Effect of Spin Clustering on Basic and Relaxometric Properties of Magnetic Nanoparticles.

An increasing awareness about novel medical applications of smaller, inorganic-based nanoparticles, possessing unique properties at the nanoscale, has led to a burst of research activities in the development of "nanoprobes" for diagnostic medicine and agents for novel, externally activated therapies. In this research field magnetic nanoparticles are prominent due to fundamental peculiar properties particularly appealing for their use in materials and biomedical applications. Aiming to study the relationship between the topology of the magnetic nanoparticles and their efficacy as MRI contrast agents (relaxometric properties), we prepared three different stable colloidal suspension (ferrofluid) of magnetic nanobeads (MNBs) constituted by a discrete number of maghemite nanoparticles, arranged in disordered clusters or ordered in a polymeric matrix. An accurate morpho-dimensional and magnetic characterization displays the close correlation between the magnetic fundamental properties and the topology of our spin systems. The NMR relaxometry profiles confirmed the nature of the physical mechanisms inducing the increase of nuclear relaxation rates at low (magnetic anisotropy) and high (Curie relaxation) magnetic fields. Moreover the transverse relaxivity (r2) values for all the MNBs are higher than those of common contrast agents and the differences between the three MNBs are suggested to be due to the spin topology effect.

Local spin dynamics of iron oxide magnetic nanoparticles dispersed in different solvents with variable size and shape: A 1H NMR study.

Colloidal magnetic nanoparticles (MNPs) based on a nearly monodisperse iron oxide core and capped by oleic acid have been used as model systems for investigating the superparamagnetic spin dynamics by means of magnetometry measurements and nuclear magnetic resonance (1H NMR) relaxometry. The key magnetic properties (saturation magnetization, coercive field, and frequency dependent "blocking" temperature) of MNPs with different core size (3.5 nm, 8.5 nm, and 17.5 nm), shape (spherical and cubic), and dispersant (hexane and water-based formulation) have been determined. 1H NMR dispersion profiles obtained by measuring the r1 (longitudinal) and r2 (transverse) nuclear relaxivities in the frequency range 0.01-60 MHz confirmed that in all samples the physical mechanisms that drive the nuclear relaxation are the Néel reversal at low temperature and the Curie relaxation at high frequency. The magnetization reversal time at room temperature extracted from the fitting of NMR data falls in the typical range of superparamagnetic systems (10-9-10-10 s). Furthermore, from the distance of minimum approach we could conclude that water molecules do not arrive in close vicinity of the magnetic core. Our findings contribute to elucidate the local spin dynamics mechanisms in colloidal superparamagnetic nanoparticles which are useful in biomedical application as, e.g., contrast agents for magnetic resonance imaging.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells.

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

A possible novel objective intraoperative measurement of maxillary bone density.

AIM: Implant survival and success rates are strictly related to the density of the bone they are placed in. Bone density, in fact, affects both implant primary stability and implant micromovements after implant positioning. Current bone density classifications rely on subjective, scarcely reproducible evaluations. A novel implant micro motor featuring a bone density measurement probe has been recently introduced. The objective of this study was to test such bone density measurement system for its capability of distinguishing different bone density areas in the upper and in the lower jaw. METHODS: 1254 implant placement sites had their bone density measured during standard implant placement at a single clinical facility. After data collection bone density distribution was statistically analyzed in order to test the hypothesis of a non-homogeneous distribution in four different predefined anatomical maxillary zones, namely pre-antral (between teeth from 14 to 24) and sub-antral (more distally) in the upper maxilla and interforaminal (between and including teeth from 34 to 44) and retroforaminal (more distally) zone. RESULTS: Measured bone density values, organized according the named four anatomical zones, produced a statistically significant inhomogeneous pattern (P<0.001). Density distribution was consistent with data from literature, but not always corresponding with the one achieved by applying the well known Misch classification. CONCLUSION: The measuring system we tested allowed to distinguish different and clinically significant anatomical zones according to their different bone density, and can represent a fundamental diagnostic tool to plan the proper implant placement steps.

Multifunctional nanoparticles for dual imaging.

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

Intermittent contact mode AFM investigation of native plasma membrane of Xenopus laevis oocyte.

Intermittent contact mode atomic force microscopy (AFM) was used to visualize the native plasma membrane of Xenopus laevis oocytes. Oocyte membranes were purified via ultracentrifugation on a sucrose gradient and adsorbed on mica leaves. AFM topographs and the corresponding phase images allowed for visualization and identification of both oocyte plasma membrane patches and pure lipid bilayer regions with a height of about 5 nm within membrane patches. The quantitative analysis showed a normal distribution for the lateral dimension and height of the protein complexes centered on 16.7 +/- 0.2 nm (mean +/- SE, n = 263) and 5.4 +/- 0.1 nm (n = 262), respectively. The phase signal, providing material-dependent information, allowed for the recognition of structural features observed in AFM topographs.

Flow cytometry evaluation of erythroid dysplasia in patients with myelodysplastic syndrome.

Erythroid dysplasia is the pathologic hallmark of myelodysplastic syndromes (MDS). To develop a quantitative flow-cytometry approach to its evaluation, we analyzed the expression of CD71, CD105, cytosolic H-ferritin (HF), cytosolic L-ferritin (LF) and mitochondrial ferritin (MtF) in erythroblasts from 104 MDS patients, 69 pathologic control patients and 19 healthy subjects. Six-parameter, 4-color flow cytometry was employed, and data were expressed as mean fluorescence intensity. Compared with pathologic and healthy controls, MDS patients had higher expression of HF (P < 0.001) and CD105 (P < 0.001), and lower expression of CD71 (P < 0.001). MtF was specifically detected in MDS with ringed sideroblasts, and there was a close relationship between its expression and Prussian blue staining (r = 0.89, P < 0.001). In vitro cultures of myelodysplastic hematopoietic progenitors showed that both HF and MtF were expressed at a very early stage of erythroid differentiation, and that MtF expression is specifically related to mitochondrial iron loading. A classification function based on expression levels of HF, CD71 and CD105 allowed us to correctly classify > 95% of MDS patients. This flow-cytometry approach provides an accurate quantitative evaluation of erythroid dysplasia and allows a reliable diagnosis of sideroblastic anemia, and may therefore be a useful tool in the work-up of patients with MDS.

An immunoglobulin-like fold in a major plant allergen: the solution structure of Phl p 2 from timothy grass pollen.

BACKGROUND: Grass pollen allergens are the most important and widespread elicitors of pollen allergy. One of the major plant allergens which millions of people worldwide are sensitized to is Phl p 2, a small protein from timothy grass pollen. Phl p 2 is representative of the large family of cross-reacting plant allergens classified as group 2/3. Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein. RESULTS: We have solved the solution structure of recombinant Phl p 2 by means of nuclear magnetic resonance techniques. The three-dimensional structure of Phl p 2 consists of an all-beta fold with nine antiparallel beta strands that form a beta sandwich. The topology is that of an immunoglobulin-like fold with the addition of a C-terminal strand, as found in the C2 domain superfamily. Lack of functional and sequence similarity with these two families, however, suggests an independent evolution of Phl p 2 and other homologous plant allergens. CONCLUSIONS: Because of the high homology with other plant allergens of groups 1 and 2/3, the structure of Phl p 2 can be used to rationalize some of the immunological properties of the whole family. On the basis of the structure, we suggest possible sites of interaction with IgE antibodies. Knowledge of the Phl p 2 structure may assist the rational structure-based design of synthetic vaccines against grass pollen allergy.

Structural and immunological relationships of isoferritins in normal and malignant cells.

Ferritins from normal adult human liver and heart were compared with ferritins from a lung carcinoma metastatic to liver and from HeLa cells on the basis of their isoferritin profiles, subunit composition, and immunological relationships. Each ferritin preparation gave different isoferritin profiles, but several contained common isoferritins. All of the tumor isoferritins had counterparts in the normal tissues. All ferritins contained similar subunits but in different proportions. Qualitative differences were demonstrable in some ferritins with antibodies to different tissue ferritins. These differences correlated with the subunit composition of the ferritins. By appropriate absorption, an antibody population was obtained that was apparently specific for one subunit type. Heart ferritin gave lines of apparent identity with the tumor ferritins with these antibodies. It is concluded that tumor ferritins are not tumor-specific antigens but correspond to isoferritins in normal adult heart.

Characterization of magnetic nanoparticles from Magnetospirillum Gryphiswaldense as potential theranostics tools.

We investigated the theranostic properties of magnetosomes (MNs) extracted from magnetotactic bacteria, promising for nanomedicine applications. Besides a physico-chemical characterization, their potentiality as mediators for magnetic fluid hyperthermia and contrast agents for magnetic resonance imaging, both in vitro and in vivo, are here singled out. The MNs, constituted by magnetite nanocrystals arranged in chains, show a superparamagnetic behaviour and a clear evidence of Verwey transition, as signature of magnetite presence. The phospholipid membrane provides a good protection against oxidation and the MNs oxidation state is stable over months. Using an alternate magnetic field, the specific absorption rate was measured, resulting among the highest reported in literature. The MRI contrast efficiency was evaluated by means of the acquisition of complete NMRD profiles. The transverse relaxivity resulted as high as the one of a former commercial contrast agent. The MNs were inoculated into an animal model of tumour and their presence was detected by magnetic resonance images two weeks after the injection in the tumour mass.

Electrophoretic analysis of horse tissue ferritins at different pH values.

Electrophoretic mobilities of ferritins from horse heart, liver and spleen were compared in the pH range 3.5-10. Electrophoretic titrations and continuous buffer electrophoreses were used. The order of anionic mobilities was heart > liver > spleen at alkaline pH and this order was reversed in the neutral to acidic range. This order of mobilities, and the intermediate behavior of liver in respect to the other two ferritins correlate with the known subunit composition of the three ferritins, and strongly support the idea of different amino acid residues being exposed on the protein shell surfaces. From the analyses of differential mobility around the pK values of the various ionizable groups it was concluded that heart and spleen ferritins have a very similar number of acidic amino acid residues on their surfaces, whereas they differ in basic residues. Heart seems to have about 15% more Lys and Arg, and twice as many His as spleen.

Selective crystallization of horse isoferritins.

Various precipitating agents were examined in order to crystallize horse heart and spleen ferritins. Cadmium sulfate induced the crystallization of the spleen ferritin, while 2-methyl-2,4-pentanediol and poly(ethylene glycol) only induced that of the heart ferritin. Isoelectric focusing analysis showed that the crystals grown from cadmium sulfate contained only the more acidic isoferritins, and those grown from methyl pentanediol only the less acidic isoferritins. Heart ferritin crystallizes in a cubic space group, as previously reported for spleen ferritin crystals grown from cadmium sulfate.

The ferritins: molecular properties, iron storage function and cellular regulation.

The iron storage protein, ferritin, plays a key role in iron metabolism. Its ability to sequester the element gives ferritin the dual functions of iron detoxification and iron reserve. The importance of these functions is emphasised by ferritin's ubiquitous distribution among living species. Ferritin's three-dimensional structure is highly conserved. All ferritins have 24 protein subunits arranged in 432 symmetry to give a hollow shell with an 80 A diameter cavity capable of storing up to 4500 Fe(III) atoms as an inorganic complex. Subunits are folded as 4-helix bundles each having a fifth short helix at roughly 60 degrees to the bundle axis. Structural features of ferritins from humans, horse, bullfrog and bacteria are described: all have essentially the same architecture in spite of large variations in primary structure (amino acid sequence identities can be as low as 14%) and the presence in some bacterial ferritins of haem groups. Ferritin molecules isolated from vertebrates are composed of two types of subunit (H and L), whereas those from plants and bacteria contain only H-type chains, where 'H-type' is associated with the presence of centres catalysing the oxidation of two Fe(II) atoms. The similarity between the dinuclear iron centres of ferritin H-chains and those of ribonucleotide reductase and other proteins suggests a possible wider evolutionary linkage. A great deal of research effort is now concentrated on two aspects of ferritin: its functional mechanisms and its regulation. These form the major part of the review. Steps in iron storage within ferritin molecules consist of Fe(II) oxidation, Fe(III) migration and the nucleation and growth of the iron core mineral. H-chains are important for Fe(II) oxidation and L-chains assist in core formation. Iron mobilisation, relevant to ferritin's role as iron reserve, is also discussed. Translational regulation of mammalian ferritin synthesis in response to iron and the apparent links between iron and citrate metabolism through a single molecule with dual function are described. The molecule, when binding a [4Fe-4S] cluster, is a functioning (cytoplasmic) aconitase. When cellular iron is low, loss of the [4Fe-4S] cluster allows the molecule to bind to the 5'-untranslated region (5'-UTR) of the ferritin m-RNA and thus to repress translation. In this form it is known as the iron regulatory protein (IRP) and the stem-loop RNA structure to which it binds is the iron regulatory element (IRE). IREs are found in the 3'-UTR of the transferrin receptor and in the 5'-UTR of erythroid aminolaevulinic acid synthase, enabling tight co-ordination between cellular iron uptake and the synthesis of ferritin and haem. Degradation of ferritin could potentially lead to an increase in toxicity due to uncontrolled release of iron. Degradation within membrane-encapsulated "secondary lysosomes' may avoid this problem and this seems to be the origin of another form of storage iron known as haemosiderin. However, in certain pathological states, massive deposits of "haemosiderin' are found which do not arise directly from ferritin breakdown. Understanding the numerous inter-relationships between the various intracellular iron complexes presents a major challenge.

Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies.

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate that heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.

A mutational analysis of the epitopes of recombinant human H-ferritin.

Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli. They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin. Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E. coli, altered in different accessible areas of the molecule. They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region. Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized. The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin. Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity.

Influence of site-directed modifications on the formation of iron cores in ferritin.

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.

The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers.

Mammalian ferritins are 24-meric proteins composed of variable proportions of H and L-subunits. The L-chain, in contrast to the H-chain, lacks detectable ferroxidase activity, and its role in ferritin iron incorporation is unclear. In this study, apoferritins were subjected to iron loading with large iron increments to favour spontaneous iron hydrolysis. The homopolymers of the wild-type H-chain, and of a mutant H-chain with an inactivated ferroxidase centre, formed massive protein aggregates, while the L-chain homopolymers remained mostly soluble. The difference between H and L-ferritins was not related to the rate of iron oxidation or to the presence of preformed iron cores. Heteropolymers were constructed in vitro by co-renaturing different proportions of the H-chain with the L-chain or mutant H-chain with an inactivated ferroxidase centre. After loading with high iron increments, protein aggregation of the heteropolymers was reduced when the L-chain content was above 70 to 80%, either in combination with the wild-type H-chain or with the inactivated mutant H-chain. Under acidic conditions (pH 5.5, 1000 Fe atoms per molecule) the heteropolymers with about 20% H and 80% L-chains incorporated three to fourfold more iron into soluble 24-mers than the homopolymers. The data indicate that ferritins with more than 18 L-chains per molecule have the capacity to lower non-specific iron hydrolysis in bulk solution. This property is possibly due to a specific attraction of the incoming oxidized iron into the cavity and may be related to an effect of the L-chain on the cavity microenvironment. It is concluded that under high iron increments the ferritins with high L:H-chain ratios are the most efficient in incorporating iron, and this goes some way to explain why iron storage tissues contain L-rich isoferritins.

Recombinant allergen Lol p II: expression, purification and characterization.

Pollen from perennial rye grass (Lolium perenne) is a major cause of type I allergies worldwide. It contains complex mixtures of proteins, among which Lol p II is a major allergen. Previously, we have reported the cloning and sequencing of Lol p II and its expression in fusion with the heavy chain of human ferritin as carrier polypeptide (Sidoli et al., 1993, J. biol. Chem. 268, 21819-21825). Here, we describe the expression, purification and characterization of a recombinant Lol p II overproduced as a non-fusion protein in the periplasm of E. coli. The recombinant allergen was expressed in high yields and was easily purified in milligram amounts. It competed with the natural Lol p II for binding to specific IgE, and it induced allergic responses in skin prick tests, indicating to be immunologically analogous to the natural protein. Biochemical analyses indicate that recombinant Lol p II is a highly stable and soluble monomeric molecule which behaves like a small globular protein.

Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications.

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Characterization of human ferritin H chain synthetized in Escherichia coli.

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.