Effect of ultrasound-irradiation combined pretreatment on the foamability of liquid egg white.

This study aims to optimize the ultrasound-irradiation combined pretreatment conditions to enhance the liquid egg white (LEW) foamability and investigate the changing mechanism about the physical and structural properties of LEW during the processing. Results indicated that the foamability of the LEW was increased by ultrasound-irradiation combined pretreatment to the highest value of 92.6% (irradiation dose = 33 kGy, ultrasound time = 6 min, and ultrasound power = 300 W). Three significant proteins in LEW (ovalbumin, ovotransferrin, and lysozyme) were chosen to explore the change law on their physical and structural properties. Results indicated that ultrasound-irradiation combined pretreatment increased the solubility and reduced the pH and particle size of ovalbumin and lysozyme, thus enhancing the foamability of LEW. Furthermore, the fluorescence spectra changes implied the un-folding and destruction of ovalbumin, ovotransferrin, and lysozyme during the ultrasound-irradiation combined pretreatment. Moreover, circular dichroism spectroscopy analysis revealed that the pretreatment decreased α-helix and ß-sheet of the ovalbumin, ovotransferrin, and lysozyme, which bring out the improvement of LEW foamability. The present study indicated that ultrasound-irradiation combined pretreatment had the potential to be implemented to enhance the foamability of LEW.

Visible light neutralizes the effect produced by ultraviolet radiation in proteins.

The damage produced by UV-C radiation (100-280nm) in organisms and cells is a well known fact. The main reactions of proteins to UV-C radiation consist in the alteration of their secondary structures, exposure of hydrophobic residues, unfolding and aggregation. Furthermore, it has been found that electromagnetic radiation of lower energy (visible light, where wavelengths are between 400 and 750nm) also induces different disturbances in biomolecules. For instance, it has been observed that blue visible light from emitting diodes (LEDs) produces severe damage in murine cone photoreceptor-derived cells, and it can be even more harmful for some organisms than UV radiation. Recently, it has been found that the exposure of proteins to green and red light produces conformational changes, considerably increasing their cohesion enthalpies. This is presumably due to the strengthening of the hydrogen bonds and the formation of new ones. Therefore, it seems that visible light acts contrary to what it is observed for UV-C: instead of unfolding the proteins it folds them further, halting the damage produced by UV-C. This can be understood if we consider the modification of the folding energy-landscape; visible light induces the descent of the proteins into deeper states impeding the unfolding produced by UV-C.

The immunogenicity of thin-film freeze-dried, aluminum salt-adjuvanted vaccine when exposed to different temperatures.

Insoluble aluminum salts such as aluminum oxyhydroxide have been used for decades as adjuvants in human vaccines, and many vaccines contain aluminum salts as adjuvants. Aluminum salt-adjuvanted vaccines must be managed in cold-chain (2-8° C) during transport and storage, as vaccine antigens in general are too fragile to be stable in ambient temperatures, and unintentional slowing freezing causes irreversible aggregation and permanent damage to the vaccines. Previously, we reported that thin-film freeze-drying can be used to convert vaccines adjuvanted with an aluminum salt from liquid suspension into dry powder without causing particle aggregation or decreasing in immunogenicity following reconstitution. In the present study, using ovalbumin (OVA)-adsorbed Alhydrogel® (i.e. aluminum oxyhydroxide, 2% w/v) as a model vaccine, we showed that the immunogenicity of thin-film freeze-dried OVA-adsorbed Alhydrogel® vaccine powder was not significantly changed after it was exposed for an extended period of time in temperatures as high as 40° C or subjected to repeated slow freezing-and-thawing. It is expected that immunization programs can potentially benefit by integrating thin-film freeze-drying into vaccine preparations.

CW/pulsed NIR irradiation of gold nanorods: effect on transdermal protein delivery mediated by photothermal ablation.

Transdermal delivery is a useful and attractive method for drug delivery, even though the stratum corneum is a major barrier of protein translocation into the skin. To achieve protein delivery through the stratum corneum, we first cast gold nanorods, acting as a heating device in response to near-infrared light irradiation, onto the skin surface. After applying an aqueous solution of ovalbumin to the skin, the skin was irradiated by near-infrared laser light. Irradiation of the skin using a continuous-wave laser increased the skin temperature resulting in an efficient translocation of ovalbumin into the skin. Furthermore, migration of inflammation cells and induction heat shock protein 70 (HSP70) were observed. Irradiation of the skin using a pulsed laser caused an enhanced permeability of the stratum corneum without an increase in skin temperature, migration of inflammation cells, or HSP70 induction. This effect is due to the pulsed-laser irradiation increasing the temperature of a limited part of the skin surface. Thus, the physiological response of skin is dependent on the type of laser light used. It is anticipated that this phenomenon will find wide application in such applications as, for example, general transdermal protein delivery and transdermal vaccination.

Ovalbumin modified by gamma irradiation alters its immunological functions and allergic responses.

It is well known that gamma (gamma)-ray irradiation results in the alteration of biological function of bioactive materials such as proteins, saccharides and lipids. In this study the effect of gamma-irradiation on the chemical and immunological property of an allergen, ovalbumin (OVA), was investigated. Irradiation of more than 10 kGy resulted in the alteration of the structure of OVA. However, OVA treated with 10 kGy irradiation (10 kGy-OVA), but not 100 kGy-OVA, fully maintained immunological reactivity to a monoclonal antibody specific to the intact allergen (clone 14). Mice immunized with 10 kGy- as well as 100 kGy-OVA showed significantly lower antibody response to the allergen than those with intact OVA in a gamma-ray dosage-dependent manner. Especially immunization of both 10 kGy- and 100 kGy-OVA induced a significant decrease of OVA-specific IgE. Splenocytes of mice immunized with irradiated OVA showed a significant reduction in OVA-specific T cell proliferation and the secretion of Th1-type (IFN-gamma and IL-2) and Th2-type cytokines (IL-4 and IL-6). The expression of T cell activation markers such as CD25 and CD44 was also down-regulated in T cells of mice immunized with irradiated OVAs. These results suggest that gamma-ray irradiation of OVA suppress humoral and cellular immune responses specific to the allergen OVA, and the modification method with gamma-irradiation may be available for the control of allergy.

Surface characterization by atomic force microscopy of sterilized PLGA microspheres.

Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by gamma-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to gamma-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards gamma-irradiation.

The effect of gamma-irradiation on PLGA/PEG microspheres containing ovalbumin.

Poly(ethylene glycol) (PEG) and sodium chloride (NaCl) are excipients used in PLGA microsphere preparation to stabilize proteins and reduce their burst release. No information is till now available in the literature on the effect due to the use of such excipients on the biopharmaceutical performance of gamma-irradiated microparticulate systems. On this purpose, different batches of microspheres containing ovalbumin (OVA) were prepared by using a PLGA 50:50 (average Mr: 13000), different amounts of PEG (Mr: 400 or 4000) and/or sodium chloride. The non-irradiated and irradiated microspheres were characterized in terms of morphology (SEM, particle size distribution), OVA and PEG content and in vitro OVA release. Radiolysis mechanisms of OVA and OVA loaded microspheres were investigated by EPR analysis. Gamma irradiation affects either microsphere morphology or the release of OVA as a function of the amount of PEG, and the use of NaCl. Irradiation significantly reduces release rate of protein from the microspheres containing 15% and 30% of PEG and from controls (microspheres without additives), while no significative effect on protein release rate is highlighted on microspheres containing lower amounts of PEG. EPR investigation shows that increasing amounts of PEG up to 30% have a perturbation effect on OVA radiolysis path.

Changes in the antigenic and immunoglobulin E-binding properties of hen's egg albumin with the combination of heat and gamma irradiation treatment.

This study was carried out to evaluate the changes in the allergenic and antigenic properties of hen's egg albumin (ovalbumin [OVA]) with the combination of heat and gamma irradiation treatment. OVA solution samples were treated by (i) heating (sample 1), (ii) irradiation after heating (sample 2), and (iii) heating after irradiation (sample 3). Samples were isothermally heated and irradiated at the absorption dose of 10 kGy. Competitive indirect enzyme-linked immunosorbent assays (ELISAs) were performed with blood serum to test the ability of treated OVA to bind to immunoglobulin E (IgE) and mouse murine monoclonal antibody (IgG). OVA's ability to bind to mouse IgG changed upon heating at 75 degrees C, and its ability to bind to egg-allergic IgE changed upon heating at 80 degrees C. The ELISAs showed that egg-allergic IgE did not recognize OVA very well when heated at > or = 80 degrees C, while mouse IgG retained better activity under these conditions. Egg-allergic IgE binding was low both for OVA samples treated by heating and for samples treated by irradiation followed by heating. These results show that allergies induced by OVA could be effectively reduced by the combination of heat and gamma irradiation treatment.

Effects of pulsed electric fields on ovalbumin solutions and dialyzed egg white.

Ovalbumin solutions (2%, pH 7.0, 200 ohm.cm) and dialyzed fresh egg white (pH 9.2, 200-250 ohm.cm) were subjected to 50-400 exponential decay pulses with an electric field strength of 27-33 kV/cm. The pulse width was ca. 0.3 micros (at a capacitance of 20 nF) or 0.9 micros (at 80 nF), and the corresponding dissipated energy was 0.7 or 2.3 J/(pulse.mL) of solution. The sample temperature was maintained below 29 degrees C. While the four sulfhydryl groups of native ovalbumin did not react with DTNB, they became reactive immediately after pulse processing, indicating either partial protein unfolding or enhanced SH ionization. The extent of SH reactivity increased with dissipated energy, 3.7 SH groups becoming reactive after 100 or 200 pulses at 31.5 kV/cm and 80 nF. However, SH reactivity was reversible, since only 0.79 or 0.2 SH group was found to remain reactive 30 min or 8 h after pulse processing. The fourth derivatives of UV spectra of ovalbumin were determined, before and 15-30 min after pulse processing, to assess possible polarity and conformation changes in the environment of tyrosine and tryptophan. No differences were observed. Thermal gels prepared from fresh or dialyzed egg white had markedly different mechanical and water retention characteristics. Pulse processing of dialyzed egg white (200 pulses, 30 kV/cm, 80 nF) only slightly reduced its gelling properties. Thus electric pulses known to induce significant microbial inactivation did not cause notable changes in the proteins investigated.

Monitoring the irradiation-induced conformational changes of ovalbumin by using monoclonal antibodies and surface plasmon resonance.

Two types of conformationally specific anti-irradiated ovalbumin monoclonal antibodies were prepared in order to study and monitor irradiation-induced structural changes in the ovalbumin molecule. Surface plasmon resonance (SPR) detection was used to investigate the kinetic parameters of the reaction between antibodies and ovalbumin which had been administered with different doses of irradiation (0, 1.5, 2.0, 5.0, 10, 20, 50, and 100 kGy). The results demonstrate that the combination of monoclonal antibodies and the SPR method can be used to characterize the irradiation-induced conformational change with an unlabelled reagent.

Heating of cardiovascular stents in intense radiofrequency magnetic fields.

We consider the heating of a metal stent in an alternating magnetic field from an induction heating furnace. An approximate theoretical analysis is conducted to estimate the magnetic field strength needed to produce substantial temperature increases. Experiments of stent heating in industrial furnaces are reported, which confirm the model. The results show that magnetic fields inside inductance furnaces are capable of significantly heating stents. However, the fields fall off very quickly with distance and in most locations outside the heating coil, field levels are far too small to produce significant heating. The ANSI/IEEE C95.1-1992 limits for human exposure to alternating magnetic fields provide adequate protection against potential excessive heating of the stents.

Radiolysis of alpha, alpha-trehalose in concentrated aqueous solution; the effect of co-irradiated proteins and lipids.

gamma-Radiolysis (dose-rate: 0 X 89 Gy/s) or electron (e)-radiolysis (dose-rate: 5 X 10(7) Gy/s) of unbuffered aqueous solutions of alpha, alpha-trehalose (concentration: 60 mg/ml, radiation dose: 20 kGy) at 0 degree C yielded glucose (Ggamma = 1 X 7; Ge = 0 X 63) and 5-deoxyxylohexodialdose (Ggamma = 0 X 21; Ge = 0 X 05). Buffering at pH-values of 5 X 0 or 5 X 5 and irradiation caused increased formation of these monomeric products, particularly of the deoxy-compound. On addition of increasing amounts of bovine serum albumin or ovalbumin (10-30 mg/ml) and irradiation the yields of products were markedly reduced. The decrease in glucose formation was less pronounced when sperm whale myoglobin was present during gamma- or electron-irradiation. The G-values of 5-deoxyxylohexodialdose, however, were increased by 45 per cent (gamma-irradiation) and 70 per cent (electron-irradiation) at approximately 10 mg/ml of admixed myoglobin. Further increase in myoglobin concentration led to a gradual decrease in the yields of the deoxy-product. The observed effects are explained by scavenging of water radicals and by interactions of the added substrates with sugar radicals. Emulsified lipids (palmitic acid methylester or trilinoleic glycerol) did not affect the radiation-induced formation of products from trehalose.

[Luminescence quenching of UV-irradiated proteins]. / Tushenie liuminestsentsii belkov pri UF-obluchenii

Kinetic curves of paramagnetic centres accumulation and quenching of the luminescence of UV-irradiated proteins at 77 degrees K are compared. Initiation of luminescence of UV-irradiated proteins at the annealing of paramagnetic centres was studied. The luminescence of UV-irradiated proteins was shown to be quenched with the paramagnetic centres formed. It was shown that protein absorption spectrum was not changed when the limiting concentration of paramagnetic centres was obtained in it.

[Mechanism of the formation of paramagnetic centers in proteins exposed to UV-radiation]. / Mekhanizm obrazovaniia paramagnitnykh tsentrov v belkakh pri deistvii UF-sveta

It is shown that the process of formation of paramagnetic centres in UV-irradiated at 77degreesK protein solutions in a biquantum one. The protein molecule in triplet state is an intermediate product.

[Accumulation of paramagnetic centers in protein solutions exposed to UV-radiation]. / Issledovanie nakopleniia paramagnitnykh tsentrov v rastvorakh belkov pri deistvii UF-sveta

It is shown that concentration of paramagnetic centres (PC) in UV-irradiated protein solutions at 77degreesK approximates the limiting value. The limiting number of PC (n) per one molecule is in direct proportion to that of aromatic amino acid residues in it n(sigma)=2+0,1 sigma. The formation of PC slopps because all the energy absorbed by aromatic amino acid residues is transfered to the radicals formed.