Effect of Phospholipid Bilayer Phase Asymmetry on Phospholipase D Reaction-Induced Vesicle Rupture

Spherical phospholipid bilayers, vesicles, were formed with respect to phase of each layer via a double emulsion technique. At the outer layer of the vesicles, phospholipase D catalyzed for the conversion of phosphatidylcholine (PC) to phosphatidic acid (PA). The reaction caused by phospholipase D (PLD) induced a curvature change in the vesicles, which eventually led them to rupture. Response time from the PLD injection to the rupture was monitored for the phase of each layer by using fluorescence intensity changes of pH-sensitive dye encapsulated in the vesicles. It was found that low ionic strength and asymmetric phase retarded response time. The retardation seems to be related to the stability of the vesicles, which is due to the interaction between the lipid molecules. In the liquid phases of the outer lipid layers, the unexpected slow response time may be attributed either to the fast lateral diffusion, which relieves the curvature change of the vesicles, or to the low concentration of PCs, which are less for the reaction compared to the solid phase of the outer lipid layer, rather than the stability.     

Interpretation of atypical patterns encountered when using a flow cytometry-based method to detect residual leukocytes in leukoreduced red blood cell components

BACKGROUND: Universal leukoreduction of blood components is becoming the standard of care. Flow cytometry methods are being used for quality control of the leukoreduction process. METHODS: We provide an atlas of atypical flow cytograms generated by a commercial LeucoCOUNT assay that was used to enumerate residual leukocytes in leukoreduced red blood cell components. Numeric results are derived from a flow cytogram generated by the assay. RESULTS: Three types of atypical flow cytogram patterns were observed during process validation or routine quality control of leukoreduced red blood cell components. (a) Fixation artifact: Fixation of control or test samples can alter the staining intensity compared with fresh cells. (b) "Rain" pattern: Flow cytometry methods count slightly damaged leukocytes not removed during leukoreduction. Slightly damaged leukocytes appear on a flow cytogram like "rain" falling from a well-defined "cloud" of intact residual leukocytes. Discrepancies between automated flow cytometry results and subjective manual counting methods can occur. (c) Autofluorescence-debris pattern: Cell debris and age-related changes in the sample can cause shifts in the fluorescence staining pattern, resulting in erroneous test results. CONCLUSION: Review of flow cytograms is essential for accurate reporting of flow cytometry-based methods for enumerating residual leukocytes in leukoreduced blood components.     

Thermodynamic and kinetic studies of the interaction of vesicular dipalmitoylphosphatidylcholine with Agkistrodon piscivorus piscivorus phospholipase A2

The tryptophan fluorescence emission intensity at 340 nm of monomeric phospholipase A2 from Agkistrodon piscivorus piscivorus increased about 70% upon addition of dipalmitoylphosphatidylcholine small unilamellar vesicles (DPPC SUV) at 25 degrees C. The emission spectrum was also blue-shifted 6-8 nm, suggesting that the environment of 1 or more tryptophan residues had become less polar. This effect of SUV on the phospholipase A2 fluorescence was independent of Ca2+ at 25 degrees C, and the apparent association constant for the interaction was approximately 1.7 x 10(4) M-1. The apparent Km for hydrolysis of DPPC SUV was equal to the inverse of the estimated association constant. In the absence of Ca2+, the change in fluorescence intensity decreased with increasing temperature. Thermodynamic analysis of this reversible, temperature-dependent fluorescence change indicated that the A. p. piscivorus monomer phospholipase A2 interacts only with SUV in the true gel phase existing below the pretransition of gel to "ripple" phase lipid in the absence of Ca2+. In contrast, the fluorescence intensity change upon addition of SUV in the presence of Ca2+ was independent of temperature over the range of 25-48 degrees C. Under these conditions, hydrolysis of the lipid occurred concomitantly with the change in fluorescence which could not be reversed by the addition of EDTA. With a nonhydrolyzable analog of DPPC, however, the fluorescence changes upon mixing of SUV, Ca2+, and phospholipase A2 were reversible and temperature-dependent. Thus, the apparent irreversibility of the change in fluorescence observed with Ca2+ and DPPC SUV was correlated with hydrolysis of the vesicles. These results indicate that the magnitude of the initial interaction of enzyme with substrate is reversible, is Ca2+-independent, depends upon the lipid state, and is quantitatively correlated to the maximum rate of hydrolysis.     

Effects of low-intensity extremely high frequency electromagnetic radiation on chromatin structure of lymphoid cells in vivo and in vitro

Using a comet assay technique, it was shown for the first time that low-intensity extremely high-frequency electromagnetic radiation (EHF EMR) in vivo causes oppositely directed effects on spatial organization of chromatin in cells of lymphoid organs. In 3 hrs after single whole-body exposure of NMRI mice for 20 min at 42.0 GHz and 0.15 mW/cm2, an increase by 16% (p < 0.03 as compared with control) and a decrease by 16% (p < 0.001) in fluorescence intensity of nucleoids stained with ethidium bromide were found in thymocytes and splenocytes, respectively. The fluorescence intensity of stained nucleoids in peripheral blood leukocytes was not changed after the exposure. The exposure of cells of Raji hunan lymphoid line and peripheral blood leukocytes to the EHF EMR in vitro induced a decrease in fluorescence intensity by 23% (p < 0.001) and 18% (p < 0.05), respectively. These effects can be determined by changes in a number of physiological alkali-labile sites in DNA of exposed cells. We suggested that the effects of low-intensity EHF EMR on the immune system cells are realized with the participation of neuroendocrine and central nervous systems.     

A role for phospholipids in the binding and metabolism of drugs by hepatic microsomes. Use of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulphonate

1. The pretreatment of rat liver microsomes with phospholipase C or D decreased the N-demethylation of (+)-benzphetamine. The hydroxylation of aniline was essentially unchanged by pretreatment of microsomes with phospholipase C. 2. Some components of the microsomal mixed-function oxidase system were impaired by phospholipases. 3. The fluorescence of 1-anilinonaphthalene-8-sulphonate (ANS) was greatly enhanced by microsomes. Phospholipase C or D markedly decreased ANS–microsome fluorescence. Quantum yield of ANS–microsome fluorescence appeared to be related directly to phospholipid content of microsomes. 4. Most of the drugs studied enhanced ANS–microsome fluorescence. Warfarin, however, displaced ANS fluorescence competitively from microsomes. The latter effect was postulated as being due to warfarin competing with ANS for the cationic site on microsomal phosphatidylcholine. 5. ANS fluorescence was also increased by the presence of phospholipid micelles. The fluorescence of ANS–phosphatidylcholine micelles was modified by warfarin and (+)-benzphetamine in a manner similar to that observed with microsomes. Warfarin decrease of fluorescence was absent when ANS was bound to phosphatidic acid, which lacks a cationic site. 6. Trypsin pretreatment of microsomes did not modify ANS–microsome fluorescence, including drug-induced changes. 7. It was postulated that phospholipids have a permissive role in the metabolism of most drugs by hepatic microsomes and that the ANS probe might reflect interactions of compounds with microsomal membrane phospholipids.     

The information value of clinico-hematologic criteria for the early diagnosis of acute radiation sickness in pig-tailed macaques

Ten pig-tailed monkeys (Macaca nemestrina) were subjected to 60Co radiation at a dose of 6.0-6.5 Gy and a dose rate of 1.2 Gy/min. Acute radiation sickness has developed in the monkeys causing their death on the 16-20 day. In spite of this, the initial reaction was weakly expressed and according to its manifestation it was impossible to evaluate severity and possible outcome of the lesion. At an early stage of the disease (6-24 hours) insufficient was uranin fluorescence in blood plasma, but more informative were the changes in adhesive properties of leukocytes the dynamics of lymphocytes (lymphopenia), reticulocytes (reticulocytopenia) and shifts in reticulograms (increased per cent of juvenile forms).     

Effect of phospholipase D on micellar phospholipids and the role of calcium ions]

The rates of the reaction products formation under simultaneous phospholipase D effect on phosphatidyl ethanolamine and phosphatidyl choline were studied. The hydrolysis of cephalin, unlike the phospholipase D effect on lecithin, does not require Ca2+ ions. Ca2+ does not affect the enzymatic degradation of lecithin and inhibits the reaction with cephalin in "inorganized" phospholipid emulsions. The hydrolysis of micellar phospholipids by phospholipase D (in the presence of the anionic detergent sodium dodecyl sulfate) is accelerated by Ca2+ ions for both substrates. The apparent Km value is equal to 1.5 mM and does not depend on the phospholipid type. In contrast, the value of kcat for lecithin is twice as high as that for cephalin. It was demonstrated that the phase state of the phospholipids and the chemical nature of the alcohol residue in the phospholipid molecule are essential for the substrate specificity of phospholipase D.     

Association of the ACE I/D gene polymorphism with DNA damage in hypertensive men

The aim of the study was to evaluate the association between the angiotensin-converting enzyme ACE I/D (rs 4340) polymorphism and DNA damage in patients with essential hypertension (EH). The I/D polymorphism of ACE was determined by polymerase chain reaction in 170 male hypertensive patients and 64 normotensive blood donors. We used flow cytometry to determine the levels of cell death, micronuclei and accumulation of peripheral blood leukocytes in G1/G0, S, G2/M phases of the cell cycle. Additionally, the whole blood samples were incubated in vitro at 4°C for 24 h to investigate the genotype effects on the susceptibility of cells to DNA damage. We found lower frequency of cells in DNA synthesis S phase and higher levels of micronuclei in the hypertensive compared to normotensive group (p < 0.05); increased formation of micronuclei was seen due to elevated micronuclei frequencies in patients with the ACE II genotype (p < 0.05), but not in ID or DD genotype carriers. Incubation of whole blood samples of normotensive individuals lead to the most active cell death (p < 0.05) and micronuclei formation (p > 0.05) in the II genotype carriers too. However, hypertensive patients displayed different cellular response to incubation-induced DNA damages in the ACE I/D genotype groups; after incubation, the frequencies of micronuclei were significantly higher in the DD genotype carriers (p < 0.05). To conclude, the study suggests that the ACE I/D polymorphism may contribute to mechanisms and intensity of DNA damages in hypertensive and normotensive individuals.     

Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate

A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of supported pyrenephospholipid monolayers by phospholipase A2

Hydrolysis by pancreatic and snake venom (Crotalus atrox) phospholipase A2 of fluorescent monolayers of pyrene-labelled phosphatidylglycerol on solid support was studied. We used a fluorescence microscope equipped with video camera, video recorder and an image analyzer to monitor changes in fluorescence. Decrease in pyrene excimer emission was evident when pyrene phosphatidylglycerol monolayers transferred onto quartz glass slides (at a surface pressure of 15 mN m-1) were subjected to enzymatic hydrolysis. Snake venom phospholipase A2 could hydrolyze the monolayers almost completely while pancreatic phospholipase A2 could cause only 50% decrease in fluorescence intensity. EDTA totally inhibited the action of both A2 phospholipases. When monolayers were transferred onto solid supports at a surface pressure of 31 mN m-1 C. atrox phospholipase A2 could still exert activity whereas porcine pancreatic phospholipase A2 was inactive.     

gamma-Glutamyltranspeptidase activity in intact leukocytes: flow cytometric analysis and sorting

A fluorescent method developed for visualizing gamma-glutamyltranspeptidase (GGT) in intact liver cells was adapted to leukocytes and used in a multiparameter flow cytometric study of blood and bone marrow cells from rats with subcutaneous implants of mammary carcinoma 5A. The severe granulocytosis caused by this non-metastatic tumor was preceded by a progressive rise in the percentage of leukocytes with high GGT fluorescence. Both granulocytes and small, immature cells of bone marrow showed increased GGT expression, whereas in blood this increase was attributable entirely to mature granulocytes. At 28 days (but not yet at 14 days) after carcinoma implantation, 20-30% of blood or bone marrow granulocytes constituted a distinct subpopulation in that their GGT fluorescence intensity range was much higher and did not overlap with the range for the rest of the population. The results indicate that fluorescent GGT assay of intact leukocytes provides a useful probe for flow cytometric analysis of population heterogeneity in leukoproliferative disorders.     

Influence of coprecipitation and interactions with anionic surfactants on the activity of plant phospholipase D.

The effects of two surfactants (sodium dodecyl sulfate and sodium dodecyl dioxyethylene sulfate) of similar structure but differing water solubility (of their calcium salts) on the enzymatic activity of cabbage phospholipase D have been studied. The solubility difference is insignificant because the two surfactants activate phospholipase D similarly. To elucidate the mechanism of their influence on the enzyme, the phase behavior in the reaction media and the interactions of the surfactants with the enzyme were investigated by potentiometry and by light scattering and UV spectroscopy. Calcium dodecyl dioxyethylene sulfate (which is more soluble in water than calcium dodecyl sulfate) precipitates in the presence of phosphatidylcholine, the substrate of the enzymatic reaction. In the reaction media phospholipase D was involved into a precipitate consisting of calcium salts of the surfactants and phosphatidylcholine that might be interpreted as its immobilization. In addition, the surfactants were adsorbed on the enzyme, unfolding the globular enzyme molecule due to electrostatic repulsion between adsorbed surfactant anions. The observed increase in the functional activity of phospholipase D is accounted for by transfer to an optimal tertiary structure for the enzyme molecule in the course of consecutive conformational transitions induced by the surfactants.     

Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.     

Photosynthesis-related infrared light transmission changes in spinach leaf segments

The time courses of infrared light transmission changes and fluorescence induced by light in spinach leaf segments were measured. The illumination by red light exhibited a complex wave pattern. The transmission approached the baseline after repeating decreases and increases. Illumination by far-red light decreased the transmission. One of the differences between the two responses was the difference between the two amplitudes of the first increasing component. The component in the red light response was larger than the component in the far-red light response. The transmission decrease by far-red light is supposed to correspond to "red drop." The transmission decrease by far-red light was suppressed by red light. This is due to an activation of a transmission-increasing component. This probably corresponds to "enhancement." A proportional correlation existed between the intensity of far-red light and the minimum intensity of red light that suppressed the transmission decrease induced by far-red light. The component which made Peak D in the time course of fluorescence yield and the first increasing component in the transmission changes were suppressed by intense light.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

Increased proliferation of activated T-lymphocytes in response to a monoclonal antibody (anti-p68).

A series of monoclonal antibodies was produced by immunization of mice with cells of the human promonocytic cell line CM-S; one of these recognized a membrane antigen (MW 68,000) constitutively expressed by these cells. Antigen p68 was also found to be expressed on all granulocytic cells and most mononuclear leukocytes from normal human peripheral blood, but not on hemopoietic precursor cells from bone marrow. Various types of leukemic cells also expressed antigen p68 as did various transformed human cell lines whether derived from hemopoietic cells or from other tissues. Antigen p68 is involved in T-lymphocyte regulation. In fact, the antibody anti-p68 has a strong synergistic effect increasing the proliferative response of peripheral blood T-lymphocytes both in the mixed lymphocyte reaction and when the lymphocytes are stimulated by suboptimal doses of lectin (phytohemagglutinin), tumor promoter phorbol esters, or tetanus toxoid. The anti-p68 antibody synergizes with the active metabolite of vitamin D3, 1,25-dehydroxyvitamin D3, to induce monocyte to macrophage maturation and enhances the function of mature granulocytes stimulated with the granulocyte-macrophage colony-stimulating factor in vitro.     

Studies on the characterization of the Rho(D) antigen.

The Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rho(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment. The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity.     

Eicosanoid generation from antigen-primed mast cells by extracellular mammalian 14-kDa group II phospholipase A2.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.     

Immunocytochemical diagnosis of acute leukemia with pleural involvement

Cytologic examination of the pleural effusion from a patient with acute leukemia, leukocytosis and bleeding revealed the presence of many leukemic cells, "lymphocytes" and erythrocytes. The significance of these cellular changes was investigated by simultaneous study of blood and effusion leukocytes by morphologic, cytochemical and immunochemical methods. Both the leukemic blasts and the "lymphocytes" in the effusion and the blood were found to be neoplastic and contained antigens characteristic of both myeloid cells (OKM-1) and lymphoblasts (C-ALLA, common acute lymphoblastic leukemia antigen). These results, when analyzed in the context of the clinical findings, were indicative of acute leukemia with pleural involvement. Such a clinically oriented approach may further enhance the potential of cytodiagnosis in patients with serous effusions.     

Fluorescence signals in ANS-stained squid giant axons during voltage-clamp

Summary An analysis is presented of the changes in fluorescence intensity, associated with nerve stimulation, of 1-anilinonaphthalene-8-sulfonate (ANS) injected in squid axons. A preliminary and qualitative account of the physiological modifications produced by the ANS injection is also given. The time course of the fluorescence intensity during the first 300 sec following the onset of voltage-clamp is shown to be exponential with a time constant of about 35 msec, fairly independent of the amplitude and sign of the applied voltage, the intensity increasing during hyperpolarizations and decreasing during depolarizations. Data are presented on the relationship between the amplitude of the changes in fluorescence intensity and the voltage applied, the amplitude of the changes associated with depolarizations being measured at the time of occurence of the peak inward current. The interpretation of the changes in fluorescence intensity in terms of electrophoretic effects or as being due to a direct effect of the electric field upon the quantum yield of ANS fluorescence, is hardly compatible with the results of our present analysis.