Neutrophil priming mechanisms of sulfolipid-I and n-formyl-methionyl-leucyl-phenylalanine

The principal sulfatide of virulentMycobacterium tuberculosis, sulfolipid-I (SL-I), both directly stimulates neutrophil superoxide (O ₂ ^– ) release and, at substimulatory concentrations, primes these cells for markedly enhanced oxidative responsiveness to other stimuli. The present study was undertaken to clarify the priming mechanisms by comparing cellular events following priming doses of SL-I with those following priming with N-formyl-methionyl-leucyl-phenylalanine (FMLP). We compared the involvement of the calcium cation (Ca²⁺), as well as membrane protein kinase C (PKC) activity and the translocation of NADPH oxidase-cytosolic cofactor effected by priming levels of the two agonists. The investigation led to two important conclusions. First, we clearly demonstrate that priming by both SL-I and FMLP results from activation of cellular processes that are not involved in direct oxidative activation. For example, whereas direct induction of O ₂ ^– generation by FMLP and SL-I required increases in intracellular Ca²⁺, an increase in intracellular calcium concentration ([Ca²⁺]_i) above basal levels was not required for priming. Second, we identified key differences in the cellular responses to priming doses of SL-I and FMLP. Whereas increased membrane PKC activity caused by priming doses of FMLP was only partially blocked by chelation of intracellular Ca²⁺, Ca²⁺ chelation completely inhibited the increase in membrane PKC activity caused by SL-I. NADPH oxidase-cytosolic factor translocation to plasma membranes was completely blocked by pertussis toxin when priming doses of SL-I were used. This guanine-nucleotide-binding protein inhibitor had no effect on FMLP-dependent translocation of the oxidase cofactors. The comparative approach introduced in this report provides a valuable and novel method to discern the complex interactions of various cellular processes that regulate the state of activation of stimulated cells.     

Kin recognition and cannibalism in polyphenic salamanders

We investigated kin discrimination among larvae of Arizona tigersalamanders (Ambystoma tigrinum nebulosum) which occur as "typical"morphs that feed mostly on invertebrate prey and occasionallyon conspecifics, and as "cannibal" morphs that feed primarilyon conspecifics. When housed with smaller larvae that differedin relatedness, both cannibals and typicals preferentially consumedless-related individuals. Cannibals ate typicals much quickerwhen the choice was between nonkin and siblings than when thechoice was between nonkin and cousins, indicating that cannibalscould distinguish different categories of relatives. Cannibalswere less likely to eat a larval sibling that was a cannibalmorph than a sibling that was a typical morph. Occluding animals'nares temporarily eliminated kin discrimination, implying thatolfaction is important in recognition. Larvae from differentsibships varied considerably in their ability to discriminatekin, and the greater the probability that a larva from a givensibship would develop into a cannibal morph, the more likelythe members of that sibship were to discriminate kin. Our resultsenable us to infer the functional significance of kin recognitionin this species and to develop an evolutionary model of themechanisms underlying the joint control of kin recognition andcannibalistic polyphenism.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Simple electromyographic biofeedback treatment for chronic pediatric constipation/encopresis: Preliminary report

Pediatric constipation/encopresis is thought to be due, in part, to paradoxical constriction of the external anal sphincter (EAS) muscle during attempted defecation. This inappropriate contraction can lead to delayed, impacted, painful, and infrequent bowel movements. Standard Medical Care (SMC) involves disimpaction with enemas, followed by laxative therapy and diet modification, to maintain frequent soft stools. Using the case control method, the efficacy of SMC alone was compared with SMC plus EAS electromyographic biofeedback aimed at eliminating paradoxical contraction. Thirteen consecutive chronically constipated children received SMC plus biofeedback, and were compared with 13 age- and sex-matched children who received only SMC. Biofeedback subjects demonstrated post-treatment elimination of EAS paradoxical constriction. At 16 months follow-up parents of biofeedback children reported significantly greater improvement in constipation, encopresis, laxative use, and painful bowel movements compared to SMC.This research report was supported by the NIH under grant No. RO1 HD 28160.     

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diiso-propylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.     

Mechanism and regulation of neutrophil priming by platelet-activating factor

Although a weak direct stimulus of superoxide anion (O₂^?) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley-Liss, Inc.     

K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [³H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [³H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.