RESUMEN
BACKGROUND: Hydroxyapatite (HAp) presents similarities with the human bone structure and presents properties such as biodegradability, biocompatibility, and osteoconductivity, which favors its use in prostheses implants and enables its use as a vehicle for the delivery of photosensitizers (PS) from systems of release (DDS) for photodynamic therapy applications Methods: In this work was to synthesized hydroxyapatite microspheres (meHAp), encapsulated with chloroaluminium phthalocyanine (ClAlPc), for DDS. meHAp was synthesized using vaterite as a template. The drug was encapsulated by mixing meHAp and a 50.0 mg.mL-1 ClAlPc solution. Photochemical, photophysical, and photobiological studies characterized the system. RESULTS: The images from the SEM analysis showed the spherical form of the particles. All spectroscopic results showed excellent photophysical parameters of the drug studied when served in the meHAp system. The incorporation efficiency was 57.8 %. The trypan blue exclusion test results showed a significant reduction (p < 0.05) in cell viability for the groups treated with PDT at all concentrations above 250 µg.mL-1. In 9 L/lacZ gliosarcoma cells, PDT mediated at concentrations from 250 to 62.5 µg.mL-1 reduced cell viability by more than 98 %. In the cell internalization study, it was possible to observe the internalization of phthalocyanines at 37 °C, with the accumulation of PS in the cytoplasm and inside the nucleus in the two tested concentrations. CONCLUSIONS: From all the results presented throughout the article, the meHAp system shows promise for use as a modified release system (DSD) in photodynamic therapy.
Asunto(s)
Gliosarcoma , Fotoquimioterapia , Humanos , Fármacos Fotosensibilizantes , Fotoquimioterapia/métodos , Durapatita , Operón Lac , Microesferas , Sistemas de Liberación de MedicamentosRESUMEN
Development delivery systems, such as nanoparticles, represent a growing area in biomedical research. Nanoparticles (NP) were prepared using a double-emulsion method to load zinc(II) phthalocyanine (ZnPc). NP were obtained using poly (lactic acid) (PLA). ZnPc is a second generation of photosensitizer used in photodynamic therapy (PDT). ZnPc loaded PLA nanoparticles (NPLA-ZnPc) were prepared by double-emulsion method, characterized and available in cellular culture. The mean nanoparticle size presented particle size was 384.7 ± 84.2 nm with polydispersity index (PDI) of 0.150 ± 0.015, and the encapsulation efficiency was of 83%. The nanoparticle formulations presented negative zeta potential values (-27.5 ± 1.0 mV), explaining their colloidal stability. ZnPc loaded nanoparticles maintain its photophysical behavior after encapsulation. Photosensitizer release from nanoparticles was sustained over 168 h with a biphasic ZnPc release profile. An in vitro phototoxic effect in range of 80% was observed in 9 L/LacZ gliosarcoma cells at laser light doses (10 J cm-2) with 3.0 µg mL-1 of NPLA-ZnPc. All the physical-chemical, photophysical and photobiological measurements performed allow us to conclude that ZnPc loaded PLGA nanoparticles is a promising drug delivery system for PDT.
Asunto(s)
Gliosarcoma , Nanopartículas , Compuestos Organometálicos , Fotoquimioterapia , Emulsiones , Humanos , Operón Lac , Ácido Láctico , Fármacos Fotosensibilizantes , Poliésteres , Zinc , Compuestos de ZincRESUMEN
Gliosarcoma is an aggressive brain tumor. Photodynamic Therapy (PDT) is a treatment that can be used for various cancers of the CNS. The aim of this study was to analyze the effects of PDT with Photodithazine (PDZ) in the treatment of gliosarcoma, using 9â¯L/lacZ cells and serial concentrations of 200⯵g/mL to 3.1⯵g/mL of PDZ. The samples were divided into two groups: dark and light (10â¯J/cm²). The PDZ was internalized along all the cytoplasmic extension. Viability tests demonstrated a reduction in viable cells after PDT. The production of ROS was concentration-dependent and PDZ was found in mitochondria and lysosomes, presenting a discrete connection with α-tubulin. However, this structure is likely damaged, evidenced by changes in the morphological analysis. Thus, according to the parameters of this study, PDZ proved to be an interesting PS in PDT for the treatment of gliosarcoma, with the inherent limitations of an in vitro study.
Asunto(s)
Gliosarcoma , Fotoquimioterapia , Línea Celular Tumoral , Gliosarcoma/tratamiento farmacológico , Glucosamina/análogos & derivados , Humanos , Operón Lac , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
BACKGROUND: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. RESULTS: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. CONCLUSIONS: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.
Asunto(s)
Cromosomas Bacterianos , Ingeniería Genética/métodos , Integrasas/genética , Rhizobium etli/enzimología , Rhizobium etli/genética , Conjugación Genética , ADN , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Replicación del ADN , Escherichia coli/genética , Citometría de Flujo , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Operón Lac , Plásmidos/genética , Regiones Promotoras Genéticas , Recombinación GenéticaRESUMEN
Natural transformation is one mechanism of horizontal gene transfer (HGT) in Vibrio cholerae, the causative agent of cholera. Recently, it was found that V. cholerae isolates from the Haiti outbreak were poorly transformed by this mechanism. Here, we show that an integrating conjugative element (ICE)-encoded DNase, which we name IdeA, is necessary and sufficient for inhibiting natural transformation of Haiti outbreak strains. We demonstrate that IdeA inhibits this mechanism of HGT in cis via DNA endonuclease activity that is localized to the periplasm. Furthermore, we show that natural transformation between cholera strains in a relevant environmental context is inhibited by IdeA. The ICE encoding IdeA is globally distributed. Therefore, we analyzed the prevalence and role for this ICE in limiting natural transformation of isolates from Bangladesh collected between 2001 and 2011. We found that IdeA(+) ICEs were nearly ubiquitous in isolates from 2001 to 2005; however, their prevalence decreased to â¼40% from 2006 to 2011. Thus, IdeA(+) ICEs may have limited the role of natural transformation in V. cholerae. However, the rise in prevalence of strains lacking IdeA may now increase the role of this conserved mechanism of HGT in the evolution of this pathogen.
Asunto(s)
Proteínas Bacterianas/genética , Transferencia de Gen Horizontal , Secuencias Repetitivas Esparcidas , Transformación Bacteriana , Vibrio cholerae/genética , Proteínas Bacterianas/fisiología , Bangladesh , Quitina/química , Cólera/genética , Cólera/microbiología , Conjugación Genética , ADN/metabolismo , ADN Bacteriano/genética , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas/química , Evolución Molecular , Haití , Humanos , Operón Lac , Modelos Genéticos , Mutación , Periplasma/metabolismo , Filogenia , Prevalencia , Vibrio cholerae/metabolismo , beta-Lactamasas/metabolismoRESUMEN
The developmental brain gene NPAS3 stands out as a hot spot in human evolution because it contains the largest number of human-specific, fast-evolving, conserved, non-coding elements. In this paper we studied 2xHAR142, one of these elements that is located in the fifth intron of NPAS3. Using transgenic mice, we show that the mouse and chimp 2xHAR142 orthologues behave as transcriptional enhancers driving expression of the reporter gene lacZ to a similar NPAS3 expression subdomain in the mouse central nervous system. Interestingly, the human 2xHAR142 orthologue drives lacZ expression to an extended expression pattern in the nervous system. Thus, molecular evolution of 2xHAR142 provides the first documented example of human-specific heterotopy in the forebrain promoted by a transcriptional enhancer and suggests that it may have contributed to assemble the unique properties of the human brain.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Evolución Molecular , Regulación de la Expresión Génica/genética , Proteínas del Tejido Nervioso/genética , Prosencéfalo/metabolismo , Factores de Transcripción/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biología Computacional , Secuencia Conservada/genética , Cartilla de ADN/genética , Galactósidos , Humanos , Inmunohistoquímica , Hibridación in Situ , Indoles , Operón Lac/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Pan troglodytes/genética , Elementos de Nucleótido Esparcido Corto/genética , Especificidad de la Especie , Factores de Transcripción/metabolismoRESUMEN
Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (P(trc)), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacI(q) gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including P(trc)melA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110P(tr)(c)melA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110P(tr)(c)melA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems.
Asunto(s)
Cromosomas Bacterianos/genética , Escherichia coli/genética , Melaninas/metabolismo , Medios de Cultivo/metabolismo , Electroporación , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Fermentación , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Vectores Genéticos/genética , Operón Lac , Melaninas/genética , Plásmidos/genética , Regiones Promotoras Genéticas , Rhizobium etli/genética , Transformación Genética , Tirosina/genética , Tirosina/metabolismoRESUMEN
BACKGROUND: DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. RESULTS: Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML-/-, Daxx-/-, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. CONCLUSION: Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Línea Celular , Herpesvirus Humano 1/genética , Papillomavirus Humano 11/genética , Humanos , Operón Lac , Regiones Operadoras Genéticas , Unión Proteica , Virus 40 de los Simios/genéticaRESUMEN
Reporter gene assays are important tools for evaluating gene expression. A frequently used assay measures the activity of ß-galactosidase (ß-gal) expressed from lacZ in plasmid or genomic constructions. Such constructions are often used to interrogate the ability of DNA (query DNA), potentially encoding a transcription factor, to regulate in trans the expression of a promoter fused to the reporter lacZ. Query DNA is frequently inserted into a second plasmid within the α-subunit of ß-gal, interrupting its function. However, this plasmid can induce up-expression of ß-gal even when void of query DNA, leading to confusion between artifact and authentic regulation.
Asunto(s)
Regulación de la Expresión Génica , Genes Reporteros , beta-Galactosidasa/genética , Artefactos , Escherichia coli/enzimología , Escherichia coli/genética , Técnicas Genéticas , Operón Lac , Modelos Genéticos , Plásmidos/genética , Regiones Promotoras Genéticas , Proteobacteria , beta-Galactosidasa/químicaRESUMEN
A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cupriavidus/genética , Proteínas de Unión al ADN/genética , Silenciador del Gen , Metales/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Activación Transcripcional , Citometría de Flujo , Fluorescencia , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Operón LacRESUMEN
In this paper, the history and importance of the lac operon in the development of molecular and systems biology are briefly reviewed. We start by presenting a description of the regulatory mechanisms in this operon, taking into account the most recent discoveries. Then we offer a survey of the history of the lac operon, including the discovery of its main elements and the subsequent influence on the development of molecular and systems biology. Next the bistable behaviour of the operon is discussed, both with respect to its discovery and its molecular origin. A review of the literature in which this bistable phenomenon has been studied from a mathematical modelling viewpoint is then given. We conclude with some brief remarks.
Asunto(s)
Escherichia coli/genética , Operón Lac , Escherichia coli/enzimología , Inestabilidad Genómica , Matemática , Proteínas de Transporte de Membrana/genética , Modelos Genéticos , Biología Molecular , Biología de SistemasRESUMEN
Malpighian tubules constitute the main excretion organ of insects. Infection by egt(-) recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt(-), and used another two recombinants (one egt(-)) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt(-) viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.
Asunto(s)
Lepidópteros/anatomía & histología , Lepidópteros/virología , Túbulos de Malpighi/patología , Túbulos de Malpighi/virología , Nucleopoliedrovirus/patogenicidad , Animales , Muerte Celular , Línea Celular , Regulación Viral de la Expresión Génica , Genes de Insecto/genética , Genes Virales/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Operón Lac/genética , Larva/anatomía & histología , Larva/virología , Túbulos de Malpighi/ultraestructura , Microscopía Electrónica de Transmisión , Plásmidos/genéticaRESUMEN
Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.
Asunto(s)
Adhesinas Bacterianas/inmunología , Anticuerpos Antibacterianos/inmunología , Bordetella pertussis/inmunología , Hemaglutinación/inmunología , Lacticaseibacillus casei/genética , Factores de Virulencia de Bordetella/inmunología , Tos Ferina/prevención & control , Adhesinas Bacterianas/genética , Adyuvantes Inmunológicos , Animales , Anticuerpos Antibacterianos/genética , Femenino , Inmunidad Mucosa , Operón Lac , Ratones , Ratones Endogámicos BALB C , Vacuna contra la Tos Ferina/inmunología , Estructura Terciaria de Proteína , Proteínas Recombinantes/inmunología , Transformación Bacteriana , Vacunas Sintéticas/inmunología , Factores de Virulencia de Bordetella/genéticaRESUMEN
Cheese whey (CW) is the major subproduct from cheese manufacturing and it is considered as a waste pollutant since its high content of lactose. In this work a fermentation process for the production of penicillin acylase (PA) by a recombinant Escherichia coli and using CW as unique carbon source and inducer was developed. A design factorial 3(2) was used to evaluate the influence of independent variables (dissolved oxygen and CW concentration) on the ability of E. coli W3110/pPA102 to produce PA. Maximum specific PA activity of 781 U g(-1) was attained at 5 g L(-1) of CW and 3% dissolved oxygen. The results showed that CW can be used successfully as unique carbon source and inducer for the production of recombinant proteins using constructions driven by the lac promoter and this way reducing the discharges of that pollutant to the environment.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Penicilina Amidasa/biosíntesis , Proteínas Recombinantes/biosíntesis , Queso , Medios de Cultivo , Escherichia coli/enzimología , Escherichia coli/genética , Operón Lac , Lactosa/metabolismo , Penicilina Amidasa/genética , Proteínas Recombinantes/genéticaRESUMEN
The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.
Asunto(s)
Codón/genética , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Secuencia de Bases , Codón de Terminación/genética , Escherichia coli/genética , Operón Lac/genética , Terminación de la Cadena Péptídica Traduccional/genética , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
We have shown that apotransferrin (aTf) promotes the differentiation of two oligodendroglial cell (OLGc) lines, N19 and N20.1, representing different stages of OLGc maturation. Although in both cell lines aTf promoted myelin basic protein (MBP) expression, an increase in cAMP levels and CREB phosphorylation was observed only in the less mature cells (N19), suggesting that the maturation induced by aTf is achieved probably through different signaling pathways. We transfected both cell lines with the proximal region of the human MBP promoter fused to the lacZ reporter gene. In both transfected cell lines, addition of aTf produced an activation of the promoter. To elucidate the mechanisms involved in this action, Western blot analysis, EMSAs, and RT-PCR were performed for different transcription factors involved in mbp regulation. In the N20.1 line, treatment with aTf increased the expression and the DNA-binding capacity of thyroid hormone (TH) receptors, Sp1, and nuclear factor-kappaB (NFkappaB). For these cells we found that an inductor of NFkappaB (tumor necrosis factor-alpha) promoted MBP messenger synthesis, whereas mithramycin, a specific inibitor of Sp1, and a cAMP analog (db-cAMP) inhibited its transcription. In the N19 cell line, aTf stimulated NF-I and NFkappaB activation, but, aside from aTf, only db-cAMP induced mbp transcription. These data suggest that, depending on the OLGc maturational stage, aTf modulates MBP expression and OLGc differentiation through different signaling pathways and different transcription factors.
Asunto(s)
Apoproteínas/fisiología , Proteína Básica de Mielina/biosíntesis , Oligodendroglía/metabolismo , Factores de Transcripción/fisiología , Transferrina/fisiología , Western Blotting , Línea Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , ADN Complementario/biosíntesis , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Operón Lac/genética , FN-kappa B/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Plásmidos/genética , Regiones Promotoras Genéticas/genética , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/genética , Sales de Tetrazolio , Tiazoles , Hormonas Tiroideas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The previous report from our laboratory has recently identified a new trpE gene (termed trpE2) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE1(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is the first report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE1(G) while these sequence features did not exist in front of trpE2. The beta-galactosidase activity of an A. brasilense strain carrying a trpE2-lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE1(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE1(G) is regulated at the transcriptional level by attenuation while trpE2 is constantly expressed. The anthranilate synthase assays with trpE1(G)- and trpE2- mutants demonstrated that TrpE1(G) fusion protein is feedback inhibited by tryptophan while TrpE2 protein is not. We also found that both trpE1(G) and trpE2 gene products were involved in IAA synthesis.
Asunto(s)
Antranilato Sintasa/genética , Azospirillum brasilense/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Antranilato Sintasa/biosíntesis , Clonación Molecular , Relación Dosis-Respuesta a Droga , Regulación Bacteriana de la Expresión Génica , Marcadores Genéticos , Genotipo , Operón Lac , Datos de Secuencia Molecular , Mutación , Péptidos/química , Plásmidos/metabolismo , Señales de Clasificación de Proteína , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
The putative nifB promoter region of Herbaspirillum seropedicae contained two sequences homologous to NifA-binding site and a -24/-12 type promoter. A nifB::lacZ fusion was assayed in the backgrounds of both Escherichia coli and H. seropedicae. In E. coli, the expression of nifB::lacZ occurred only in the presence of functional rpoN and Klebsiella pneumoniae nifA genes. In addition, the integration host factor (IHF) stimulated the expression of the nifB::lacZ fusion in this background. In H. seropedicae, nifB expression occurred only in the absence of ammonium and under low levels of oxygen, and it was shown to be strictly dependent on NifA. DNA band shift experiments showed that purified K. pneumoniae RpoN and E. coli IHF proteins were capable of binding to the nifB promoter region, and in vivo dimethylsulfate footprinting showed that NifA binds to both NifA-binding sites. These results strongly suggest that the expression of the nifB promoter of H. seropedicae is dependent on the NifA and RpoN proteins and that the IHF protein stimulates NifA activation of nifB promoter.
Asunto(s)
Proteínas Bacterianas/genética , Herbaspirillum/genética , ARN Polimerasa Sigma 54/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Ensayo de Cambio de Movilidad Electroforética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Herbaspirillum/efectos de los fármacos , Herbaspirillum/metabolismo , Factores de Integración del Huésped/genética , Factores de Integración del Huésped/metabolismo , Operón Lac , Datos de Secuencia Molecular , Oxígeno/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Compuestos de Amonio Cuaternario/farmacología , ARN Polimerasa Sigma 54/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Light and stereomicroscopy examinations of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV)-infected insects were performed in order to follow infection in its host, A. gemmatalis. Fourth-instar A. gemmatalis larvae were infected by administration of occluded virus (polyhedra) from two recombinant AgMNPV viruses (vAgEGTDelta-lacZ or vAgGalA2) directly into the larvae foregut. The recombinant virus vAgEGTDelta-lacZ has the beta-galactosidase gene (lac-Z) of Escherichia coli under the control of a constitutive promoter (hsp70 from Drosophila melanogaster). The vAgGalA2 virus has the reporter gene lac-Z under the control of the AgMNPV very late polyhedrin gene promoter. At different times post-infection (p.i.) the infected larvae were dissected, fixed, and the product of the expression of the lac-Z gene detected by incubating the insects in a buffer containing X-gal. This allowed us to follow the infection through the blue cells (due to the degradation of X-gal by the enzyme Lac-Z). Insect larvae inoculated with polyhedra from the recombinant viruses showed midgut cells to be infected first, followed by tracheal cells, hemolymph, fat body, Malpighian tubules and brain cells. The infection was similar for the two recombinant viruses, with blue cells appearing earlier in insects infected with the vAgEGTDelta-lacZ virus when compared to the vAgGalA2 virus.