Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N-formyl-methionyl-leucyl-phenylalanine (fMLF) -stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) -primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2-) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L-arginine, the physiological substrate for NO synthase (NOS), and NG-nitro-L-arginine methyl ester, an inhibitor of NOS, both caused a 40-50% inhibition of LPS-induced priming of O2- generation in PMNs in stirred suspension, but not in LPS-primed PMNs under static or adherent conditions. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, completely abrogated the LPS-induced priming of 02- generation in PMNs in suspension, while causing only a 40-50% inhibition in PMNs under static or adherent conditions. The Ca2+ ionophore, A23187, prevented the LPS-induced priming of 02-generation without affecting 02- generation in unprimed PMNs. LPS priming of PMNs induced about a twofold increase in fMLF-stimulated homotypic aggregation, exocytosis of secondary granules, and rises in [Ca2+]i. In related studies, we also provide definitive evidence for enzymatic formation of NO in human PMNs and demonstrate a significant decrease in NO levels in LPS-primed PMNs. Taken together, these findings indicate that NO modulates PMN inflammatory responses and plays a protective role in priming and activation processes of inflammatory PMNs.
In the present Article, a reversible transition behavior from Jaggregates to excimer of an indocarbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI) in Langmuir−Blodgett (LB) films was reported. Surface pressure−area (π−A) isotherms, UV−vis, and fluorescence spectroscopies as well as atomic force microscopy (AFM) were used for characterizations of the films. π−A isotherms suggest a balance of interactions between DiI and fatty acids in the mixed monolayer at DiI mole fraction X DiI = 0.4, resulting in a stable and ideally mixed monolayer. It has been observed that pure DiI formed excimer in LB films, whereas both J aggregates and excimer were formed in LB films when DiI was mixed with long chain fatty acids, viz., stearic acid or arachidic acid. In fatty acid matrix at X DiI = 0.4, only J aggregates were formed in the LB films. This has been confirmed using deconvolution of spectroscopic results as well as using excitation spectroscopy. The coherent size of the J aggregate was found to be a maximum for the mixed film at the mole fraction 0.4 of DiI in fatty acid matrix. The J-aggregate domain in the LB film contains approximately (20 ± 5) coherent sizes. However, J aggregates were totally absent when DiI was mixed with cationic surfactant, polymer, or nanoclay.
Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs, This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP § and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD § and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+-kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.Our earlier studies have shown that neither cAMP nor its butyrated derivatives, at concentrations up to 2 • 10 -~ M, affect early cleavage of sea urchin or clam eggs, at least through the late gastrulation and feeding stages (47). Incorporation experiments with both radioactive cAMP and dibutyryl cAMP demonstrated uptake of exogenously added nucleotides in sea urchin eggs. Identification and quantitation of the intracellular compounds showed that intracellular levels of cAMP or N emonobutyryl cAMP (the converted intracellular product of dibutyryl cAMP) reached at least 10 -8 M by first cleavage (at an external concentration of 10 -8 M), two orders of magnitude higher 440
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