RESUMO
Prostate cancer is frequently treated with radiotherapy. Unfortunately, aggressive radioresistant relapses can arise, and the molecular underpinnings of radioresistance are unknown. Modern clinical radiotherapy is evolving to deliver higher doses of radiation in fewer fractions (hypofractionation). We therefore analyzed genomic, transcriptomic and proteomic data to characterize prostate cancer radioresistance in cells treated with both conventionally fractionated and hypofractionated radiotherapy. Independent of fractionation schedule, resistance to radiotherapy involved massive genomic instability and abrogation of DNA mismatch repair. Specific prostate cancer driver genes were modulated at the RNA and protein levels, with distinct protein subcellular responses to radiotherapy. Conventional fractionation led to a far more aggressive biomolecular response than hypofractionation. Testing pre-clinical candidates identified in cell lines, we revealed POLQ (DNA Polymerase Theta) as a radiosensitizer. POLQ-modulated radioresistance in model systems and was predictive of it in large patient cohorts. The molecular response to radiation is highly multi-modal, and sheds light on prostate cancer lethality.
RESUMO
PURPOSE: Improved antitumor responses have been observed in patients after combination radiation therapy (RT) and immune checkpoint blockade (ICB). Whether these clinical responses are linked to the host systemic immune system has not been elucidated. METHODS AND MATERIALS: In this single-institution prospective observational study, peripheral blood was longitudinally collected from 10 patients with metastatic disease who had responded to anti-PD-1/anti-PD-L1 ICB and received RT (8-50 Gy in 1-5 fractions) upon disease progression at the following timepoints: baseline (pre-RT), 1 to 2 weeks post-RT, and post-ICB (cycle 1) on reintroduction post-RT. To thoroughly characterize the interaction between combined RT-ICB and the host immune system, we performed high-dimensional, mass cytometry-based immunophenotyping of circulating lymphocytes using a 40-marker panel addressing lineage, differentiation, activation, trafficking, cytotoxicity, and costimulatory and inhibitory functions. Phenotypic expression of circulating lymphocytes was compared across patients and time points and correlated with post-RT tumor responses. RESULTS: Foremost, we demonstrated excellent posttreatment clinical responses, including 4 local responses with >50% reduction in radiated tumor size, 1 out-of-field response, and 4 patients who resumed ICB for >1 year. Baseline and post-RT immune states were highly heterogeneous among patients. Despite this interindividual heterogeneity in baseline immune states, we observed a systemic immune reaction to RT-ICB common across patients, histology, and radiation sites; a subset of pre-existing Ki-67+ CD8+ T cells were increased post-RT and further expanded upon reintroduction of ICB post-RT (2.3-fold increase, P = .02). Importantly, RT did not alter the phenotypic profile of these Ki-67+ CD8+ T cells, which was characterized by a distinct activated and differentiated effector phenotype. CONCLUSIONS: Collectively, these findings point toward a sustained reinvigoration of host antitumor immunity after RT-ICB and suggest an expansion in activated Ki-67+ CD8+ T cells as a possible demonstration of this synergy, thereby providing new insights that may support the development of optimal sequencing strategies.
Assuntos
Inibidores de Checkpoint Imunológico/farmacologia , Radioterapia , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Sobrevivência Celular/efeitos da radiação , Terapia Combinada , Humanos , Imunofenotipagem , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/efeitos da radiação , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/efeitos da radiaçãoRESUMO
Upconversion nanoparticles (UCNPs) are the preferred choice for deep-tissue photoactivation, owing to their unique capability of converting deep tissue-penetrating near-infrared light to UV/visible light for photoactivation. Programmed photoactivation of multiple molecules is critical for controlling many biological processes. However, syntheses of such UCNPs require epitaxial growth of multiple shells on the core nanocrystals and are highly complex/time-consuming. To overcome this bottleneck, we have modularly assembled two distinct UCNPs which can individually be excited by 980/808 nm light, but not both. These orthogonal photoactivable UCNPs superballs are used for programmed photoactivation of multiple therapeutic processes for enhanced efficacy. These include sequential activation of endosomal escape through photochemical-internalization for enhanced cellular uptake, followed by photocontrolled gene knockdown of superoxide dismutase-1 to increase sensitivity to reactive oxygen species and finally, photodynamic therapy under these favorable conditions. Such programmed activation translated to significantly higher therapeutic efficacy in vitro and in vivo in comparison to conventional, non-programmed activation.
Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Processos Fotoquímicos/efeitos da radiação , Animais , Compostos de Cálcio/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/efeitos da radiação , Desenho de Fármacos , Endossomos/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HeLa , Humanos , Indóis/administração & dosagem , Indóis/farmacocinética , Raios Infravermelhos , Isoindóis , Camundongos , Nanopartículas/efeitos da radiação , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/farmacocinética , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Silicatos/química , Superóxido Dismutase-1/genética , Distribuição Tecidual , Raios Ultravioleta , Compostos de ZincoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) overexpression is characteristic in head and neck cancers and is associated with tumour regrowth following photodynamic therapy (PDT). PURPOSE: We investigated vandetanib, which selectively blocks EGFR and vascular endothelial growth factor receptor-2 (VEGFR-2), to enhance the efficacy of PDT. METHODS: We assessed the in vitro therapeutic efficacy of: 1) vandetanib; 2) PDT with the photosensitizer Chlorin e6 (Fotolon®); and 3) combined PDTâ¯+â¯vadetanib treatment in CAL-27 oral squamous cell carcinoma (OSCC) cell line by cell viability, γH2AX foci immunostaining, cell cycle arrest and western blot. We also performed in vivo tumour regression study and immunohistochemical staining of formalin-fixed paraffin-embedded (FFPE) regressed and regrown tumour tissues. RESULTS: First, we observed significantly higher cytotoxicity and residual DNA damage in vandetanibâ¯+â¯PDT-treated CAL-27 OSCC cells than tumour cells treated with PDT alone. This is due to impaired DNA DSB repair caused by downregulation of EGFR-mediated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activation. Next, combined vandetanib and PDT resulted in significant tumour growth delay in vivo that is linked to reduction of PDT-induced EGFR phosphorylation and cellular proliferation, along with loss of tumour vasculature. In particular, we observed significant revascularisation of the microenvironment that is associated with upregulated ERK1/2 phosphorylation in regrown tumours post-vandetanibâ¯+â¯PDT, thereby corroborating the importance of microenvironmental modification for the observed drug-PDT synergistic interaction. CONCLUSION: Taken together, our data suggests that vandetanib enhances the efficacy of PDT through both direct and indirect effects on the cellular DNA repair machinery and tumour microenvironment, respectively.
Assuntos
Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Piperidinas/farmacologia , Porfirinas/farmacologia , Quinazolinas/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Clorofilídeos , Dano ao DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/metabolismo , Regulação para Baixo , Quimioterapia Combinada , Receptores ErbB/antagonistas & inibidores , Humanos , Camundongos , Camundongos Nus , Microambiente Tumoral/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Marine litter pollution is a global environmental problem. Beach litter is a part of this problem, and is widely monitored in Europe. The European Marine Strategy Framework Directive (MSFD) requires a reduction of beach litter. A reduction of 30% has been proposed in the European Plastics Strategy. The aims of this study are to develop (a) a method to calculate sufficiently stable and precise baseline values for beach litter, and (b) to derive a method of power analysis to estimate the number of beach litter surveys, necessary to detect a given reduction, using these baseline values. Beach litter data from the OSPAR (Oslo Paris Convention) region were used, and tailor-made statistical methods were implemented in open source software, litteR. Descriptive statistics and Theil-Sen and Mann-Kendall trend analyses were calculated for the most abundant beach litter types, for 14 survey sites. The length of a baseline period necessary to obtain a specified precision of the mean baseline value, expressed as Coefficient of Variation (CV), was calculated. Power analyses were performed using Monte Carlo simulations combined with Wilcoxon tests to determine significant deviations of the simulated datasets from the baseline mean values. For most survey sites, the mean length of monitoring periods necessary to achieve a CV < 10% amounts to four to five years with four surveys a year. The mean number of surveys necessary to detect a statistically significant reduction of 30% with 80% power ranges from 14 to 20. Power analyses show that a reduction of 10% is difficult to detect, because more than 24 surveys are needed. In contrary, a reduction of 40-50% can be detected easily with a small (<12) number of surveys. The new methods could also be applied to other areas where similar beach litter surveys are performed.
Assuntos
Praias/estatística & dados numéricos , Monitoramento Ambiental/métodos , Poluição Ambiental/estatística & dados numéricos , Plásticos/análise , Resíduos/estatística & dados numéricos , Ecossistema , Europa (Continente) , SoftwareRESUMO
AIMS: To determine the feasibility of formulating and aerosolizing powders containing bacteriophages KS4-M and ΦKZ for lung delivery and treatment of pulmonary Burkholderia cepacia complex and Pseudomonas aeruginosa infections. METHODS AND RESULTS: Endotoxin-removed bacteriophages KS4-M and ΦKZ were lyophilized in lactose/lactoferrin 60 : 40 w/w matrix and deagglomerated in a mixer mill (without beads) to formulate respirable powders. The powders were then aerosolized using an Aerolizer(®) capsule inhaler. Mass median aerodynamic diameter (MMAD) of this inhalable aerosol was determined using Andersen cascade impactor at 60 l min(-1). Measured MMAD for both types of powders was 3·4 µm, and geometric standard deviation was 1·9-2·0. Viability of bacteriophages delivered distal to an idealized mouth-throat replica was determined from bioassays of samples collected on filters placed after the idealized replica. As a percentage of inhaler load, amount of powder delivered distal to the mouth-throat replica, which is a measure of lung delivery, was 33·7 ± 0·3% for KS4-M and 32·7 ± 0·9% for ΦKZ. Titres collected downstream of the mouth throat were (3·4 ± 2·5) × 10(6) PFU for KS4-M with an Aerolizer capsule load of (9·8 ± 4·8) × 10(6) and (1·9 ± 0·6) × 10(7) for ΦKZ with an Aerolizer capsule load of (6·5 ± 1·9) × 10(7). CONCLUSIONS: Bacteriophages KS4-M and ΦKZ can be lyophilized without significant loss of viability in a lactose/lactoferrin 60 : 40 w/w matrix. The resulting powders can be aerosolized to deliver viable bacteriophages to the lungs. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of lactoferrin-based bacteriophage aerosol powders solidifies the ground for future research on developing novel formulations as an alternative to inhaled antibiotic therapy in patients with cystic fibrosis.
Assuntos
Bacteriófagos , Infecções por Burkholderia/terapia , Complexo Burkholderia cepacia , Fibrose Cística/complicações , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa , Aerossóis , Bacteriófagos/ultraestrutura , Inaladores de Pó Seco , Liofilização , Humanos , Lactoferrina/análise , Pulmão , Nebulizadores e VaporizadoresRESUMO
The fission yeast Schizosaccharomyces pombe CBS 356 exhibits extracellular maltase activity. This activity may be of commercial interest as it exhibited a low pH optimum (3.5) and a high affinity for maltose (Km of 7.0+/-1.8 mM). N-terminal sequencing of the protein indicates that it is the product of the AGL1 gene. Regulation of this gene occurs via a derepression/repression mechanism. In sugar- or nitrogen-limited chemostat cultures, the specific rate of enzyme production (q(p)) was independent of the nature of the carbon source (i.e. glucose or maltose), but synthesis was partially repressed by high sugar concentrations. Furthermore, q(p) increased linearly with specific growth rate (mu) between 0.04 and 0.10 h(-1). The enzyme is easily mass-produced in aerobic glucose-limited fed-batch cultures, in which the specific growth rate is controlled to prevent alcoholic fermentation. In fed-batch cultures in which biomass concentrations of 83 g L(-1) were attained, the enzyme concentration reached 58,000 Units per liter culture supernatant. Extracellular maltase may be used as a dough additive in order to prevent mechanisms such as maltose-induced glucose efflux and maltose-hypersensitivity that occur in maltose-consuming Saccharomyces cerevisiae.
Assuntos
Schizosaccharomyces/enzimologia , alfa-Glucosidases/metabolismo , Aerobiose , Sequência de Aminoácidos , Biomassa , Indução Enzimática , Fermentação , Glucose/metabolismo , Maltose/metabolismo , Dados de Sequência Molecular , Análise de Sequência de Proteína , Homologia de Sequência , alfa-Glucosidases/biossíntese , alfa-Glucosidases/química , alfa-Glucosidases/genéticaRESUMO
Amendment of soil with Trichoderma koningii strain Tr5 grown on autoclaved white millet grain provided between 63 and 79% control of white rot of onion when added to soil containing 10, 25, 50, or 100 sclerotia of Sclerotium cepivorum per kilogram of soil at the time of onion seed sowing. There was no significant difference in the proportion of S. cepivorum infections suppressed among the different sclerotial density treatments. Rhizosphere colonization by T. koningii Tr5 was assessed by incubating onion roots sampled from plants growing in soil with the appropriate density of sclerotia, on a Trichoderma selective medium (Rose bengall-Allisan-streptomycin-Previcur agar) developed for the purpose of the study. Trichoderma spp. isolated were typed by comparison of culture morphology as well as polygalacturonase (PG) (EC 3.2.1.15) and pectinesterase (PE) (EC 3.1.1.11) isozyme profiles to the series of one PG and two PE isozymes known to be produced by T. koningii Tr5. The method was used successfully to assess rhizosphere colonization. Three rates of a millet grain formulation colonized by T. koningii Tr5 were added to soil (1,590, 3,180, and 4,770 kg/ha). At the lowest of these rates, 97% of roots were found to be colonized by isolates which could not be distinguished from T. koningii Tr5, whereas 8% of the roots from nontreated controls were colonized by such isolates. An objective of the study was to determine whether the ability of T. koningii Tr5 to suppress S. cepivorum infections was influenced by increased concentrations of both S. cepivorum sclerotia and T. koningii Tr5-colonized millet grain, and it was found that no further improvements in the percentage of disease suppression were recorded as a result of adding T. koningii Tr5-colonized millet to the soil at more than 1,590 kg/ha at any of the sclerotium concentrations tested.
RESUMO
The distribution of quorum-sensing genes among strains from seven genomovars of the Burkholderia cepacia complex was examined by PCR. cepR and cepI were amplified from B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis. cepR was also amplified from B. multivorans and B. cepacia genomovar VI. bviIR were amplified from B. vietnamiensis. All genomovars produced N-octanoyl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. B. vietnamiensis and B. cepacia genomovar VII produced additional N-acyl-L-homoserine lactones.
Assuntos
4-Butirolactona/análogos & derivados , Proteínas de Bactérias/genética , Burkholderia cepacia/genética , Genes Bacterianos , Ligases , 4-Butirolactona/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Burkholderia/genética , Homosserina/análogos & derivados , Homosserina/biossíntese , Lactonas , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A gene was cloned from Burkholderia cepacia DBO1 that is homologous with Escherichia coli cysH encoding 3'-phosphoadenylylsulfate (PAPS) reductase. The B. cepacia gene is the most recent addition to a growing list of cysH homologs from a diverse group of sulfate-assimilating bacteria whose products show greater homology to plant 5'-adenylylsulfate (APS) reductase than they do to E. coli CysH. The evidence reported here shows that the cysH from one of the species, Pseudomonas aeruginosa, encodes APS reductase. It is able to complement an E. coli cysH mutant and a cysC mutant, indicating that the enzyme is able to bypass PAPS, synthesized by the cysC product. Insertional knockout mutation of P. aeruginosa cysH produced cysteine auxotrophy, indicating its role in sulfate assimilation. Purified P. aeruginosa CysH expressed as a His-tagged recombinant protein is able to reduce APS, but not PAPS. The enzyme has a specific activity of 5.8 micromol. min(-1). mg of protein(-1) at pH 8.5 and 30 degrees C with thioredoxin supplied as an electron donor. APS reductase activity was detected in several bacterial species from which the novel type of cysH has been cloned, indicating that this enzyme may be widespread. Although an APS reductase from dissimilatory sulfate-reducing bacteria is known, it shows no structural or sequence homology with the assimilatory-type APS reductase reported here. The results suggest that the dissimilatory and assimilatory APS reductases evolved convergently.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia cepacia/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Oxirredutases/genética , Oxirredutases/metabolismo , Adenosina Fosfossulfato/metabolismo , Sequência de Aminoácidos , Bactérias/enzimologia , Bactérias/genética , Burkholderia cepacia/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Oxirredutases/classificação , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Sulfatos/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismoRESUMO
Induction of the membrane-associated organic solvent efflux system SrpABC of Pseudomonas putida S12 was examined by cloning a 312-bp DNA fragment, containing the srp promoter, in the broad-host-range reporter vector pKRZ-1. Compounds that are capable of inducing expression of the srpABC genes include aromatic and aliphatic solvents and alcohols. General stress conditions such as pH, temperature, NaCl, or the presence of organic acids did not induce srp transcription. Although the solvent efflux pump in P. putida S12 is a member of the resistance-nodulation-cell division family of transporters, the srpABC genes were not induced by antibiotics or heavy metals.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Pseudomonas putida/genética , Solventes , Antibacterianos/farmacologia , Transporte Biológico Ativo , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Concentração de Íons de Hidrogênio , Óperon Lac , Metais Pesados , Plasmídeos , Regiões Promotoras Genéticas , Pseudomonas putida/crescimento & desenvolvimento , Pseudomonas putida/metabolismo , Temperatura , Ativação TranscricionalAssuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos/genética , Mutagênese Sítio-Dirigida , Sítios de Ligação/genética , Cloranfenicol/farmacologia , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Polimerase Dirigida por DNA , Canamicina/farmacologia , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , Deleção de Sequência , Trimetoprima/farmacologiaRESUMO
A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon's site of insertion has been developed. The basic plasposon, named TnMod, consists of the Tn5 inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn5 transposase, in combination with the flexibility afforded by the modular arrangement of TnMod, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.
Assuntos
DNA Bacteriano/análise , Bactérias Gram-Negativas/genética , Elementos de DNA Transponíveis/genética , Resistência Microbiana a Medicamentos/genética , Genoma Bacteriano , Dados de Sequência Molecular , Mutagênese , Origem de ReplicaçãoRESUMO
Bacteria able to grow in aqueous:organic two-phase systems have evolved resistance mechanisms to the toxic effects of solvents. One such mechanism is the active efflux of solvents from the cell, preserving the integrity of the cell interior. Pseudomonas putida S12 is resistant to a wide variety of normally detrimental solvents due to the action of such an efflux pump. The genes for this solvent efflux pump were cloned from P. putida S12 and their nucleotide sequence determined. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins known to be involved in proton-dependent multidrug efflux systems. Transfer of the genes for the solvent efflux pump to solvent-sensitive P. putida strains results in the acquisition of solvent resistance. This opens up the possibilities of using the solvent efflux system to construct bacterial strains capable of performing biocatalytic transformations of insoluble substrates in two-phase aqueous:organic medium.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/genética , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Pseudomonas putida/efeitos dos fármacos , Solventes/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/metabolismo , Elementos de DNA Transponíveis , DNA Bacteriano , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Fenótipo , Pseudomonas putida/genética , Pseudomonas putida/metabolismoRESUMO
The tolQ, tolR, and tolA genes from Pseudomonas aeruginosa PAO were cloned using degenerate oligonucleotide PCR primers designed based on conserved transmembrane regions of Escherichia coli TolQ and TolR and E. coli and Pseudomonas putida ExbB and ExbD. The resulting PCR product was used as a probe to isolate a 6.5-kb DNA fragment containing P. aeruginosa tolQ, tolR, and tolA. The nucleotide sequence of a 2.9-kb DNA fragment containing the tolQ, tolR, and tolA genes was determined. The DNA sequence predicts TolQ to be a 25,250-Da protein exhibiting 53% identity to E. coli TolQ. TolR is predicted to be a 15,788-Da protein, sharing 38% identity with the E. coli TolR protein. The P. aeruginosa tolA sequence predicts a 37,813-Da protein with 27% identity to the E. coli TolA. The P. aeruginosa TolQRA proteins were expressed in E. coli minicells. Analysis of plasmid-encoded tolQ::lacZ and tolA::lacZ promoter fusions in E. coli indicated that these genes are expressed at different levels, suggesting transcription from different promoters. Transcriptional analysis of the tol genes in P. aeruginosa revealed that the tolQ and tolR genes are cotranscribed as an approximately 1.5-kb transcript and that tolA is transcribed from its own promoter as an approximately 1.2-kb transcript. The P. aeruginosa Tol proteins were functionally unable to complement E. coli tol mutants, although P. aeruginosa TolQ was able to complement the iron-limited growth of an E. coli exbB mutant. Introduction of the tolQRA genes in the tol-like mutant PAO 1652 restored pyocin AR41 killing, indicating that the Tol proteins are involved in the uptake of pyocin AR41 in P. aeruginosa. Attempts to inactivate the chromosomal copy of the tolA or tolQ gene in the parent strain PAO proved to be unsuccessful, and we propose that inactivation of these genes in P. aeruginosa results in a lethal phenotype.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Proteínas de Membrana/genética , Pseudomonas aeruginosa/genética , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Sequência de Bases , Cromossomos Bacterianos , Clonagem Molecular , DNA Bacteriano , Escherichia coli/genética , Escherichia coli/metabolismo , Teste de Complementação Genética , Óperon Lac , Dados de Sequência Molecular , Mutação , Plasmídeos , Pseudomonas putida/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Gênica , beta-Galactosidase/metabolismoAssuntos
Eletroporação/métodos , Plasmídeos , Pseudomonas/genética , Transformação Bacteriana , Vetores Genéticos , Indicadores e Reagentes , Magnésio , Pseudomonas/crescimento & desenvolvimento , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Soluções , SacaroseRESUMO
A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.
Assuntos
Burkholderia cepacia/química , Ionóforos/isolamento & purificação , Quelantes de Ferro/isolamento & purificação , Salicilatos , Tiazóis , Transporte Biológico/efeitos dos fármacos , Burkholderia cepacia/efeitos dos fármacos , Burkholderia cepacia/crescimento & desenvolvimento , Cromatografia em Gel , Cromatografia em Camada Fina , Meios de Cultura , Humanos , Ionóforos/química , Ionóforos/farmacologia , Ferro/metabolismo , Ferro/farmacologia , Quelantes de Ferro/química , Quelantes de Ferro/farmacologia , Fenóis/farmacologia , SideróforosRESUMO
The gene for exoenzyme S, an ADP-ribosyl transferase, was cloned from Pseudomonas aeruginosa strain DG1 using an oligonucleotide probe based on the partial N-terminal amino acid sequence to screen a library of DG1 SstI fragments inserted into pKT230 in Escherichia coli DH1. A positive clone, designated pPD3, hybridized with the oligonucleotide probe and contained a 15 kb SstI insert. In E. coli minicells pPD3 expressed a single protein of Mr 68,000. This protein was localized primarily in the periplasm in E. coli. A 3.6 kb HindIII-BamHI fragment was subcloned into the vector pT7-4 which contains the promoter from bacteriophage T7 to construct pT7-4HB. In E. coli strains expressing the T7 RNA polymerase on a second plasmid, the Mr 68,000 protein was expressed and shown to react with antibodies to exoenzyme S. No enzymatic activity was detected in cell sonicates or culture supernatants of E. coli (pPD3). Cell sonicates of E. coli (pT7-4HB) however were cytotoxic to HeLa cells and this cytotoxicity was neutralizable with anti-exoenzyme S antiserm. Thus, exoenzyme S expressed in E. coli is toxic but not enzymatically active. When plasmids carrying the exoenzyme S gene were introduced into P. aeruginosa, there was a significant increase in ADP-ribosyl transferase activity, indicating that the plasmid encoded protein is enzymatically active in P. aeruginosa.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Regulação Bacteriana da Expressão Gênica , Poli(ADP-Ribose) Polimerases/genética , Pseudomonas aeruginosa/genética , Southern Blotting , Clonagem Molecular , Escherichia coli/enzimologia , Genes Bacterianos , Poli(ADP-Ribose) Polimerases/biossíntese , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/toxicidade , Pseudomonas aeruginosa/enzimologia , Mapeamento por RestriçãoRESUMO
The photochemical interaction between intravenously injected rose bengal dye and 514.5-nm argon laser irradiation was employed to initiate permanent thrombotic occlusions in the corneal neovasculature of rabbit eyes with experimentally induced lipid keratopathy. This photothrombotic procedure did not produce corneal edema or polymorphonuclear leukocyte infiltration, as was found in previous studies employing laser-induced photocoagulation. Thus, by means of the new technique, we avoided the formation of additional neovascularization and lipid deposition. Vascular occlusion by photothrombosis, effected with 8.5 times less incident intensity and 27.5 times less total light exposure than with photocoagulation, yielded an average reduction of corneal cholesterol content of 36% as opposed to an increase of 24% found with previous argon laser photocoagulation.
Assuntos
Córnea/irrigação sanguínea , Hipercolesterolemia/complicações , Neovascularização Patológica/tratamento farmacológico , Fotoquimioterapia , Animais , Neovascularização Patológica/etiologia , CoelhosRESUMO
A comparison was made of the accuracy of the Tono-Pen, Pneumatonometer, and Perkins hand-held tonometers by measuring the hydrostatically controlled intracular pressure from 10 to 50 mm Hg in human eye bank eyes. The open stopcock method was used in which the cannulated eye, the hydrostatic pressure controlling column, and a manometer were continuously open to each other. The Pneumatonometer gave accurate results at 50 mm Hg but overestimated the lower intraocular pressures; whereas, the Perkins gave satifactory results at 10 and 20 mm Hg but underestimated higher intraocular pressures. The Tono-Pen displayed the least deviation from the controlled pressure over the entire range studied. Another aspect of this study involved determining that a therapeutic soft contact lens did not have any noticeably adverse effect on the accuracy of the Pneumatonometer and Tono-Pen tonometers.