A systematic approach to the complete study of a signaling molecule: ribosomal p90rsk as an example.

Ribosomal p90rsk is a kinase of central importance in transducing mitogenic signals from an activated receptor to the cell nucleus and for protein synthesis. Here, we analyze the optimal steps to fully describe this kinase in both normal neutrophils and leukemic cell lines. These are: (i) immunological analyses (immunoblotting and immunoprecipitation); (ii) enzyme activity assays (in vitro and "in-gel"); and (iii) immunobiochemical combination methods (immunoprecipitation/kinase assay, immunoprecipitation/"in-gel" assay and ion exchange chromatography/immunoblotting). For the enzyme assays, we describe a novel method to measure ribosomal p90rsk kinase activity "in-gel", based on a renatured-protein method that allows for the direct quantitation of enzyme activity. Finally, we present an algorithm that can be readily implemented to the quantification of the extent of stimulation of a kinase in response to a particular extracellular stimuli. In our case, it was found that activation of p90rsk was higher in proliferating leukemic cells than in mature neutrophils, indicating that a suppression of key signal transduction links could contribute to the maturational arrest typical of acute leukemia. All the techniques and strategies described here for p90rsk could be easily extrapolated to the study of any signal transduction molecule, provided it has a phosphotransferase activity.

Binding of GM-CSF to adherent neutrophils activates phospholipase D.

When the hematopoietic growth factor granulocyte-macrophage colony-stimulating factor was incubated with neutrophils adherent to plastic tissue culture plates or plates coated with extracellular matrix proteins, a rapid (3 min) but transient formation of phosphatidic acid was observed. This stimulation was dependent on the dose of GM-CSF, with an EC50 of 140 pM, and was further enhanced (up to 350%) with the PA phosphatase inhibitor propranolol in a dose-dependent manner. Conversely, GM-CSF was unable to trigger any PA formation in neutrophils maintained in suspension, even in the presence of soluble fibronectin. However, GM-CSF did prime the cells for enhanced PA formation in the presence of a secondary stimulus (fMet-Leu-Phe or PAF). GM-CSF also caused a time-dependent stimulation of diacylglycerol formation in adherent, but not suspended, cells and elicited a time-dependent stimulation of phosphatidylethanol formation, with a concomitant decrease in the formation of PA only at early (< 7 min) times. These observations were consistent with a rapid activation of the enzyme phospholipase D in adherent cells stimulated with GM-CSF. Additional data indicated that the source of DAG was PLD coexisting with PLC, especially at later times ( > 7 min) of stimulation with GM-CSF. Finally, the formation of PA and PEt, and to a minor extent, DAG, were inhibited by the protein tyrosine kinase inhibitor erbstatin in conditions in which tyrosine phosphorylation occurred. Taken together the data indicate that GM-CSF rapidly activates PLD in adherent cells, which is responsible for the generation of PA. Thus, PLD activation is an early event in neutrophil signal transduction following exposure of adherent cells to GM-CSF.

p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation.

Mitogen-activated protein (MAP) kinase-mediated signal-transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the roles of MAP kinases in neutrophils are not well understood yet. Protein phosphorylation analysis of cellular MAP kinases indicates that exposure of human neutrophils to chemotactic factor FMLP as well as granulocyte-macrophage CSF, PMA, or ionomycin rapidly induced the activation of p38 and p44/42 MAP kinases, but stimulation with inflammatory cytokine TNF-alpha triggered the activation of p38 MAP kinase only. To study the cellular functions of these MAP kinases, the inhibitor SB20358, which specifically inhibited enzymatic activity of cellular p38 MAP kinase, and the inhibitor PD98059, which specifically blocked the induced protein phosphorylation and activation of p44/42 MAP kinase in intact neutrophils, were utilized. Inhibition of the cellular p38 MAP kinase activation almost completely abolished the TNF-alpha-stimulated IL-8 production and superoxide generation of human neutrophils. In addition, the FMLP-induced neutrophil chemotaxis as well as superoxide generation were suppressed markedly by inhibiting the activation of cellular p38 MAP kinase, but not p44/42 MAP kinase. Moreover, RIA indicates that the activation of cellular p38 MAP kinase was required for the neutrophil IL-8 production stimulated by granulocyte-macrophage CSF or LPS as well as TNF-alpha, but not for that induced by PMA or ionomycin. These results demonstrate that the activation of cellular p38 MAP kinase is indispensable for the TNF-alpha- or FMLP-mediated cellular functions in human neutrophils, and suggest that p38 MAP kinase may play a different role in response to distinct stimulation.

Tyrosine phosphorylation and activation of a new mitogen-activated protein (MAP)-kinase cascade in human neutrophils stimulated with various agonists.

The presence of a novel 38 kDa protein that is tyrosine phosphorylated in human neutrophils, a terminally differentiated cell, upon stimulation of these cells with low concentrations of lipopolysaccharide (LPS) in combination with serum has been demonstrated. This 38 kDa protein was identified as the mammalian homologue of HOG1 in yeast, the p38 mitogen-activated protein (MAP) kinase. This conclusion is based on the experimental findings that anti-phosphotyrosine (anti-PY) antibody immunoprecipitates a 38 kDa protein that is recognized by anti-p38 MAP kinase antibody, and conversely, anti-p38 MAP kinase antibody immunoprecipitates a 38 kDa protein that can be recognized by anti-PY antibody. Moreover, this tyrosine phosphorylated protein is found associated entirely with the cytosol. It was also found that this p38 MAP kinase is activated following stimulation of these cells with low concentrations of LPS in combination with serum. This conclusion is based on three experimental findings. First, soluble fractions isolated from LPS-stimulated cells phosphorylate heat shock protein 27 (hsp27) in an in vitro assay, and this effect is not inhibited by protein kinase C and protein kinase A inhibitor peptides. This effect is similar to the effect produced by the commercially available phosphorylated and activated MAPKAP kinase-2 (MAP kinase activated protein kinase-2). Secondly, a 27 kDa protein that aligns with a protein recognized by anti-hsp27 antibody is phosphorylated upon LPS stimulation of intact human neutrophils prelabelled with radioactive phosphate. Lastly, immune complex protein kinase assays, using [gamma-32P]ATP and activating transcription factor 2 (ATF2) as substrates, showed increased p38 MAP kinase activity from LPS-stimulated human neutrophils. The phosphorylation and activation of this p38 MAP kinase can be affected by both G-protein-coupled receptors such as platelet-activating factor (PAF) and non-G-protein-coupled receptors such as the cytokine-coupled receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor alpha (TNF-alpha). The effect of low concentrations of PAF is greatly increased in cells pretreated with LPS. The tyrosine phosphorylation of the p38 MAP kinase is not restricted to stimuli that mediate their actions through membrane-associated receptors, but it can be affected by agents that bypass membrane-associated receptors such as the protein translation blocker anisomycin. While anisomycin is known to increase the tyrosine phosphorylation of the 54 kDa SAPK (stress-activated protein kinase), this is the first report that shows that anisomycin also tyrosine phosphorylates the p38 MAP kinase. Cytokine receptors that increase the tyrosine phosphorylation and activation of the erk1 and erk2 MAP kinases have less effect on this p38 MAP kinase than those that do not affect the erk1 and erk2 MAP kinases. The possible role of the p38 MAP kinase in the phosphorylation of cytosolic phospholipase A2 is discussed.

Differential prediction of young adult arrests for property and personal crimes: findings of a cohort follow-up study of violent boys from North Carolina's Willie M Program.

Which characteristics of violent youth predict adult arrest for property crimes (e.g. burglary), and for personal crimes (e.g. assault)? We addressed this question by focusing on a group of particularly violent and assaultive boys. Separate logistic regression analyses were conducted to predict property and personal crime arrests as a function of adolescent psychiatric diagnosis, behavior problem history, race and IQ. Property crime arrest was predicted by an adolescent history of property offenses, conduct disorder diagnosis, and race (more arrests among non-whites than whites). Personal crime arrest was predicted by adolescent history of property offenses and adolescent history of substance abuse.

Arrests among emotionally disturbed violent and assaultive individuals following minimal versus lengthy intervention through North Carolina's Willie M Program.

Time to first arrest after termination of Willie M Program services was compared in 2 groups of former clients. All Ss had met program criteria and "aged out" after their 18th birthday, but the two groups differed in duration and extent of intervention received: (a) A short-certification group (n = 21), because they turned 18 near the 1981 program start date, had received Willie M services for a mean of only 26 days (all cases less than 3 months); (b) a long-certification group (n = 147) averaged 896 days in the program (all cases greater than 1 year). The groups did not differ significantly in gender or race; geographic region; IQ; diagnosis according to the Diagnostic and Statistical Manual of Mental Disorders, 3rd ed. (DSM-III, American Psychiatric Association, 1980); or age at earliest antisocial acts. A survival analysis compared the short and long groups on proportion avoiding arrest as a function of time since aging out. The long group showed slightly better arrest survival, but survival curves for the 2 groups did not differ reliably. Thus the program was not found to significantly reduce the risk of young adult arrests.

Impact of involuntary outpatient commitment on the revolving-door syndrome in North Carolina.

Involuntary outpatient commitment has been used as a means of treating so-called revolving-door patients in the community and allowing them to experience the positive aspects of social and community life that stability can bring. To study the impact of North Carolina's revised outpatient commitment law on institutionalization rates, the authors measured state hospital admissions and days of hospitalization within a three-year period for all patients committed to outpatient treatment during that period. Statistically significant decreases occurred in both measures; the biggest percent reduction occurred in admission rates.

Thyroid function in hemidecorticate rats.

1. Thyroid function was evaluated in hemidecorticate (HD) and control (C) rats by determining serum T3 and T4 levels and the development of incisors and mandibles and through analysis of various histological features of the thyroid such as follicle size, colloid droplet content and [3H]-glycine uptake by follicular cells. 2. HD animals presented normal levels of circulating T3 but significantly lower T4 levels. 3. There was slight atrophy of the gland in HD animals and fewer colloid droplets were present in the cytoplasm of the follicular cells in this group, indicating a reduction in the breakdown of thyroglobulin. [3H]-glycine uptake by HD indicated that the rate of thyroglobulin biosynthesis was not altered in the experimental animals. 4. The growth of mandibles (weight) and incisors (weight and length) was reduced in HD compared to the control animals. 5. These results suggest that hemidecortication causes mild hypothyroidism (trophoprivic type) probably by affecting hypothalamic function.