Phenotypic characterization and analysis of allogeneic T cell responses in ZAP-70 dominant negative transgenic mice.

Antigen stimulation of T cells results in a series of biochemical events including the interaction of both SH2 domains of ZAP-70 with phosphorylated ITAMS on the T cell receptor. In order to study the physiological relevance of decreasing native ZAP-70-SH2 interaction in vivo, we generated transgenic mice expressing a T cell-specific, dominant negative form of ZAP-70 consisting of only the tandem SH2 domains (ZAP-NC). Phenotypically, these animals had a comparable distribution of lymphocyte subsets in the thymus and spleen compared with the wild-type (WT) controls. However, examination of peripheral blood revealed a slow but progressive decrease in the number of lymphocytes, particularly CD4+ cells, with age (17% reduction by 3 months, 58% reduction by 6 months). Allogeneic responses were then evaluated in vitro as well as in vivo using a subcutaneous sponge matrix implant. Although spleen cells cultured for 4 days in vitro with alloantigen developed normal functional responses, allogeneic responses generated in vivo within a subcutaneous sponge matrix were impaired. This was characterized by a depression in cytotoxic T lymphocyte (CTL) activity, a 82% reduction in the frequency of helper T cells, and a 78% reduction in the capacity of sponge-infiltrating lymphocytes to produce IL-2 in response to secondary antigen stimulation. These results indicate that although overt lymphocyte development and in vitro function were unremarkable, expression of a truncated ZAP-70 affected the in vivo survival of peripheral lymphocytes and altered the in vivo generation of functional activity to alloantigen.

Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation.

Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

"Spot transfer", elution and comigration with known proteins allows accurate transferral of protein identifications between distinct two-dimensional electrophoretic systems.

We report a simple method, designated "spot transfer", where known proteins are excised and eluted from a two-dimensional (2-D) gel run on one gel system to transfer identification of the proteins to a different 2-D gel system by comigration with a comparable sample. In one experiment, 8 of 16 proteins eluted from an isoelectric focusing (IEF) 2-D gel, of the format described for the Celis human keratinocyte database (Celis, J. E. et al., Electrophoresis 1993, 14, 1091-1198), were found to comigrate with proteins in a human T lymphoma (JURKAT) whole cell lysate run using the Millipore Investigator 2-D system. The method should have general utility in allowing the exchange of protein identifications between investigators and could be used to standardize gel loci used in 2-D gel protein databases.

The role of individual Fc gamma receptors in aggregated IgG-stimulated protein tyrosine phosphorylation in the human neutrophil.

Having previously shown that heat-aggregated IgG stimulates a significant increase in tyrosine phosphorylation in the human neutrophil, we next sought to determine the role of individual Fc gamma receptors in this response. Specific cross-linking of Fc gamma RII reproduced the phosphorylation observed with heat-aggregated IgG treatment and monoclonal antibodies recognizing the ligand binding domain of Fc gamma RII efficiently blocked the heat-aggregated IgG induced response. Thus engagement of Fc gamma RII alone appears sufficient to mimic the heat-aggregated IgG stimulation. Similar experiments carried out with antibodies specific for Fc gamma RIII suggested that Fc gamma RII and Fc gamma RIII may cooperate in the phosphorylation response. The activation of a tyrosine kinase by Fc gamma R engagement was demonstrated by the specific immunoprecipitation of several tyrosine phosphorylated proteins from lysates of neutrophils following treatment with aggregated IgG.

C5a as a model for chemotactic factor-stimulated tyrosine phosphorylation in the human neutrophil.

Human neutrophils were exposed to the chemotactic factors C5a, FMLP, IL-8, leukotriene B4, and PAF, for 30 s, and subsequently analyzed for protein tyrosine phosphorylation by immunoblotting whole cell lysates with a polyclonal antiphosphotyrosine Ab. All chemotactic factors caused the rapid de novo tyrosine phosphorylation of a broad band of approximately 120 kDa and increased the phosphotyrosine content of several other proteins, including two with molecular masses of 60 and 56 kDa that were present in the unstimulated neutrophil. Tyrosine phosphorylation was evident as early as 10 s after stimulation and was maintained for 1 to 3 min before dephosphorylation occurred. The extent of tyrosine phosphorylation was dependent on the concentration of chemotactic factor, with stimulation observed at concentrations as low as 10 to 100 nM. To investigate the pathway used by chemotactic factors to transduce this signal, neutrophils were treated with PMA. PMA also stimulated tyrosine phosphorylation in the neutrophil but with a slower response time and a different pattern of affected proteins. Additional experiments suggested that tyrosine phosphorylation is involved in the regulation of the neutrophil respiratory burst because the tyrosine kinase inhibitor, herbimycin A, inhibited C5a-induced protein tyrosine phosphorylation and also prevented C5a- and FMLP-induced superoxide anion production. Herbimycin A also inhibited PMA-induced tyrosine phosphorylation and superoxide anion production. To confirm that the ability to stimulate tyrosine phosphorylation was intrinsic to the C5a receptor, tyrosine phosphorylation was examined in both undifferentiated U937 cells (C5a receptor negative) and cAMP differentiated U937 cells (C5a receptor positive). C5a induced tyrosine phosphorylation only in differentiated U937 cells. Analysis of the C5a receptor mRNA using the PCR confirmed its presence in differentiated and its absence in undifferentiated U937 cells. Therefore, C5a stimulates tyrosine phosphorylation via a receptor-mediated mechanism and U937 cells provide a system in which G-coupled receptor-mediated tyrosine phosphorylation can be investigated.

Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3).

Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL-2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells.

The epidermal growth factor receptor and the product of the neu protooncogene are members of a receptor tyrosine phosphorylation cascade.

The protein product of the neu protooncogene, p185, is a tyrosine kinase with a high degree of sequence homology to the epidermal growth factor (EGF) receptor. Although p185 does not bind EGF, EGF stimulates tyrosine phosphorylation of p185. To determine the mechanism of this interaction we have used a vaccinia virus/bacteriophage T7-based transient gene expression system to induce production of normal and kinase-deficient forms of p185 in the absence and presence of EGF receptors. Tyrosine phosphorylation of kinase-deficient p185 was observed, but only in the presence of the EGF receptor. These findings strongly support the hypothesis that p185 is a substrate for the EGF receptor tyrosine kinase in a tyrosine kinase cascade.

Evidence for the activation of the multifunctional Ca2+/calmodulin-dependent protein kinase in response to hormones that increase intracellular Ca2+.

The phosphorylation state of six cytoplasmic proteins is increased following treatment of isolated rat hepatocytes with hormones that elevate free intracellular Ca2+ levels (Garrison, J. C. and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Tryptic 32P-phosphopeptide maps of two of the substrates, pyruvate kinase and a 49,000-dalton protein, the major 32P-labeled protein in hepatocytes, were prepared following stimulation of cells with vasopressin, a Ca2+-linked hormone. Peptide maps of the 49,000-dalton protein phosphorylated in vitro with the recently identified multifunctional Ca2+/calmodulin-dependent protein kinase contained phosphopeptides identical to those observed in the intact cell, suggesting that this kinase is activated in response to Ca2+-mobilizing hormones. Similar in vitro phosphorylation experiments with pyruvate kinase suggested that the Ca2+/calmodulin-dependent protein kinase can phosphorylate not only the serine residues observed following vasopressin stimulation of the intact cell but also additional threonine residues. Both pyruvate kinase and the 49,000-dalton protein are also phosphorylated in the hepatocyte in response to glucagon and in vitro by the cAMP-dependent protein kinase. Both vasopressin and glucagon appear to stimulate the phosphorylation of identical serine residues in pyruvate kinase but only vasopressin enhances the phosphorylation of certain sites in the 49,000-dalton protein. Comparison of the tryptic phosphopeptide maps of these substrates phosphorylated in vitro with either the Ca2+/calmodulin-dependent protein kinase or the cAMP-dependent protein kinase suggests that the Ca2+-dependent kinase can phosphorylate unique sites in both substrates. It appears to share specificity at other sites with the cAMP-dependent protein kinase. Overall, the results suggest that the multifunctional Ca2+/calmodulin-dependent protein kinase plays an important role in the response of the hepatocyte to a Ca2+ signal.

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate.

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.

Blood pressure and physical fitness in children.

The association between physical fitness and blood pressure was studied in 2061 children selected from all fourth graders in 44 elementary schools in the New York City area. Their blood pressure and physical fitness were measured on two consecutive examinations 1 year apart. Systolic and diastolic blood pressure were highest in children with poor physical fitness. The change in physical fitness between the 2 examination years was related to the change in systolic and diastolic blood pressure (i.e., children with a decline in physical fitness showed the largest rise in blood pressure). These observations suggest that the level of systolic and diastolic blood pressure in children is associated with the level of physical fitness. They also indicate that change in blood pressure in childhood may be related to change in physical fitness.

Coronary heart disease prevention in childhood: one-year results of a randomized intervention study.

A six-year intervention study of the feasibility and effectiveness of a program aimed at the primary prevention of coronary heart disease (CHD) has been initiated among children in six school districts in Westchester County, New York. Schools randomly were assigned either to the intervention program or to a control group. The intervention program consists of a curriculum focusing on nutrition, physical fitness, and cigarette smoking prevention. The study population at baseline comprised 1,822 fourth-graders. This paper presents the findings at baseline and at one-year follow-up for the following target risk factors: systolic and diastolic blood pressure, plasma total and high-density lipoprotein (HDL) cholesterol, serum thiocyanate, ponderosity index, triceps skinfold thickness, and postexercise pulse recovery rate. After one year of intervention, the program was found to be acceptable to school administrators, teachers, parents, and children. Small net changes in the favorable direction were observed for diastolic blood pressure and thiocyanate. Intervention programs in schools may, after sufficient duration, prove to be effective in lowering CHD risk.

Pertussis toxin or phorbol 12-myristate 13-acetate can distinguish between epidermal growth factor- and angiotensin-stimulated signals in hepatocytes.

Epidermal growth factor (EGF) causes rapid increases in free intracellular Ca2+ and stimulates the phosphorylation of 11 cytosolic proteins in hepatocytes. Ten of the 11 cytosolic proteins altered by EGF are identical to those affected by angiotensin II, a hormone that stimulates the breakdown of phosphatidylinositol 4,5-bisphosphate. An increase in the phosphorylation of the other protein, spot c (Mr = 36,000, pI = 5.5), is observed only with EGF. Treatment of intact rats with pertussis toxin to ADP-ribosylate Ni, the inhibitory GTP-binding protein of the adenylate cyclase complex, abolished the effect of EGF on Ca2+ mobilization and on the phosphorylation of the 10 proteins affected in common with angiotensin II. This treatment had minimal effects on the ability of EGF to stimulate the phosphorylation of its unique substrate, spot c. In marked contrast, modification of Ni did not block the ability of angiotensin II to stimulate Ca2+ mobilization or protein phosphorylation. Pretreatment of normal hepatocytes with 4 beta-phorbol 12-myristate 13-acetate blocked all responses to EGF, including the increased phosphorylation of spot c, but had no effect on the responses to angiotensin II. These results imply that Ni or a similar pertussis toxin substrate may mediate the apparent effects of EGF on phosphatidylinositol breakdown and that protein kinase C may regulate a site in the transduction pathway. Angiotensin II appears to use a different signal transduction mechanism to stimulate phosphatidylinositol metabolism in hepatocytes.

Identification of a ternary complex between cAMP and a trimeric form of cAMP-dependent protein kinase.

One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C.

Primary prevention of chronic disease in childhood: changes in risk factors after one year of intervention.

A five-year intervention study of the feasibility and effectiveness of a program aimed at the primary prevention of chronic disease was initiated in 1980 among children in 22 elementary schools in the Bronx, New York. Schools randomly were assigned either to the intervention program or to a control group. The intervention program consists of a curriculum focusing on nutrition, physical fitness, and cigarette smoking prevention. The study population at baseline comprised 2,283 fourth-graders. Subjects were eligible at baseline and at one-year follow-up for participation in a medical examination in which the following target risk factors were measured: systolic and diastolic blood pressures, plasma total and high-density lipoprotein (HDL) cholesterol, serum thiocyanate, ponderosity index, triceps skinfold thickness, and postexercise pulse recovery rate. After one year of intervention, systolic pressure increased less in the intervention group than among controls. Diastolic pressure decreased in both groups, but more in the intervention subjects than in controls. Total cholesterol decreased in the intervention group while increasing among controls. Significant net changes in the favorable direction also were observed for total cholesterol/HDL cholesterol ratio and for thiocyanate. These observations indicate that it is feasible to implement a school-based program aimed at the primary prevention of chronic disease. The intervention program appears to have had a favorable effect on several target risk factors. Although the effects were relatively small, intervention programs in schools may prove to be effective in lowering chronic disease risk.

Screening for risk factors as a component of a chronic disease prevention program for youth.

Screening for risk factors within the framework of a chronic disease prevention program for youth can enhance both the educational and evaluative impact of the program. Screening results can provide the focus for curriculum development and the impetus for health-related behavior change. Program evaluation is facilitated by the use of screening results as objective outcome data regarding health status. The implementation of medical screening as a component of a chronic disease prevention program is described, and issues related to educational and evaluative impact are discussed.

Physicians' patient referrals to a nurse practitioner in a primary care medical clinic.

A survey of interns and residents in a Primary Care Medical Care Medical Clinic disclosed that these physicians have a very favorable impression of nurse practitioners' potential contributions to health care delivery, yet refer patients to the nurse practitioner to a much lesser degree than would have been expected on the basis of their attitudes.