scholarly journals Characterization of IS1245 for Strain Typing of Mycobacterium avium

1998 ◽  
Vol 36 (7) ◽  
pp. 1859-1863 ◽  
Author(s):  
Martine Pestel-Caron ◽  
Robert D. Arbeit
Keyword(s):  
Mycobacterium Avium ◽  
Strain Typing ◽  
Environmental Isolates ◽  
Pfge Analysis ◽  
Insertion Element ◽  
Copy Numbers ◽  
Immunodeficiency Virus

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.

scholarly journals Characterization of aMycobacterium aviumsubsp.aviumOperon Associated with Virulence and Drug Detoxification

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Mariana Noelia Viale ◽  
Kun Taek Park ◽  
Belén Imperiale ◽  
Andrea Karina Gioffre ◽  
María Alejandra Colombatti Olivieri ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Efflux Pump ◽  
Mice Model ◽  
Toxic Compounds ◽  
Drug Detoxification ◽  
Insight Into ◽  
Knockout Mutation

ThelprG-p55operon ofMycobacterium tuberculosisandMycobacterium bovisis involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in theMycobacterium aviumcomplex, in this study, we assayed the effect of the deletion oflprG gene in the D4ER strain ofMycobacterium aviumsubsp.avium. The replacement oflprG gene with a hygromycin cassette caused a polar effect on the expression ofp55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophagesin vitroand in a mice modelin vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAAin vitroandin vivo.

Physiological, morphological, and immunochemical parameters used for the characterization of clinical and environmental isolates ofAcanthamoeba

Parasitology ◽  
2012 ◽  
Vol 140 (3) ◽  
pp. 396-405 ◽  
Author(s):  
A. BECKER-FINCO ◽  
A. O. COSTA ◽  
S. K. SILVA ◽  
J. S. RAMADA ◽  
C. FURST ◽  
...  
Keyword(s):  
Comparative Evaluation ◽  
Polyclonal Antibodies ◽  
Protein Profiles ◽  
Environmental Isolates ◽  
Pathogenic Potential ◽  
Cytopathic Effects ◽  
Specific Proteins

SUMMARYThe factors that characterizeAcanthamoebastrains as harmless or potentially pathogenic have not been elucidated. Analysing thein vitroandin vivoparameters ofAcanthamoebasamples, including heat tolerance at temperatures close to that of the human body, cytopathic effects, and their ability to cause infections in animals, has been proposed to identify their pathogenic potential. Another promising criterion for differentiating strains is the analysis of their biochemical and immunochemical properties. In this study, a comparative evaluation between clinical and environmentalAcanthamoebaisolates was performed on the basis of physiological, morphological, and immunochemical criteria. Crude antigens were used to characterize the protein profiles by electrophoresis and immunize mice to produce polyclonal and monoclonal antibodies. The antibodies were characterized using ELISA, Western blotting, and immunofluorescence techniques. The results obtained with polyclonal antibodies suggest the presence of specific proteins for each studied isolate and co-reactive immunochemical profiles among conserved components. Ten monoclonal antibody clones were obtained; mAb3 recognized 3 out of 4 samples studied. The results of this study may help standardize criteria for identifying and characterizingAcanthamoebastrains. Taken together, our results support the view that a set of features may help differentiateAcanthamoebaspecies and isolates.

scholarly journals Regulation of interleukin 12 p40 expression through an NF-kappa B half-site.

1995 ◽  
Vol 15 (10) ◽  
pp. 5258-5267 ◽  
Author(s):  
T L Murphy ◽  
M G Cleveland ◽  
P Kulesza ◽  
J Magram ◽  
K M Murphy
Keyword(s):  
Leishmania Major ◽  
Interleukin 12 ◽  
Cell Mediated Immunity ◽  
Nf Kappa B ◽  
Binding Interaction ◽  
Ifn Gamma ◽  
Immunodeficiency Virus

Interleukin 12 (IL-12) is an inducible cytokine composed of 35- and 40-kDa subunits that is critical for promoting T helper type 1 development and cell-mediated immunity against pathogens. The 40-kDa subunit, expressed by activated macrophages and B cells, is induced by several pathogens in vivo and in vitro and is augmented or inhibited by gamma interferon (IFN-gamma) or IL-10, respectively. Control of IL-12 p40 expression is therefore important for understanding resistance and susceptibility to a variety of pathogens, including Leishmania major and perhaps human immunodeficiency virus. In this report, we provide the first characterization of IL-12 p40 gene regulation in macrophages. We localize inducible activity of the promoter to the sequence -122GGGGAATTTTA-132 not previously recognized to bind Rel family transcription factors. We demonstrate binding of this sequence to NF-kappa B (p50/p65 and p50/c-Rel) complexes in macrophages activated by several p40-inducing pathogens and provide functional data to support a role for NF-kappa B family members in IL-12 p40 activation. Finally, we find that IFN-gamma treatment of cells enhances this binding interaction, thus potentially providing a mechanism for IFN-gamma augmentation of IL-12 production by macrophages.

scholarly journals Characterization of the Pseudomonas putida Mobile Genetic Element ISPpu10: an Occupant of Repetitive Extragenic Palindromic Sequences

2006 ◽  
Vol 188 (1) ◽  
pp. 37-44 ◽  
Author(s):  
María Isabel Ramos-González ◽  
María Jesús Campos ◽  
Juan Luis Ramos ◽  
Manuel Espinosa-Urgel
Keyword(s):  
Pseudomonas Putida ◽  
Insertion Element ◽  
Left And Right ◽  
First Time ◽  
Free Strain ◽  
Palindromic Sequences ◽  
Repetitive Extragenic Palindromic

ABSTRACT We have characterized the Pseudomonas putida KT2440 insertion element ISPpu10. This insertion sequence encodes a transposase which exhibits homology to the transposases and specific recombinases of the Piv/Moov family, and no inverted repeats are present at the borders of its left and right ends, thus constituting a new member of the atypical IS110/IS492 family. ISPpu10 was found in at least seven identical loci in the KT2440 genome, and variants were identified having an extra insertion at distinct loci. ISPpu10 always appeared within the core of specific repetitive extragenic palindromic (REP) sequences TCGCGGGTAAACCCG CT CCTAC, exhibiting high target stringency. One intragenic target was found associated with the truncation of a GGDEF/EAL domain protein. After active in vitro transposition to a plasmid-borne target, a duplication of the CT (underlined above) at the junction as a consequence of the ISPpu10 insertion was experimentally demonstrated for the first time in the IS110/IS492 family. The same duplication was observed after transposition of ISPpu10 from a plasmid to the chromosome of P. putida DOT-T1E, an ISPpu10-free strain with REPs similar to those of strain KT2440. Plasmid ISPpu10-mediated rearrangements were observed in vivo under laboratory conditions and in the plant rhizosphere.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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