Does field hockey increase morphofunctional asymmetry? A pilot study.

Common practice in field hockey requires athletes to adopt a semi-crouched posture, so players have a greater risk of musculoskeletal disorders than non-athletes. The aim of the present study was to assess how field hockey determines asymmetry in morphological and functional characteristics of the body by comparing athletes to control participants. The sample consisted of 15 male field hockey players from the Polish Youth National Team and 14 male university students. Antimeric differences in the chosen variables between body sub-regions were assessed. All morphological characteristics (bone mineral density, fat mass, and lean mass) were estimated using a dual energy X-ray absorptiometry. Additionally, the range of motion in transverse and frontal planes of the cervical, thoracic and lumbar spine was measured by using an electrogoniometric system. The results showed that the values of all morphological characteristics were higher in the left body segments, both in athletes and controls. However, the differences between sides were much more pronounced in the field hockey players. With regard to functional traits, higher values were obtained for the right body side in athletes but for the left side of the body among the controls. The difference between right and left side bending increased from the cervical spine (2.7%) through thoracic spine (7.8%) to lumbar spine (16.5%) in athletes. Rotational asymmetry in the thoracic spine was the largest in both groups. These findings indicate that it is important to monitor all athletes to prevent injury and health problems connected with strong morphological asymmetry.

Management of pregnancy-related emergencies: what do Polish anesthesiologists know?

Antecedentes. Las emergencias pueden ocurrir en cualquier momento del embarazo. Además de obstetras y matronas, los anestesiólogos también deberían familiarizarse con las emergencias relacionadas con el embarazo. El objetivo del presente estudio es valorar los conocimientos básicos y avanzados de los anestesistas en lo que a la gestión de las emergencias relacionadas con el embarazo se refiere. Métodos. Durante dos congresos se distribuyó un cuestionario a anestesiólogos (S1, n=87, S2, n=35) y a otros grupos entre los que se encontraban médicos residentes en proceso de especialización (DS, n=28) y médicos que no habían comenzado la residencia (PD, n=130). En la encuesta participaron un total de 280 doctores. En la primera parte del cuestionario se recopilaban datos demográficos y en la segunda se evaluaban sus conocimientos básicos y avanzados conforme a la clasificación de los grupos. Resultados. Los conocimientos básicos acerca de la gestión de emergencias relacionadas con el embarazo del grupo analizado fueron más pobres en comparación con los conocimientos avanzados. El grupo DS presentó mejores habilidades de gestión que los anestesiólogos y que el grupo PD. Se consiguieron unos resultados considerablemente peores en las preguntas sobre maniobras contra la asfixia en embarazadas y sobre el tiempo para la cesárea durante la reanimación cardiopulmonar. Los resultados de los especialistas y el grupo DS en las preguntas de nivel avanzado fueron mejores que los del grupo PD. Conclusiones. Los anestesiólogos más mayores no sabían cómo gestionar correctamente las emergencias relacionadas con el embarazo de tipo básico; no obstante, sí estaban familiarizados con la gestión de nivel avanzado. No se encontró relación entre el conocimiento y la aplicación de dichos conocimientos en situaciones complicadas. Se debe mejorar el proceso de aprendizaje de las emergencias obstétricas agudas realizando cursos nacionales obligatorios y comprobando los conocimientos cada pocos años(AU)

Background. Emergencies can occur at any time during pregnancy. In addition to obstetricians and midwives, anesthesiologists should also be familiar with pregnancy-related emergencies. The aim of this study was to assess the basic and advanced knowledge regarding the management of pregnancy-related emergencies of anesthesiologists. Methods. An anonymous questionnaire was distributed to anesthesiologists at two conferences (S1, n=87; S2, n=35), and to other groups comprising doctors during specialization (DS, n=28) and postgraduate doctors (PD, n=130). Ultimately, 280 doctors were included in the survey. The first part of the questionnaire collected demographics, and a second one evaluated both their basic and advanced knowledge by taxonomy. Results. Basic knowledge regarding the management of pregnancy-related emergencies of the tested group was poorer compared with advanced knowledge. The DS group had better basic management skills than anesthesiology specialists and the PD group. Significantly worse results of the tested group were obtained on the questions about maneuvers for choking pregnant women and time to cesarean section during cardiopulmonary resuscitation. The specialists and the DS group had results on advanced level questions better than the PD group. Conclusions. Older specialists in anesthesiology did not know how to properly manage pregnancy-related emergencies at the basic level; however, anesthesiologists were familiar with advanced management. No relationship between recalling and using such knowledge in difficult situations was observed. The teaching process of acute obstetric emergencies must be improved through implementation of compulsory nationwide courses and verification of knowledge every few years(AU)

Management of pregnancy-related emergencies: what do Polish anesthesiologists know?

BACKGROUND: Emergencies can occur at any time during pregnancy. In addition to obstetricians and midwives, anesthesiologists should also be familiar with pregnancy-related emergencies. The aim of this study was to assess the basic and advanced knowledge regarding the management of pregnancy-related emergencies of anesthesiologists. METHODS: An anonymous questionnaire was distributed to anesthesiologists at two conferences (S1, n = 87; S2, n = 35), and to other groups comprising doctors during specialization (DS, n = 28) and postgraduate doctors (PD, n = 130). Ultimately, 280 doctors were included in the survey. The first part of the questionnaire collected demographics, and a second one evaluated both their basic and advanced knowledge by taxonomy. RESULTS: Basic knowledge regarding the management of pregnancy-related emergencies of the tested group was poorer compared with advanced knowledge. The DS group had better basic management skills than anesthesiology specialists and the PD group. Significantly worse results of the tested group were obtained on the questions about maneuvers for choking pregnant women and time to cesarean section during cardiopulmonary resuscitation. The specialists and the DS group had results on advanced level questions better than the PD group. CONCLUSIONS: Older specialists in anesthesiology did not know how to properly manage pregnancy-related emergencies at the basic level; however, anesthesiologists were familiar with advanced management. No relationship between recalling and using such knowledge in difficult situations was observed. The teaching process of acute obstetric emergencies must be improved through implementation of compulsory nationwide courses and verification of knowledge every few years.

Absence of minor groove monovalent cations in the crosslinked dodecamer C-G-C-G-A-A-T-T-C-G-C-G.

The structure of a crosslinked B -DNA dodecamer of sequence C-G-C-G-A-A-T-T-C-G-C-G has been solved to a resolution of 1.43 A. The dithiobis-propane crosslink, -CH2-CH2-CH2-S-S-CH2-CH2-CH2-, bridges N7 atoms of adenine bases 6 and 18 in the two central base-pairs within the major groove. The crosslink is sufficiently long that no bending is induced in the helix, which is essentially isostructural with the native unlinked dodecamer at 1.9 A. A constellation of solvent peaks tentatively fitted as a spermine molecule in that earlier analysis is now seen at higher resolution to be a well-defined octahedral magnesium hexahydrate complex in the major groove. One end of the duplex curves around that complex to produce a roll-bend near base-pairs 3-5, and an overall bend in helix axis, as has long been noted. Two other magnesium complexes connect the helices and help to knit the crystal lattice together. No evidence exists for partial sodium or potassium ion substitution for solvent water molecules within the minor groove spine of hydration, as had been suggested previously: not coordination geometry and environment, nor B values, nor calculated valence values, nor difference map analyses. Indeed, the very numbers that have been claimed in support of partial substitution by sodium or potassium ions are reproduced with the present crystals, which by chemical analysis contains only one trace sodium ion per 160 bp, and one potassium ion per 41 bp. In contrast, our crystals contain one Mg2+ per base-pair, meaning that phosphate group charge neutrality is accomplished by divalent cations, not monovalent ions. Three of these magnesium cations per duplex are localized and visible in the X-ray analysis, and nine are disordered and invisible. Hence although binding of monovalent cations within the minor groove of A -tracts on occasion may be a consequence of groove narrowing, it cannot be the cause of that narrowing. Cations, contrary to what has been claimed, are not in charge.

NarL dimerization? Suggestive evidence from a new crystal form.

The structure of the Escherichia coli response regulator NarL has been solved in a new, monoclinic space group, and compared with the earlier orthorhombic crystal structure. Because the monoclinic crystal has two independent NarL molecules per asymmetric unit, we now have three completely independent snapshots of the NarL molecule: two from the monoclinic form and one from the orthorhombic. Comparison of these three structures shows the following: (a) The pairing of N and C domains of the NarL molecule proposed from the earlier analysis is in fact correct, although the polypeptide chain connecting domains was, and remains, disordered and not completely visible. The new structure exhibits identical relative orientation of N and C domains, and supplies some of the missing residues, leaving a gap of only seven amino acids. (b) Examination of corresponding features in the three independent NarL molecules shows that deformations in structure produced by crystal packing are negligible. (c) The "telephone receiver" model of NarL activation is confirmed. The N domain of NarL blocks the binding of DNA to the C domain that would be expected from the helix-turn-helix structure of the C domain. Hence, binding can only occur after significant displacement of N and C domains. (d) NarL monomers have a strong tendency toward dimerization involving contacts between helixes alpha 1 in the two monomers, and this may have mechanistic significance in DNA binding. Analogous involvement of helix alpha 1 in intermolecular contacts is also found in UhpA and in the CheY/CheZ complex.

Structure of the Escherichia coli response regulator NarL.

The crystal structure analysis of the NarL protein provides a first look at interactions between receiver and effector domains of a full-length bacterial response regulator. The N-terminal receiver domain, with 131 amino acids, is folded into a 5-strand beta sheet flanked by 5 alpha helices, as seen in CheY and in the N-terminal domain of NTRC. The C-terminal DNA-binding domain, with 62 amino acids, is a compact bundle of 4 alpha helices, of which the middle 2 form a helix-turn-helix motif closely related to that of Drosophila paired protein and other H-T-H DNA-binding proteins. The 2 domains are connected by an alpha helix of 10 amino acids and a 13-residue flexible tether that is not visible and presumably disordered in the X-ray structure. In this unphosphorylated form of NarL, the C-terminal domain is turned against the receiver domain in a manner that would preclude DNA binding. Activation of NarL via phosphorylation of Asp59 must involve transfer of information to the interdomain interface and either rotation or displacement of the DNA-binding C-terminal domain. Docking of a B-DNA duplex against the isolated C-terminal domain in the manner observed in paired protein and other H-T-H proteins suggests a stereochemical basis for DNA sequence preference: T-R-C-C-Y (high affinity) or T-R-C-T-N (low affinity), which is close to the experimentally observed consensus sequence: T-A-C-Y-N. The NarL structure is a model for other members of the FixJ or LuxR family of bacterial transcriptional activators, and possibly to the more distant OmpR and NtrC families as well.

The crystal structure of C-C-A-T-T-A-A-T-G-G. Implications for bending of B-DNA at T-A steps.

The single-crystal X-ray analysis of trigonal C-C-A-T-T-A-A-T-G-G, and its comparison with orthorhombic C-G-A-T-T-A-A-T-C-G, have shown that the A-T-T-A-A-T sequence has limited polymorphism under the influence of packing forces from neighboring molecules in the crystal. The T-A step is intrinsically variable. It is not inconsistent with a large propeller twist, a narrow minor groove, and a single spine of hydration, as has sometimes been claimed on theoretical grounds. The T-A step does show a persistent positive roll, in a direction that compresses the major groove, and this may be a significant factor in macroscopic DNA curvature induced by phased A-tracts. A-tracts, as understood in this paper, include A-A and A-T steps, but not the T-A step, which is disruptive. Three conclusions regarding A-tract-induced curvature can be drawn from this and other X-ray crystal structure analyses, and from key gel retardation experiments: (1) The A-tract bending model is disqualified on two grounds: (i) tilt-wedge bending within A-tracts is incompatible with the observed direction of curvature; (ii) roll-wedge bending within A-tracts is contradicted by every crystal structure analysis, and is inconsistent with gel retardation results for (G-C-A-A-A-A-T-T-T-T)n and for (A-A-A-A-A-T-T-T-T-T)n. (2) The junction bend model is contradicted by crystallography because: (i) the inclination of base-pairs does not change between A-tract and non-A-tract regions of helix; and (ii) the observed bends at GC/AT junctions are roll-wedge bends, not tilt-wedge as the junction bend model demands. (3) The non-A-tract bending model is consistent with both gel retardation data and with X-ray crystallography, and must be regarded as the only consistent model for A-tract bending.

Formation of inositol (1,4,5) trisphosphate and increase of cytosolic Ca2+ mediated by Fc receptors in human neutrophils.

The correlation between the increase of [Ca2+]i and the activation of hydrolysis of phosphoinositide-4,5-bisphosphate and formation of inositol(1,4,5)trisphosphate in neutrophils treated with Fc receptor-binding agonists is still under discussion. In this communication evidence is presented supporting the conclusion that, as it is widely accepted for the activation of other receptors, also upon the activation of Fc receptors the stimulation of the production of inositol(1,4,5) trisphosphate is involved in the increase in [Ca2+]i. In fact: i) treatment of neutrophils with immune complexes induced a very rapid phosphoinositide hydrolysis measured as [3H]inositol phosphates production from [3H]phosphoinositides and as inositol(1,4,5) trisphosphate formation measured with radioreceptor assay, ii) immune complexes caused a dose-dependent increase of [Ca2+]i; iii) the increase of [Ca2+]i correlated with the production of inositol(1,4,5) trisphosphate with respect to time course, dose dependence and pertussis toxin insensitivity.

Crystallographic analysis of C-C-A-A-G-C-T-T-G-G and its implications for bending in B-DNA.

Stacked B-DNA double helices of sequence C-C-A-A-G-C-T-T-G-G exhibit the same 23 degrees bend at -T-G-G C-C-A- across the nonbonded junction between helices that is observed in the middle of the decamer helix of sequence C-A-T-G-G-C-C-A-T-G, even though the space group (hexagonal vs orthorhombic), crystal packing, and connectedness at the center of the bent segment are quite different. An identical bend occurs across the interhelix junction of every monoclinic crystal structure of sequence C-C-A-x-x-x-x-T-G-G, suggesting that T-G-G-C-C-A constitutes a natural bending element in B-DNA. The bend occurs by rolling stacked base pairs about their long axes; there is no "tilt" component. Of the three possible models for A-tract bending--bent-A-tract, junction bends, or bent-non-A--which cannot be distinguished by solution measurements, all crystallographic evidence over the past 10 years unanimously supports the non-A regions as the actual bending loci.

The potentiation by TNF-alpha and PMA of Fc receptor-mediated phagocytosis in neutrophils is independent of reactive oxygen metabolites produced by NADPH oxidase and of protein kinase C.

The results presented in this paper demonstrate that the potentiation of phagocytosis of erythrocyte (E) IgG by TNF-alpha or PMA is not due to an oxygen-dependent mechanism. In fact, the potentiation of phagocytosis occurs normally in human neutrophils 1) when the respiratory burst is inhibited by diphenyleneiodonium, 2) in conditions where the reactive oxygen metabolites produced by the activation of NADPH oxidase, that accompanies the phagocytosis, were removed by catalase or superoxide dismutase, 3) of a patient lacking NADPH oxidase activity due to a genetic defect of p67-phox, 4) treated with staurosporine which allowed PMA to potentiate the ingestion of E-IgG at concentrations which inhibited the activation of the respiratory burst. Evidence is also presented that staurosporine not only did not inhibit, but amplified the potentiation of phagocytosis by PMA and TNF-alpha. This last finding suggests that the activation of protein kinase C plays a modulatory rather than a positive role in the mechanism of potentiation of phagocytosis.

Transmembrane signaling pathways involved in phagocytosis and associated activation of NADPH oxidase mediated by Fc gamma Rs in human neutrophils.

We have previously shown that in neutrophils classical transmembrane signaling consisting of increased [Ca2+]i and hydrolysis of phospholipids was not essential for phagocytosis mediated by more than one receptor (yeast-IgG, yeast-C3b/bi, yeast-Con A). This work deals with the role of this transmembrane signaling in phagocytosis of erythrocyte (E) IgG, which is mediated only by receptors for IgG (Fc gamma Rs). The ingestion of E-IgG was associated with an increase in [Ca2+]i and production of inositol phosphates, phosphatidic acid, diacylglycerol, and arachidonic acid, via activation of phospholipases C, D and A2. Related to the same number of particles ingested, the respiratory burst and the transmembrane signaling during phagocytosis of E-IgG were much smaller than during phagocytosis of yeast-IgG. In Ca(2+)-depleted neutrophils, where the increase in [Ca2+]i and hydrolysis of phospholipids were lacking, the phagocytosis of E-IgG was depressed by about 60%; the respiratory burst was also depressed due to the decrease of ingestion and of stimulation of NADPH oxidase by residual phagocytosis. Pertussis toxin (PT) did not inhibit the phagocytosis of E-IgG but depressed by about 40% the stimulation of lipidic transmembrane signaling and the respiratory burst in normal neutrophils. In Ca(2+)-depleted neutrophils the toxin was without effect on ingestion and respiratory burst. Staurosporine did not inhibit the ingestion of E-IgG in normal and Ca(2+)-depleted neutrophils but depressed by 30-40% the respiratory burst in normal and not in Ca(2+)-depleted neutrophils. Genistein, an inhibitor of tyrosine kinase, did not inhibit the ingestion of E-IgG but depressed by 30-40% the respiratory burst both in normal and Ca(2+)-depleted neutrophils. These results demonstrate the following findings in human neutrophils. (1) Contrary to the phagocytosis mediated by more than one receptor (yeast-IgG, yeast-Con A, yeast-C3b/bi), the transmembrane signaling involving increase in [Ca2+]i and hydrolysis of phospholipids plays a role in the phagocytosis and respiratory burst mediated by Fc gamma Rs alone. Thus, different signal transduction pathways can be involved in phagocytosis and associated respiratory burst depending on the receptor or combination of receptors activated. (2) Fc gamma Rs alone promote phagocytosis with two signaling pathways independent of and dependent on [Ca2+]i changes and phospholipid hydrolysis and insensitive to PT, staurosporine, and genistein. (3) The signaling pathways promoting phagocytosis triggered by Fc gamma Rs alone are in some way, or at some step, different from those that activate the respiratory burst.

Structure of the B-DNA decamer C-C-A-A-C-I-T-T-G-G in two different space groups: conformational flexibility of B-DNA.

For the first time, the same B-DNA oligomer has been crystallized and its structure solved in two different space groups. Crystallization of C-C-A-A-C-I-T-T-G-G with Ca2+ yields monoclinic space group C2 with a = 31.87 A, b = 25.69 A, c = 34.21 A, beta = 114.1 degrees, and five base pairs per asymmetric unit. The 5026 2 sigma data to 1.3 A refine to R = 0.152 with 72 waters, one heptavalent hydrated calcium complex, and one cacodylate ion per asymmetric unit. In contrast, crystallization with Mg2+ yields trigonal space group P3(2)21 with a = b = 33.23 A, c = 94.77 A, gamma = 120 degrees, and 10 base pairs per asymmetric unit. The 1725 2 sigma data to 2.2 A refine to R = 0.164 with 36 water molecules and one octahedral magnesium complex per asymmetric unit. The monoclinic form is virtually isostructural with previously solved monoclinic decamers, including twist angles of ca. 50 degrees at C-A and T-G steps. In contrast, the trigonal structure has quite different local helix parameters, with twist angles of ca. 36 degrees at the corresponding steps. These local parameter differences can only be attributed to crystal packing, suggesting that certain sequences of B-DNA are more flexible and influenced by their surroundings than had previously been thought. Such deformability may be important for interaction of B-DNA with control proteins, where both static structure and dynamic deformability comprise components of the recognition process. The crossing of two helices at an angle of 120 degrees in the trigonal cell is a model for an antiparallel, uncrossed Holliday junction, as has been noted earlier by Timsit and Moras [Timsit, Y., & Moras, D. (1991) J. Mol. Biol. 221, 919-940] from a rhombohedral DNA dodecamer structure analysis.

Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca(2+)-depleted human neutrophils.

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding.

The 98-amino acid Fis protein from Escherichia coli functions in a variety of reactions, including promotion of Hin-mediated site-specific DNA inversion when bound to an enhancer sequence. It is unique among site-specific DNA-binding proteins in that it binds to a large number of different DNA sequences, for which a consensus sequence is difficult to establish. X-ray crystal structure analyses have been carried out at 2.3 A resolution for wild-type Fis and for an Arg-89----Cys mutant that does not stimulate DNA inversion. Each monomer of the Fis dimer has four alpha-helices, A-D; the first 19 residues are disordered in the crystal. The end of each C helix is hydrogen bonded to the beginning of helix B' from the opposite subunit in what effectively is one long continuous, although bent, helix. The four helices, C, B', C', and B, together define a platform through the center of the Fis molecule: helices A and A' are believed to be involved with Hin recombinase on one side, and helices D and D' interact with DNA lying on the other side of the platform. Helices C and D of each subunit comprise a helix-turn-helix (HTH) DNA-binding element. The spacing of these two HTH elements in the dimer, 25 A, is too short to allow insertion into adjacent major grooves of a straight B-DNA helix. However, bending the DNA at discrete points, to an overall radius of curvature of 62 A, allows efficient docking of a B-DNA helix with the Fis molecule. The proposed complex explains the experimentally observed patterns of methylation protection and DNase I cleavage hypersensitivity. The x-ray structure accounts for the effects of mutations in the Fis sequence. Those that affect DNA inversion but not DNA binding are located within the N-terminal disordered region and helix A. This inversion activation domain is physically separated in the Fis molecule from the HTH elements and may specify a region of contact with the Hin recombinase. In contrast, mutations that affect HTH helices C and D, or interactions of these with helix B, have the additional effect of decreasing or eliminating binding to DNA.

Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils.

The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: 1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; 2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; 3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; b) these two processes were insensitive to staurosporine.

De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.

Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of the NADPH oxidase by FMLP in human neutrophils.

It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.

Studies on molecular regulation of phagocytosis and activation of the NADPH oxidase in neutrophils. IgG- and C3b-mediated ingestion and associated respiratory burst independent of phospholipid turnover and Ca2+ transients.

The role of messengers derived from hydrolysis of phosphoinositides and other phospholipids, of the basal level of [Ca2+]i and of the increase in [Ca2+]i in phagocytosis and respiratory burst was investigated, using normal neutrophils and neutrophils Ca2(+)-depleted by pretreatment with Quin2/AM and EGTA. 1) Phagocytosis and respiratory burst in control neutrophils challenged with yeast opsonized with IgG or C3b/bi were associated with a stimulation of the production of inositol phosphates, diacylglycerol, phosphatidic acid, arachidonic acid, and rise in [Ca2+]i. 2) In Ca2(+)-depleted neutrophils (basal [Ca2+]i 10 to 20 nM) the phagocytosis of yeast-IgG was similar to that in control neutrophils, the respiratory burst was slightly depressed (-30%), while the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, and phosphatidic and arachidonic acid did not occur. 3) In Ca2(+)-depleted neutrophils the phagocytosis of yeast-C3b/bi was slightly lower than that in control neutrophils, and the respiratory burst, related to the same number of particles ingested, was depressed by about 60%, whereas the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, phosphatidic acid, and arachidonic acid release did not occur. These findings demonstrate that transmembrane signaling pathways involving the hydrolysis of phosphoinositides by phospholipase C and D and of other phospholipids by phospholipase C and Az, and the rise in [Ca2+]i are not essential processes for triggering the ingestion of yeast particles opsonized with IgG and C3b/bi and the activation of the NADPH oxidase.

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release.

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid.

Phosphatidic acid (PA), a molecule that is rapidly produced by the stimulated turnover of phospholipids in a variety of cells including blood neutrophils, elicited NADPH-dependent superoxide anion (O2-) production in detergent extracts from membranes of resting pig neutrophils. The stimulatory effect of PA was independent of cytosolic factors, differing from arachidonic acid and sodium dodecyl sulfate which, on the contrary, absolutely required the presence of cytosol to elicit the same result. The O2(-)-forming activity of the detergent extract activable by PA, as that by sodium dodecyl sulfate and arachidonic acid plus cytosol, was found in the chromatographic fractions containing cytochrome b558 and presented a chromatographic profile identical to that of the activated NADPH oxidase, which was obtained from neutrophils prestimulated with phorbol 12-myristate 13-acetate. The PA-induced NADPH-dependent O2(-)-forming activity showed kinetic properties and sensitivity to the inhibitors similar to the classical ones of the activated neutrophil NADPH oxidase. The data suggest that, in this cell-free system, PA may stimulate O2- formation by direct interaction with latent NADPH oxidase of neutrophils or with some of its regulatory components.