scholarly journals In vivoR–plasmid transfer in a patient with a mixed infection of shigella dysentery

1994 ◽  
Vol 112 (2) ◽  
pp. 247-252 ◽  
Author(s):  
M. P. Bratoeva ◽  
J. F. John.
Keyword(s):  
Antimicrobial Agents ◽  
Shigella Flexneri ◽  
Plasmid Transfer ◽  
Shigella Dysenteriae ◽  
Type I ◽  
E Coli ◽  
R Plasmids ◽  
Short Time

SUMMARYTransfer of shigella R–plasmidsin vivohas seldom been demonstrated. Strains ofShigella dysenteriaetype 1 andShigella flexneritype 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains ofS. sonneipredominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48–kilobase plasmid that conferred the entire resistance phenotype to a susceptibleEscherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonicE. coli. Transconjugants hybridized to a dihydrofolate reductase type I–DNA probe. These studies support the hypothesis that R–plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.

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.

scholarly journals Ectopic Expression of Inhibitors of Protein Phosphatase Type 1 (PP1) Can Be Used to Analyze Roles of PP1 in Drosophila Development

Genetics ◽  
2003 ◽  
Vol 164 (1) ◽  
pp. 235-245
Author(s):  
Daimark Bennett ◽  
Balázs Szöőr ◽  
Sascha Gross ◽  
Natalia Vereshchagina ◽  
Luke Alphey
Keyword(s):  
Protein Phosphatase ◽  
Muscle Development ◽  
Ectopic Expression ◽  
High Specificity ◽  
Type I ◽  
Protein Phosphatase Type 1 ◽  
Mitotic Figures ◽  
Protein Phosphatase Type

Abstract We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGFβ superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

1991 ◽  
Vol 37 (5) ◽  
pp. 407-410
Author(s):  
Mônica A. M. Vieira ◽  
Beatriz E. C. Guth ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Sensitivity And Specificity ◽  
Dna Probes ◽  
Type I ◽  
Enteropathogenic Escherichia Coli ◽  
Key Words Dna ◽  
E Coli ◽  
Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

scholarly journals Evaluation In Vivo and In Vitro of the Antioxidant, Antinociceptive, and Anti-Inflammatory Activities of Biflavonoids From Ouratea hexasperma and O. ferruginea

2019 ◽  
Vol 14 (6) ◽  
pp. 1934578X1985680 ◽  
Author(s):  
Poliana de Araujo Oliveira ◽  
Queli Cristina Fidelis ◽  
Thayane Ferreira da Costa Fernandes ◽  
Milene Conceição de Souza ◽  
Dayane Magalhães Coutinho ◽  
...  
Keyword(s):  
Type I Collagen ◽  
In Vivo Model ◽  
Type I ◽  
Anti Inflammatory ◽  
Vitro Model ◽  
Factor Α ◽  
Necrosis Factor

Ouratea species are used for the treatment of inflammation-related diseases such as rheumatism and arthritic disorders. The Ouratea genus is a rich source of flavonoids and bioflavonoids and for this reason we evaluated the effects of the biflavonoid fractions from the leaves of O. hexasperma (OHME) and O. ferruginea (OFME) in the in vivo model of complete Freund’s adjuvant (CFA)-induced arthritis and in the in vitro model of oxidative stress and cellular viability. The CFA-induced arthritis model in rats was followed by paw volume, articular incapacitation and Randall-selitto models, as well as quantification of cytokines and serum C-terminal telopeptide of type I collagen levels. OHME and OFME demonstrated antinociceptive and anti-inflammatory activities, as well as improvement in articular incapacity and reduction in levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α, and type 1 collagen, and increased cell viability. No adverse effects were observed. The results suggest that OHME and OFME can reduce inflammation and bone resorption besides their antioxidant action.

scholarly journals In Vivo Dynamics of Type 1 Fimbria Regulation in UropathogenicEscherichia coli during Experimental Urinary Tract Infection

2001 ◽  
Vol 69 (5) ◽  
pp. 2838-2846 ◽  
Author(s):  
Nereus W. Gunther ◽  
Virginia Lockatell ◽  
David E. Johnson ◽  
Harry L. T. Mobley
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Invertible Element ◽  
Molecular Techniques ◽  
E Coli ◽  
Type 1 Fimbriae ◽  
Invertible Elements

ABSTRACT Escherichia coli is the primary cause of uncomplicated infections of the urinary tract including cystitis. More serious infections, characterized as acute pyelonephritis, can also develop. Type 1 fimbriae of E. coli contribute to virulence in the urinary tract; however, only recently has the expression of the type 1 fimbriae been investigated in vivo using molecular techniques. Transcription of type 1 fimbrial genes is controlled by a promoter that resides on a 314-bp invertible element capable of two orientations. One places the promoter in the ON orientation, allowing for transcription; the other places the promoter in the OFF orientation, preventing transcription. A PCR-based assay was developed to measure the orientation of the invertible element during an experimental urinary tract infection in mice. Using this assay, it was found that the percentage of the population ON in urine samples correlated with the respective CFU per gram of bladder (P = 0.0006) but not with CFU per gram of kidney (P > 0.069). Cystitis isolates present in the urine of mice during the course of infection had a higher percentage of their invertible elements in the ON orientation than did pyelonephritis isolates (85 and 34%, respectively, at 24 h; P < 0.0001). In general, cystitis isolates, unlike pyelonephritis isolates, were more likely to maintain their invertible elements in the ON orientation for the entire period of infection. E. coli cells expressing type 1 fimbriae, expelled in urine, were shown by scanning electron microscopy to be densely packed on the surface of uroepithelial cells. These results suggest that expression of type 1 fimbriae is more critical for cystitis strains than for pyelonephritis strains in the early stages of an infection during bladder colonization.

Molecular Strategies for the Detection, Identification, and Differentiation between Enteroinvasive Escherichia coli and Shigella spp.

2005 ◽  
Vol 68 (2) ◽  
pp. 239-245 ◽  
Author(s):  
CESAR I. BIN KINGOMBE ◽  
MARIA-LUCIA CERQUEIRA-CAMPOS ◽  
JEFFREY M. FARBER
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shigella Flexneri ◽  
Shigella Dysenteriae ◽  
Shigella Boydii ◽  
Epidemiological Tool ◽  
Pcr Rflp ◽  
Shigella Spp

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc−/ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH−) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella flexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.

scholarly journals In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism

2011 ◽  
Vol 11 (1) ◽  
pp. 147 ◽  
Author(s):  
Srilatha Kuntumalla ◽  
Quanshun Zhang ◽  
John C Braisted ◽  
Robert D Fleischmann ◽  
Scott N Peterson ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Shigella Dysenteriae ◽  
Protein Abundance ◽  
Shigella Dysenteriae Type ◽  
Vitro Protein

scholarly journals Interactions with Commensal and Pathogenic Bacteria Induce HIV-1 Latency in Macrophages through Altered Transcription Factor Recruitment to the LTR

2021 ◽  
Author(s):  
Gregory A. Viglianti ◽  
Vicente Planelles ◽  
Timothy M. Hanley
Keyword(s):  
Adaptor Protein ◽  
Type I ◽  
Major Barrier ◽  
E Coli ◽  
Tissue Microenvironment ◽  
Viral Spread ◽  
Latent Hiv ◽  
Hiv 1

Macrophages are infected by HIV-1 in vivo and contribute to both viral spread and pathogenesis. Recent human and animal studies suggest that HIV-1-infected macrophages serve as a reservoir that contributes to HIV-1 persistence during anti-retroviral therapy. The ability of macrophages to serve as persistent viral reservoirs is likely influenced by the local tissue microenvironment, including interactions with pathogenic and commensal microbes. Here we show that the sexually transmitted pathogen Neisseria gonorrhoeae (GC) and the gut-associated microbe Escherichia coli (E. coli), which encode ligands for both Toll-like receptor 2 (TLR2) and TLR4, repressed HIV-1 replication in macrophages and thereby induced a state reminiscent of viral latency. This repression was mediated by signaling through TLR4 and the adaptor protein TRIF and was associated with increased production of type I interferons. Inhibiting TLR4 signaling, blocking type 1 interferon, or knocking-down TRIF reversed LPS- and GC-mediated repression of HIV-1. Finally, the repression of HIV-1 in macrophages was associated with the recruitment of interferon regulatory factor 8 (IRF8) to the interferon stimulated response element (ISRE) downstream of the 5’ HIV-1 long terminal repeat (LTR). Our data indicate that IRF8 is responsible for repression of HIV-1 replication in macrophages in response to TRIF-dependent signaling during GC and E. coli co-infection. These findings highlight the potential role of macrophages as HIV-1 reservoirs as well as the role of the tissue microenvironment and co-infections as modulators of HIV-1 persistence. IMPORTANCE The major barrier toward the eradication of HIV-1 infection is the presence of a small reservoir of latently infected cells, which include CD4+ T cells and macrophages that escape immune-mediated clearance and the effects of anti-retroviral therapy. There remain crucial gaps in our understanding of the molecular mechanisms that lead to transcriptionally silent or latent HIV-1 infection of macrophages. The significance of our research is in identifying microenvironmental factors, such as commensal and pathogenic microbes, that can contribute to the establishment and maintenance of latent HIV-1 infection in macrophages. It is hoped that identifying key processes contributing to HIV-1 persistence in macrophages may ultimately lead to novel therapeutics to eliminate latent HIV-1 reservoirs in vivo.

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