Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.     

Positron emission tomography detects metabolic viability in myocardium with persistent 24-hour single-photon emission computed tomography 201Tl defects.

BACKGROUND. Four-hour 201Tl redistribution images underestimate myocardial viability in patients with coronary artery disease (CAD). Because 4-hour defects often redistribute late, delayed imaging may enhance assessment of tissue viability. Myocardial metabolic activity was therefore assessed with positron emission tomography (PET) in 26 CAD patients with impaired ventricular function (ejection fraction, 32.1 +/- 13.9%) and 24-hour single-photon emission computed tomography (SPECT) 201Tl defects. METHODS AND RESULTS. On circumferential profile analysis, PET ischemia was defined by preserved glucose metabolism in hypoperfused myocardium, and PET infarction was defined by concordant reductions in perfusion and metabolism. On 19 stress-redistribution and seven rest-redistribution SPECT studies, four observers visually scored 201Tl activity in eight segments on a scale from 0 (normal) to 3 (complete defect). Using an improvement in visual score > or = 0.75 to define redistribution, there were 100 fixed, 17 partially reversible, and 12 completely reversible defects. PET identified tissue metabolic activity in 51 (51%) segments with fixed defects (21 PET ischemia, 30 PET normal) and nine (53%) segments with partially reversible defects (five PET ischemia, four PET normal). When grouped by 24-hour score, the proportion of fixed defects with metabolic activity varied from 84% (scores < or = 1.4) to 15% (scores > 2.6). For partially reversible defects, only 53% with scores < 2.0 and one of two with scores > or = 2.0 were considered metabolically viable on PET. Of 12 completely reversible defects, six (50%) were normal, five (42%) had PET ischemia, and one (8%) had PET infarction. The proportion of fixed defects with metabolic activity did not depend on whether a rest or stress study was performed or on the change in visual score used to define 201Tl redistribution (0.25, 0.50, 0.75, and 1.00). CONCLUSIONS. In CAD patients, PET identifies glucose metabolic activity in the majority of fixed 24-hour 201Tl defects. However, very severe (near-complete) 24-hour 201Tl defects are less likely to exhibit metabolic activity on PET imaging than are defects with less-pronounced reductions in segmental 201Tl activity.     

Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated intravascular coagulation

Concentrations of plasma fibrinopeptide A (FPA) were measured by radioimmunoassay in 50 patients with venous thromboembolism or disseminated intravascular coagulation or both. A consistent discrepancy was observed in values obtained with two anti-FPA antisera. Analysis of extracts from plasma of these patients by high-performance liquid chromatography (HPLC) revealed the presence of a phosphorylated and an unphosphorylated form of the A peptide. Differences in concentrations of FPA measured with the two antisera could be accounted for by their different reactivity with phosphorylated FPA (FPA-P). The differences were abolished by treatment with alkaline phosphatase. A good correlation was observed between the FPA-P content of free A- peptide material and of fibrinogen in plasma as determined by HPLC (r = .88, P less than .001, n = 11). In patients with elevated FPA levels, the mean FPA-P content of fibrinogen was significantly higher (P less than .002, n = 13) than in patients with normal FPA levels (n = 8) and in healthy controls (n = 14). Phosphorus in fibrinogen did not correlate with fibrinogen degradation products or fibrinogen levels and became normal on adequate anticoagulation. Therefore, blood-clotting activation may lead to a high phosphate content of fibrinogen and of free FPA in plasma.     

High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.     

Morphologic characteristics of aortic atresia: implications for fetal hemodynamics

We studied hearts with aortic atresia to determine the relationship between cardiac morphology and fetal cardiac blood flow. We compared measurements of aortic, pulmonary, and tricuspid valve circumference and right ventricular wall thickness among 37 hearts with aortic atresia and mitral hypoplasia, 14 hearts with aortic atresia together with mitral atresia and 24 normal hearts. Right ventricular free wall thickness and tricuspid and pulmonary valve annular circumferences were greater and aortic circumferences were smaller in hearts with aortic atresia than in normal hearts (P less than 0.05). Between the subgroups of aortic atresia, the aortic circumference was 5 +/- 1 mm in those with mitral atresia compared to 7 +/- 2 mm in association with mitral hypoplasia (P less than 0.05). The morphologic differences between the subgroups of aortic atresia are consistent with differences in fetal blood flow. Left heart blood flow was likely to be greater during development in those hearts with aortic atresia and mitral hypoplasia than in those with the combination of aortic and mitral atresia.     

Increasing the on-task homework behavior of youth with behavior disorders using functional behavioral assessment

Research has shown a positive correlation between time spent on homework and learning. However, students often engage in off-task behaviors to escape the demands of homework. Youth with emotional or behavioral disorders (EBD) are especially likely to engage in off-task behaviors. Effective interventions to increase on-task behavior during homework are needed to increase students' academic success. Functional behavioral assessment (FBA) procedures may be helpful for intervention planning; however, there has been limited research on use of FBA with youth with EBD experiencing poor academic performance or task completion problems. In the current study, FBA methods were used to identify the contingencies maintaining the off-task behavior of four youth with behavior problems. Effects of interventions based on functional hypotheses were compared to the effects of interventions not linked to such hypotheses. Discussion focuses on utility of FBA procedures for developing and implementing effective interventions for youth with EBD.     

Effects of N-methyl-D-aspartate antagonists and spantide on spinal reflexes and responses to substance P and capsaicin in isolated spinal cord preparations from mouse and rat.

Electrical stimulus intensity, capsaicin, excitatory amino acid antagonists and the substance P antagonist, spantide, have been used to investigate the roles of primary afferent C fibres and excitatory amino acid receptors in the generation of long duration (half time 9.1 s +/- 1.1 S.E.M., N = 24) contralateral reflexes recorded in ventral roots of immature rat spinal cords in vitro. The relationship between C fibre compound action potentials recorded in the dorsal root and duration of the dorsal root-evoked contralateral ventral root potential appeared to be coincidental rather than causal. Dorsal root-evoked contralateral ventral root potentials of greater than 2 s in duration could not be evoked in mature mouse spinal preparations. Application of capsaicin (1 microM for 15-120 min) produced a long lasting increase in spontaneous activity of ventral roots as well as blockade of C fibre conduction in dorsal roots. The dorsal root potential evoked following stimulation of adjacent dorsal roots at intensities insufficient for activation of C fibres was depressed by capsaicin. Dorsal root-evoked contralateral ventral root potentials were abolished by kynurenate (EC50 56 +/- 13 microM, N = 3) and depressed to 38.2 +/- 6.9% S.E.M. (N = 7) of pre-drug levels by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonopentanoate (20 microM) or to 51.8 +/- 9.0% (N = 7) by the substance P analogue spantide (33 microM). Spantide consistently antagonised substance P-induced, but not capsaicin-induced, depolarizations recorded in ventral roots (+-)-2-Amino-5-phosphonopentanoic acid (10-50 microM) depressed both substance P- and capsaicin-induced depolarizations. The depressant effect of spantide, unlike that of (+/-)-2-amino-5-phosphonopentanoic acid, was associated with a long lasting excitatory action. In the presence of tetrodotoxin (0.1 microM), spantide (33 microM) failed to antagonize substance P-induced depolarizations. It is suggested that long duration of the dorsal root-evoked contralateral ventral root potential is a consequence of the activation of the N-methyl-D-aspartate receptor operated ion channels by excitatory amino acid transmitters.     

Assessment of rheumatoid activity based on clinical features and blood and synovial fluid analysis.

Joint inflammation in rheumatoid arthritis has been assessed, and the most useful guides to disease activity were determined by analysis of synovial fluid and blood together with the history of joint disability. The patient's own evaluation of the amount of pain suffered was the most useful clinical assessment. Differential cell count and glucose estimations were the most helpful guides in the synovial fluid, while C-reactive protein in the serum most accurately reflected disease activity. The effects of systemic steroids on these indices were studied, and the differences between seronegative and seropositive patients noted.     

Optofluidic wavelength division multiplexing for single-virus detection

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine.The ability to overlay multiple electromagnetic waves in the same physical space by virtue of linear superposition is arguably at the root of modern communication as we know it. Originally implemented in the radiofrequency regime, this wavelength division multiplexing (WDM) principle was transferred to optical wavelengths in the visible and near-infrared range, which can be carried by a single, low-loss silica fiber (1). Available in both coarse and dense varieties, terabits of data are now shuttled between a source and their destination using anywhere from 4 to over 100 wavelengths (2, 3). Here, we transfer the WDM principle from data communications into a different realm, that of chip-based biomedical analysis, where much can be gained by superimposing multiple colors in an optical waveguide, albeit for different reasons.First, one of the key requirements for diagnostic test panels, aside from high sensitivity and specificity, is the ability to multiplex, i.e., detect and identify multiple biomarkers simultaneously. A standard influenza test, for example, simultaneously screens for eight pathogen types, enabling differential diagnosis of diseases with similar early symptoms (www.questdiagnostics.com/testcenter/testguide.action?dc=TS_RespVirusPanel). Current gold-standard techniques for nucleic acid and protein detection such as PCR and ELISA use fluorescent organic dyes as a means of signal reporting (4). Optical detection, by fluorescence or “label-free,” is extremely sensitive, allowing for single-molecule and even single-dye detection under appropriate conditions (5–11). The availability of over a dozen dyes across the visible spectrum opens the door to implementing the desired multiplexing capability with multiple dyes, i.e., spectral channels (12–14). Secondly, diagnostic tests are rapidly transitioning toward integrated laboratory-on-chip platforms on which small volumes of biological or chemical samples can be rapidly analyzed. The WDM principle of routing all spectral channels through the same physical space is, therefore, ideal for increasing compactness of an analytic device. Finally, chip-scale integration has recently been advanced by the advent of optofluidic devices in which both fluidic and optical components are miniaturized in the same system (15–17).Here, we implement WDM on an optofluidic platform for on-chip analysis of single influenza viruses. In place of silica fiber as the physical carrier, we create a single waveguide structure that combines multiple spectral channels for fluorescence excitation of biological targets. Instead of temporally modulating each channel to transport information, we use this waveguide to produce wavelength-dependent spatial patterns in an intersecting fluidic channel. The spatial encoding of spectral information then allows for direct identification of multiple labeled targets with extremely high sensitivity and fidelity. The technique is demonstrated in two implementations for direct counting and identification of individual virus particles from three different influenza A subtypes––H1N1, H2N2, and H3N2––at clinically relevant concentrations.