The in vitro O2′-methylation of RNA in the ribonucleoprotein precursor-particles from a Relaxed mutant of Escherichia coli

1968 ◽  
Vol 46 (1) ◽  
pp. 109-115 ◽  
Author(s):  
J. L. Nichols ◽  
B. G. Lane
Keyword(s):  
Escherichia Coli ◽  
Sodium Chloride ◽  
Snake Venom ◽  
Chloride Solution ◽  
Sodium Chloride Solution ◽  
E Coli ◽  
Adenosyl Methionine

Ribonucleoprotein "precursor-particles" were isolated from a methionine auxotroph of E. coli (RCrelaxed), which had been cultured under conditions of methionine deprivation. Following incubation of the "particles" in vitro with [methyl-14C]-S-adenosyl methionine, the ribonucleates were extracted from the particles, and precipitated from aqueous 2 M sodium chloride solution at 0°, before analysis for the incorporation of O2′-[methyl-14C]-substituents into the RNA. It was found that two of the four principal alkali-stable dinucleotides, known to be present in alkali hydrolysates of E. coli rRNA, contained O2′-[methyl-14C]-substituents: O2′Me-GpGp and O2′Me-CpCp, whereas the two remaining dinucleotides, N4,O2′diMe-CpCp and O2′Me-UpGp, did not contain significant radioactivity. It has been concluded that highly specific enzymic O2′-methylation of E. coli RNA can occur subsequent to polynucleotide synthesis. Allied studies are reported concerning the characterization of N4,O2′-[dimethyl-14C]-cytidine 5′-phosphate that was recovered from whole snake venom hydrolysates of E. coli rRNA, which had been labeled in vivo with [methyl-14C].

scholarly journals Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

2005 ◽  
Vol 389 (2) ◽  
pp. 541-548 ◽  
Author(s):  
Rajesh K. Soni ◽  
Parul Mehra ◽  
Gauranga Mukhopadhyay ◽  
Suman Kumar Dhar
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
Temperature Sensitive ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dnab Helicase ◽  
H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

Comparison of Vehicles for Vancomycin Drug Delivery in Extemporaneously Prepared Ophthalmic Solutions

2012 ◽  
Vol 506 ◽  
pp. 481-484 ◽  
Author(s):  
A. Khangtragool
Keyword(s):  
Sodium Chloride ◽  
Chloride Solution ◽  
Sodium Chloride Solution ◽  
Inhibitory Concentration ◽  
Tear Film ◽  
Eye Drops ◽  
Ophthalmic Delivery ◽  
Ophthalmic Solutions

This work compares the ophthalmic delivery of vancomycin 50 mg/ml eye drops using 5 different vehicles, namely: 0.3% w/v chitosan, 0.3% and 0.4% w/v HPMC (Methocel E4M), Tears NaturaleTMII and 0.9% w/v sodium chloride solution.In vitroandin vivostudies were carried out and the results evaluated in terms of viscosity, compatibility, stability, clarity, minimum inhibitory concentration (MIC) and pharmacokinetics. The viscosity of Tears NaturaleTMII was comparable with that of HPMC (0.3% pH 7.1) but was higher than 0.3% w/v chitosan. The percent labeled amounts and MIC of vancomycin 50 mg/ml in all of the vehicles were stable for 30 days at 2-8°C, while the clarity in 0.3% w/v chitosan, 0.3% and 0.4% HPMC (pH 7.1), Tears NaturaleTMII and 0.9% sodium chloride solution was stable for 30, 14, 1 and 3 days respectively at 2-8°C.In vivopharmacokinetic determinations of the AUC of tear film reciprocal of minimum inhibitory titer showed that vancomycin 50 mg/ml in 0.3% w/v chitosan, 0.3% and 0.4% w/v HPMC pH 7.1 and Tears NaturaleTMII were significantly different from 0.9% sodium chloride solution. At the present time, chitosan is undergoing clinical trials in Thailand with a view to its use in ophthalmology, while HPMC (0.3% w/v) in pH 7.1 has already been approved for use as a vehicle in ophthalmology for the delivery of vancomycin 50 mg/ml in extemporaneous eye drops.

scholarly journals Discovery and Characterization of Three New Escherichia coli Septal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA

2009 ◽  
Vol 192 (1) ◽  
pp. 242-255 ◽  
Author(s):  
S. J. Ryan Arends ◽  
Kyle Williams ◽  
Renada J. Scott ◽  
Silvana Rolong ◽  
David L. Popham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Caulobacter Crescentus ◽  
Negative Regulator ◽  
Aquifex Aeolicus ◽  
E Coli ◽  
Division Site ◽  
Genes Encoding

ABSTRACT SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

scholarly journals Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

2006 ◽  
Vol 72 (9) ◽  
pp. 6405-6410 ◽  
Author(s):  
Raul R. Raya ◽  
Peter Varey ◽  
Rebecca A. Oot ◽  
Michael R. Dyen ◽  
Todd R. Callaway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Oral Dose ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Isolation And Characterization ◽  
Unit Reduction

ABSTRACT Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.

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 Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

Characterization of in vitro and in vivo murine models of human enteropathogenic E. coli (EPEC) infection

2003 ◽  
Vol 124 (4) ◽  
pp. A558
Author(s):  
Suzana D. Savkovic ◽  
Farol L. Tomson ◽  
Michelle Muza ◽  
Gail Hecht
Keyword(s):  
Murine Models ◽  
E Coli

scholarly journals Biological Cost of Single and Multiple Norfloxacin Resistance Mutations in Escherichia coli Implicated in Urinary Tract Infections

2005 ◽  
Vol 49 (6) ◽  
pp. 2343-2351 ◽  
Author(s):  
Patricia Komp Lindgren ◽  
Linda L. Marcusson ◽  
Dorthe Sandvang ◽  
Niels Frimodt-Møller ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Infection Model ◽  
Resistance Mutations ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Tract Infections ◽  
Subsequent Selection

ABSTRACT Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

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