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Biomedical subjects

E L Becker

Publications and source records attributed to E L Becker.

At least 19 recordsLinked to original sources
PubMed1992

Granulocyte-macrophage colony-stimulating factor-induced protein tyrosine phosphorylation of microtubule-associated protein kinase in human neutrophils.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionylleucylphenylalanine, tumor necrosis factor alpha, platelet-activating factor, phorbol ester (phorbol 12-myristate 13-acetate), and calcium ionophore A23187 are able to increase the level of tyrosine phosphorylation of different protein substrates, as demonstrated by Western blotting with anti-phosphotyrosine antibody (anti-PY). A protein of 41 kDa (p41) consistently showed more intense reactivity to anti-PY than controls. Blots treated with anti-PY, stripped of the antibody, and reblotted with microtubule-associated protein kinase (MAPK, p42MAPK) antibody show only one band. The molecular mass of that band exactly matches that of p41. MAPK-reactive protein is present in control and stimulated cells, although the intensity of the band is greater in the latter. GM-CSF-stimulated phosphorylation of p41 is time- and dose-dependent. Anti-MAPK antibody detects a single band of 41 kDa, whose intensity increases with time of incubation and concentration of the agonist. Thus, the anti-MAPK antibody appears to react better to the phosphorylated form of p41 from GM-CSF-stimulated cells than to the dephosphorylated form. The p41 and MAPK proteins are localized in the cytosol. Finally, MAPK immunoprecipitates were probed with anti-PY in Western blots and a band of 41 kDa was found. In summary, these results suggest that this 41-kDa protein in neutrophils that is tyrosine phosphorylated in response to GM-CSF and other stimuli is MAPK. Its phosphorylation may represent an early and crucial signal associated with the GM-CSF neutrophil stimulation cascade.

Amino Acid Sequence↗
PubMed1992

Lipopolysaccharide and serum cause the translocation of G-protein to the membrane and prime neutrophils via CD14.

Lipopolysaccharide (LPS) in combination with human serum, in the absence of a second stimulus, causes an increase in the amount of the alpha -subunit (Gi alpha 2) of the guanine nucleotide binding protein Gi2 associated with the membrane. The LPS-serum complex also primes human neutrophils for O2- production in response to stimulation by the chemotactic factor fMet-Leu-Phe. Added serum factor is essential for priming at low concentrations of LPS. In the presence of serum, significant potentiation can be observed at LPS concentration as low as 0.1 ng/ml. The priming is dose and time dependent. Furthermore, the observed actions of the LPS-serum complex are not reversible since they cannot be overcome by washing. Monoclonal antibody against CD14 inhibits both the direct and priming actions of the LPS-serum complex. On the other hand, neither the antibody against CD11b nor the antibody against TNF-alpha inhibits the action of this complex.

Antibodies, Monoclonal↗
PubMed1992

Phorbol ester inhibits granulocyte-macrophage colony-stimulating factor binding and tyrosine phosphorylation.

Exposure of human polymorphonuclear neutrophils to phorbol 12-myristate 13-acetate (PMA) results in a 70-75% reduction in the specific binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) to its receptors. The PMA-induced reduction in 125I-GM-CSF binding is due to a decrease in the number of available GM-CSF receptors, as derived from Scatchard analysis of the binding data. On the other hand, the phorbol ester 4-alpha-phorbol 12,13-didecanoate (4 alpha-PDD) fails to affect 125I-GM-CSF binding. PMA promotes phosphorylation on tyrosine residues of several proteins, as demonstrated by Western blotting analysis using antiphosphotyrosine antibodies. The molecular masses of those proteins are 41, 55, 66, 78, 85, 104, and 115 kDa. GM-CSF increases the levels of the tyrosine phosphorylation of several proteins, the majority of which have similar Mr to those found in PMA-stimulated neutrophils. This increase, on all but the 41-kDa protein, is partially prevented by treatment of the cells with PMA. The inhibition by PMA of GM-CSF binding to its receptors and its phosphorylated effects is partially prevented by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and, to a greater extent, by staurosporine. It is suggested that PMA, through the activation of protein kinase C, interrupts the excitation-response sequence initiated by GM-CSF, which includes tyrosine phosphorylation, and that the earliest altered step is the binding of GM-CSF to its receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

The significance of functional receptor heterogeneity in the biological responses of the rabbit neutrophil to stimulation by chemotactic formyl peptides.

The characteristics of binding to the chemotactic receptors on rabbit peritoneal neutrophils were examined for seven formyl peptide analogues. These receptor-binding characteristics were compared with the abilities of the analogues to induce the biological responses of degranulation and chemotaxis. Five of the analogues showed distinct functional heterogeneity in their receptor-binding patterns, whereas the two most potent compounds displayed homogeneous binding patterns. The relative potencies of the formyl peptide analogues for stimulation of degranulation correlated well with their relative potencies for high-affinity, but not low-affinity, binding. The biphasic patterns for stimulation of chemotactic migration were similar for the less potent analogues, and their potencies paralleled those for both degranulation and receptor binding. In contrast, the most potent analogues induced a greater maximal extent of chemotactic migration than the other compounds, but displayed a lower than expected potency (i.e. they required higher than expected concentrations). These anomalies in the patterns of the chemotactic response cannot be reconciled with a simple receptor model comprising two independent classes of receptors. Instead, a model comprising interconvertible states of different affinities is proposed. The state of higher affinity appears to play a central role in initiation of both degranulation and chemotaxis. The more potent formyl peptide analogues are thought to stabilize an activated, higher-affinity, state of the receptor; this can explain their greater efficacy in stimulating chemotaxis. The proposed model may also be applicable to other receptors that are coupled by a guanine-nucleotide-binding regulatory protein to their associated effector.

Amino Acid Sequence↗
PubMed1991

Modulation of leukotriene B4 and platelet-activating factor binding to neutrophils.

Preincubation of human neutrophils with the human hormone granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibits the specific binding of leukotriene B4 ([3H]LTB4) but not the nonmetabolizable bioactive platelet-activating factor ([3H]C-PAF) to intact cells. This inhibition requires that the GM-CSF interacts with intact cells. The action of GM-CSF is not prevented by pertussis toxin. Moreover, the rise in calcium produced by LTB4 but not by PAF is also inhibited in human neutrophils pretreated with GM-CSF. Interestingly, neither the inhibitory action of GM-CSF on [3H]LTB4 binding or LTB4-induced calcium rise nor the potentiation of superoxide production by GM-CSF is reduced by inhibitors of arachidonic acid metabolism by the lipoxygenase pathway. In contrast, preincubation of human neutrophils with either the chemotactic factor formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) or the active phorbol ester, phorbol 12-myristate 13-acetate (PMA), inhibits the binding of both [3H]LTB4 and [3H]C-PAF to intact cells. The inhibitory actions of GM-CSF, PMA, and fMet-Leu-Phe require that they interact with the intact cells; their actions cannot be reproduced in plasma membrane preparations. The effects of both GM-CSF and fMet-Leu-Phe cannot be prevented by the protein kinase C inhibitor staurosporine. The mechanisms of fMet-Leu-Phe and GM-CSF actions are probably not mediated through the release of LTB4 by the cells. Interestingly, this new action, unlike other reported effects of GM-CSF, is not mediated through a pertussis toxin-sensitive G protein (Gi alpha 2). This indicates that not all GM-CSF receptors are coupled to Gi alpha 2.

Alkaloids↗
PubMed1990

The short and happy life of neutrophil activation.

This is largely a history of attempts from approximately 1960 until about 1980-81 to understand the mechanisms by which neutrophils are activated by chemotactic factors to induce chemotaxis and granule secretion. As such, it deals with the growth of our knowledge of neutrophil chemotactic factors and their receptors; the importance and role of cation fluxes, especially Ca2+, microfilaments and microtubules, membrane potential, cyclic nucleotides, and the start of our recognition of the importance of arachidonic acid and phospholipid metabolism and protein phosphorylation. In a very real sense this is a history of the origins of our present realization that reactions and functions which had been considered specific to the neutrophil are to be thought of as similar or identical to general biological and physiological processes such as muscle contraction, cellular secretion, etc.

Animals↗
PubMed1990

Comparison of stimulation by chemotactic formyl peptide analogs between GTPase activity in neutrophil plasma membranes and granule enzyme release from intact neutrophils.

Stimulation by fMet-Leu-Phe analogs of GTPase activity in plasma membranes from rabbit neutrophils was compared with the stimulation of degranulation in intact neutrophils. All four formyl peptides examined (fMet-Leu-Phe-Phe, fMet-Leu-Phe, fNle-Leu-Phe, and fVal-Leu-Phe) were full agonists for both responses. Their ED50 values for the two responses correlated well, although those for GTPase stimulation were uniformly about tenfold greater. The specific antagonist tBoc-Phe-Leu-Phe-Leu-Phe competitively inhibited both GTPase activity and degranulation stimulated by fMet-Leu-Phe; its Ki values were similar for the two responses. Pertussis toxin treatment, in contrast, inhibited the maximal stimulation of both responses by fMet-Leu-Phe with minimal shift in ED50. The inhibitory actions of tBoc-Phe-Leu-Phe-Leu-Phe and pertussis toxin on GTPase activity thus paralleled the effects on degranulation. These observations substantiate the hypothesis that a guanine nucleotide-binding protein that is a pertussis toxin substrate couples the formyl peptide receptors to physiological function in the neutrophil.

Animals↗
PubMed1990

Purification and characterization of soluble inositol 1,4,5-trisphosphate 5-phosphomonoesterase from rabbit peritoneal neutrophils.

A specific soluble inositol phosphate 5-phosphomonoesterase has been purified approximately 2,700-fold from a 120,000g supernatant of rabbit neutrophil homogenate. The specific enzyme represented 25-50% of the total hydrolytic activity toward inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3) with the remaining activity hydrolyzing both Ins-1,4,5-P3 as well as inositol 1,4-bisphosphate (Ins-1,4-P2). However, the enzyme could not be identified on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue, indicating that it represents a minor protein in the purified enzyme preparations. The purified enzyme has an apparent molecular mass of 43,000-47,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphomonoesterases. The enzyme hydrolyzes Ins-1,4,5-P3 with an apparent Km of 18 microM and a Vmax of 1.2 mumol/min/mg. The 5-phosphomonoesterase requires Mg2+ for activity and is not affected by physiological concentrations of Ca2+ or calmodulin. The pH optimum for activity is 7.5. Inositol 1,3,4,5-tetrakisphosphate is a potent competitive inhibitor of Ins-1,4,5-P3 hydrolysis (Ki = microM), whereas Ins-1,4-P2 is a weak inhibitor (Ki = 173 microM). Ins-1,4,5-P3 hydrolysis is relatively unaffected by monophosphorylated substrates, however, bisphosphorylated substrates are potent inhibitors. Comparisons of neutrophil 5-phosphomonoesterase characteristics with those of platelet and rat brain enzymes support the idea that each 5-phosphomonoesterase may be a unique enzyme and play a different role dependent upon the cell or tissue in which it acts.

Animals↗
PubMed1990

Replacement of the N alpha-blocking group in the formyl-methionyl tripeptide chemoattractant: an insight into the mode of binding at the receptor on rabbit neutrophils.

The tripeptide N alpha-carbamoyl-L-methionyl-L-leucyl-L-phenylalanine methyl ester has been synthesized in solution by classical methods and fully characterized. This compound, prepared in order to obtain a deeper insight into the mode of binding at the formyl peptide chemotactic receptor, has been tested for its ability to induce granule enzyme secretion from rabbit peritoneal neutrophils and found to be a complete agonist. These results confirm the hypothesis that a proton on the N alpha-blocking group of the tripeptide forms a hydrogen bond with an acceptor in the binding site.

Amino Acid Sequence↗
PubMed1979

Nuclear magnetic resonance conformational studies on the chemotactic tripeptide formyl-L-methionyl-L-leucyl-L-phenylalanine. A small beta sheet.

Previous work by several groups has shown that the combination of spin--spin coupling constants and spectral density components (derived from spin--lattice relaxation and/or nuclear Overhauser measurements) may aid in the task of conformational determination of peptides in solution. Using the peptide formyl-L-methionyl-L-leucyl-L-phenylalanine, which is a potent specific chemotactic agent for leucocytes, we show the following: (a) that 3JNHCH coupling constants are consistent with a high degree of rigidity in the peptide backbone in solution, (b) that 3H isotopic substitution in combination with relaxation data taken at different Larmor frequencies enables spectral density, and thence conformational, information to be obtained, (c) that side-chain conformations for this molecule mirror, in some aspects, those found in the solid state for other peptides containing the same residues, and (d) that temperature dependence of amide chemical shifts does not have direct implication concerning the existence of intramolecular hydrogen bonds in peptides. We are able to propose a family of conformations which appear to interchange rapidly on the NMR time scale and are characterized by a distribution of side-chain rotamers. The basic backbone conformation is, or closely approximates, a small beta antiparallel pleated sheet and as such suggests a possible mode of receptor--chemotactic peptide interaction.

Amino Acid Sequence↗
PubMed1979

Chemotactic factor-induced release of membrane calcium in rabbit neutrophils.

The interaction of chemotactic factors (fMet-Leu-Phe and C5a) with rabbit neutrophils leads to rapid and specific release of membrane calcium, as evidenced by changes in the fluorescence of cell-associated chlorotetracycline. These two structurally different stimuli appear to interact with the same pool of membrane calcium.

Animals↗
PubMed1979

The chemotactic factors induced movement of calcium and sodium across rabbit neutrophil membranes: effect of densensitization to cytochalasin B.

The preincubation of rabbit neutrophils with the chemotactic factor F-Met-Leu-Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Ca and 22Na was found upon preincubation with F-Met-Leu-Phe. In the presence of extracellular Ca2+, this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F-Met-Leu-Phe. In the absence of added extracellular Ca2+ an initial and rapid (less than 1 minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of the neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils.

Animals↗
PubMed1979

Arachidonic acid aggregates neutrophils.

Arachidonic acid, but not several structurally similar fatty acids, stimulated neutrophils in suspension to aggregate; this effect was blocked by 5, 8, 11, 14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism. Analagous to platelets, arachidonate may be a precursor of active metabolites which mediate neutrophil responses.

Arachidonic Acids↗
PubMed1979

Role of arachidonic acid derivatives in neutrophil aggregation: a hypothesis.

Chemotactic substances stimulate neutrophils in suspension to aggregate. Arachidonic acid (but not several structurally related fatty acids) induces a similar neutrophil response. We now report that two blockers of arachidonic acid metabolism, indomethacin and 5,8,11,14-eicosatetraynoic acid, inhibit this arachidonic acid-mediated response. Moreover, both blockers also inhibit aggregation stimulated by a synthetic chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, and their potency in doing so parallels their potency in inhibiting the response to arachidonic acid. These results suggest that metabolites of arachidonic acid may stimulate certain neutrophil functions and be involved in cellular responses to at least some chemotactic substances.

5,8,11,14-Eicosatetraynoic Acid↗