电泳和质谱技术研究4种胰脏铁蛋白的亚基类型和等电点特性

采用电泳和质谱技术对所制备的鸡、鸭、牛和猪胰脏铁蛋白的亚基类型和等电点特性进行了研究。采用天然聚丙烯酰胺凝胶电泳(PAGE)技术研究的结果表明,上述4种铁蛋白呈现不同的迁移率,据此可知鸡胰铁蛋白的相对分子质量(Mr)>鸭胰铁蛋白的Mr>黄牛胰铁蛋白的Mr>猪胰铁蛋白的Mr,而且均大于马脾铁蛋白(HSF)的Mr。采用十二烷基硫酸钠(SDS)-PAGE技术研究的结果表明,上述4种铁蛋白均由H(heavy chain)和L(light chain)类型的亚基组成,但H和L亚基的相对数量（即H/L亚基数量的比值）有差别。采用肽指纹图谱技术分别鉴定各铁蛋白的H和L亚基。选用变性等电聚焦方法研究发现,上述4种铁蛋白分别由3～6种不同等电点的亚基聚合体组成,说明铁蛋白的H和L亚基之间呈现复杂的相互作用和不同的聚合体。不同陆生动物胰脏铁蛋白亚基之间相互作用的强度和聚合态存在着差异,这一差异特性可能与调控铁蛋白释放铁的速率有关,也与动物对铁的需求和铁解毒速率有关。     

Mössbauer studies of iron incorporation into ferritins

Iron(III) monomers, dimers and clusters have been identified by Mössbauer spectroscopy during the initial stages of iron incorporation into ferritins, following Fe(II) oxidation. Iron(III) monomers seem to arise from dimer dissociation. Some of the monomers are transferred from iron poor to iron rich ferritin molecules, where they join the iron core clusters. Horse spleen ferritin, several variants of human H chain ferritin andEscherichia coli ferritin (Ec-FTN) can all accept the iron from human H chain ferritin. The small iron cores of Ec-FTN are different from those of mammalian ferritins, which indicates that the structure of the iron core depends on the protein shell.     

Separation of Multiple Ferritin Subunit Types by Reversed-Phase HPLC

Abstract

Subjection of ferritin subunits to reversed-phase HPLC effected a separation of them into three chromatographic species. These three putative subunit species were found in varying proportions from porcine spleen, liver, and heart ferritins as well as human liver, spleen, and placental ferritins. Detailed chemical-physical characterization of each of the isolated species indicate them to be two H-type and one L-type ferritin subunits. Thus, reversed-phase HPLC presently stands as the only available means to separate preparatively the various subunit types of ferritin.     

Dynamic interfacial properties of poly(ethylene glycol)-modified ferritin at the solid/liquid interface

Poly(ethylene glycol)-modified ferritins (PEG-ferritins) with various molecular weights were synthesized by the grafting method, and their dynamic interfacial properties at the solid/liquid interface were investigated. The number of PEG grafted to ferritins was controlled by the amount of 1,1'-carbonyldiimidazole-modified PEG adding to the reaction solution. The adsorption kinetics and energy dissipation of PEG-ferritins onto bare Si substrate and amino-modified Si substrate were investigated with a quartz crystal microbalance (QCM) in 10 mM bis-Tris/HCl buffer (pH 5.8), while their morphologies were characterized by scanning electron microscopy (SEM). The adsorption dynamics of PEG-ferritins onto amino-modified Si substrate were quite different from those of unmodified ferritin, which can be reasonably interpreted by the desorption capability of PEG-ferritins on the surface attributed to amphiphilicity and the high-chain mobility of PEG chains.     

Tuning band gap of holoferritin by metal core reconstitution with Cu, Co, and Mn

Utility of ferritin in molecular electronics, especially in single molecule electronics based devices, has recently been proposed, since the iron core of holoferritin is semiconducting in nature. However, the practical aspects, e.g., how its electronic properties can be varied/tuned, need to be better addressed. In this direction, we have performed direct tunneling experiments using scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) on several metal core reconstituted ferritins, where the reconstitution has been carried out using biocompatible metals like copper, cobalt, and manganese that are found naturally in the human body. We show, for the first time, that, by metal core reconstitution of the ferritin protein, the band gap of the protein can be tuned to different values (here, within the range 1.17-0.00 eV, considering iron-containing holoferritin and apoferritin as well). From the respective current-voltage curves and the well-defined band gaps, clear distinction can be made among the five different ferritins indicating that the metal core has direct contribution in the observed electrical conductivities of ferritins. It is further revealed that the electrical conductivities of the reconstituted ferritins are of the same order as that for the free metal conductivities, meaning that the relative changes in the free metal conductivities are reflected in the contributions of the metals in protein shell-confinement (i.e., the ～8 nm core of ferritin). This finding could lead to a strategy for fine-tuning ferritin band gap by preselecting a metal on the basis of the free metal conductivity values.     

Realizing a two-dimensional ordered array of ferritin molecules directly on a solid surface utilizing carbonaceous material affinity peptides

A two-dimensional hexagonally close-packed (2D-HCP) array of ferritin molecules with a nanoparticle core was fabricated directly on a carbonaceous solid substrate by genetically modifying the outer surface of the ferritin with carbonaceous materials-specific binding peptides. The displayed peptides endowed ferritins with a specific protein-substrate interaction and masked the strong nonspecific interaction. The specific interaction was weak enough to allow ferritins to be rearranged as well as an attractive protein-protein interaction that could be adjusted by selecting the buffer conditions. This method not only produced 2D-HCP arrays of ferritin but also 2D-ordered arrays of independent inorganic nanoparticles after protein elimination that can be applied to floating gate memories.     

Ferritin immobilization on patterned poly(2-hydroxyethyl methacrylate) brushes on silicon surfaces from colloid system

In this paper, we describe a graft polymerization/solvent immersion method for generating poly(2-hydroxyethyl methacrylate) (PHEMA) brushes in various patterns. We used a novel fabrication process, involving very-large-scale integration and oxygen plasma treatment, to generate well-defined patterns of polymerized PHEMA on patterned Si(100) surfaces. We observed brush- and mushroom-like regions for the PHEMA brushes, with various pattern resolutions, after immersing wafers presenting lines of these polymers in MeOH and n-hexane, respectively. The interaction between PHEMA and ferritin protein sheaths in MeOH and n-hexane (good and poor solvent for PHEMA, respectively) was used to capture and release ferritins from fluidic system. The “tentacles” behaver for PHEMA brushes was found through various solvents in fluidic system. Using high-resolution scanning electron microscopy, we observed patterned ferritin Fe cores on the Si surface after pyrolysis of the patterned PHEMA brushes and ferritin protein sheaths, which verify the “tentacles” behaver for PHEMA brushes.     

A Ferritin Reactor for Entrapping Various Heavy Metal Ions in Flowing Seawater

Recent studies showed that the ferritins had three tunnels across the protein shell with 0.7~0.9 nm diameter for iron metabolism^[1-2]. However, very little was known the mechanism(s) that the ferritin reactor entrapped various heavy metal ions (M²⁺) and other small organic molecules in the flowing seawater directly.     

Antibacterial effect,efflux pump inhibitory (NorA,TetK and MepA) of Staphylococcus aureus and in silico prediction of α, β and δ-damascone compounds

ObjectiveThe present study aimed to evaluate the antibacterial effect and inhibitory capacity against NorA, TetK and MepA efflux pump of Staphylococcus aureus multiresistant by in vitro and in silico approach of α, β and δ-damascone compounds.ResultsThe compounds α, β and δ-damascone showed a clinically relevant effect against S. aureus ATCC 6538 standard strain. A modulating effect was also observed in association with antibiotics against MDR strains. Regarding the inhibition of the efflux pump, the compounds showed a modulating effect with antibiotics, against SA-1199, SA-1199B, SA IS-58 and K2068. Only δ-damascone demonstrated an efflux pump inhibitory effect. Regarding ADME properties, α, β and δ-damascone showed similar physicochemical properties with good pharmacokinetic characteristics, such as lipophilicity, oral bioavailability and low toxicity. In addition, they exhibited the binding energy and Ligand Efficiency (LE) ?81.17 (5.4), ?77.48(-5.4) and ?64.55 (-5.1), which shows good interactions with the critical residues of the MepA receptor.ConclusionsFrom the results it is concluded that the compounds α, β and δ-damascone do not have antibacterial activity, but show a modulating effect in association with antibiotics, as well as not showing direct activity on the efflux pump, but they do have a modulating effect with antibiotics and with EtBr (α and β-damascone).     

Spectroscopic evidence for and characterization of a trinuclear ferroxidase center in bacterial ferritin from Desulfovibrio vulgaris Hildenborough

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 ? in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by M?ssbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a μ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.     

Effect of growth on surface charge and hydrophobicity of Staphylococcus aureus

Modifications in the surface charge and hydrophobicity of Staphylococcus aureus Oxford during growth were studied by analysing electrophoretic mobility and adherence to hydrocarbons (hexadecane), respectively. Bacterial concentration had no effect upon the measurements. Both surface charge and hydrophobicity varied during the exponential phase of growth (1 to 4 h): surface charge decreased significantly (p < 0.001), while hydrophobicity increased (p < 0.001). In the stationary phase (4 to 9 h), the surface charge increased significantly (p < 0.001), whereas hydrophobicity showed no change. Cationized ferritin decreased the surface charge and had no effect on hydrophobicity. These results suggest that in S. aureus, different structures could be responsible for their surface charge and hydrophobic properties.     

Four‐fold Channel‐Nicked Human Ferritin Nanocages for Active Drug Loading and pH‐Responsive Drug Release

Human ferritins are emerging platforms for non‐toxic protein‐based drug delivery, owing to their intrinsic or acquirable targeting abilities to cancer cells and hollow cage structures for drug loading. However, reliable strategies for high‐level drug encapsulation within ferritin cavities and prompt cellular drug release are still lacking. Ferritin nanocages were developed with partially opened hydrophobic channels, which provide stable routes for spontaneous and highly accumulated loading of Fe^II‐conjugated drugs as well as pH‐responsive rapid drug release at endoplasmic pH. Multiple cancer‐related compounds, such as doxorubicin, curcumin, and quercetin, were actively and heavily loaded onto the prepared nicked ferritin. Drugs on these minimally modified ferritins were effectively delivered inside cancer cells with high toxicity.     

Preparation, characterization and dielectric properties of 4,4-diphenylmethane diisocyanate (MDI) based cross-linked polyimide films

Four different types of cross-linked polyimides based on 4,4-diphenylmethane diisocyanate (MDI) were prepared by the reaction of different types of conventional poly(amic acid) intermediates with MDI as a cross-linking agent. Subsequently, they were thermally imidized in order to obtain corresponding cross-linked polyimide structure. The results of FTIR-ATR showed that MDI can effectively react with carboxylic acid groups of PAA to form cross-linked polyimide films. TGA, FTIR-ATR and SEM analyses were carried out for characterization of cross-linked polyimide (CPI) films. Moreover, the electrical properties such as dielectric breakdown strength, dielectric constant, I-V characteristics and loss factor of MDI based cross-linked polyimides have been checked. In addition, some physical properties such as water uptake, adhesion, hardness and solubility properties of the films were investigated.The results showed that all CPI films have good insulating properties such as high dielectric breakdown voltage, low loss factor (tan δ), leakage density and excellent physical properties.     

Mass spectrometric characteristics and kinetics of iron release in visceral mass of Saccostrea cucullata

Ferritins with electrophoretic homogeneity were prepared from the visceral mass of Saccostrea cucullata in batch. The native PAGE approach showed similar electrophoretic mobility among pig pancreatic ferritin, liver ferritin of Dasyatis akajei, and visceral mass ferritin of Saccostrea cucullata. SDS-PAGE indicated that the Saccostrea cucullata visceral ferritin (SCVF) consisted of a single subunit type and had a molecular weight (MW) of approximately 20 kDa, suggesting that the protein shell in SCVF was composed of a single subunit. In addition, peptide mass fingerprinting and transmission electron microscopy were used to identify SCVF further, and to observe its molecular structure. We found that the molecular structure in SCVF was similar to that of most mammalian ferritins, which are composed of a protein shell and an iron core. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry under the assistance of an acidic matrix, sinapic acid, also showed that SCVF was composed of a single subunit type and its subunit MW was calculated to be 19871.042 Da in the absence of heme. Kinetics analysis revealed that the complete process of iron release fitted the law of a first-order reaction, which is similar to that of most ferritins in mammals. Similar to bacterial ferritin, studies indicated that the shell consisted of a single subunit type and showed similar kinetics of iron release, suggesting that this subunit plays two important roles in iron release and storage, and that it shows different stability and intensity of interaction in carrying out its physiological functions in SCVF.     

Behaviour of a desolvation system based on microwave radiation heating for use in inductively coupled plasma atomic emission spectrometry

The present paper describes the preliminary results obtained with a desolvation system for inductively coupled plasma atomic emission spectrometry that incorporates a heating unit based on microwave (MW) radiation. This system has been called Microwave Desolvation System (MWDS). The results have proved that MW radiation can be considered as a good choice for aerosol heating in a sample introduction system. MW radiation seems to be a more uniform way of aerosol desolvation than conductive/convective heating (i.e. lower radial temperature gradients), the degree of vaporization of the droplets is less dependent on the liquid flow rate (Q_l), and also the background noise associated with the vaporization of droplets is reduced. As regards the results obtained with MWDS, in comparison with a conventional desolvation system (CDS), they are very dependent on Q_l. When heating is applied, the amount of analyte that leaves the heating step increases by 30–60% with the MWDS, irrespective of Q_l, whereas for the CDS this increase is very high (up to 300%) at low Q_l values (0.4 ml min⁻¹), but almost negligible at high Q_l values (2.4 ml min⁻¹). In agreement with this, the analytical figures of merit are favourable to the CDS at low flow rates, and to the MWDS at high liquid flows. Under all the conditions studied, the amount of solvent that leaves the condensation unit are lower for MWDS than for CDS.     

In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees—one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed.     

Visualization and quantification of chaotic mixing for helical-type micromixers

The paper presents a micro mixer structures fabricated by an interesting technique of embedded twisted threads in polydimethylsiloxane (PDMS), which is polymerized and cured. After removing the threads carefully, the remaining channel structure is studied concerning the flows and mixing characteristics. Three-, four-, and six-strand-helical fluid microchannels (d _h ?=?100?μm, L?=?3?mm) with a specified helix angle (θ?=?22°) were used to conduct the experiments. The electroosmotic flow, inlet velocity, local velocity at specified positions, and sample concentration distribution along a downstream direction were measured via microparticle image velocimetry and laser-induced fluorescence techniques, respectively. Local mixing efficiency and mixing length were obtained at very low Reynolds numbers (≤0.0242) and low Peclet numbers (≤65.8). Results show that four-strand micromixer has the best mixing performance. Finally, a correlation of mixing length with Pe was developed, which might be applicable to a microbiomedical device design.     

Thermodynamic properties of acetone calculated from accurate experimental speed of sound measurements at low temperatures and high pressures

Thermodynamic properties of fluids are both required for designing and implementing industrial processes and in different research fields; in particular they play a fundamental role in the development of equations of state (EoS). This paper describes very accurate speed of sound measurements in liquid-phase acetone along eleven isotherms in the temperature range of (248.15 and 298.15) K and over a wide range of pressure (up to 100 MPa). Since very accurate direct measurements of the fluids properties (like density and isobaric heat capacity) are relatively easy at atmospheric pressure, but difficult at elevated pressures, a combination of speed of sound measurements and numerical integration offers a well balanced approach to determine the thermodynamic properties of liquids. In this case, density and heat capacity of the liquid at high pressures are calculated by numerical integration of u^?2(p, T), using, as initial values, the same quantities (density and heat capacity) at atmospheric pressure as a function of temperature. The experimental values of speed of sound are subjected to an overall estimated uncertainty of about 0.1%.     

Facile preparation of Au nanoparticles mediated by Foeniculum Vulgare aqueous extract and investigation of the anti-human breast carcinoma effects

This experiment evaluated antioxidant, anti-human breast cancer activities, and cytotoxicity effects of green synthesis of Au nanoparticles (AuNPs) containing Foeniculum Vulgare aqueous extract. Mixing Foeniculum Vulgare aqueous with Au chloride solution produced Au nanoparticles. The characteristics of Au nanoparticles determined using Fourier Transformed Infrared Spectroscopy (FT‐IR), Transmission Electron Microscopy (TEM), UV–Visible Spectroscopy (UV–Vis), and Field Emission Scanning Electron Microscopy (FE‐SEM). To check the cytotoxicity and anti-breast cancer effects of Au chloride, Foeniculum Vulgare aqueous extract, and AuNPs on common breast cancer cell lines i.e., ZR-75-30, T47D, and HCC1187 was used MTT assay. AuNPs showed no cytotoxicity and the most effective anti-breast cancer features compared to other items that were tested. They had no cytotoxic effects on normal cell line (HUVEC) and had very low cell viability, high anti-breast cancer activities dose-dependently against ZR-75-30, T47D, and HCC1187 cell lines. In the presence of butylated hydroxytoluene as the positive control, the DPPH test was used to evaluate the antioxidant features of Au chloride, Foeniculum Vulgare aqueous extract, and Au nanoparticles. AuNPs showed the best antioxidant properties compared to other items that were tested. Perhaps remarkable anti-human breast cancer activities of Au nanoparticles synthesized by Foeniculum Vulgare aqueous extract due to its antioxidant properties. After clinical trial and confirmation of results, this formulation can be used as an effective drug to treat breast cancer.     

Film formation of polymeric emulsions: Structure set-up and the pinhole effect characterized by microscopic techniques

Drying of emulsions of special polymeric core-shell latexes results in structured films and coatings with advantageous material properties. Here, we focus on so-called “container particles”, consisting of a low viscosity core with a low glass transition (poly(2-ethylhexyl methacrylate), PEtHMA), covered by a thin shell of a cross-linked rubber (poly(n-butyl acrylate), PBA). These particles can be regarded as model emulsions of reactive polymeric oils with a very high colloidal stability. The film formation of these latexes was studied by atomic force microscopy (AFM) in the tapping mode as well as by transmission electron microscopy (TEM). It is shown that the films stay nanostructured after the drying process, i. e. they exhibit both a controlled topography as well as a network superstructure originating from the characteristics of the original dispersions.

TEM allows to detect the whereabouts of the polar stabilizer. Both continous surfactant films as well as inverted micelles are found. A geometrically induced demixing phenomenon is found which enriches the polar components and might be the molecular reason for the so-called pinhole-effect, the failure of water-born coatings in contact with water.