Activation of the p21-activated protein kinases from neutrophils with an antibody that reacts with the N-terminal region of Pak 1

AbstractNeutrophils contain two renaturable p21-activated protein kinases (Paks) with molecular masses of ca. 69 and 63 kDa that undergo rapid activation upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. We now report that these kinases undergo a massive, ATP-dependent activation in lysates of unstimulated neutrophils during immunoprecipitation with an antibody generated to residues 2–21 of the N-terminal region of Pak1. This activation was specific as it was completely blocked by a peptide that corresponds to residues 2–21 of Pak1 and was not observed with an antibody generated to the C-terminal region of Pak 1. The properties of the Paks activated with the antibody were virtually identical to those observed for these kinases from stimulated neutrophils, or activated in vitro with Rac-GTPγS plus ATP. These data indicate that perturbation of the N-terminal region of Pak can trigger activation of this enzyme, and that both the 69 and 63 kDa kinases may represent forms of Pak 1 that differ in their content of phosphate.©1997 Federation of European Biochemical Societies

Antagonists of Calcium Fluxes and Calmodulin Block Activation of the p21-Activated Protein Kinases in Neutrophils

Neutrophils stimulated with fMLP or a variety of other chemoattractants that bind to serpentine receptors coupled to heterotrimeric G proteins exhibit rapid activation of two p21-activated protein kinases (Paks) with molecular masses of ~63 and 69 kDa (y- and a-Pak). Previous studies have shown that products of phosphatidylinositol 3-kinase and tyrosine kinases are required for the activation of Paks. We now report that a variety of structurally distinct compounds which interrupt different stages in calcium/calmodulin (CaM) signaling block activation of the 63- and 69-kDa Paks in fMLP-stimulated neutrophils. These antagonists included selective inhibitors of phospholipase C (1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole- 2,5-dione), the intracellular Ca^(2+) channel (8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), CaM (N-(6-aminohexyl)-5- chloro-1-naphthalenesulfonamide; N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), and CaM-activated protein kinases (N-[2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide). This inhibition was dose-dependent with IC50 values very similar to those that interrupt CaM-dependent reactions in vitro. In contrast, less active analogues of these compounds (1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; N-(6-aminohexyl)-1-naphthalenesulfonamide; N-(4-aminobutyl)-1-naphthalenesulfonamide; promethazine; 2-[N-(4- methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzyl-amine]) did not affect activation of Paks in these cells. CaM antagonists (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide; trifluoperazine), but not their less-active analogues (N- (6-aminohexyl)-1-naphthalenesulfonamide; promethazine), were also found to block activation of the small GTPases Ras and Rac in stimulated neutrophils along with the extracellular signal-regulated kinases. These data strongly suggest that the Ca^(2+)/CaM complex plays a major role in the activation of a number of enzyme systems in neutrophils that are regulated by small GTPases

Structural and biosynthetic features of the Mo5 human myeloid differentiation antigen

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65573/1/j.1399-0039.1993.tb02007.x.pd

Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions

To clarify the biochemical behavior of 2′-deoxyribonucleoside 5′-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm values of modified DNA–DNA duplexes incorporating 2′-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo−) and Vent (exo−) suggested that Co and Ao selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo−) toward Ao on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator

Reactive oxygen species in phagocytic leukocytes

Phagocytic leukocytes consume oxygen and generate reactive oxygen species in response to appropriate stimuli. The phagocyte NADPH oxidase, a multiprotein complex, existing in the dissociated state in resting cells becomes assembled into the functional oxidase complex upon stimulation and then generates superoxide anions. Biochemical aspects of the NADPH oxidase are briefly discussed in this review; however, the major focus relates to the contributions of various modes of microscopy to our understanding of the NADPH oxidase and the cell biology of phagocytic leukocytes

Lysophosphatides enhance superoxide responses of stimulated human neutrophils

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidytcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma- O -hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly- L -histidine, phytohemagglutinin, and N -formyl methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O 2 − ). None of the lyso compounds by themselves caused generation of O 2 − . The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O 2 − generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O 2 − . However, in the presence of cytochalasin B, both lysophosphatidyiethanolamine and lysophosphatidylserine also markedly enhanced O 2 − generation induced by immune complexes. O 2 − generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A 2 , phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O 2 − . The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O 2 − in stimulated PMNs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44499/1/10753_2004_Article_BF00924787.pd

Highly efficient and irreversible removal of cadmium through the formation of a solid solution

Sulfur-containing materials are very attractive for the efficient decontamination of some heavy metals. However, the effective and irreversible removal of Cd2+, coupled with a high uptake efficiency, remains a great challenge due to the relatively low bond dissociation energy of CdS. Herein, we propose a new strategy to overcome this challenge, by the incorporation of Cd2+ into a stable ZnxCd1-xS solid solution, rather than into CdS. This can be realised through the adsorption of Cd2+ by ZnS nanoparticles, which have exhibited a Cd2+ uptake capacity of approximate 400 mg g−1. Through this adsorption mechanism, the Cd2+ concentration in a contaminated solution could effectively be reduced from 50 ppb to 80% uptake capacity for Cd2+, compared with only 9% uptake capacity for similarly-aged FeS particles. This work reveals a new mechanism for Cd2+ removal with ZnS and establishes a valuable starting point for further studies into the formation of solid solutions for hazardous heavy metal removal applications

A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis

Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage in sepsis. Interleukin-8 (IL-8), a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. In this study a mechanistic computational model was used to evaluate and deploy an extracorporeal sepsis treatment which modulates CXCR-1/2 levels. First, a simplified mechanistic computational model of IL-8 mediated activation of CXCR-1/2 receptors was developed, containing 16 ODEs and 43 parameters. Receptor level dynamics and systemic parameters were coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils which reduce pathogen load, and/or primed neutrophils which cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a two-hour infusion of E coli and followed for a maximum of 28 days. Ensembles of parameters were generated using a Bayesian parallel tempering approach to produce model fits that could recreate experimental outcomes. Stepwise logistic regression identified seven model parameters as key determinants of mortality. Sensitivity analysis showed that parameters controlling the level of killer cell neutrophils affected the overall systemic damage of individuals. To evaluate rescue strategies and provide probabilistic predictions of their impact on mortality, time of onset, duration, and capture efficacy of an extracorporeal device that modulated neutrophil phenotype were explored. Our findings suggest that interventions aiming to modulate phenotypic composition are time sensitive. When introduced between 3–6 hours of infection for a 72 hour duration, the survivor population increased from 31% to 40–80%. Treatment efficacy quickly diminishes if not introduced within 15 hours of infection. Significant harm is possible with treatment durations ranging from 5–24 hours, which may reduce survival to 13%. In severe sepsis, an extracorporeal treatment which modulates CXCR-1/2 levels has therapeutic potential, but also potential for harm. Further development of the computational model will help guide optimal device development and determine which patient populations should be targeted by treatment