Study of paramagnetic defect centers in as-grown and annealed TiO2 anatase and rutile nanoparticles by a variable-temperature X-band and high-frequency (236 GHz) EPR.

Detailed EPR investigations on as-grown and annealed TiO2 nanoparticles in the anatase and rutile phases were carried out at X-band (9.6 GHz) at 77, 120-300 K and at 236 GHz at 292 K. The analysis of EPR data for as-grown and annealed anatase and rutile samples revealed the presence of several paramagnetic centers: Ti3+, O-, adsorbed oxygen (O2-) and oxygen vacancies. On the other hand, in as-grown rutile samples, there were observed EPR lines due to adsorbed oxygen (O2-) and the Fe3+ ions in both Ti4+ substitutional positions, with and without coupling to an oxygen vacancy in the near neighborhood. Anatase nanoparticles were completely converted to rutile phase when annealed at 1000° C, exhibiting EPR spectra similar to those exhibited by the as-grown rutile nanoparticles. The high-frequency (236 GHz) EPR data on anatase and rutile samples, recorded in the region about g = 2.0 exhibit resolved EPR lines, due to O- and O2- ions enabling determination of their g-values with higher precision, as well as observation of hyperfine sextets due to Mn2+ and Mn4+ ions in anatase.

ESR Microscopy for Biological and Biomedical Applications.

We report on electron-spin resonance microscopy (ESRM) providing sub-micron resolution (~700nm) with a high spin concentration sample, i.e. lithium phthalocyanine (LiPc) crystal. For biomedical applications of our ESRM, we have imaged samples containing rat basophilic leukemia (RBL) cells as well as cancerous tissue samples with a resolution of several microns using a water soluble spin probe, Trityl_OX063_d24. Phantom samples with the nitroxide spin label, (15)N PDT, were also imaged to demonstrate that nitroxides, which are commonly used as spin labels, may also be used for ESRM applications. ESRM tissue imaging would therefore be valuable for diagnostic or therapeutic purposes. Also, ESRM can be used to study the motility or the metabolism of cells in various environments. With further modification and/or improvement of imaging probe and spectrometer instrumentation sub-micron biological images should be obtainable, thereby providing a useful tool for various biomedical applications.

Joint analysis of ESR lineshapes and 1H NMRD profiles of DOTA-Gd derivatives by means of the slow motion theory.

The "Swedish slow motion theory" [Nilsson and Kowalewski, J. Magn. Reson. 146, 345 (2000)] applied so far to Nuclear Magnetic Relaxation Dispersion (NMRD) profiles for solutions of transition metal ion complexes has been extended to ESR spectral analysis, including in addition g-tensor anisotropy effects. The extended theory has been applied to interpret in a consistent way (within one set of parameters) NMRD profiles and ESR spectra at 95 and 237 GHz for two Gd(III) complexes denoted as P760 and P792 (hydrophilic derivatives of DOTA-Gd, with molecular masses of 5.6 and 6.5 kDa, respectively). The goal is to verify the applicability of the commonly used pseudorotational model of the transient zero field splitting (ZFS). According to this model the transient ZFS is described by a tensor of a constant amplitude, defined in its own principal axes system, which changes its orientation with respect to the laboratory frame according to the isotropic diffusion equation with a characteristic time constant (correlation time) reflecting the time scale of the distortional motion. This unified interpretation of the ESR and NMRD leads to reasonable agreement with the experimental data, indicating that the pseudorotational model indeed captures the essential features of the electron spin dynamics.

Variable Coupling Scheme for High Frequency Electron Spin Resonance Resonators Using Asymmetric Meshes.

The sensitivity of a high frequency electron spin resonance (ESR) spectrometer depends strongly on the structure used to couple the incident millimeter wave to the sample that generates the ESR signal. Subsequent coupling of the ESR signal to the detection arm of the spectrometer is also a crucial consideration for achieving high spectrometer sensitivity. In previous work, we found that a means for continuously varying the coupling was necessary for attaining high sensitivity reliably and reproducibly. We report here on a novel asymmetric mesh structure that achieves continuously variable coupling by rotating the mesh in its own plane about the millimeter wave transmission line optical axis. We quantify the performance of this device with nitroxide spin-label spectra in both a lossy aqueous solution and a low loss solid state system. These two systems have very different coupling requirements and are representative of the range of coupling achievable with this technique. Lossy systems in particular are a demanding test of the achievable sensitivity and allow us to assess the suitability of this approach for applying high frequency ESR to the study of biological systems at physiological conditions, for example. The variable coupling technique reported on here allows us to readily achieve a factor of ca. 7 improvement in signal to noise at 170 GHz and a factor of ca. 5 at 95 GHz over what has previously been reported for lossy samples.

A 236-GHz Fe EPR STUDY OF NANO-PARTICLES OF THE FERRO-MAGNETIC ROOM-TEMPERATURE SEMICONDUCTOR Sn(1-x)Fe(x)O(2)(x=0.005).

High frequency (236 GHz) electron paramagnetic resonance (EPR) studies of Fe(3+) ions at 255 K are reported in a Sn(1-x)Fe(x)O(2) powder with x = 0.005 which is a ferromagnetic semiconductor at room temperature. The observed EPR spectrum can be simulated reasonably well as overlap of spectra due to four magnetically inequivalent high-spin (HS) Fe(3+) ions (S = 5/2). The spectrum intensity is calculated, using the overlap I(BL) + (I(HS1)+I(HS2)+I(HS3)+I(HS4))×e(-0.00001×B), where B is the magnetic field intensity in Gauss, I represents the intensity of an EPR line (HS1, HS2, HS3, HS4), and BL stands for the base line. (The exponential factor, as found by fitting to the experimental spectrum, is related to the Boltzmann population distribution of energy levels at 255 K, which is the temperature of the sample in the spectrometer.) These high-frequency EPR results are significantly different from those at X-band. The large values of the zero-field splitting parameter (D) observed here for the four centers at the high frequency of 236 GHz are beyond the capability of X-band, which can only record spectra of ions only with much smaller D values than those reported here.

A structural mode-coupling approach to 15N NMR relaxation in proteins.

The two-body Slowly Relaxing Local Structure (SRLS) model was applied to (15)N NMR spin relaxation in proteins and compared with the commonly used original and extended model-free (MF) approaches. In MF, the dynamic modes are assumed to be decoupled, local ordering at the N-H sites is represented by generalized order parameters, and internal motions are described by effective correlation times. SRLS accounts for dynamical coupling between the global diffusion of the protein and the internal motion of the N-H bond vector. The local ordering associated with the coupling potential and the internal N-H diffusion are tensors with orientations that may be tilted relative to the global diffusion and magnetic frames. SRLS generates spectral density functions that differ from the MF formulas. The MF spectral densities can be regarded as limiting cases of the SRLS spectral density. SRLS-based model-fitting and model-selection schemes similar to the currently used MF-based ones were devised, and a correspondence between analogous SRLS and model-free parameters was established. It was found that experimental NMR data are sensitive to the presence of mixed modes. Our results showed that MF can significantly overestimate order parameters and underestimate local motion correlation times in proteins. The extent of these digressions in the derived microdynamic parameters is estimated in the various parameter ranges, and correlated with the time scale separation between local and global motions. The SRLS-based analysis was tested extensively on (15)N relaxation data from several isotropically tumbling proteins. The results of SRLS-based fitting are illustrated with RNase H from E. coli, a protein extensively studied previously with MF.

ADP ribosylation factor 6 binding to phosphatidylinositol 4,5-bisphosphate-containing vesicles creates defects in the bilayer structure: an electron spin resonance study.

The effects of binding of myristoylated ADP ribosylation factor 6 (myr-ARF6), an activator of phospholipase D (PLD), to a model membrane were investigated using an electron spin resonance (ESR) labeling technique. Initial studies were conducted in vesicles composed of 1-palmitoyl-2-oleoyl phosphatidylethanolamine, dipalmitoylphosphatidylcholine, phosphatidylinositol 4,5-biphosphate (PIP(2)), and cholesterol. Recombinant ARF6 binding significantly enhances defects in both the headgroup and acyl-chain regions of the membrane, which are revealed by the emergence of sharp components in the spectra from a headgroup label, 1,2-dipalmitoylphosphatidyl-2,2,6,6-tetramethyl-1-piperidinyloxy-choline (DPPTC), and a chain label, 10PC, after myr-ARF6 binding. Binding of non-myristoylated ARF6 (non-ARF6) shows markedly reduced effects. Interestingly, no change in spectra from DPPTC was observed upon myr-ARF6 binding when PIP(2) in the vesicles was replaced by other negatively charged lipids, including phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol, even when normalized for charge. The production of the sharp peak appears to be a specific event, because another GTP binding protein, CDC42, which binds PIP(2) and activates PLD, fails to induce changes in vesicle structure. These results suggest a previously unappreciated role for ARF in mediating a protein/lipid interaction that produces defects in lipid bilayers. This function may serve as an initial event in destabilizing membrane structure for subsequent membrane fusion or biogenesis of vesicles.

Electron spin resonance in studies of membranes and proteins.

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.

New technologies in electron spin resonance.

New electron spin resonance (ESR) technologies have been developed, which have led to new and improved applications. (a) The development of two-dimensional Fourier transform (FT) ESR required spectrometers providing intense pi/2 microwave pulses of very short (3-5 ns) duration, wide bandwidths, and very short dead times. It has enabled studies that resolve sophisticated details of molecular dynamics in complex fluids. (b) Methods that produce multiple quantum coherences by pulsed ESR now enable accurate measurements of large distances (>12A). (c) One of the most important advances has been the extension of ESR to high magnetic fields and high frequencies. This has benefited from the utilization of quasi-optical methods, especially above 150 GHz. The greatly improved orientational resolution and the faster "snapshot" of motions that are provided by ESR at high frequencies enhance studies of molecular dynamics. The use of both high and lower frequencies enables one to unravel faster and slower modes from the complex dynamics of fluids and macromolecules. (d) The development of FT-ESR imaging required substantial pulsed field gradients lasting only 50-100 ns. ESR imaging is effective in studying diffusion in fluids. Areas for further development are also described.

Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP).

Although the Src homology 2 domain-containing 5' inositol phosphatase (SHIP) is a well-known mediator of inhibitory signals after B cell antigen receptor (BCR) coaggregation with the low affinity Fc receptor, it is not known whether SHIP functions to inhibit signals after stimulation through the BCR alone. Here, we show using gene-ablated mice that SHIP is a crucial regulator of BCR-mediated signaling, B cell activation, and B cell development. We demonstrate a critical role for SHIP in termination of phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P(3)) signals that follow BCR aggregation. Consistent with enhanced PI(3,4,5)P(3) signaling, we find that splenic B cells from SHIP-deficient mice display enhanced sensitivity to BCR-mediated induction of the activation markers CD86 and CD69. We further demonstrate that SHIP regulates the rate of B cell development in the bone marrow and spleen, as B cell precursors from SHIP-deficient mice progress more rapidly through the immature and transitional developmental stages. Finally, we observe that SHIP-deficient B cells have increased resistance to BCR-mediated cell death. These results demonstrate a central role for SHIP in regulation of BCR signaling and B cell biology, from signal driven development in the bone marrow and spleen, to activation and death in the periphery.

MHC class II-bound self peptides from autoimmune MRL/lpr mice reveal potential T cell epitopes for autoantibody production in murine systemic lupus erythematosus.

The systemic lupus erythematosus-like syndrome in MRL/lpr mice involves high-titered IgG autoantibodies, particularly antinuclear Abs that target histones, DNA, and RNA particles. Although T cell help is required for the generation of antinuclear Abs, the epitopes recognized by such helper T cells are unknown. To address this question, we isolated and sequenced self peptides bound by MHC class II molecules from MRL/lpr mice. We identified a number of peptides that are not seen in similar preparations from nonautoimmune C3H animals. The "abnormal" peptide donors include histone, a protein component of a small nuclear ribonucleoprotein, ribosomal proteins, and RNA processing enzymes. We postulate that the peptides from these donors are T cell epitopes required for the generation of the most frequent antinuclear Abs specificities seen in MRL/lpr mice.

Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains.

CD84, a new member of the immunoglobulin superfamily, shows high homology with several molecules belonging to the CD2 family of differentiation antigens. By screening a peripheral blood leukocyte cDNA library four CD84 isoforms were obtained differing in their 3' sequence. A reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that these isoforms were normally found on leukocytes and a new isoform was identified. To establish the nature of the five isoforms obtained (CD84a, CD84b, CD84c, CD84d and CD84e) the genomic structure of the CD84 gene was determined. Our results show that it is composed of at least eight exons, with an exon coding for the 5' UTR and the leader peptide, two exons coding for each of the two immunoglobulin-like domains, an exon encoding the transmembrane portion and four exons coding for the cytoplasmic domains. The isoforms are generated by several mechanisms: alternative use of exons, reading frame shift, use of a cryptic splice site or absence of splicing. The differential expression of several potentially phosphorylatable residues on the different isoforms could be a way to regulate its possible activity in signal transduction.

Surface interleukin-10 inhibits listericidal activity by primary macrophages.

Interleukin-10 (IL-10) down-regulates multiple functions of monocytes and macrophages, including the ability of macrophages to kill many intracellular microorganisms. The experiments presented here test the hypothesis that IL-10 expressed on the cell surface inhibits the ability of primary mouse macrophages to kill the facultative, intracellular bacterium Listeria monocytogenes. We show that, in contrast to macrophages from normal mice, both bone marrow-derived macrophages (BMDM) and thioglycollate-elicited macrophages obtained from IL-10-/- mice can kill L. monocytogenes. Treatment with anti-IL-10 monoclonal antibody (mAb) enables BMDM from normal mice and thioglycollate-elicited macrophages from RAG-2-/- mice (which lack T or B cell-derived IL-10) to kill L. monocytogenes, and concurrently down-regulates the expression of surface IL-10. Surface IL-10 on paraformaldehyde-fixed cells can inhibit nitric oxide (NO) production by interferon-gamma (IFN-gamma)-stimulated macrophages from IL-10-/- mice, thus directly showing functional activity of surface IL-10. Taken together, these studies indicate that macrophage surface IL-10 is biologically active and down-regulates macrophage bactericidal activity.

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.

Increased expression of CD40 on thymocytes and peripheral T cells in autoimmunity: a mechanism for acquiring changes in the peripheral T cell receptor repertoire.

CD40, a cell surface molecule found on B lymphocytes and other antigen presenting cells, can, when engaged by CD40 ligand (CD40L), induce gene rearrangements and isotype switching. We report here that CD40 is also expressed on thymocytes and on up to 50% of peripheral T cells from autoimmune prone strains of mice. In normal animals, CD40 is present on a small population of T cells and thymocytes. CD40 is expressed on most T cell hybridomas. We demonstrate that CD40 engagement on peripheral T cells, T cell hybridomas and thymocytes results in altered TCRValpha expression. That induced expression of different Valpha's results from the activity of the recombinase gene is implied by the observation that CD40 does not induce TCR changes in RAG knock-out mice. Total cell numbers remained unchanged between anti-CD40 treated and untreated populations of thymocytes or T cells indicating that treatment does not induce cell proliferation or cell death. The data presented here suggest a mechanism by which self reactive T cells accumulate peripherally and independently of selective processes of the thymus.

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.

A multifrequency electron spin resonance study of T4 lysozyme dynamics.

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C to 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic ordering with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time scale" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for the 9-GHz ESR spectra, the overall rotational diffusion must be accounted for in the analysis. This is accomplished by using a slowly relaxing local structure model (SRLS) for the dynamics, wherein the tether motion and the overall motion are both included. In this way a simultaneous fit is successfully obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motional/ordering modes of the probe are found for both lysozyme derivatives, indicating that the tether exists in two distinct conformations on the ESR time scale. The probe diffuses more rapidly about an axis perpendicular to its tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.

Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event.

Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.

Electron-spin resonance study of aggregation of gramicidin in dipalmitoylphosphatidylcholine bilayers and hydrophobic mismatch.

The effect of aggregation of gramicidin A' (GA) on the phase structure of dipalmitoylphosphatidylcholine (DPPC) multilamellar vesicles was studied by cw-ESR using a chain-labeled lipid (16PC) at temperatures between 30 degrees and 45 degreesC that span the main phase transition of DPPC. Boundary lipids were observed only in dispersions with GA/DPPC molar ratios >1:15, where GA aggregates. Detailed fits by nonlinear least squares (NLLS) methods are consistent with the boundary lipid being characterized by a large negative order parameter ( approximately -0.4), indicative of a dynamic bending of the end of the acyl chain, and a substantially reduced motion, about an order of magnitude slower than that of the bulk lipid. The NLLS analysis compares favorably with a recent two-dimensional Fourier transform ESR study on DPPC/GA vesicles, which accurately discerned the bulk lipid. The detailed ESR observables are discussed in terms of the ordering effect of GA at low concentration of GA, the dissociation of the GA channel and the dynamic bending of the end chain segment of boundary lipid at high concentration of GA, and of HII phase formation induced by GA. It is suggested that these phenomena can be interpreted in terms of the combined effects of partial dehydration of the lipid headgroup by the GA and of the hydrophobic mismatch between GA and DPPC molecules. Substantial hysteresis is observed for heating versus cooling cycles, but only for a GA/DPPC molar ratio >1:15. This is consistent with the aggregation of GA molecules at high concentrations.

A 50-kDa variant form of human surfactant protein D.

The dominant form of human surfactant protein D (SP-D) is a multimeric collagenous glycoprotein composed of monomeric subunits that have a molecular mass of 43 kDa under reducing conditions. However, in evaluating monoclonal antibodies to human SP-D, an additional monomeric subunit was identified with a reduced molecular mass of 50 kDa. This 50-kDa variant was detected in approximately half of the samples evaluated and was found in lavage fluid from normal subjects, patients with alveolar proteinosis or idiopathic pulmonary fibrosis and in amniotic fluid. This 50-kDa variant had the same amino-terminal sequence, amino acid composition and apparent size of the carboxy-terminal collagenase-resistant fragment (20 kDa) as the 43-kDa subunit. The major difference was in the amino-terminal portion of the molecule and was due to altered glycosylation, as determined by carbohydrate staining, chemical deglycosylation, treatment with N-glycanase and neuraminidase and reduced signals for threonine at positions 5, 9 and 10 during amino-terminal sequencing. After gel filtration chromatography, the 50-kDa form was not present in the high molecular weight fraction, which is commonly used in purification of SP-D, but was found only in the smaller molecular weight fraction of monomers and trimers of SP-D. In conclusion, the 50 kDa-form of surfactant protein D is produced by post-translational glycosylation and does not form higher ordered oligomers, but its precise physiological function remains to be determined.