Genome-Scale Characterization of Fungal Phytases and a Comparative Study Between Beta-Propeller Phytases and Histidine Acid Phosphatases.

This work intended to prospect new phytase-producing organisms. In silico genomic analyses allowed the selection of twelve potential phytase-producing fungi. Based on gene sequence, it was possible to identify four well-defined groups of phytate-degrading enzymes: esterase-like, ß-propeller phytases (ßPP), phosphoglycerate mutase-like, and phytases of the histidine acid phosphatases (HAP) family. Analysis of the predicted genes encoding phytases belonging to the HAP family and ßPP phytases and in silico characterization of these enzymes indicated divergence among the catalytic activities. Predicted fungal ßPP phytases exhibited higher molecular mass (around 77 kDa) probably due to the epidermal growth factor-like domain. Twelve sequences of phytases contained signal peptides, of which seven were classified as HAP and five as ßPP phytases, while ten sequences were predicted as phytases secreted by non-classical pathways. These fungi were grown in liquid or semi-solid medium, and the fungal enzymatic extracts were evaluated for their ability to hydrolyze sodium phytate at 50 °C and pH ranging from 2.0 to 9.0. Seven fungi were identified as phytase producers based on phosphate release under enzyme assay conditions. Results obtained from in silico analyses combining experimental enzymatic activities suggest that some selected fungi could secrete ßPP phytases and HAP phytases.

FTIR study of secondary structure changes in Epidermal Growth Factor by gold nanoparticle conjugation.

Conformation of protein is vital to its function, but may get affected when processing to manufacture products. It is therefore important to understand structural changes during each step of production. In this study, we investigate secondary structure changes in the targeting protein Epidermal Growth Factor (EGF) during synthesis of theranostic bifunctional nanoparticle, devised for Photodynamic therapy of breast cancer. We acquired FTIR spectra of EGF; unconjugated, post treatment with α-lipoic acid, attached to gold nanoparticle, and bound to the bifunctional nanoprobe. We observed decreasing disordered structures and turns, and increasing loops, as the synthesis process progressed. There was an overall increase in ß-sheets in final product compared to pure EGF, but this increase was not linear and fluctuated. Previous crystal structure studies on EGF-EGFR complex have shown loops and ß-sheets to be important in the binding interaction. Since our study found increase in these structures in the final product, no adverse effect on binding function of EGF was expected. This was confirmed by functional assays. Such studies may help modify synthesis procedures, and thus secondary structures of proteins, enabling increased functionality and optimum results.

Translocation of Epidermal Growth Factor (EGF) to the nucleus has distinct kinetics between adipose tissue-derived mesenchymal stem cells and a mesenchymal cancer cell lineage.

Nuclear Epidermal Growth Factor Receptor (EGFR) has been associated with worse prognosis and treatment resistance for several cancer types. After Epidermal Growth Factor (EGF) binding, the ligand-receptor complex can translocate to the nucleus where it functions in oncological processes. By three-dimensional quantification analysis of super-resolution microscopy images, we verified the translocation kinetics of fluorescent conjugated EGF to the nucleus in two mesenchymal cell types: human adipose tissue-derived stem cells (hASC) and SK-HEP-1 tumor cells. The number of EGF clusters in the nucleus does not change after 10 min of stimulation with EGF in both cells. The total volume occupied by EGF clusters in the nucleus of hASC also does not change after 10 min of stimulation with EGF. However, the total volume of EGF clusters increases only after 20 min in SK-HEP-1 cells nuclei. In these cells the nuclear volume occupied by EGF is 3.2 times higher than in hASC after 20 min of stimulation, indicating that translocation kinetics of EGF differs between these two cell types. After stimulation, EGF clusters assemble in larger clusters in the cell nucleus in both cell types, which suggests specific sub-nuclear localizations of the receptor. Super-resolution microscopy images show that EGF clusters are widespread in the nucleoplasm, and can be localized in nuclear envelope invaginations, and in the nucleoli. The quantitative study of EGF-EGFR complex translocation to the nucleus may help to unravel its roles in health and pathological conditions, such as cancer.

Role of EGF on in situ culture of equine preantral follicles and metabolomics profile.

The effects of epidermal growth factor (EGF) concentrations (0, 10, 50, and 100ng/ml) on in vitro culture (IVC) of equine preantral follicles were evaluated using histology, estradiol and reactive oxygen species (ROS) production and metabolomics. After IVC, the percentage of normal follicles was lower (P<0.05) for all treatments when compared to non-cultured control. EGF 50ng/ml treatment had more (P<0.05) normal follicles at Day 7 of culture when compared with EGF 0 and 100ng/ml. EGF 50ng/ml had more (P<0.05) developing follicles than the 0ng/ml and 10ng/ml EGF treatments. Follicular and oocyte diameters were greater (P<0.05) with EGF 50ng/ml than the other cultured treatments, but similar (P>0.05) to the non-cultured control. From Day 1 to Day 7 estradiol production increased (P<0.05) in all EGF treatments. EGF 50ng/ml was the only treatment that maintained ROS production through IVC. Metabolomics profiles of the spent media indicated that eleven ions from variable influence in the projection (VIP) scores were higher represented in the EGF 50ng/ml treatment. In conclusion, EGF 50ng/ml treatment maintained follicle survival and ROS production, and promoted activation of cultured equine preantral follicles enclosed in ovarian tissue.

Neuroprotective effect of epidermal growth factor in experimental acrylamide neuropathy: an electrophysiological approach.

The neuroprotective effect of epidermal growth factor (EGF) has been documented in different contexts, but its potential benefits in peripheral neuropathies have been little studied. We investigated the neuroprotective action of EGF in experimental neuropathy induced by acrylamide (ACR). Mice and rats were treated chronically with acrylamide for 6 and 8 weeks, respectively. Concurrently they received EGF in daily doses of 1 and 5 mg/kg in mice and 3 mg/kg in rats, or saline (PBS). ACR severely affected the neurological score, the muscle strength, and the muscle potential M, in mice, as well as F-waves (F-Wii), sensory potentials (SPii), and apomorphine-induced penile erection, in rats. EGF reduced the ACR effects in both species. A dose-dependent effect of EGF was manifested in the proportion of diseased animals at the end of treatments, as well as in the reduction of M amplitude throughout the treatment. F-Wii parameters were less protected by EGF than SP. The results show a protective effect of EGF in acrylamide-induced neuropathy and support previous studies concerning the neuroprotective action of this peptide.

Layer-by-layer polysaccharide-coated liposomes for sustained delivery of epidermal growth factor.

A three-dimensional layer-by-layer (LbL) structure composed by xanthan and galactomannan biopolymers over dioctadecyldimethylammonium bromide (DODAB) liposome template was proposed and characterized for protein drug delivery. The polymers and the surfactant interaction were sufficiently strong to create a LbL structure up to 8 layers, evaluated using quartz crystal microbalance (QCM) and zeta potential analysis. The polymer-liposome binding enthalpy was determined by isothermal titration calorimetry (ITC). The bilayer of biopolymer-coated liposomes with diameters of 165 (±15)nm, measured by dynamic light scattering (DLS), and ζ-potential of -4 (±13)mV. These bilayer-coated nanoparticles increased up to 5 times the sustained release of epidermal growth factor (EGF) at a first order rate of 0.005min(-1). This system could be useful for improving the release profile of low-stability drugs like EGF.

A combinatorial mutagenesis approach for functional epitope mapping on phage-displayed target antigen: application to antibodies against epidermal growth factor.

Although multiple different procedures to characterize the epitopes recognized by antibodies have been developed, site-directed mutagenesis remains the method of choice to define the energetic contribution of antigen residues to binding. These studies are useful to identify critical residues and to delineate functional maps of the epitopes. However, they tend to underestimate the roles of residues that are not critical for binding on their own, but contribute to the formation of the target epitope in an additive, or even cooperative, way. Mapping antigenic determinants with a diffuse energetic landscape, which establish multiple individually weak interactions with the antibody paratope, resulting in high affinity and specificity recognition of the epitope as a whole, is thus technically challenging. The current work was aimed at developing a combinatorial strategy to overcome the limitations of site-directed mutagenesis, relying on comprehensive randomization of discrete antigenic regions within phage-displayed antigen libraries. Two model antibodies recognizing epidermal growth factor were used to validate the mapping platform. Abrogation of antibody recognition due to the introduction of simultaneous replacements was able to show the involvement of particular amino acid clusters in epitope formation. The abundance of some of the original residues (or functionally equivalent amino acids sharing their physicochemical properties) among the set of mutated antigen variants selected on a given antibody highlighted their contributions and allowed delineation of a detailed functional map of the corresponding epitope. The use of the combinatorial approach could be expanded to map the interactions between other antigens/antibodies.

Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: effect of pH, buffers, and excipients.

A successful parenteral formulation can be developed by studying stability and compatibility of biopharmaceuticals as a function of solution composition. Here, we evaluate the influence of pH, buffers, ionic strength, protein concentration and presence of excipients on recombinant human epidermal growth factor (rhEGF) stability. The stability was accessed by reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), enzyme-linked immunosorbent assay (ELISA), Far-UV circular dichroism (CD) and light scattering. The overall maximal stability was obtained in pH near to 7.0 in phosphate, Tris and histidine buffers as the results of the different methods revealed. The CD results revealed that this protein is stable in an extensive pH range. Aggregation of rhEGF was minimized at pH values ranged from 6.0 to 8.0 as indicated the SEC-HPLC and light scattering results. Nor the ionic strength neither the rhEGF concentration had significant effect on the reaction rate constants. Most rhEGF-excipient instability occurs among this protein and reducing sugars. Polymers like poly(ethylene glycol) (PEG) and polysorbates increased methionine oxidation. The rhEGF oxidation and deamidation were the most common degradation pathways. This research identified critical solution factors to be considered for the development of a successful rhEGF parenteral formulation.

Extraction of PLGA-microencapsulated proteins using a two-immiscible liquid phases system containing surfactants.

PURPOSE: The extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated. METHODS: First, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry. RESULTS: Without surfactants, protein recovery was only 27-43% for IFN-α2b and 58-73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins. CONCLUSION: Proteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

Plasmodium vivax merozoite surface protein 8 cloning, expression, and characterisation.

Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasite's partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N- and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This gene's transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patients' sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multi-antigen P. vivax vaccine.

Interaction of a monoclonal antibody against hEGF with a receptor site for EGF.

Epidermal growth factor (EGF) has been detected by radioimmunoassay (RIA) in different body fluids such as serum, amniotic fluid, and urine. Human tumor tissues with EGF receptors (EGF-Rc) may be saturated with EGF, which may be of prognostic value. An RIA was envisaged to measure human epidermal growth factor (hEGF) levels using EGF-Rc as capture agent and a monoclonal antibody anti-hEGF (MAb anti-hEGF) labeled with 125Iodine as a marker for this binding. The purpose of this work was to study the feasibility of MAb anti-hEGF to detect the receptor binding sites and to investigate the interaction between MAb anti-hEGF and the EGF-Rc. Various binding experiments were performed to study possible interference and interactions in the complex MAb anti-hEGF and the receptor. Affinity constants were determined by means of Scatchard plot analysis to interpret the complex stability challenged with other compounds for a better understanding of the interaction process. Binding constants were of the same order for all the ligands tested separately involving the EGF-Rc, but were significantly higher (t = 15.7, p < 0.05) for hEGF in its binding to MAb anti-hEGF. It was possible with equilibrium studies and competition experiments to evaluate the interaction of EGF and MAb anti-hEGF with the EGF receptor. This observation makes the MAb anti-hEGF a potential tracer for the quantitation of receptors in vitro, and possibly for the detection of membrane receptors on tumor cells in vivo.

MHC class I-hormone receptor associations: still a physiological enigma?

Besides the important role of the Major Histocompatibility Complex (MHC) class I molecules n physiologically restricting immune responses, they seem to play a non-immunological function by the molecular association with several hormone receptors (R). Among the hormone R and class I antigen (Ag) interactions, insulin R, epidermal growth factor (EGF) R, interleukin-2 (IL-2) R, luteinizing (LH) hormone R, beta adrenergic R and muscarinic cholinergic R were described in several human and animal models. Evidences from immunoprecipitation assays, binding assays and immunofluorescence techniques pointed to the molecular association of all of these R with class I molecules on cellular surfaces. Only for beta adrenergic R, muscarinic cholinergic R and LH-R, antibodies directed against class I products were described to exert the activation of these R leading to the production of intracellular second messengers and consequently modifying the physiology of the corresponding cell. This was also obtained on insulin R with peptides derived from class I molecules. The selectivity or R involved in all studied cellular types, should find its explanation in the physiological importance of the considered R for the indicated cell. The participation of cytoskeletal proteins on these interactions and the proximity on cell surfaces between both molecules, probably managed by ligand-mediated microaggregation, are also facts to be taken into account to better understand the biological consequences of these interactions.

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).