Finite-Size Effect in Phonon-Induced Elliott-Yafet Spin Relaxation in Al.

The Elliott-Yafet theory of spin relaxation in nonmagnetic metals predicts proportionality between spin and momentum relaxation times for scattering centers such as phonons. Here, we test this theory in Al nanowires over a very large thickness range (8.5-300 nm), finding that the Elliott-Yafet proportionality "constant" for phonon scattering in fact exhibits a large, unanticipated finite-size effect. Supported by analytical and numerical modeling, we explain this via strong phonon-induced spin relaxation at surfaces and interfaces, driven in particular by enhanced spin-orbit coupling.

Experimental Realization of the 1D Random Field Ising Model.

We have measured magnetic-field-induced avalanches in a square artificial spin ice array of interacting nanomagnets. Starting from the ground state ordered configuration, we imaged the individual nanomagnet moments after each successive application of an incrementally increasing field. The statistics of the evolution of the moment configuration show good agreement with the canonical one-dimensional random field Ising model. We extract information about the microscopic structure of the arrays from our macroscopic measurements of their collective behavior, demonstrating a process that could be applied to other systems exhibiting avalanches.

Mass spectrometric characterization of proteins extracted from Jurkat T cell detergent-resistant membrane domains.

Plasma membranes of most cell types are thought to contain microdomains commonly referred to as lipid rafts, biochemically distinct from bulk plasma membrane, apparently enriched for proteins involved in signal transduction. In T cells, it is believed that lipid rafts aggregate at the site of T cell receptor engagement and act as foci for initiation of the signaling process. In order to gain insight into the possible functioning of lipid rafts, we applied microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (microLC-ESI-MS/MS) methodologies to the identification of proteins which copurified with lipid rafts. Following isolation of lipid rafts as Triton-insoluble, low-density membrane fractions from Jurkat T cells, tryptic digests were generated of individual protein bands resolved electrophoretically. Alternatively, cysteine-containing peptides were isolated from total tryptic digests of unseparated lipid raft proteins following labeling with a cysteine-specific biotinylation reagent and avidin affinity purification. In both cases, protein identifications were made by comparison of tandem MS spectra generated by microLC-ESI-MS/MS to both protein and DNA sequence databases using Sequest software. Proteins identified essentially fell into two groups: cytoskeletal proteins, and proteins involved in signal transduction. These findings are discussed in the light of the current understanding of both lipid raft biology and signal transduction.

CD28 stimulation regulates its association with N-ethylmaleimide-sensitive fusion protein and other proteins involved in vesicle sorting.

CD28 delivers a co-stimulatory signal for T cell antigen receptor induced activation of T cells through a mechanism which remains mostly elusive to date. In order to try and gain insight into CD28 function, we therefore applied state-of-the-art mass spectrometric protein identification technology to the analysis of CD28 immunoprecipitates prepared from Jurkat T cells. We found that N-ethylmaleimide-sensitive fusion protein (NSF) and other proteins with sequence similarities to proteins part of or implicated in vesicular protein sorting pathways, were associated with CD28 in a CD28 stimulation-dependent manner. Furthermore, N-ethylmaleimide treatment abolished the NSF/CD28 interaction completely, and blocked CD28 association with a tyrosine phosphorylated 103 kDa protein in the activated cells. These results are suggestive of a potential model for CD28 co-stimulation regulated by an NSF-catalyzed mechanism.

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

A systematic approach to the analysis of protein phosphorylation.

Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically requiring the purification to homogeneity of the phosphoprotein of interest before analysis. Thus, there has been a substantial need for a more rapid and general method for the analysis of protein phosphorylation in complex protein mixtures. Here we describe such an approach to protein phosphorylation analysis. It consists of three steps: (1) selective phosphopeptide isolation from a peptide mixture via a sequence of chemical reactions, (2) phosphopeptide analysis by automated liquid chromatography-tandem mass spectrometry (LC-MS/MS), and (3) identification of the phosphoprotein and the phosphorylated residue(s) by correlation of tandem mass spectrometric data with sequence databases. By utilizing various phosphoprotein standards and a whole yeast cell lysate, we demonstrate that the method is equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins, and is capable of selectively isolating and identifying phosphopeptides present in a highly complex peptide mixture.

A comprehensive characterization of the T-cell antigen receptor complex composition by microcapillary liquid chromatography-tandem mass spectrometry.

It has become apparent that many intracellular signaling processes involve the dynamic reorganization of cellular proteins into complex signaling assemblies that have a specific subunit composition, function, and subcellular location. Since the elements of such assemblies interact physically, multiprotein signaling complexes can be isolated and analyzed. Recent technical advances in highly sensitive protein identification by electrospray-tandem mass spectrometry have dramatically increased the sensitivity with which such analyses can be performed. The T-cell antigen receptor (TCR) is an oligomeric transmembrane protein complex that is essential to T-cell recognition and function. The extracellular protein domains are responsible for ligand binding while intracellular domains generate and transduce signals in response to specific receptor-ligand interactions. We used microbore capillary chromatography-tandem mass spectrometry to investigate the composition of the TCR protein complex isolated from resting and activated cells of the murine T-cell line CD11.3. We identified all the previously known subunits of the TCR/CD3 complex as well as proteins previously not known to associate with the TCR. The catalytic activities of some of these proteins could potentially be used to interfere pharmacologically with TCR signaling.

Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry.

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS.

On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction.

The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously. We quantitiated ceramides ranging from C14 to C26, finding that their basal levels and relative distribution varied significantly, both within and between different cell types. However, we were not able to detect any significant changes in either total ceramide content or species distribution until 1 h or more post-stimulation with any of these treatments, by which time the cells were in an advanced stage of apoptosis. Differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis. These data indicate that in vivo ceramide generation occurs as a consequence of apoptosis rather than as an essential second messenger involved in its induction. They also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis, and demonstrate a clear need to utilize the resolving power of mass spectrometry-based assays in any future investigations into the biological function of ceramides.

Sensitivity of various radiographic methods for detection of oral cancellous bone lesions.

OBJECTIVE: The purpose of this study was to determine the effectiveness in diagnosing cancellous bone defects of the following radiographic methods: conventional film, digitized film, enhanced digitized film, direct digital imaging, enhanced direct digital imaging, digital subtraction, and enhanced digital subtraction. STUDY DESIGN: Mechanical lesions of varying depths were generated beneath cadaver molar and premolar mandibular tooth roots. A portfolio of radiographic images of random types and lesion sizes was presented to 20 clinicians, and their diagnoses were evaluated. RESULTS: Positive identification of lesions was significantly improved by enhanced subtraction radiography over all other forms of radiography for the 4-mm lesions and was better than all forms except enhanced digital radiography and film for the 6-mm lesions. Subtraction radiography and enhanced subtraction radiography significantly reduced false positive diagnoses at all lesion sizes in comparison with the other radiographic methods except enhanced digital radiography at the 6-mm lesion size. CONCLUSIONS: For the methods evaluated, only subtraction radiography and enhanced subtraction radiography can significantly improve the clinician's diagnostic abilities for detection of oral cancellous bone lesions through increased rates for detection of existing defects and, even more importantly, through decreased rates of defect misdiagnosis.

Fas-induced apoptosis of T cells occurs independently of ceramide generation.

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.

Characterization of Lnk. An adaptor protein expressed in lymphocytes.

Stimulation of the T cell antigen receptor (TCR) activates a set of non-receptor protein tyrosine kinases that assist in delivering signals to the cell interior. Among the presumed substrates for these kinases, adaptor proteins, which juxtapose effector enzyme systems with the antigen receptor complex, figure prominently. Previous studies suggested that Lnk, a 38-kDa protein consisting of a single SH2 domain and a region containing potential tyrosine phosphorylation sites, might serve to join Grb2, phospholipase C-gamma1, and phosphatidylinositol 3-kinase to the TCR. To elucidate the physiological roles of Lnk in T cell signal transduction, we isolated the mouse Lnk cDNA, characterized the structure of the mouse Lnk gene, and generated transgenic mice that overproduce Lnk in thymocytes. Here we report that although Lnk becomes phosphorylated during T cell activation, it plays no limiting role in the TCR signaling process. Moreover, we have distinguished p38(Lnk) from the more prominent 36-kDa tyrosine phosphoproteins that appear in activated T cells. Together these studies suggest that Lnk participates in signaling from receptors other than antigen receptors in lymphocytes.

Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry.

Ceramides and sphingoid bases are important intracellular second messengers that play a role in the regulation of cell growth, differentiation, and programmed cell death. Until now, quantitative and qualitative analysis of ceramide second messengers has been limited by a lack of analytical methods capable of detecting endogenous levels of, and differentiating between, individual ceramide species. Here we report the use of electrospray ionization tandem mass spectrometry for the qualitative and quantitative analysis of ceramides. Collision-induced fragmentation resulted in characteristic product ions for the sphingosine and dihydrosphingosine (sphinganine) head groups at m/z 264 and 282 and m/z 266 and 284, respectively, regardless of the length of the fatty acyl chains, with spectra being reproducible at concentrations as low as 25 nM (25 fmol/microliter). These reporter ions were used to detect both sphingosine- and sphinganine-based ceramides in complex mixtures using precursor ion scan analysis. We demonstrated the application of this method for profiling the composition of ceramides in a commercial preparation of bovine brain ceramides and, following minimal chromatographic separation, a lipid extract of cultured T cells. Furthermore, we easily detected relative differences in individual ceramide species levels by comparing the profiles of three related lymphocyte cell lines, Jurkat, U937, and WEHI 231. Finally, by the addition of a nonnaturally occurring internal standard, we show that the technique can be used to measure quantitative changes in ceramide levels in such biologically derived lipid preparations.

An alternative method of nitrous oxide delivery into a minimal-flow circle breathing system.

We have sought to define a way in which nitrous oxide can be safely and universally used at minimal to low flows by utilising a circle system with a controlled leak provided by a standard gas analyser sampling line and a fresh gas supply of 50% nitrous oxide in oxygen, entering from a trunk interposed between the ventilator and the circle system. Although preliminary calculations suggested that this arrangement was likely to work, it was found that 13 of 23 patients studied prospectively developed an inspired oxygen fraction below 0.3. We conclude that, although this arrangement provides a new means of introducing nitrous oxide into the circle breathing system, it does not appear inherently safer or more convenient than the conventional route.

Identification and characterization of a substrate specific for the T cell protein tyrosine kinase ZAP-70.

ZAP-70 is a protein tyrosine kinase (PTK) that plays a critical role in T cell activation. To study the role of ZAP-70 catalytic activity in this process, a substrate capable of distinguishing between the activities of ZAP-70 and other PTKs would be useful, especially since it has recently been shown that ZAP-70 interacts with another T cell PTK, Lck. We have thus identified a site of phosphorylation on the cytoplasmic fragment of the erythrocyte band 3 protein that is recognized by ZAP-70, but not Lck. A synthetic peptide based on this site has been demonstrated to be a good in vitro substrate for ZAP-70 and a poor substrate for the T cell PTKs Lck and Itk. This peptide molecule should thus prove useful to many investigators working in the field of T cell activation.

Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system.

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent Km of ZAP-70 for ATP was approximately 3.0 microM. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Several proteins were tested as potential in vitro substrates of ZAP-70. Only alpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting Kmvalues of approximately 3.3 and approximately 2.5 microM, respectively ([ATP] = 50 microM). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCRzeta chain or short peptides corresponding to the CD3epsilon or the TCRzeta immunoreceptor tyrosine-based activation motifs. Our studies suggest that ZAP-70 exhibits a high degree of substrate specificity.

Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro.

p56lck is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase alpha. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56lck (residues 34-150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56lck, bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56lck to CD45, and when added together worked synergistically to compete for p56lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85 alpha subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction.

Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70.

ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.