RESUMEN
BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5'GCTCTTC N1/N4 3'. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN de Cadena Simple/metabolismo , Secuencia de Aminoácidos , Bacteriófago T7/genética , Secuencia de Bases , Dominio Catalítico , Clonación Molecular , Roturas del ADN de Cadena Simple , Enzimas de Restricción del ADN/genética , ADN de Cadena Simple/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The aims of this study were to investigate the effects of photodynamic therapy (PDT) on endodontic pathogens in planktonic phase as well as on Enterococcus faecalis biofilms in experimentally infected root canals of extracted teeth. Strains of microorganisms were sensitized with methylene blue (25 microg/ml) for 5 minutes followed by exposure to red light of 665 nm with an energy fluence of 30 J/cm2. Methylene blue fully eliminated all bacterial species with the exception of E. faecalis (53% killing). The same concentration of methylene blue in combination with red light (222 J/cm2) was able to eliminate 97% of E. faecalis biofilm bacteria in root canals using an optical fiber with multiple cylindrical diffusers that uniformly distributed light at 360 degrees. We conclude that PDT may be developed as an adjunctive procedure to kill residual bacteria in the root canal system after standard endodontic treatment.
Asunto(s)
Infecciones Bacterianas/tratamiento farmacológico , Enfermedades de la Pulpa Dental/microbiología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/uso terapéutico , Biopelículas/efectos de los fármacos , Recuento de Colonia Microbiana , Cavidad Pulpar/efectos de los fármacos , Cavidad Pulpar/microbiología , Enfermedades de la Pulpa Dental/tratamiento farmacológico , Desinfección/métodos , Enterococcus faecalis/efectos de los fármacos , Fusobacterium nucleatum/efectos de los fármacos , Humanos , Terapia por Láser , Azul de Metileno/uso terapéutico , Microscopía Electrónica de Rastreo , Peptostreptococcus/efectos de los fármacos , Fotoquimioterapia/instrumentación , Porphyromonas endodontalis/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Prevotella intermedia/efectos de los fármacosRESUMEN
The primary objective of this study was to determine whether or not Spiroplasma mirum would be capable of producing lesions of transmissible spongiform encephalopathy (TSE) when inoculated in raccoons (Procyon lotor) and, if that was possible, to compare the clinicopathological findings with those of transmissible mink encephalopathy (TME) in the same experimental model. For this purpose, 5 groups (n = 5) of raccoon kits were inoculated intracerebrally with either S. mirum and/or TME. Two other groups (n = 5) of raccoon kits served as sham-inoculated controls. All animals inoculated with TME, either alone or in combination, showed clinical signs of neurologic disorder and were euthanized within 6 mo post-inoculation (MPI). None of the carcasses revealed gross lesions. Spongiform encephalopathy was observed by light microscopy and the presence of abnormal disease-causing prion protein (PrP(d)) was detected by immunohistochemistry (IHC) and Western blot (WB) techniques in only the raccoons administered TME. Raccoons inoculated with Spiroplasma, but not administered TME agent, were euthanized at 30 MPI. They did not show clinical neurologic signs, their brains did not have lesions of spongiform encephalopathy, and their tissues were negative for S. mirum by polymerase chain reaction (PCR) and for PrP(d) by IHC and WB techniques. The results of this study indicate that Spiroplasma mirum does not induce TSE-like disease in raccoons.