Specificity of the SH2 domains of SHP-1 in the interaction with the immunoreceptor tyrosine-based inhibitory motif-bearing receptor gp49B

Inhibitory receptors on hemopoietic cells critically regulate cellular function. Despite their expression on a variety of cell types, these inhibitory receptors signal through a common mechanism involving tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif (ITIM), which engages Src homology 2 (SH2) domain-containing cytoplasmic tyrosine or inositol phosphatases. In this study, we have investigated the proximal signal-transduction pathway of an ITIM-bearing receptor, gp49B, a member of a newly described family of murine NK and mast cell receptors. We demonstrate that the tyrosine residues within the ITIMs are phosphorylated and serve for the association and activation of the cytoplasmic tyrosine phosphatase SHP-1. Furthermore, we demonstrate a physiologic association between gp49B and SHP-1 by coimmunoprecipitation studies from NK cells. To address the mechanism of binding between gp49B and SHP-1, binding studies involving glutathione S-transferase SHP-1 mutants were performed. Utilizing the tandem SH2 domains of SHP-1, we show that either SH2 domain can interact with phosphorylated gp49B. Full-length SHP-1, with an inactivated amino SH2 domain, also retained gp49B binding. However, binding to gp49B was disrupted by inactivation of the carboxyl SH2 domain of full-length SHP-1, suggesting that in the presence of the phosphatase domain, the carboxyl SH2 domain is required for the recruitment of phosphorylated gp49B. Thus, gp49B signaling involves SHP-1, and this association is dependent on tyrosine phosphorylation of the gp49B ITIMs, and an intact SHP-1 carboxyl SH2 domain.     

Air-gaps in 0.3 /spl mu/m electrical interconnections

A copper/air-gap interconnection structure using a sacrificial polymer and SiO/sub 2/ in a damascene process has been demonstrated. The air-gap occupies the entire intralevel volume with fully densified SiO/sub 2/ as the planar interlevel dielectric. The copper was deposited by physical vapor deposition and planarized by chemical-mechanical planarization. The Ta/Cu barrier/seed layer was deposited by physical vapor deposition; the bulk copper was electrochemically deposited. The resulting structure has an effective intralevel dielectric constant of 2.19.     

A dynamical investigation of acrylodan-labeled mutant phosphate binding protein

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy. As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine. This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity. Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed. Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction. Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe. The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free. This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes). Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein. Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees. In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197. Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.     

Intra-accumbens infusions of antisense oligodeoxynucleotides to one isoform of glutamic acid decarboxylase mRNA, GAD65, but not to GAD67 mRNA, impairs sustained attention performance in the rat

The effects of bilateral infusions of antisense oligodeoxynucleotides (ODNs) for the two isoforms of glutamic acid decarboxylase (GAD65; GAD67) into the nucleus accumbens on the performance of intact rats in a task designed to assess sustained attention were tested. The task required the animals to discriminate between signal and non-signal events. Signals and non-signals were presented randomly and unpredictably. The task generated all four response types of a sustained attention task, i.e., hits, misses, correct rejections, false alarms. Infusions of the scrambled sequence ODNs did not affect performance. Likewise, infusions of the GAD67 ODNs failed to produce any effect. However, infusions of the GAD65 ODNs into the nucleus accumbens resulted in a robust and reliable decrease in the relative number of hits. Similarly, the combined infusion of GAD65+67 ODNs impaired the hit rate but did not affect the animals' ability to reject non-signals. Following each treatment series, performance rapidly returned to baseline, further indicating the specificity and reversibility of the effects of the infusions of the ODNs. While these data suggest that translation arrest of specifically the GAD65 isoform of the enzyme in the nucleus accumbens impairs attentional performance, the neuronal mechanisms mediating these effects remain unsettled.     

Termination of translation in eukaryotes: new results and new hypotheses

The viscoelastic properties of the human and canine pharyngeal tissue in tension were evaluated, based on both an experimental protocol-consisting of cyclic load, tensile stress relaxation, and incremental step load tests-and the quasi-linear viscoelastic theory. The reduced stress relaxation function and the elastic response of the pharyngeal tissues were derived from the experimental results specifically obtained from those tissues. The characteristic features of viscoelastic property were obtained for both human and canine pharyngeal tissues by applying the quasi-linear viscoelastic theory and compared with each other. The material properties of the pharyngeal tissue were sought to facilitate the three-dimensional biomechanical model of the pharyngeal function by using the finite element method.     

Fathers and mothers of school-age children with developmental disabilities: parental stress, family functioning, and social support

Thirty pairs of fathers and mothers who had school-age children with mental retardation and other disabilities were compared with each other and with 32 father and mother pairs of parents of children without disabilities. Responses to family scales indicated that fathers and mothers of children with developmental disabilities did not differ from each other nor from fathers and mothers of children without disabilities in parental stress, family social support, or family functioning. However, parents of children with disabilities experienced a disproportionately greater level of stress relating to their children than did those of children without disabilities. Fathers' and mothers' stress was associated with aspects of family functioning as perceived by themselves and their spouses.     

Genetic evaluation by BLUP in two-breed terminal crossbreeding systems under dominance

To obtain estimates of breeding values by BLUP using Henderson's mixed-model equations, it is necessary to invert the covariance matrix for each random effect in the model. In a model in which the genotypic value is included as a random effect (genotypic model), it is necessary to invert the genotypic covariance matrix. Under additive inheritance, the inverse of the genotypic covariance matrix can be computed efficiently. Under dominance inheritance, however, an efficient method to invert the genotypic covariance matrix has not yet been developed, especially for crossbred populations. Thus, the use of a genotypic model for BLUP is not suitable for genetic evaluation in large, crossbred populations. We present an equivalent model in which the genotypic effect is partitioned into additive and dominance effects. With this equivalent model, methods used for within-breed genetic evaluation by BLUP can be used for a two-breed terminal cross under dominance.     

Endotoxin temporarily impairs canine jejunal absorption of water, electrolytes, and glucose

OBJECTIVE: To evaluate the usefulness of a portable, computer-based, oscillometric, noninvasive blood pressure monitor for the automatic surveillance of postoperative infrainguinal bypasses. DESIGN: Prospective unblinded study. SETTING: Regional tertiary care Veterans Affairs medical center. PATIENTS: A total of 60 limbs were examined in 3 groups (n = 20 in each group): normal, asymptomatic volunteers; older patients with symptomatic peripheral vascular disease; and patients who had undergone infrainguinal bypass procedures. INTERVENTIONS: Noninvasive blood pressure monitor pressures, complete lower extremity arterial studies with Doppler ankle-brachial index (ABI), segmental pressures, and pulse volume recordings were performed in all groups. The patients who had undergone surgery also had hourly noninvasive blood pressure measurements taken. MAIN OUTCOME MEASURES: Ankle-brachial indices obtained with the noninvasive blood pressure monitor were compared with simultaneous manual Doppler ABIs and pulse volume recordings. RESULTS: There was a significant overall correlation between the Doppler-derived ABIs and automatic oscillometric values (r = 0.89, P < .001). Although there was a slight difference in absolute values (+/-SEM) (0.90 +/- 0.03 vs 0.84 +/- 0.03, respectively), an excellent correlation was found between the 2 methods in the clinically relevant range of ABI values between 0.60 and 1.10. The oscillometric method, however, overestimated the ABI when it was less than 0.60 by the Doppler method (0.61 +/- 0.02 vs 0.44 +/- 0.03, P < .001) and underestimated it when it was more than 1.10 (1.05 +/- 0.02 vs 1.16 +/- 0.01, P < .001). (All values given as +/-SEM.) Oscillometric overestimation correlated with depressed pulse volume recording waveforms (P < .001) or diminished pulse volume recording amplitudes of less than 10 mm (P < .001). CONCLUSION: The automatic oscillometric monitor provides a useful, accurate, objective, and quantitative method for postoperative bypass graft surveillance.