RESUMEN
Magnetotactic bacteria (MTB) have the unique ability to produce magnetic particles surrounded by a biomembrane to form the magnetosome organelle. Therefore, MTB have novel physical and magnetic properties and have consequently been used in several biotechnological applications. The magnetic properties of these micro-organisms and their magnetosomes have, however, never been used for the generation of electricity as described in this letter. Comparisons were made between, firstly, the electricity generated from purified magnetosomes, MTB culture (bacterial cells with magnetosomes) and sterile, liquid growth medium (control). Secondly, the electricity generated by a dilution series of purified magnetosomes were compared. A statistically significant difference was found between the voltage measured from the purified magnetosomes (highest voltage), MTB culture (lower voltage) and liquid growth medium (lowest voltage). In the dilution series, the voltage measured increased as the magnetosome concentration increased, but only up to an optimum concentration (0·0376 mg ml-1 ). In this study, we have demonstrated that a significantly higher voltage than that of the control could be measured when MTB or purified magnetosomes were pumped through a solenoid by applying Faraday's law of electromagnetic induction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides proof-of-concept of electromagnetic induction using magnetosomes or magnetotactic bacteria in an experimental setup based on the law of Faraday. The concept of using these bacteria or their biomineralized magnetic nanoparticles as a biological alternative in low voltage electricity generation has the potential to be further explored and developed.
Asunto(s)
Electricidad , Fenómenos Electromagnéticos , Magnetosomas/metabolismo , Magnetospirillum/metabolismo , Nanopartículas de Magnetita , Prueba de Estudio ConceptualRESUMEN
We investigate here the potential of single step production of genetically engineered magnetosomes, bacterial biogenic iron-oxide nanoparticles embedded in a lipid vesicle, as a new tailorable magnetic resonance molecular imaging probe. We demonstrate in vitro the specific binding and the significant internalization into U87 cells of magnetosomes decorated with RGD peptide. After injection at the tail vein of glioblastoma-bearing mice, we evidence in the first 2 h the rapid accumulation of both unlabeled and functionalized magnetosomes inside the tumor by Enhanced Permeability and Retention effects. 24 h after the injection, a specific enhancement of the tumor contrast is observed on MR images only for RGD-labeled magnetosomes. Post mortem acquisition of histological data confirms MRI results with more magnetosomes found into the tumor treated with functionalized magnetosomes. This work establishes the first proof-of-concept of a successful bio-integrated production of molecular imaging probe for MRI.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Mejoramiento Genético/métodos , Magnetosomas/química , Magnetosomas/genética , Imagen Molecular/métodos , Oligopéptidos/farmacocinética , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Medios de Contraste/química , Imagen por Resonancia Magnética/métodos , Magnetosomas/ultraestructura , Ratones , Técnicas de Sonda Molecular , Sondas Moleculares/química , Nanoconjugados/química , Nanoconjugados/ultraestructura , Oligopéptidos/química , Distribución TisularRESUMEN
The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Butionina Sulfoximina/farmacología , Glutatión/metabolismo , Metales/toxicidad , Radioisótopos/toxicidad , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Transportadoras de Casetes de Unión a ATP/genética , Inactivación Metabólica , Cloruro de Mercurio/toxicidad , Plásmidos , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The virion-associated Vpr protein of human immunodeficiency virus type 1 (HIV-1) alters cell cycle progression from the G2 phase, influences the virus in vivo mutation rate, and participates in the nuclear translocation of viral DNA. While many Vpr-interacting proteins have been identified, the functional relevance of these interactions remains to be thoroughly documented. We have explored the contribution of the interaction of HIV-1 Vpr with HHR23A, a cellular protein implicated in DNA repair, to the known phenotypes of Vpr. The association of Vpr with HHR23A required the core region of Vpr, which encompasses the two alpha-helical structures of the protein. No binding of HHR23A was detected with the Vpr and Vpx proteins of other primate lentiviruses. HIV-1 Vpr variants containing single amino acid substitutions in each alpha-helix and deficient for binding to HHR23A were isolated. The functional characterization of these Vpr variants indicated that binding to HHR23A did not correlate with the ability of Vpr to induce cell cycle arrest, even though it was previously proposed that HHR23A is a mediator of the Vpr-induced G2 arrest. Also, the Vpr-HHR23A interaction did not influence the HIV-1 in vivo mutation rate. Finally, Vpr and HHR23A are both localized in the nucleus, but no correlation was observed between the nuclear targeting of Vpr and the interaction with HHR23A. Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Productos del Gen vpr/metabolismo , VIH-1/metabolismo , Proteínas de los Retroviridae/metabolismo , Sustitución de Aminoácidos , Animales , Células COS , Ciclo Celular , Núcleo Celular/metabolismo , Enzimas Reparadoras del ADN , Vectores Genéticos , Células HeLa , Humanos , Transfección , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.
Asunto(s)
ADN Glicosilasas , Productos del Gen vpr/metabolismo , VIH-1/genética , Mutación , N-Glicosil Hidrolasas/metabolismo , Animales , Secuencia de Bases , Células COS , Núcleo Celular/enzimología , Productos del Gen vpr/genética , Prueba de Complementación Genética , Vectores Genéticos , VIH-1/fisiología , Células HeLa , Humanos , Datos de Secuencia Molecular , Técnicas del Sistema de Dos Híbridos , Uracil-ADN Glicosidasa , Virión/metabolismo , Replicación Viral , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Hexitol nucleic acid (HNA) is a new steric blocking oligonucleotide, hybridizing sequence selectively with RNA. The biological activity of HNA was evaluated in an in vitro translation arrest system targeting Ha-ras mRNA and in a cellular system targeting intracellular adhesion molecule-1 (ICAM-1) expression. HNA very efficiently and selectively inhibited Ha-ras mRNA translation (IC(50) of 50 nM) when targeted at the translation initiation region. When targeting at the 12th codon region, a gap-mer approach was needed to inhibit mRNA translation. Similarly, HNA inhibited ICAM-1 expression in keratinocytes when targeting at codon sequences. In this test system, HNA is less active but more selective than phosphorothioates, but needs lipofection to become active in keratinocytes. This new steric blocker may be an efficient antisense agent providing that enough material can be brought into cells.
Asunto(s)
Genes ras/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/efectos de los fármacos , Oligonucleótidos/farmacología , Células Cultivadas , Citocinas/farmacología , Expresión Génica/efectos de los fármacos , Genes ras/genética , Humanos , Molécula 1 de Adhesión Intercelular/genética , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Oligonucleótidos/química , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/efectos de los fármacosRESUMEN
Vpr and Vpx proteins from human and simian immunodeficiency viruses (HIV and SIV) are incorporated into virions in quantities equivalent to those of the viral Gag proteins. We demonstrate here that Vpr and Vpx proteins from distinct lineages of primate lentiviruses were able to bind to their respective Gag precursors. The capacity of HIV type 1 (HIV-1) Vpr mutants to bind to Pr55(Gag) was correlated with their incorporation into virions. Molecular analysis of these interactions revealed that they required the C-terminal p6 domain of the Gag precursors. While the signal for HIV-1 Vpr binding lies in the leucine triplet repeat region of the p6 domain reported to be essential for incorporation, SIVsm Gag lacking the equivalent region still bound to SIVsm Vpr and Vpx, indicating that the determinants for Gag binding are located upstream of this region of the p6 domain. Binding to Gag cleavage products showed that HIV-1 Vpr interacted directly with the nucleocapsid protein (NC), whereas SIVsm Vpr and Vpx did not interact with NC but with the p6 protein. These results (i) reveal differences between HIV-1 and SIVsm for the p6 determinants required for Vpr and Vpx binding to Gag and (ii) suggest that HIV-1 Vpr and SIVsm Vpr and Vpx interact with distinct cleavage products of the precursor following proteolytic processing in the virions.