RESUMEN
Marbofloxacin (MB) is a newly developed fluoroquinolone antibiotic used especially as a veterinary drug. It may be regarded as the improved version of enrofloxacin owing to its antibacterial activity, enhanced bioavailability, and pharmacokinetic-pharmacodynamic (PK-PD) properties. In this study, nine heavy rare-earth ions (Y, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) were selected in light of their potential antibacterial activity and satisfactory biosafety to afford the corresponding rare-earth metal complexes of MB: the MB-Ln series. Their chemical structures and coordination patterns were characterized using IR spectroscopy, HRMS, TGA, and X-ray single-crystal diffraction analysis. Our results confirmed that all the MB-Ln complexes yielded the coincident coordination modes with four MB ligands coordinating to the Ln(III) center. In vitro antibacterial screening on five typical bacteria strains revealed that the MB-Ln complexes exhibited antibacterial activities comparable with MB, as indicated by the MIC/MBC values, in which Escherichia coli and Salmonella typhi were the most sensitive ones to MB-Ln. Furthermore, the MB-Ln complexes were found to be much less toxic in vivo than MB, as suggested by the evaluated LD50 (50% lethal dose) values. All the MB-Ln series complexes fell in the LD50 range of 5000-15 000 mg kg-1, while the LD50 value of MB was only 1294 mg kg-1. Furthermore, MB-Lu, as the selected representative of MB-Ln, could effectively inhibit the activity of DNA gyrase, the same as MB, suggesting the primary antibacterial mechanism of the MB-Ln series. The results demonstrated the good prospects and potential of metal-based veterinary drugs with better drug performance.
Asunto(s)
Metales de Tierras Raras , Drogas Veterinarias , Estructura Molecular , Metales de Tierras Raras/farmacología , Metales de Tierras Raras/química , Fluoroquinolonas/farmacología , Antibacterianos/farmacología , Iones/químicaRESUMEN
In Arabidopsis thaliana, a family of four genes (HY1, HO2, HO3 and HO4) encode haem oxygenase (HO), and play a major role in phytochrome chromophore biosynthesis. To characterize the contribution of the various haem oxygenase isoforms involved in salt acclimation, the effects of NaCl on seed germination and primary root growth in Arabidopsis wild-type and four HO mutants (hy1-100, ho2, ho3 and ho4) were compared. Among the four HO mutants, hy1-100 displayed maximal sensitivity to salinity and showed no acclimation response, whereas plants over-expressing HY1 (35S:HY1) exhibited tolerance characteristics. Mild salt stress stimulated biphasic increases in RbohD transcripts and production of reactive oxygen species (ROS) (peaks I and II) in wild-type. ROS peak I-mediated HY1 induction and subsequent salt acclimation were observed, but only ROS peak I was seen in the hy1-100 mutant. A subsequent test confirmed the causal relationship of salt acclimation with haemin-induced HY1 expression and RbohD-derived ROS peak II formation. In atrbohD mutants, haemin pre-treatment resulted in induction of HY1 expression, but no similar response was seen in hy1-100, and no ROS peak II or subsequent salt acclimatory responses were observed. Together, the above findings suggest that HY1 plays an important role in salt acclimation signalling, and requires participation of RbohD-derived ROS peak II.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Hemo Oxigenasa (Desciclizante)/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Aclimatación , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Hemo Oxigenasa (Desciclizante)/genética , Mutagénesis Insercional , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Salinidad , Plantas Tolerantes a la Sal/enzimología , Plantas Tolerantes a la Sal/genéticaRESUMEN
In animals and recently in plants, heme oxygenase-1 (HO1) has been found to confer protection against a variety of oxidant-induced cell and tissue injuries. In this study, a wheat (Triticum aestivum) HO1 gene TaHO1 was cloned and sequenced. It encodes a polypeptide of 31.7 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of TaHO1 was found to be 78% similar to that of maize HO1. Phylogenetic analysis revealed that TaHO1 clusters together with the HO1-like sequences in plants. The purified recombinant TaHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXa (BV), and showed that the V(max) was 8.8 U·mg(-1) protein with an apparent K(m) value for hemin of 3.04 µM. The optimum Tm and pH were 35 °C and 7.4, respectively. The result of subcellular localization of TaHO1 showed that the putative transit peptide was sufficient for green fluorescent protein (GFP) to localize in chloroplast and implied that TaHO1 gene product is at least localized in the chloroplast. Moreover, we found that TaHO1 mRNA could be differentially induced by the well-known nitric oxide (NO) donor sodium nitroprusside (SNP), gibberellin acid (GA), abscisic acid (ABA), hydrogen peroxide (H(2)O(2)) and NaCl treatments. Therefore, the results suggested that TaHO1 might play an important role in abiotic stress responses.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hemo-Oxigenasa 1/genética , Estrés Fisiológico , Triticum/genética , Ácido Abscísico/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Hemo/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hemina/metabolismo , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Óxido Nítrico/metabolismo , Nitroprusiato/metabolismo , Filogenia , Alineación de Secuencia , Triticum/enzimología , Zea mays/genéticaRESUMEN
Marbofloxacin (MB) is a newly developed veterinary drug with broad-spectrum antibacterial activity. In this study, a new calcium(II)-based complex of marbofloxacin, MB-Ca, was synthesized and structurally characterized by IR, ESI-MS, UV-Vis and single crystal X-ray diffraction analysis. The characterization of this complex in solution state indicated that the coordinated MB-Ca was partly retained, along with the monomeric and dimeric forms of MB. It also showed satisfactory water solubility (1.89â¯mg/mL), comparing with MB (2.82â¯mg/mL) at 35⯰C. The in vitro antibacterial activity of MB-Ca was also screened towards a series of typical pathogenic bacteria, and determined by the methods of turbidimetry and disc diffusion. The results indicated it showed comparable antibacterial activity to MB. However, it exhibited higher inhibitive ability in vitro on DNA gyrase than MB alone. Furthermore, MB-Ca showed significantly lower acute toxicity (LD50, 3186â¯mg/kg) than MB (LD50, 1294â¯mg/kg) in mice, based on the in vivo acute toxicity test. The histopathological examination on the major organs of the mice by the oral administration of MB-Ca did not show obvious organic lesions, which is similar to those treated by MB. The research results suggest that MB-Ca could be further developed into a new promising metal-based veterinary drug and a better substitute of MB, showing unabated antibacterial activity along with lower toxicity.
Asunto(s)
Antibacterianos/síntesis química , Calcio/química , Complejos de Coordinación/síntesis química , Fluoroquinolonas/química , Hepatocitos/efectos de los fármacos , Compuestos Organometálicos/síntesis química , Animales , Antibacterianos/toxicidad , Bacterias/efectos de los fármacos , Complejos de Coordinación/toxicidad , Ratones , Compuestos Organometálicos/toxicidadRESUMEN
Both nitric oxide (NO) and reactive oxygen species (ROS) are versatile molecules that mediate a variety of cellular responses in plants. In this chapter, methods for imaging NO and ROS using laser scanning confocal microscopy (LSCM) are presented. Arabidopsis roots, dyed with DAF-FM or H(2)DCF, are observed using the Leica TCS-SP2 LSCM. NO or ROS production are imaged and their kinetic changes monitored with the laser excitation and emission wavelengths at 488 nm and between 500 and 530 nm, respectively. In addition, Leica software is employed to visualize and calculate the fluorescence intensity data.
Asunto(s)
Microscopía Confocal , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Plantones/metabolismo , Arabidopsis/fisiología , Microscopía Fluorescente , Imagen Molecular/métodos , Plantones/fisiología , Espectrometría de FluorescenciaRESUMEN
Previous results revealed that haem oxygenase-1 (HO-1)/carbon monoxide (CO) system is involved in auxin-induced adventitious root formation. In this report, a cDNA for the gene ZmHO-1, encoding an HO-1 protein, was cloned from Zea mays seedlings. ZmHO-1 has a conserved HO signature sequence and shares highest homology with rice SE5 (OsHO-1) protein. We further discovered that N-1-naphthylacetic acid (NAA), haemin, and CO aqueous solution, led to the induction of ZmHO-1 expression as well as the thereafter promotion of lateral root development. These effects were specific for ZmHO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) differentially blocked the above actions. The addition of haemin and CO were able to reverse the auxin depletion-triggered inhibition of lateral root formation as well as the decreased ZmHO-1 transcripts. Molecular evidence showed that the haemin- or CO-mediated the modulation of target genes responsible for lateral root formation, including ZmCDK and ZmCKI2, could be blocked by ZnPPIX. Overexpression of ZmHO-1 in transgenic Arabidopsis plants resulted in promotion of lateral root development as well as the modulation of cell cycle regulatory gene expressions. Overall, our results suggested that a maize HO-1 gene is required for the lateral root formation.
Asunto(s)
Genes de Plantas/genética , Hemo-Oxigenasa 1/genética , Proteínas de Plantas/genética , Raíces de Plantas/enzimología , Raíces de Plantas/crecimiento & desarrollo , Zea mays/enzimología , Zea mays/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Monóxido de Carbono/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Hemo-Oxigenasa 1/metabolismo , Hemina/farmacología , Ácidos Indolacéticos/farmacología , Datos de Secuencia Molecular , Ácidos Naftalenoacéticos/farmacología , Fenotipo , Filogenia , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Protoporfirinas/farmacología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/crecimiento & desarrollo , Análisis de Secuencia de ADN , Zea mays/crecimiento & desarrolloRESUMEN
It has been documented that plant heme oxygenase-1 (HO-1; EC 1.14.99.3) is both development- and stress-regulated, thus it plays a vital role in light signalling and stress responses. In this study, an alfalfa (Medica sativa L.) HO-1 gene MsHO1 was isolated and sequenced. It contains four exons and three introns within genomic DNA sequence and encodes a polypeptide with 283 amino acids. MsHO1 had a conserved HO signature sequence and showed high similarity to other HOs in plants, especially HO-1 isoform. The MsHO1:GFP fusion protein was localized in the chloroplast. Further biochemical activity analysis of mature MsHO1, which was expressed in Escherichia coli, showed that the Vmax was 48.78 nmol biliverdin-IXα (BV) h⻹ nmol⻹ protein with an apparent Km value for hemin of 2.33 µM, and the optimum Tm and pH were 37 °C and 7.2, respectively. Results of semi-quantitative RT-PCR and western blot showed that the expressions of MsHO1 were higher in alfalfa stems and leaves than those in germinating seeds and roots. Importantly, MsHO1 gene expression and protein level were induced significantly by some pro-oxidant compounds, including hemin and nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, MsHO1 may play an important role in oxidative responses.