DNA. Furthermore, Southern hybridizations with several actinomycete strains, belonging to different genera, indicate that most strains contain multiple hybridizing bands well in excess of the number expected from the structure of the oligopeptides produced by these strains. Even strains not reported to produce oligopeptides gave clear positive signals when examined with the probes. These results strongly suggest that actinomycetes devote a notable fraction of their genomes to the non-ribosomal synthesis of peptides, and that most strains have the genetic potential to produce more oligopeptides than are currently described.
Asunto(s)
Actinomycetales/genética , Familia de Multigenes , Péptido Sintasas/biosíntesis , Péptido Sintasas/genética , Southern Blotting , Mapeo Contig , Cósmidos , Biblioteca de Genes , Modelos Genéticos , Hibridación de Ácido Nucleico , Oligonucleótidos/genética , Mapeo Físico de Cromosoma , Plásmidos/metabolismoRESUMEN
The effects of pH on the different kinds of currents occurring at the lepidopteran amino acid cotransporter KAAT1 have been examined using heterologous expression in Xenopus oocytes and voltage clamp. Acidic pH (5.5-4.5) caused a slight depression of the uncoupled current and a complete inhibition of the coupled and transient currents, in the presence of either Na+ or K+, the two ions physiologically relevant to the transport process. Conversely, at alkaline pH (9) no statistically significant effects could be observed on uncoupled, coupled and transient currents compared to the effects at pH 7.6. These effects of pH indicate that operation of the transporter is maximal in the physiologically alkaline native environment. The dose-response curves for the inhibition of coupled and transient currents were similar, with respective pKa values of 6. 29 +/- 0.05 and 6.40 +/- 0.03 and respective Hill coefficient values (nH) of 0.93 +/- 0.07 and 1.08 +/- 0.08, suggesting that the two effects can be explained by a single proton binding to the same site in the transporter.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Portadoras/metabolismo , Proteínas de Insectos , Glicoproteínas de Membrana/metabolismo , Animales , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/genética , Concentración de Iones de Hidrógeno , Manduca , Glicoproteínas de Membrana/genética , Microinyecciones , Oocitos/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , ARN Mensajero/metabolismo , Sodio/metabolismo , XenopusRESUMEN
Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC) that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.
Asunto(s)
Actinomycetales/genética , Actinomycetales/metabolismo , Antibacterianos/biosíntesis , Cromosomas Bacterianos , Southern Blotting , Escherichia coli/genética , Biblioteca de Genes , Ingeniería Genética/métodos , Modelos Genéticos , Plásmidos/genética , Streptomyces/genéticaRESUMEN
The transport properties of the intestinal amino acid cotransporter KAAT1, heterologously expressed in Xenopus oocytes, were studied using simultaneous voltage-clamp and tritiated leucine uptake measurements. While addition of 1 mM leucine to oocytes kept at -80 mV in presence of Na(+) or K(+) caused an increase in holding current, in presence of Li(+) the current was reduced. Uptake measurements in voltage-clamp conditions showed that a comparable accumulation of amino acid occurred in all three ionic conditions and irrespective of the direction and amount of the current change. The ratio of moles of transferred charge to moles of transported amino acid ranges from 1.45 for K(+) to 3.52 for Li(+). A hypothetical interpretation involving the coexistence of two populations of transporters, one operating in the uncoupled mode and the other in the substrate transport mode is discussed.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Proteínas Portadoras/metabolismo , Proteínas de Insectos , Leucina/metabolismo , Glicoproteínas de Membrana/metabolismo , Xenopus laevis/metabolismo , Animales , Transporte Biológico , Cationes Monovalentes , Membrana Celular/metabolismo , Transporte de Electrón , Electrofisiología , Microinyecciones , Oocitos/metabolismo , TritioRESUMEN
The genetic determinants for the biosynthesis of the glycopeptide antibiotic teicoplanin were identified. In order to isolate the corresponding gene cluster, oligonucleotides derived from highly conserved motifs in peptide synthetases were used. These synthetic probes, and gene fragments derived from the balhimycin gene cluster of Amycolatopsis mediterranei, led to the identification of the likely teicoplanin gene cluster centered on a region of ca. 110 kb from the genome of Actinoplanes teichomyceticus, the teicoplanin producer. Partial nucleotide sequences identified partial ORFs likely to encode two glycosyltransferases, three P-450 monooxygenases and one ABC transporter. The corresponding genes have been found in other glycopeptide gene clusters. Furthermore, upstream to the peptide synthetase region a segment was identified with a remarkable similarity to the vanHAX operon, conferring resistance to glycopeptides in enterococci. Thus, in contrast to the other glycopeptide producers thus far analyzed, in A. teichomyceticus the genes for teicoplanin biosynthesis are closely linked to homologs of glycopeptide resistance commonly found in vancomycin-resistant enterococci.
Asunto(s)
Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Teicoplanina/biosíntesis , Actinomycetales/genética , Antibacterianos/biosíntesis , Pared Celular/metabolismo , Mapeo Cromosómico , Conformación Molecular , Estructura Molecular , Familia de Multigenes , Teicoplanina/químicaRESUMEN
The ability of the intestinal amino acid co-transporter KAAT-1 expressed in Xenopus oocytes to transport different cations in either amino acid coupled or uncoupled manner was studied using voltage-clamp conditions. KAAT1-expressing oocytes exhibit a transporter-related current in the absence of organic substrate (uncoupled current). In the presence of various alkali cations the amplitude of this current follows the sequence: ILi > INa > IK approximately equal to IRb approximately equal to ICs. Addition of 1 mM leucine causes large increases in K+ and Na+ currents, while the Li+ current undergoes a more complex change and Rb+ and Cs+ currents are only marginally affected. Pre-steady-state currents in the absence of organic substrate are apparent when Na+, K+, or Li+ are the bathing ions; analysis of these currents in terms of charge movement reveals that Na+, K+, and Li+ interact differently with the transporter. The uncoupled current in mixtures of Na+ and Li+ fails to exhibit anomalous mole-fraction behavior. Kinetic analysis of ion binding and uncoupled permeation argues against a multi-ion single-file mechanism in the KAAT1 cotransporter.