More than the sum of the parts: annual partitioning within spatial guilds underpins community regulation.

To withstand the pressures of a rapidly changing world, resilient ecosystems should exhibit compensatory dynamics, including uncorrelated temporal shifts in population sizes. The observation that diversity is maintained through time in many systems is evidence that communities are indeed regulated and stabilized, yet empirical observations suggest that positive covariance in species abundances is widespread. This paradox could be resolved if communities are composed of a number of ecologically relevant sub-units in which the members compete for resources, but whose abundances fluctuate independently. Such modular organization could explain community regulation, even when the community as a whole appears synchronized. To test this hypothesis, we quantified temporal synchronicity in annual population abundances within spatial guilds in an estuarine fish assemblage that has been monitored for 36 years. We detected independent fluctuations in annual abundances within guilds. By contrast, the assemblage as a whole exhibited temporal synchronicity-an outcome linked to the dynamics of guild dominants, which were synchronized with each other. These findings underline the importance of modularity in explaining community regulation and highlight the need to protect assemblage composition and structure as well as species richness.

Spontaneous chirality through mixing achiral components: a twist-bend nematic phase driven by hydrogen-bonding between unlike components.

The spontaneous formation of a chiral phase via molecular recognition in a system consisting of achiral components is reported. Specifically, the liquid crystalline behaviour of two molecular complexes assembled by hydrogen bonding between a stilbazole-based template and alkoxybenzoic acids has been characterised. The complexes exhibit the heliconical twist-bend nematic phase (NTB) over a broad temperature range despite the hydrogen-bond acceptor not being liquid crystalline and the donor exhibiting the conventional achiral nematic phase.

Fish and macro-crustacean communities and their dynamics in the Severn Estuary.

The species of fish and macro-crustacean living within the Severn Estuary are reviewed. The fish community is notably species rich and exceeds 100 species in total for the estuary. Standardised long-term sampling at Hinkley Point in Bridgwater Bay gives a total complement of 83 for a single locality and this number is increasing by about one new species every two years. Most of these new species are moving in from centres of population lying to the south of the estuary. Almost all species of fish and macro-crustacean living within the estuary undertake regular migrations so that they tend to move seasonally in waves up and down the estuary. For fish, both species richness and the total abundance reach a maximum in late summer and autumn. The timing of this peak varies between the upper and lower estuary. This seasonal maximum is primarily caused by the arrival of the new recruits which use the estuary as a nursery. In contrast, crustaceans tend to be at their most diverse and abundant in early to mid summer. Using a 30-year time series of fish and crustacean abundance collected at Hinkley Point it is shown that major changes in the structure of the community are now underway and there are considerable recent changes in the abundance. However, some abundant species, including sand goby, Pomatoschistus spp., whiting, Merlangius merlangus and sprat, Sprattus sprattus, the three most abundant species in the estuary, have shown no long-term trend. At present, approximately 20% of the fish and macro-crustaceans observed in Bridgwater Bay are undergoing rapid, typically exponential, change in abundance. For a numerically abundant, diverse, fauna composed of approximately 90 species such levels of change are unexpected and suggest that the system is presently far from equilibrium. In some cases, the observed changes can be related to recent warming and the North Atlantic Oscillation. The overall increase in fish abundance observed may reflect a general improvement in water quality and a reduction in other anthropogenic impacts such as mortality in cooling-water intakes. The potential impacts of tidal power generation in the Severn Estuary are reviewed. There is considerable potential for any major installation to impact the fish and crustacean populations as they migrate and also alter the nature of the habitat resulting in changes in community composition. A particular difficulty in predicting the future impact of harnessing tidal energy is that the present community is already changing rapidly. The ability of fish and crustaceans to pass through the turbines unharmed will be a key issue in an assessment of the impact of tidal power generation.

Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cgamma1.

We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cgamma1 (PLCgamma1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCgamma1 were immunoprecipitated as a complex by a polyclonal antibody to PLCgamma1. The association of gelsolin and actin was specific for PLCgamma1 because immunoprecipitates of PLCs beta2, beta3, gamma2 and delta1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCgamma1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCgamma1. After stimulation with thrombin, PLCgamma1 activity in immunoprecipitates was increased 2-3-fold. This elevation in PLCgamma1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCgamma1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCgamma1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCgamma1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCgamma1 from gelsolin and actin, and the stimulation of PLCgamma1 activity.

Prevalence of factor V Leiden in a Canadian blood donor population.

OBJECTIVE: To determine the prevalence of factor V Leiden in a Canadian blood donor population. DESIGN: Cross-sectional laboratory study. SETTING: Hamilton Centre of the Canadian Red Cross Society. PARTICIPANTS: Volunteer donors who attended Hamilton Centre blood donor clinics over a 4-day period in August 1994; blood samples from 356 people were evaluable. OUTCOME MEASURES: Presence of factor V Leiden. RESULTS: Factor V Leiden was detected in 19 of the 356 people, for a prevalence rate of 5.3% (95% confidence interval 3.0% to 7.6%). All 19 people were shown to be heterozygous for the mutation. CONCLUSION: Factor V Leiden is common in the Canadian population. Its prevalence is similar to that reported in other Western countries. These data are relevant in the clinical management of patients at risk for venous thrombosis and those with recurrent thrombotic disorders.

Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC) in pancreatic acini.

We used rat pancreatic acini and measured the effects of various agents on digestive enzyme secretion, diacylglycerol (DAG) and the cellular distribution of protein kinase C (PKC) enzyme activity as well as isoforms of PKC determined by quantitative immunoblot analysis. TPA, but not CCK-8, caused translocation of PKC enzyme activity from the cytosol fraction to the membrane fraction. Immunoblot analysis detected PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-beta, PKC-gamma and PKC-eta were not detected. TPA caused translocation of all isoforms from cytosol to membrane, whereas CCK-8 caused translocation of PKC-delta and PKC-epsilon, carbachol caused translocation of PKC-epsilon, and bombesin and secretin caused no detectable translocation of any isoform. Specific receptor antagonists could prevent, as well as reverse completely, the translocation of PKC isoforms caused by CCK-8 or carbachol. Agonists added in sequence with an interposed addition of a specific receptor antagonist caused cycling of PKC-epsilon between cytosol and membrane fractions. Each receptor-mediated agonist that caused translocation of PKC also increased DAG, and with CCK-8 and carbachol cycling of PKC-epsilon between cytosol and membrane was accompanied by corresponding cyclic changes in cellular DAG. CCK-JMV-180, bombesin and secretin increased DAG but did not cause translocation of any PKC isoform. Translocation of a PKC isoform could be accounted for by whether the increased DAG originated from PIP2 (accompanied by translocation) or from phosphatidylcholine (no accompanying translocation). Thus it appeared that DAG, in pancreatic acini, is functionally compartmentalized depending on the source of the lipid. Studies using CCK-8 and CCK-JMV-180 indicated that occupation of the low affinity state of the CCK receptor by either peptide increased DAG from phosphatidylcholine, whereas occupation of the very low affinity state by CCK-8 increased DAG from PIP2 and caused translocation of PKC-delta and PKC-epsilon. TPA stimulated amylase secretion, indicating that activation of PKC can stimulate enzyme secretion; however, with the various receptor-mediated secretagogues there was no consistent, unequivocal correlation between translocation of an isoform of PKC and accompanying changes in enzyme secretion.

Translocation and downregulation of protein kinase C isoenzymes-alpha and -epsilon by phorbol ester and bryostatin-1 in human keratinocytes and fibroblasts.

Protein kinase C isoenzymes can be subdivided into two classes, based on their requirement for calcium. Protein kinase C-alpha, beta I, -beta II, and -gamma are calcium dependent, whereas protein kinase C-gamma, -epsilon, -zeta, -eta, and -theta are calcium independent. We have examined the expression, translocation, downregulation, and activation of calcium-dependent and -independent protein kinase C isoenzymes in human skin keratinocytes and fibroblasts. Human keratinocytes and fibroblasts expressed protein kinase C-alpha, -delta, -epsilon, and -zeta mRNA and protein, whereas protein kinase C-eta (L) was detected only in keratinocytes. Protein kinase C-beta I, -beta II, -gamma, and -theta were not detected in either cell type. The protein kinase C activators 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1 (50 nM, for 5 min) induced translocation of protein kinase C-alpha and -epsilon cytosol to membrane in both keratinocytes and fibroblasts. 12-0-tetradecanoylphorbol 13-acetate and bryostatin-1, for 18 h, induced complete downregulation (i.e., loss) of protein kinase C-alpha and -epsilon in keratinocytes, but only partial downregulation was observed in fibroblasts. The subcellular distribution of protein kinase C-delta, -zeta or protein kinase C-eta, in keratinocytes or fibroblasts, did not change in response to 12-0-tetradecanoylphorbol 13-acetate or bryostatin-1. These data indicate differential expression, subcellular distribution, and regulation of protein kinase C isoenzymes in human skin cells.

Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-alpha in normal human skin fibroblasts and keratinocytes.

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, 'MARCKS'). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.

Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq.

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.

Retinoic acid and phorbol ester synergistically up-regulate IL-8 expression and specifically modulate protein kinase C-epsilon in human skin fibroblasts.

Phorbol ester (TPA) and retinoic acid (RA) are two potent immunomodulatory agents whose actions are mediated through distinct signal transduction pathways involving protein kinase C (PKC) and nuclear RA receptors, respectively. We have investigated the interactions between these two pathways in the regulation of expression of the inflammatory cytokine IL-8 in human skin fibroblasts. TPA (as previously reported) and RA both induced IL-8 mRNA and protein in a time- and dose-dependent manner. IL-8 mRNA induction by TPA (10 nM) was maximal (15-fold) within 6 h, and returned to baseline within 24 h of treatment, although maximal induction (10-fold) by RA (1 microM) did not occur until 24 h posttreatment. Induction of IL-8 by TPA was blocked by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine, which inhibits PKC and cAMP-dependent protein kinases (PKA), but not by N-(2-ganidinoethyl)-5-isoquinoline sulfonamide, which preferentially inhibits PKA, consistent with the participation of PKC in the induction of IL-8 by TPA. In contrast, induction of IL-8 by RA was inhibited by both 1-(5-isoquinoline sulfonamide and N-(2-gamidinoethyl)-5-isoquinoline sulfonamide, suggesting the participation of PKA in the induction of IL-8 by RA. However, activation of PKA by addition of cAMP analogues was not sufficient to induce IL-8 expression. Induction of IL-8 by RA also did not appear to be mediated indirectly through induction of IL-1, because addition of IL-1R antagonist did not block IL-8 induction by RA. RA and TPA added in combination synergistically enhanced expression of IL-8 mRNA, measured at 6 (2-fold) and 24 h (10-fold) posttreatment. To investigate the mechanism of this synergy, the effect of TPA and RA on fibroblast PKC activation and PKC isozyme levels were determined. TPA, either alone or together with RA, but not RA alone, stimulated phosphorylation of an endogenous 80-kDa PKC substrate. Dermal fibroblasts expressed three PKC isozymes (alpha, (delta, and (epsilon). TPA, but not RA, down-regulated PKC-alpha, neither TPA or RA affected the level of PKC-delta, and both TPA and RA down-regulated PKC-epsilon. This latter effect was enhanced 2-fold by addition of RA and TPA together. These data suggest that modulation of PKC-epsilon may be a common participant in the regulation of IL-8 expression by TPA and RA.

Translocation of protein kinase C isozymes in thrombin-stimulated human platelets. Correlation with 1,2-diacylglycerol levels.

Human platelets were found by immunoblot analysis to express protein kinase C (PKC) isozymes alpha, beta, delta, and zeta, but not gamma, epsilon, or eta. Exposure of platelets to thrombin, in the presence of 1 mM calcium, induced increased membrane association of PKC-alpha, -beta, and -zeta, while the subcellular distribution of PKC-delta remained unaltered. Maximal membrane association (2-fold) of PKC-alpha, -beta, and -zeta occurred within 1 min and was sustained for at least 10 min after the addition of thrombin. Similar results were obtained in the presence of the RGDS peptide, which blocks thrombin-induced binding of fibrinogen to its receptor, which indicates that PKC translocation was independent of fibrinogen binding. In the absence of added extracellular calcium, thrombin-induced translocation of PKC-alpha, -beta, and -zeta was transient, reaching a maximum at 1 min and returning to base line by 10 min. In the presence of calcium, thrombin induced a rapid (within 15 s) 8-fold rise in inositol 1,4,5-trisphosphate, which returned to baseline levels within 1 min, and a biphasic increase in sn-1,2-diacylglycerol (DAG), with peaks at 15 s and 2 min, which remained elevated for at least 5 min. Chelation of external calcium abolished the second phase of DAG formation but had no effect on the kinetics or magnitude of the increase in inositol 1,4,5-trisphosphate or the first phase of DAG formation. Two early PKC-dependent functions, serotonin release and 40-kDa protein phosphorylation, were independent of extracellular calcium and sustained DAG. These data demonstrate that in thrombin-stimulated human platelets the duration of the increased PKC membrane association closely parallels that of increased DAG content, and sustained elevations in DAG content and PKC translocation are dependent on extracellular calcium.

Isocapnia blocks exercise-induced reductions in ocular tension.

Previous reports suggest that isometric exercise (2-min handgrip at 50% maximal voluntary contraction [MVC]) substantially lowers intraocular pressure (IOP). The authors questioned whether the mechanism for lowered IOP in exercise is secondary to hyperventilation. Accordingly, in this study 11 subjects, with elevated IOP (greater than or equal to 18 mm Hg) and otherwise healthy, did 2 min of handgrip exercise at 50% MVC with and without carbon dioxide supplementation to maintain isocapnic conditions. Compared with a control experiment that involved neither exercise nor CO2 addition, exercise induced a fall in IOP from 18.3 to 15.6 mm Hg (P less than 0.001). This statistically significant decline in IOP persisted for 15 min after the exercise session. At the point of minimum IOP (1 min after the end of exercise), the minute ventilation was elevated from 6.5-8.1 l/min (P less than 0.05), and the end-tidal partial pressure of CO2 (PCO2) was reduced from 37.0 to 33.7 mm Hg (P less than 0.05) with respect to control values. By contrast, adding CO2 sufficient to maintain isocapnic conditions (experimental end-tidal PCO2 = 38.9 versus 38.5 mm Hg in the control study; P = not significant) abolished the exercise-induced ocular hypotension (experimental IOP = 17.8 versus 18.1 mm Hg in the control study; P = not significant). It was concluded that prevention of hypocapnia during isometric handgrip exercise blocks the subsequent fall in IOP, suggesting both that isometric exercise per se has no direct influence on IOP and that therapy for ocular hypertension could involve manipulation of blood gases.

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts.

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.

Increased phospholipase C-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate and 1,2-sn-diacylglycerol content in psoriatic involved compared to uninvolved and normal epidermis.

Evidence suggests that the phospholipase C/protein kinase C signal transduction system participates in the regulation of epidermal cell growth and differentiation. Psoriatic epidermis is characterized by hyperproliferation, defective differentiation, and inflammation. In this report, we have determined the activity of phospholipase C-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) and 1,2-diacylglycerol content in normal and psoriatic involved and uninvolved epidermis. 1,2-diacylglycerol is formed from phospholipase C-catalyzed hydrolysis of PIP2 and is the physiologic activator of protein kinase C. PIP2 hydrolysis was assayed in soluble and particulate fractions prepared from keratome biopsies of normal and psoriatic skin. Total lipids were extracted from normal and psoriatic epidermis and 1,2-diradylglycerol (a mixture of 1,2-diacylglycerol and 1-ether, 2-acyl-glycerol) quantitated by enzyme assay. Because 1,2-diacylglycerol is a more potent activator of protein kinase C, the relative proportions of 1,2-diacyl and 1-ether, 2-acylglycerol in uninvolved and involved psoriatic epidermis were determined. This was accomplished by separation of acetate derivatives of 1,2-diacylglycerol and 1-ether, 2-acyl-glycerol by thin layer chromatography. Soluble and membrane-associated phospholipase C-catalyzed PIP2 hydrolysis were increased 3.7 times (p less than 0.001) and 3 times (p less than 0.004), respectively, in psoriatic involved compared to uninvolved and normal epidermis. 1,2-diradylglycerol content was also significantly elevated (3 times, p less than 0.01) in psoriatic involved versus uninvolved and normal epidermis. Analysis of the acetate derivatives of 1,2-diradylglycerol in psoriatic uninvolved and involved epidermis revealed that 1,2-diacylglycerol was the major species (86% and 95%, respectively). There were no significant differences in either phospholipase C-catalyzed PIP2 hydrolysis or 1,2-diacylglycerol content between uninvolved and normal epidermis. 1,2-diacylglycerol purified from normal and involved psoriatic epidermis was capable of activating protein kinase C from normal epidermis in vitro. In epidermal slices, activation of protein kinase C by addition of 12-0-tetradecanoylphorbol-13-acetate and 1,2-diacylglycerol (1,2-dioctanoylglycerol) resulted in subsequently decreased protein kinase C activity, a process termed down-regulation. These data are consistent with the possibility that the elevation in lesional 1,2-diacylglycerol content may account, in part, for the previously reported reduction of protein kinase C activity in psoriasis (Horn, Marks, Fisher, et al: J Invest Dermatol 88:220-222, 1987).

Phosphatidylglycerol, lecithin/sphingomyelin ratio and respiratory distress syndrome in diabetic and non-diabetic pregnancies.

Samples of amniotic fluid from 514 non-diabetic and 69 diabetic patients were analyzed for phospholipid content. Results were correlated with incidence of respiratory distress syndrome (RDS) in the neonate. The incidence of RDS was 4.5% among diabetics and 5.3% among non-diabetics. In the presence of phosphatidylglycerol (PG), no infant developed RDS while in the absence of PG the incidence of RDS was 16.7% and 14.4%, respectively. In the presence of a mature lecithin/sphingomyelin (L/S) ratio the respective incidence of RDS was 1.6 and 1.8, while with an immature L/S ratio the incidence of RDS was 28.6% and 29%. The incidence of RDS after 37 weeks gestation was 0% among diabetics and 0.6% among non-diabetics. We conclude that amniotic fluid phospholipids are equally predictive of risk for RDS in diabetics as among non-diabetic patients. We suggest that in patients with accurate gestational dating, amniotic fluid analysis for phospholipids might not be necessary to establish fetal lung maturity.

Isolation and characterization of one soluble and two membrane-associated forms of phosphoinositide-specific phospholipase C from human platelets.

Two forms (mPLC-I, mPLC-II) of phosphoinositide-specific phospholipase C have been purified, 1494- and 1635-fold, respectively, from plasma membranes of human platelets. Purified mPLC-I and mPLC-II had estimated molecular weights by gel filtration and sodium dodecyl sulfate-polyacrylamide gels of 69,000 and 63,000, respectively. Two cytosolic forms (PLC-I and PLC-II) of phosphoinositide-specific phospholipase C were also resolved on a phenyl-Sepharose column. The major cytosolic form present in outdated platelets, PLC-II, was purified to homogeneity by chromatography on Fast Q-Sepharose, cellulose phosphate, heparin-agarose, phenyl-Sepharose, Superose 12, DEAE-5PW, and hydroxylapatite. Purified PLC-II had a molecular weight of 57,000 on sodium dodecyl sulfate-polyacrylamide gels. mPLC-I, mPLC-II, and PLC-II hydrolyzed both PI and PIP2. The Vmax for PIP2 hydrolysis was similar for all three forms of PLC and was approximately 5-fold greater than for PI hydrolysis. The Km for PIP2 hydrolysis was also similar for the three enzymes. In contrast, the Km for PI hydrolysis by PLC-II was 10-fold lower than by mPLC-I and mPLC-II. In addition, antibody prepared against PLC-II did not cross-react with either mPLC-I or mPLC-II. These data indicate that platelets contain membrane-associated phosphoinositide-specific phospholipases C that are distinct from at least one cytosolic form (PLC-II) of the enzyme.

Plasma membrane associated phospholipase C from human platelets: synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5'-O-(3-thiotriphosphate).

The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein.

GTP-binding activity was fractionated into two peaks (GI and GII) by chromatography on heparin-agarose. GTP-dependent PLC activity eluted as a single peak, which co-chromatographed with GTP-binding peak GII. Rechromatography of peak GII on heparin-agarose, in the presence of 0.5% sodium cholate, resulted in separation of PLC and GTP-binding activities, and loss of GTP-dependent PLC activity. Recombining fractions containing PLC and GTP-binding activities restored GTP-dependent PLC activity. A specific GTP-binding protein of 29,000 daltons was identified in peak GII by Western blotting of column fractions with [alpha-32P]GTP. These results demonstrate that the soluble phospholipase C from human platelets is regulated by GTP S-binding protein (G29).

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy.

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.