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

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

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

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

Production and characterization of functional recombinant hybrid heteropolymers of camel hepcidin and human ferritin H and L chains

This article has been accepted for publication in Protein Engineering design and Selection Published by Oxford University Press.Hepcidin is a liver-synthesized hormone that plays a central role in the regulation of systemic iron homeostasis. To produce a new tool for its functional properties the cDNA coding for camel hepcidin-25 was cloned at the 5’end of human FTH sequence into the pASK-IBA43plus vector for expression in Escherichia coli. The recombinant fusion hepcidin–ferritin-H subunit was isolated as an insoluble iron-containing protein. When alone it did not refold in a 24-mer ferritin molecule, but it did when renatured together with H- or L-ferritin chains. We obtained stable ferritin shells exposing about 4 hepcidin peptides per 24-mer shell. The molecules were then reduced and re-oxidized in a controlled manner to allow the formation of the proper hepcidin disulfide bridges. The functionality of the exposed hepcidin was confirmed by its ability to specifically bind the mouse macrophage cell line J774 that express ferroportin and to promote ferroportin degradation. This chimeric protein may be useful for studying the hepcidin–ferroportin interaction in cells and also as drug-delivery agent.This work is partially financed by the Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB) and the Doctoral School of the National Institute of Applied Sciences and Technology (INSAT-Tunis) – University of Carthage

Chemical Composition, Antioxidant and Antimicrobial Activities of Thymus capitata

The chemical composition, antioxidant and antimicrobial activities, and the preservative effect of Thymus capitata essential oil against Listeria monocytogenes inoculated in minced beef meat were evaluated. The essential oil extracted was chemically analyzed by gas chromatography-mass spectrometry. Nineteen components were identified, of which carvacrol represented (88.89%) of the oil. The antioxidant activity was assessed in vitro by using both the DPPH and the ABTS assays. The findings showed that the essential oil exhibited high antioxidant activity, which was comparable to the reference standards (BHT and ascorbic acid) with IC50 values of 44.16 and 0.463 μg/mL determined by the free-radical scavenging DPPH and ABTS assays, respectively. Furthermore, the essential oil was evaluated for its antimicrobial activity using disc agar diffusion and microdilution methods. The results demonstrated that the zone of inhibition varied from moderate to strong (15–80 mm) and the minimum inhibition concentration values ranged from 0.32 to 20 mg/mL. In addition, essential oil evaluated in vivo against Listeria monocytogenes showed clear and strong inhibitory effect. The application of 0.25 or 1% (v/w) essential oil of T. capitata to minced beef significantly reduced the L. monocytogenes population when compared to those of control samples (P-value  <0.01)

Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 μg/mL) and vancomycin (MIC = 2 μg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry

Purification et caractérisation d'une nouvelle peroxydase extraite de l'ail et son application en tant que biocapteur électrochimique pour l'analyse alimentaire et environnementale

La nouvelle peroxydase (POX1B) extraite de l'Ail possède des propriétés biochimiques intéressantes par comparaison à HRP. En effet, elle est hautement active à pH acide et stable vis-à-vis de la température et de la conservation. Elle présente un pH optimum d'environ 5 et une température optimale de 30C. L'enzyme conserve 70 % de son activité initiale à 60C et une activité totale à 50 et 40C après 40 min. Par la suite, l'étude de la relation structure-fonction a été réalisée en analysant les propriétés spectroscopiques ainsi que leur corrélation à la structure déterminée par une nouvelle génération de spectromètre de masse hybride à haute performance. Les études de la structure chimique ont montré que le groupement héminique de cette protéine est pentacoordiné et renferme une histidine comme ligand proximal. POX1B sous-forme native et reconstituée a montré une haute affinité vis-à-vis du peroxyde d'hydrogène ainsi que vis-à-vis d'une variété de co-substrats réducteurs. En plus, une haute spécificité de l'enzyme a été démontrée. Les valeurs de kcat/KM sont 413.28, 403.81 mM-1s-1 respectivement pour le TMB et l'ABTS. Par ailleurs, la réduction des composés nitrés en présence de POX1B a été démontrée par la formation de complexes Fe(II)-nitrosoalcanes. POX1B présente un grand potentiel d'application pour le métabolisme des médicaments puisqu'elle est capable de réagir avec le 1-nitrohexane en présence du dithionite de sodium comme ça a été démontré par l'apparition d'une bande de Soret caractéristique à 411 nm. En plus, l'efficacité catalytique de la nouvelle peroxydase extraite de l'Ail pourrait être exploitée pour la détection d'une variété d'analytes et en biocatalyse. L'enzyme possède un bon potentiel d'application en tant que biorécepteur pour la détection électrochimique du peroxyde d'hydrogène et des polluants. L'immobilisation de l'enzyme a été réalisée en utilisant le chitosane comme biopolymère. Les essais électrochimiques ont montré que le biocapteur est capable de mesurer une concentration de H2O2 allant jusqu'à 100 nM en mode de détection directe. La limite de détection mesurée dans des échantillons de lait a été de 30 M avec un temps de réponse inférieur à 1 min. Par la suite, l'activité électro-catalytique d'un second biocapteur à base de POX1B a été démontrée en présence de dérivés de chlorophénols avec des concentrations variant entre 10 pM et 10 M. Le biocapteur construit avec POX1B et sans utilisation d'un médiateur a présenté une bonne sensibilité en présence du 2,6-dichlorophénol, du 4-chlorophénol et du pentachlorophénol. Une limite de détection de l'ordre de 1 pM a été mesurée dans le cas du 4-chlorophénol avec une constante cinétique Km,app de 0.42 M et un temps de réponse électrochimique rapide de l'ordre d'1 s.A new peroxidase was purified from garlic bulbs (POX1B) with interesting biochemical properties compared to HRP. ln fact, POX1B is highly active at acidic pH and stable vs. temperature and storage. The optimal pH was around 5 and the optimal temperature was 30C. Studies of the heat-stability demonstrated that almost 70 % of the initial activity was conserved at 60C and full activity was retained at 50 and 40C for 40 min. Then, the structure-function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high performance hybrid mass spectrometer. Studies of the chemical structure showed that the heme group of this protein is pentacoordinated and has a histidine as proximal ligand. Native and reconstituted POX1B exhibited high affinity towards hydrogen peroxide as well as various reducing co-substrates. ln addition, high enzym specificity was demonstrated. The kcat/KM values were 413.28, 403.81 mM-1s-1 for TMB and ABTS, respectively. Furthermore, the reduction of nitro compounds in presence of POX1B was demonstrated by iron(II)-nitrosoalkane complexes assay. POX1B showed a great potential to be a lied for drug metabolism since its ability to react with 1-nitrohexane in resence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. Besides, the high catalytic efficiency obtained in the case of the new garlic peroxidase (POX1B is suitable for different analytes monitoring and biocatalysis). The enzyme has shown a good potential for electrochemical detection of hydrogen peroxide and chlorophenols. The enzyme immobilization was carried out using chitosan as biopolymer. Electrochemical assays showed that the biosensor was able to monitor H2O2 as low as 100 nM in direct detection mode. The measured detection limit was 30 M in milk samples with a response time less than 1 min. Then, the electrocatalytic activity of a second POX1B-based biosensor towards chlorophenols derivatives in a large range from 10 pM to 10 M was demonstrated. The mediator free POX1B based biosensor exhibited high sensitivity towards 2,6-dichlorophenol, 4-chlorophenol and pentachlorophenol. A detection limit of 1 pM in the case of 4-chlorophenol was demonstrated with kinetic constant Km,app of 0.42 M and high rapidity of electrochemical response of the biosensor of 1 s.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Lycium Europaeum Fruit Extract: Antiproliferative Activity on A549 Human Lung Carcinoma Cells and PC12 Rat Adrenal Medulla Cancer Cells and Assessment of Its Cytotoxicity on Cerebellum Granule Cells

International audienc

Assessment of cyto-protective, antiproliferative and antioxidant potential of a medicinal plant Jatropha podagrica

International audienc

Optimization of extracellular xylanase production by Sclerotinia sclerotiorum S2 using factorial design

404-409The improvement of xylanase production by Sclerotinia sclerotiorum S2 using a liquid fermentation culture was investigated. The optimized process was divided into three basic steps: (i) evaluating xylanase inducers using different agricultural residues such as wheat bran, oat bran, orange peel and barley bran at 1% final concentration, and also filter paper. Among these, wheat bran showed the maximum activity (2.5 U/ml) at 12 days post-inoculation; (ii) for optimization, we determined the optimal concentration of inducer, the effect of phosphate anion (K₂HPO₄/KH₂PO₄) and culture aeration using a rotary shaker at 100 and 180 rpm. The optimal conditions for these three factors were determined in an experimental panel using factorial data, in which a mathematical model (Minitab software) was fitted; (iii) The optimized culture medium containing a high level of wheat bran (3%) without K₂HPO₄/KH₂PO₄ and submitted to a high agitation (180 rpm/min) increased the xylanase production from 2.5 U/ml to 4 U/ml (1.6-fold)