scholarly journals The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin

2020 ◽  
Author(s):  
Mohamed Boumaiza ◽  
Samia Rourou ◽  
Paolo Arosio ◽  
Mohamed Nejib Marzouki
Keyword(s):  
Mammalian Cells ◽  
Iron Homeostasis ◽  
Coli Strain ◽  
Binding Assays ◽  
Cysteine Oxidation ◽  
E Coli ◽  
Diagnostic Use ◽  
Peptide Carrier ◽  
Human Ferritin ◽  
Ferritin H

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5’end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys E. coli strain. The transformed E.coli produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7.

scholarly journals Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin–Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

2021 ◽  
Vol 44 (1) ◽  
pp. 117-127
Author(s):  
Mohamed Boumaiza ◽  
Imene Fhoula ◽  
Fernando Carmona ◽  
Maura Poli ◽  
Michela Asperti ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Functional Characterization ◽  
Wild Type ◽  
E Coli ◽  
Molar Ratios ◽  
Human Ferritin ◽  
Stable Product ◽  
Soluble Molecule ◽  
Ferritin H

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Towards Light-Regulated Living Biomaterials

2018 ◽  
Author(s):  
Shrikrishnan Sankaran ◽  
Shifang Zhao ◽  
Christina Muth ◽  
Julieta Paez ◽  
Aránzazu del Campo
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Coli Strain ◽  
Genetically Engineered ◽  
External Control ◽  
Bacterial Surface ◽  
E Coli ◽  
And Performance ◽  
Living Materials

Living materials are a rapidly emerging material class, infused with the productive, adaptive and regenerative properties of living organisms. Property regulation in living materials requires external control of the activity of the living components, in order to achieve desired functions and performance. As a first step, a light-activatable E. coli-based system that can be externally triggered to interact with mammalian cells has been genetically engineered as an active component for developing optoregulated living-biomaterials. This has been achieved by combining optogenetic activation of gene expression using a photo-activatable inducer molecule and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. The bacteria are immobilized on surfaces and in situ light-activation of the E. coli results in mammalian cells specifically responding to them. Possible delivery of a fluorescent protein from the bacteria to the mammalian cells when they are interacting is also observed, indicating the potential of such a bacterial material to deliver complex cargo to cells in a targeted manner.<br><br>

Towards Light-Regulated Living Biomaterials

2018 ◽  
Author(s):  
Shrikrishnan Sankaran ◽  
Shifang Zhao ◽  
Christina Muth ◽  
Julieta Paez ◽  
Aránzazu del Campo
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Surface Display ◽  
Coli Strain ◽  
Genetically Engineered ◽  
External Control ◽  
Bacterial Surface ◽  
E Coli ◽  
And Performance ◽  
Living Materials

Living materials are a rapidly emerging material class, infused with the productive, adaptive and regenerative properties of living organisms. Property regulation in living materials requires external control of the activity of the living components, in order to achieve desired functions and performance. As a first step, a light-activatable E. coli-based system that can be externally triggered to interact with mammalian cells has been genetically engineered as an active component for developing optoregulated living-biomaterials. This has been achieved by combining optogenetic activation of gene expression using a photo-activatable inducer molecule and bacterial surface display technology to present an integrin-specific miniprotein on the outer membrane of an endotoxin-free E. coli strain. The bacteria are immobilized on surfaces and in situ light-activation of the E. coli results in mammalian cells specifically responding to them. Possible delivery of a fluorescent protein from the bacteria to the mammalian cells when they are interacting is also observed, indicating the potential of such a bacterial material to deliver complex cargo to cells in a targeted manner.<br><br>

scholarly journals Synthesis and in Silico Modelling of the Potential Dual Mechanistic Activity of Small Cationic Peptides Potentiating the Antibiotic Novobiocin against Susceptible and Multi-Drug Resistant Escherichia coli

2020 ◽  
Vol 21 (23) ◽  
pp. 9134
Author(s):  
Ilaria Passarini ◽  
Pedro Ernesto de Resende ◽  
Sarah Soares ◽  
Tadeh Tahmasi ◽  
Paul Stapleton ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Mammalian Cells ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Coli Strain ◽  
Cell Permeability ◽  
Cationic Peptides ◽  
Drug Resistant ◽  
E Coli

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3–9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs.

scholarly journals New indicator Escherichia coli strain for rapid and accurate detection of supF mutations

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Ruriko Fukushima ◽  
Tetsuya Suzuki ◽  
Hiroyuki Kamiya
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cells ◽  
Indicator Strain ◽  
Coli Strain ◽  
Chromosomal Dna ◽  
Amber Mutation ◽  
E Coli ◽  
Accurate Detection ◽  
Mutation Spectra ◽  
Forward Mutation

Abstract Background The supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed. Results The gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01. Conclusion RF01 is a new and useful indicator E. coli strain for analyzing supF mutations.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

Transport of escherichia coli through soil to groundwater traced by randomly amplified polymorphic DNA (RAPD)

1997 ◽  
Vol 35 (11-12) ◽  
pp. 351-357 ◽  
Author(s):  
R. Rothmaier ◽  
A. Weidenmann ◽  
K. Botzenhart
Keyword(s):  
Negative Impact ◽  
Random Amplified Polymorphic Dna ◽  
Coli Strain ◽  
Soil Samples ◽  
Test Field ◽  
Liquid Manure ◽  
E Coli ◽  
Rapd Pattern ◽  
Geological Situation ◽  
Different Strains

Isolates (50) of E. coli obtained from liquid manure (20 bovine, 20 porcine) were genotyped using random amplified polymorphic DNA (RAPD). Typing revealed 9 and 14 different strains in bovine and porcine liquid manure respectively with no strains in common. One porcine strain, showing a simple RAPD pattern, was subcultured and spread on a test field (1.5l/m2 at 1010 cfu/l) in a drinking water protection zone with loamy to sandy sediments in the Donauried area, Baden-Wurttemberg. Soil samples and groundwaters were collected at monthly intervals October 1994 – June 1995 during which 114 E. coli isolates were recovered. The first occurrence and maximum concentration of E. coli in soil samples taken from more than 20cm depth was in January 1995, declining rapidly with depth and time. All isolates from soil and only one from groundwater showed the RAPD pattern of the spread E. coli strain. The results could not demonstrate a severe negative impact of the spreading of liquid manure on the bacteriological quality of the groundwater in the given geological situation. The distinct strain patterns found in different kinds of liquid manure suggest that genotyping of E. coli by RAPD may be an adequate tool for tracing sources of faecal contamination.

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