RESUMEN
Inexpensive and safe energy-storage batteries with high energy densities are in high demand (e.g., for electric vehicles and grid-level renewable energy storage). This study focused on using NaFeCl4, comprising ubiquitous elements, as an electrode material for all-solid-state sodium-ion batteries. Monoclinic NaFeCl4, expected to be the most resource-attractive Fe redox material, is also thermodynamically stable. The Fe2+/3+ redox reaction of the monoclinic NaFeCl4 electrode has a higher potential (3.45 V vs. Na/Na+) than conventional oxide electrodes (e.g., Fe2O3 with 1.5 V vs. Na/Na+) because of the noble properties of chlorine. Additionally, NaFeCl4 exhibits unusually high deformability (99% of the relative density of the pellet) upon uniaxial pressing (382 MPa) at 298 K. NaFeCl4 operates at 333 K in an electrode system containing no electrolyte, thereby realizing next-generation all-solid-state batteries with high safety. A high energy density per positive electrode of 281 Wh kg-1 was achieved using only a simple powder press.
RESUMEN
The present study was undertaken to develop a self-micellizing solid dispersion (SMSD) of tacrolimus (TAC) to improve the biopharmaceutical properties of TAC. An SMSD formulation of TAC (SMSD/TAC) and amorphous solid dispersion formulation of TAC (ASD/TAC) were prepared with Soluplus® , an amphiphilic copolymer, and hydroxypropyl cellulose, respectively. Physicochemical properties were characterized in terms of morphology, crystallinity, storage stability, interaction of TAC with Soluplus® , and micelle-forming potency; pharmacokinetic behavior was also evaluated in rats. Tacrolimus in both formulations was in an amorphous state. After storage at 40°C/75% relativity humidity for 4 weeks, there were no significant changes in the crystallinity of TAC between nonaged and aged SMSD/TAC, whereas slight recrystallization was observed in aged ASD/TAC. The results of circular dichroism (CD) and infrared spectroscopic analyses were indicative of the potent drug-polymer interaction in SMSD/TAC, possibly leading to the prevention of recrystallization. Compared with other TAC samples, SMSD/TAC exhibited significant improvement in the dissolution behavior of TAC through the immediate formation of fine micelles. After the oral administration of TAC samples (10 mg TAC/kg) to rats, there was marked enhancement in systemic exposure to TAC with both formulations; in particular, SMSD/TAC achieved an increase in bioavailability ca. 20-fold higher than crystalline TAC. The SMSD approach might provide an effective dosage form for TAC with enhanced physicochemical stability and oral absorption.
Asunto(s)
Polietilenglicoles , Tacrolimus , Ratas , Animales , Ratas Sprague-Dawley , Solubilidad , Micelas , Disponibilidad Biológica , Administración OralRESUMEN
The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use of polyethyleneoxide (PEO) and polylysine (pLys), a cationic polymer. A mixture of pLys and FZP was added to PEO to prepare an HG formulation, and the formulation was optimized by release test and viscosity measurements. Circular dichroism (CD) and infrared absorption (IR) spectral analyses were applied to clarify the intermolecular interactions between FZP and pLys. The pharmacokinetic behavior of FZP was evaluated after a subcutaneous administration of FZP samples (2.0 mg-FZP/kg) to rats. Although the immediate release of FZP was observed for the HG formulation, the addition of pLys at a 20-fold amount of FZP or higher led to the sustained release of FZP. Considering release behavior, the concentration of pLys was optimized as 100-fold that of FZP in the HG formulation. CD and IR spectroscopic analyses of FZP and/or pLys demonstrated an intermolecular interaction between FZP and pLys, as evidenced by the slight spectral transition. After a subcutaneous administration of HG formulation containing pLys to rats, compared with FZP alone, significant differences were observed in the pharmacokinetic behavior with a decrease of Cmax from 2.3 to 0.9 mg/mL and slower elimination kinetics. HG formulation using pLys might be a viable dosage option for FZP for the treatment of AP in animals.
Asunto(s)
Pancreatitis , Polilisina , Ratas , Animales , Polilisina/química , Hidrogeles , Preparaciones de Acción Retardada/química , Antígeno-1 Asociado a Función de Linfocito , Enfermedad Aguda , LeucocitosRESUMEN
PURPOSE: The aim of the present study was to develop a new multi-unit dosage formulation, Universal ORbicular Vehicle (UniORV), to improve the biopharmaceutical properties of tacrolimus (TAC). METHODS: TAC-loaded UniORV (UO/TAC) was produced by the dripping and gelling of a solution comprising TAC, gelatin, starch syrup, and triethyl citrate at 0.5 w/w% drug loading. Its microstructure was elucidated by polarized light microscopy and the Raman mapping technique. The pharmacokinetic profiles of TAC after the oral administration of UO/TAC were evaluated in rats and healthy humans. RESULTS: The dissolution behavior of UO/TAC was similar to that of commercial capsules, and the formation of nanoparticles was detected by TEM in dissolved media. In a stability study on UO/TAC, only 2.6 and 4.7% decreases in TAC concentrations were observed at 40± 2°C/75 ± 5% relative humidity for 4 months and at 50± 2°C for 2 months, respectively. A pharmacokinetic study on rats revealed a 30-fold higher AUC than that with crystalline TAC. A randomized double-blind crossover study on 8 healthy males showed that UniORV achieved a 1.4-fold increase in AUC and 34% decrease in inter-individual variation from the reference formulation. CONCLUSION: The new dosage form UniORV is a promising approach to improve the dissolution, amorphous stability, and biopharmaceutical properties of TAC, which is a poorly water-soluble drug.
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Inmunosupresores/administración & dosificación , Inmunosupresores/farmacocinética , Vehículos Farmacéuticos/química , Tacrolimus/administración & dosificación , Tacrolimus/farmacocinética , Administración Oral , Adulto , Animales , Citratos/química , Estudios Cruzados , Método Doble Ciego , Composición de Medicamentos , Liberación de Fármacos , Gelatina/química , Humanos , Inmunosupresores/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Almidón/química , Tacrolimus/sangre , Adulto JovenRESUMEN
RNA polymerases (RNAPs) transcribe genes through the barrier of nucleoproteins and site-specific DNA-binding proteins on their own or with the aid of accessory factors. Proteins are often covalently trapped on DNA by DNA damaging agents, forming DNA-protein cross-links (DPCs). However, little is known about how immobilized proteins affect transcription. To elucidate the effect of DPCs on transcription, we constructed DNA templates containing site-specific DPCs and performed in vitro transcription reactions using phage T7 RNAP. We show here that DPCs constitute strong but not absolute blocks to in vitro transcription catalyzed by T7 RNAP. More importantly, sequence analysis of transcripts shows that RNAPs roadblocked not only by DPCs but also by the stalled leading RNAP become highly error prone and generate mutations in the upstream intact template regions. This contrasts with the transcriptional mutations induced by conventional DNA lesions, which are delivered to the active site or its proximal position in RNAPs and cause direct misincorporation. Our data also indicate that the trailing RNAP stimulates forward translocation of the stalled leading RNAP, promoting the translesion bypass of DPCs. The present results provide new insights into the transcriptional fidelity and mutual interactions of RNAPs that encounter persistent roadblocks.
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Bacteriófago T7/enzimología , Bacteriófago T7/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Transcripción Genética/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Catálisis , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Mutagénesis/genética , Mutación/genéticaRESUMEN
Solution structures of DNA duplexes containing oxanine (Oxa, O) opposite a cytosine (O:C duplex) and opposite a thymine (O:T duplex) have been solved by the combined use of (1)H NMR and restrained molecular dynamics calculation. One mismatch pair was introduced into the center of the 11-mer duplex of [d(GTGACO(6)CACTG)/d(CAGTGX(17)GTCAC), X = C or T]. (1)H NMR chemical shifts and nuclear Overhauser enhancement (NOE) intensities indicate that both the duplexes adopt an overall right-handed B-type conformation. Exchangeable resonances of C(17) 4-amino proton of the O:C duplex and of T(17) imino proton of O:T duplex showed unusual chemical shifts, and disappeared with temperature increasing up to 30 °C, although the melting temperatures were >50 °C. The O:C mismatch takes a wobble geometry with positive shear parameter where the Oxa ring shifted toward the major groove and the paired C(17) toward the minor groove, while, in the O:T mismatch pair with the negative shear, the Oxa ring slightly shifted toward the minor groove and the paired T(17) toward the major groove. The Oxa mismatch pairs can be wobbled largely because of no hydrogen bond to the O1 position of the Oxa base, and may occupy positions in the strands that optimize the stacking with adjacent bases.
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Disparidad de Par Base , ADN/química , Nucleósidos de Purina/química , Citosina/química , Desoxirribonucleósidos/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Timina/químicaRESUMEN
Although a DNA-immobilized packed-column (DNA-packed column), which relies on sequence-dependent interactions of target DNA or mRNA (in the mobile phase) with DNA probes (on the silica particle) in a continuous flow process, could be considered as an alternative platform for quantitative analysis of specific DNA to DNA chip methodology, the performance in practice has not been satisfactory. In this study, we set up a more efficient quantitative analysis system based on a DNA-packed column by employing a temperature-gradient strategy and DMSO-containing mobile phase. Using a temperature-gradient strategy based on T(m) values of probe/target DNA hybridizations and DMSO (5%)-containing mobile phase, we succeeded in the quantitative analysis of a specific complementary target distinguishable from non-complementary DNA oligomers or other similar DNA samples. In addition, two different target DNA oligomers even with similar T(m) values were separated and detected quantitatively by using a packed column carrying two different DNA probes.
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ADN/análisis , Técnicas Analíticas Microfluídicas/métodos , Sondas de ADN/química , Técnicas Analíticas Microfluídicas/instrumentación , TemperaturaRESUMEN
Oxanine (Oxa, O) is one of the damaged bases produced from guanine (G) through nitrosative deamination induced by nitric oxide (NO) or nitrous acid (HNO(2)). Large-scale preparation of Oxa-containing oligodeoxynucleotide (Oxa-ODN) with the desired base sequence is a prerequisite for exploring detailed properties of Oxa in DNA. This can be accomplished by incubation of G nucleosides with NaNO(2) in acetic acid buffer (pH 3.5) to produce Oxa nucleosides (e.g., 2'-deoxyoxanosine or dOxo), conversion of dOxo to DMT-dOxo-amidite by tritylation and conventional phosphoramidation, and subsequent synthesis of Oxa-ODN. The presence of Oxa in the synthetic ODN is confirmed by enzymatic digestion. Oxa-ODN is useful for analyzing the biochemical and biophysical properties of Oxa in DNA, which is believed to be involved in NO-induced genotoxicity and cytotoxicity. In addition, since Oxa possesses the carbodiimide-activated carboxylate function (O-acylisourea structure), Oxa-ODN can be used as a functional DNA oligomer that makes covalent cross-linkages with amine or amine-containing biomolecules and amine-modified solid surfaces.
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Desoxiguanosina/química , Oligodesoxirribonucleótidos/síntesis química , Compuestos Organofosforados/química , Cromatografía Líquida de Alta Presión/métodos , Desoxirribonucleósidos/síntesis química , Desoxirribonucleósidos/química , Modelos Químicos , Estructura Molecular , Oligodesoxirribonucleótidos/química , Nucleósidos de Purina/químicaRESUMEN
The relationship of chemical structures of 6-formylpterin (6FP) and its derivatives with scavenging activity of singlet oxygen ((1)O(2)) was examined. First, effects of pterin derivatives on (1)O(2) released from activated human neutrophils were examined. The neutrophils, stimulated with opsonized zymosan, released (1)O(2) that was detected by chemiluminescence using a (1)O(2) specific probe, trans-1-(2'-methoxyvinyl)pyrene. 6FP and its derivatives suppressed the (1)O(2) release. 6FP and other commercially available pterin derivatives, such as biopterin and neopterin, which have different substitutions at the 6-position, suppressed the (1)O(2) release with similar extent. On the other hand, newly synthesized pterin derivatives, which have different substitutions at the 2- and/or 3-position, such as 2-amino-6-formyl-3-methylpteridin-4-one, suppressed the (1)O(2) release in a dose-dependent manner and more potently than 6FP. Then, the (1)O(2) scavenging activity of pterin derivatives was examined photochemically by direct analysis of near-infrared luminescence at 1270 nm, the most sensitive method for the detection of (1)O(2). When rose Bengal, a photosensitizer, in D(2)O solution, was irradiated by 514 nm laser beam, the emission spectrum of (1)O(2) was observed. 6FP suppressed this emission spectrum of (1)O(2), and the newly synthesized pterin derivatives with different substituent at the 2- and/or 3-position suppressed the spectrum more potently than 6FP. The order of potency was similar to that obtained from biological assays. These findings indicate that the substitutions at the 2- and/or 3-position play an important role in (1)O(2) scavenging activity of pterin derivatives.
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Depuradores de Radicales Libres/farmacología , Neutrófilos/efectos de los fármacos , Pterinas/farmacología , Oxígeno Singlete/metabolismo , Relación Dosis-Respuesta a Droga , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/química , Humanos , Neutrófilos/metabolismo , Pterinas/síntesis química , Pterinas/química , Rosa Bengala , Relación Estructura-Actividad , Zimosan/farmacologíaRESUMEN
Nitric oxide (NO) causes DNA damage, generating xanthine (Xan, X) and oxanine (Oxa, O) from guanine (Gua, G) and hypoxanthine (Hyp, H) from adenine (Ade, A) by nitrosative oxidation. Although these NO-induced lesions have been thought to cause mutagenic problems in cellular systems, the influence of these lesions on enzymatic functions has not yet been compared systematically. In this study, we investigated the effect of NO-induced lesions on the activities of DNA-binding/recognizing enzymes such as T4 polynucleotide kinase (T4 PNK), DNA ligases (T4 DNA ligase, Taq DNA ligase) and DNA polymerases (E. coli DNA polymerase I, Klenow fragment, T4 DNA polymerase). The phosphorylation efficiencies of T4 PNK are dependent on the base type at the 5'-end of single-stranded DNA, where Oxa congruent with Hyp congruent with Gua > Xan congruent with Ade. The enzymatic reactions efficiencies of DNA ligases or DNA polymerases were observed to be dependent on the base-pairing type bound by the enzymes, where G:C > H:C > O:C > X:C and A:T congruent with H:T > O:T > X:T. These results suggested that NO-induced lesions and their base-pairs could participate in the interaction mechanisms of the DNA-binding/recognizing enzymes in a similar manner as natural nucleobases.
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ADN Ligasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/química , ADN/efectos de los fármacos , Óxido Nítrico/farmacología , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , ADN/metabolismo , Daño del ADN , Escherichia coli/enzimologíaRESUMEN
Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa>Gua>adenine (Ade) approximately thymine (Thy)>cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.
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ADN Ligasas/química , Enzimas de Restricción del ADN/química , ADN de Cadena Simple/química , Polinucleótido 5'-Hidroxil-Quinasa/química , Nucleósidos de Purina/química , Emparejamiento Base , Citosina/química , División del ADN , ADN de Cadena Simple/genética , Fosforilación , Nucleósidos de Purina/genéticaRESUMEN
T4 DNA ligase-based mismatch detection methods have been proposed as useful strategies for single nucleotide polymorphism (SNP) analyses. However, there is a critical problem for cytosine/thymine (C/T) SNP analyses: guanine:thymine (G:T) mismatch is not distinguished from guanine:cytosine (G:C). Here we employed chemically modified nucleobases, such as oxanine and hypoxanthine, at the end of a ligation fragment and analyzed their influence on the ligation efficiency between G:C and G:T. Successful ligation for G:C and no ligation for G:T were observed when oxanine was employed adjacent to guanine in the ligation junction. This ligation method using an oxanine-containing fragment has strong potentials for the accurate analysis of C/T SNPs.
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Citosina , ADN Ligasas/química , ADN Ligasas/genética , Guanina , Polimorfismo de Nucleótido Simple , Nucleósidos de Purina/química , Disparidad de Par Base , Secuencia de BasesRESUMEN
We have developed a simple ESR spin trapping based method for hydroxyl (OH) radical scavenging-capacity determination, using iron-free OH radical source. Instead of the widely used Fenton reaction, a short (typically 5 seconds) in situ UV-photolysis of a dilute hydrogen peroxide aqueous solution was employed to generate reproducible amounts of OH radicals. ESR spin trapping was applied to quantify OH radicals; the decrease in the OH radical level due to the specimen's scavenging activity was converted into the OH radical scavenging capacity (rate). The validity of the method was confirmed in pure antioxidants, and the agreement with the previous data was satisfactory. In the second half of this work, the new method was applied to the sera of chronic renal failure (CRF) patients. We show for the first time that after hemodialysis, OH radical scavenging capacity of the CRF serum was restored to the level of healthy control. This method is simple and rapid, and the low concentration hydrogen peroxide is the only chemical added to the system, that could eliminate the complexity of iron-involved Fenton reactions or the use of the pulse-radiolysis system.
RESUMEN
Endogenous and environmental genotoxic agents produce DNA damage and induce cell death and mutations. The repair mechanisms of base lesions and single and double strand breaks have been well characterized in both prokaryotic and eukaryotic cells. However, the molecular pathways that repair or tolerate DNA-protein crosslinks (DPCs) remains to be largely elucidated. In this study, we constructed DNA substrates containing defined DPCs and assessed the incision activities of prokaryotic and eukaryotic nucleotide excision repair systems for DPCs in vitro.
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Daño del ADN , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , ADN/química , Células HeLa , Humanos , Proteínas/químicaRESUMEN
DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.
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Reactivos de Enlaces Cruzados/farmacología , Reparación del ADN , ADN/metabolismo , Desoxirribonucleótidos/genética , Proteínas/metabolismo , Recombinación Genética , Animales , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Azacitidina/análogos & derivados , Azacitidina/farmacología , Proteína BRCA2/metabolismo , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromosomas/metabolismo , Cricetinae , ADN/química , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Decitabina , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Formaldehído/farmacología , Histonas/metabolismo , Humanos , Peso Molecular , Mutación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/química , Recombinasa Rad51/metabolismoRESUMEN
High stability of the oligonucleotides immobilized on the glass is essential for the reliable DNA microarray analysis. In the present study, effect of end-capping of the unreacted silanol, remaining after the surface amine-functionalization, was explored: (1) Cy3-NHS (N-hydorxysuccinincimide) dye was spotted on the surface and change in the fluorescent intensity was measured. (2) DNA probes were immobilized by the reactivity of oxanine linked at the 5'-end, the complementary oligonucleotides with Cy5-fluorescence at the 5'-end was hybridized, and the time-dependence of the fluorescence intensity was observed. Both the systems showed improved stability of the immobilized molecules, indicative of the stabilization by end-capping.
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Sondas de ADN/química , ADN/química , Vidrio , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Silanos/química , Estructura MolecularRESUMEN
Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37 degrees C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15-20 degrees C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.
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Proteínas Bacterianas/química , División del ADN , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Hipoxantina/química , Secuencia de Bases , Enzimas de Restricción del ADN/química , Desoxirribonucleasa BamHI/química , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa EcoRI/química , Desoxirribonucleasa EcoRI/metabolismo , Electroforesis en Gel de Poliacrilamida , Enlace de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.
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D-Xilulosa Reductasa/genética , D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Madera/metabolismo , Xilosa/metabolismoRESUMEN
Several bacteria, including Azotobacter vinelandii, possess an alternative pathway of L-rhamnose metabolism, which is different from the known bacterial pathway. In a previous article, a gene cluster related to this pathway was identified, consisting of the genes encoding the four metabolic enzymes L-rhamnose-1-dehydrogenase (LRA1), L-rhamnono-gamma-lactonase (LRA2), L-rhamnonate dehydratase (LRA3) and L-2-keto-3-deoxyrhamnonate (L-KDR) aldolase (LRA4), by which L-rhamnose is converted into pyruvate and L-lactaldehyde, through analogous reaction steps to the well-known Entner-Doudoroff (ED) pathway. In this study, bioinformatic analysis revealed that Sphingomonas sp. possesses a gene cluster consisting of LRA1-3 and two genes of unknown function, LRA5 and LRA6. LRA5 catalyzed the NAD(+)-dependent dehydrogenation of several L-2-keto-3-deoxyacid-sugars, including L-KDR. Furthermore, the reaction product was converted to pyruvate and L-lactate by LRA6; this is different from the pathway of Azotobacter vinelandii. Therefore, LRA5 and LRA6 were assigned as the novel enzymes L-KDR 4-dehydrogenase and L-2,4-diketo-3-deoxyrhamnonate hydrolase, respectively. Interestingly, both enzymes were phylogenetically similar to L-rhamnose-1-dehydrogenase and D-2-keto-3-deoxyarabinonate dehydratase, respectively, and the latter was involved in the archeal nonphosphorylative d-arabinose pathway, which is partially analogous to the ED pathway. The introduction of LRA1-4 or LRA1-3, LRA5 and LAR6 compensated for the L-rhamnose-defective phenotype of an Escherichia coli mutant. Metabolic evolution and promiscuity between the alternative l-rhamnose pathway and other sugar pathways analogous to the ED pathway are discussed.
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Metabolismo de los Hidratos de Carbono , Ramnosa/metabolismo , Sphingomonas/metabolismo , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Familia de Multigenes , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Fosforilación , Homología de Secuencia de Aminoácido , Sphingomonas/genéticaRESUMEN
Reactive oxygen species produced by phagocytosing neutrophils are essential for innate host defense against invading microbes. Previous observations revealed that antibody-catalyzed ozone formation by human neutrophils contributed to the killing of bacteria. In this study, we discovered that 4 amino acids themselves were able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway, at comparable level to antibodies. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in our distinct cell-free system and in human neutrophils. The results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defense system, when the host is challenged by high doses of infectious agents. Our findings provide biological insights into the killing of bacteria by neutrophils.