scholarly journals Circulation of Fluorescently Labelled Phage in a Murine Model

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 297
Author(s):  
Zuzanna Kaźmierczak ◽  
Joanna Majewska ◽  
Magdalena Milczarek ◽  
Barbara Owczarek ◽  
Krystyna Dąbrowska
Keyword(s):  
Lymph Nodes ◽  
Murine Model ◽  
Fluorescent Protein ◽  
Rectal Administration ◽  
Intravenous Route ◽  
Gi Tract ◽  
E Coli ◽  
Body Compartments ◽  
Spleen And Lymph Nodes

Interactions between bacteriophages and mammals strongly affect possible applications of bacteriophages. This has created a need for tools that facilitate studies of phage circulation and deposition in tissues. Here, we propose red fluorescent protein (RFP)-labelled E. coli lytic phages as a new tool for the investigation of phage interactions with cells and tissues. The interaction of RFP-labelled phages with living eukaryotic cells (macrophages) was visualized after 20 min of co-incubation. RFP-labeled phages were applied in a murine model of phage circulation in vivo. Phages administered by three different routes (intravenously, orally, rectally) were detected through the course of time. The intravenous route of administration was the most efficient for phage delivery to multiple body compartments: 20 min after administration, virions were detected in lymph nodes, lungs, and liver; 30 min after administration, they were detectable in muscles; and 1 h after administration, phages were detected in spleen and lymph nodes. Oral and rectal administration of RFP-labelled phages allowed for their detection in the gastrointestinal (GI) tract only.

Expression, purification and properties of multidrug efflux proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 513-517 ◽  
Author(s):  
P.J. F. Henderson ◽  
C. K. Hoyle ◽  
A. Ward
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
General Strategy ◽  
X Rays ◽  
Multidrug Efflux ◽  
Nmr Studies ◽  
E Coli ◽  
Efflux Proteins

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H+ antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1–25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni2+-nitrilo-triacetate-affinity column chromography, yielding 5–25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of α-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with 13C or 15N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

In vitro autoradiographic and in vivo scintigraphic localization of somatostatin receptors in human lymphatic tissue

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2143-2151 ◽  
Author(s):  
JC Reubi ◽  
B Waser ◽  
U Horisberger ◽  
E Krenning ◽  
SW Lamberts ◽  
...  
Keyword(s):  
Lymph Nodes ◽  
Gamma Camera ◽  
Somatostatin Receptors ◽  
Immune Functions ◽  
Thymic Tissue ◽  
Tissue Sections ◽  
Spleen And Lymph Nodes ◽  
Neuroendocrine Functions

Abstract Receptors for the neuropeptide somatostatin (SS) were evaluated in vitro and in vivo in various human lymphatic tissues, ie, thymus, spleen, and lymph nodes; thymic carcinoids and thymomas were also tested. The receptors were measured in vitro using receptor autoradiography on tissue sections incubated with the SS analog 125I- [Tyr3]-octreotide or 125I-[Leu8,D-Trp22,Tyr25]-SS-28. All tissues were SS-receptor positive for either radioligand, except the thymomas. In thymic tissue, the receptors were diffusely located in the medulla, presumably on epithelial cells. In the spleen, the red pulp was strongly labeled. In the lymph nodes, the germinal centers were preferentially labeled. In all tissues, the receptors were of high affinity (kd thymus, 0.84 nmol/L; kd spleen, 1.6 nmol/L; kd lymph node, 0.62 nmol/L) and specific for SS. Displacement by nanomolar concentrations of SS-14, SS-28, and octreotide was observed, as was guanosine triphosphate dependency. The in vivo visualization of somatostatin receptors was performed after injection of 111In-DTPA- octreotide and gamma-camera scintigraphy. The spleen, but not thymus or lymph nodes, were visualized. These data suggest an important role for SS in regulating immune functions through SS receptors in thymus, spleen, and lymph nodes. Furthermore, SS may regulate neuroendocrine functions in the thymus.

scholarly journals Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition

2006 ◽  
Vol 72 (12) ◽  
pp. 7607-7613 ◽  
Author(s):  
Andrew C. Tolonen ◽  
Gregory B. Liszt ◽  
Wolfgang R. Hess
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
E Coli ◽  
Phage T7 ◽  
Ecological Importance ◽  
Green Fluorescent ◽  
Oceanic Gyres ◽  
Global Carbon

ABSTRACT Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

scholarly journals THE ROLE OF LYMPHOCYTES IN THE SENSITIZATION OF RATS TO RENAL HOMOGRAFTS

1965 ◽  
Vol 122 (2) ◽  
pp. 347-360 ◽  
Author(s):  
S. Strober ◽  
J. L. Gowans
Keyword(s):  
Lymph Nodes ◽  
Thoracic Duct ◽  
Femoral Vein ◽  
F1 Hybrid ◽  
Duct Cells ◽  
Degree Of Sensitization ◽  
Spleen And Lymph Nodes

In order to study the role of blood-borne small lymphocytes in the sensitization of rats to renal homografts 2 techniques for the perfusion of isolated rat kidneys were employed: (a) the in vitro perfusion of kidneys with thoracic duct cells suspended in either an artificial medium or in blood; the perfusates were then injected into rats syngeneic with the lymphocyte donors; (b) the in vivo perfusion of kidneys with blood issuing from the femoral artery and returning to the femoral vein of living rats. The degree of sensitization conferred on the recipients by the perfusates was assessed by applying a skin homograft from the kidney donor and scoring the epithelial necrosis at 6 days. The in vitro experiments indicated that parental strain thoracic duct cells, which had passed through an F1 hybrid kidney could confer upon a parental rat sensitivity to an F1 skin graft. Several perfusions with radioactively labelled lymphocytes showed that the injected cells migrated to the lymph nodes and spleen of the recipients Labelled large pyroninophilic cells were occasionally seen in the spleen and lymph nodes of recipients, and it was suggested that these had arisen from the injected cells. Although the in vitro perfusions with blood indicated that renal homografts might sensitize their hosts within 1 hour, the in vivo perfusions suggested that about 5 to 12 hours were required. The more rapid sensitization in vitro was possibly due to the more frequent opportunity for contact between lymphocytes and kidney vascular endothelium which was afforded by the conditions in vitro.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

scholarly journals Ciprofloxacin Treatment Failure in a Murine Model of Pyelonephritis Due to an AAC(6′)-Ib-cr-Producing Escherichia coli Strain Susceptible to CiprofloxacinIn Vitro

2013 ◽  
Vol 57 (12) ◽  
pp. 5830-5835 ◽  
Author(s):  
T. Guillard ◽  
E. Cambau ◽  
F. Chau ◽  
L. Massias ◽  
C. de Champs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Murine Model ◽  
Resistance Mechanism ◽  
Bacterial Count ◽  
Coli Strain ◽  
Content Type ◽  
E Coli ◽  
Fixed Concentration

ABSTRACTAAC(6′)-Ib-cr is a plasmid-mediated quinolone resistance mechanism described worldwide forEscherichia coli. Since it confersin vitroonly a low level of resistance to ciprofloxacin, we evaluated its impact on thein vivoactivity of ciprofloxacin. Isogenic strains were obtained by transferring plasmid p449, harboringaac(6′)-Ib-cr, into the quinolone-susceptible strainE. coliCFT073-RR and its D87GgyrAmutant. MICs were 0.015, 0.06, 0.25, and 0.5 μg/ml againstE. colistrains CFT073-RR, CFT073-RR/p449, CFT073-RR GyrAr, and CFT073-RR GyrAr/p449, respectively. Bactericidal activity was reduced at 1× the MIC for the three resistant derivatives, while at a fixed concentration of 0.5 μg/ml, 99.9% killing was observed for all strains exceptE. coliCFT073-RR GyrAr/p449. In the murine model of pyelonephritis, an optimal regimen of ciprofloxacin (10 mg/kg of body weight twice a day [b.i.d.]) significantly decreased the bacterial count in the kidneys of mice infected withE. coliCFT073 (1.6 versus 4.3 log10CFU/g of kidney compared to untreated controls;P= 0.0001), while no significant decrease was observed forE. coliCFT073-RR/p449 (2.7 versus 3.1 log10CFU/g;P= 0.84),E. coliCFT073-RR GyrAr(4.2 versus 4.1 log10CFU/g;P= 0.35), orE. coliCFT073-RR GyrAr/p449 (2.9 versus 3.6 log10CFU/g;P= 0.47). While pharmacokinetic and pharmacodynamic (PK/PD) parameters accounted for ciprofloxacin failure againstgyrA-containing mutants, this was not the case for theaac(6′)-Ib-cr-containing strains, suggesting anin situhydrolysis of ciprofloxacin in the latter case.

scholarly journals In Vivo Detection and Quantification of Tetracycline by Use of a Whole-Cell Biosensor in the Rat Intestine

2004 ◽  
Vol 48 (4) ◽  
pp. 1112-1117 ◽  
Author(s):  
Martin Iain Bahl ◽  
Lars Hestbjerg Hansen ◽  
Tine Rask Licht ◽  
Søren J. Sørensen
Keyword(s):  
Drinking Water ◽  
Fluorescent Protein ◽  
Significant Finding ◽  
E Coli ◽  
Standard Curve ◽  
In Vivo Detection ◽  
Biosensor Strain ◽  
Whole Cell Biosensor ◽  
Green Fluorescent

ABSTRACT An Escherichia coli biosensor strain, harboring the plasmid pTGFP2, was introduced into the gastrointestinal tract of gnotobiotic rats that continuously received drinking water containing tetracycline. Plasmid pTGFP2 contains a transcriptional fusion between a green fluorescent protein (GFP) gene and a tetracycline-regulated promoter and was shown to produce a proportional GFP signal in response to exposure to various tetracycline concentrations when harbored by an E. coli strain. The plasmid was highly unstable in the host bacteria colonizing the intestinal system of the animals, and rapid plasmid loss was observed. Reintroduction of the E. coli MC4100/pTGFP2 strain into animals already colonized by the plasmid-free E. coli strain the day before euthanasia made it possible to extract and analyze the biosensors from intestinal samples. The induction of GFP in the biosensor cells extracted from the animals was estimated on a single-cell basis by use of flow cytometry, and the mean induction of GFP in the samples was compared to a standard curve prepared from known tetracycline concentrations. The results showed that the bioavailable tetracycline concentration within the bacterial growth habitat of the intestine was proportional to the concentration of tetracycline in drinking water but represented only approximately 0.4% of the intake concentration. This is a significant finding which will help to clarify antimicrobial therapy in the intestinal environment.

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