Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

Some isoforms of secretory phospholipase A(2) (sPLA(2)) distinguish between healthy and damaged or apoptotic cells. This distinction reflects differences in membrane physical properties. Because various sPLA(2) isoforms respond differently to properties of artificial membranes such as surface charge, they should also behave differently as these properties evolve during a dynamic physiological process such as apoptosis. To test this idea, S49 lymphoma cell death was induced by glucocorticoid (6-48 h) or calcium ionophore. Rates of membrane hydrolysis catalyzed by various concentrations of snake venom and human groups IIa, V, and X sPLA(2) were compared after each treatment condition. The data were analyzed using a model that evaluates the adsorption of enzyme to the membrane surface and subsequent binding of substrate to the active site. Results were compared temporally to changes in membrane biophysics and composition. Under control conditions, membrane hydrolysis was confined to the few unhealthy cells present in each sample. Increased hydrolysis during apoptosis and necrosis appeared to reflect substrate access to adsorbed enzyme for the snake venom and group X isoforms corresponding to weakened lipid-lipid interactions in the membrane. In contrast, apoptosis promoted initial adsorption of human groups V and IIa concurrent with phosphatidylserine exposure on the membrane surface. However, this observation was inadequate to explain the behavior of the groups V and IIa enzymes toward necrotic cells where hydrolysis was reduced or absent. Thus, a combination of changes in cell membrane properties during apoptosis and necrosis capacitates the cell for hydrolysis differently by each isoform.

Who is the Teacher and Who is the Student? The Dual Service- and Engaged-Learning Pedagogical Model of Anatomy Academy.

Anatomy Academy is a simultaneous service-learning experience for preprofessional school undergraduate students and preclinical professional students acting as classroom paraprofessional teachers (Mentors), and engaged-learning experience for fourth to sixth grade elementary school children (Students). Using didactic and kinesthetic active learning teaching strategies in small-group classroom environments, Mentors taught anatomy, physiology, and nutrition concepts to Students. In this study of the program's early years (2012-2014), overall objectives of improving Mentors' pedagogical confidence; and Students' science interest, science knowledge, and exercise self-efficacy were assessed. Mentors showed (89% response of 595 surveyed) improvement in content delivery (P < .001), student engagement (P < .001), classroom management (P < .001), and professionalism (P = .0001). Postprogram Mentor reflections were categorized into 7 major themes that demonstrated personal growth through the service-learning opportunity: (1) realization of an ability to make a difference in the world now; (2) acknowledgment of the importance of listening in teaching; (3) recognition that lives can and will change with "a little love"; (4) insight into the effectiveness of guiding Students through material rather than lecturing; (5) awareness of the value of respect in the learning environment; (6) cognizance of the power of individualized attention to motivate Students; and (7) reflection of one's own personal growth through the open influence of Students. Students showed (88% response of 1259 surveyed) improvement in science knowledge (P = .014) and exercise self-efficacy (P = .038), but not science interest (P = .371). Thus, while Students are learning more science and becoming more aware of their health, we need to be more overt in our presence as scientists in the educational arena.

Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers.

The various lamellar phases of dipalmitoylphosphadtidylcholine bilayers with and without cholesterol were used to assess the versatility of the fluorescent probe merocyanine 540 through simultaneous measurements of emission intensity, spectral shape, and steady-state anisotropy. Induction of the crystalline phase (Lc') by pre-incubation at 4°C produced a wavelength dependence of anisotropy which was strong at 15 and 25°C, weak at 38°C, and minimal above the main transition (>~41.5°C) or after returning the temperature from 46 to 25°C. The profile of anisotropy values across this temperature range revealed the ability of the probe to detect crystalline, gel (Lß'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pß') and gel phases. In contrast, the shape of the emission spectrum, quantified as the ratio of merocyanine monomer and dimer peaks (585 and 621 nm), was primarily sensitive to the crystalline and gel phases because dimer fluorescence requires a highly-ordered environment. This requirement also explained the diminution of anisotropy wavelength dependence above 25°C. Repetition of experiments with vesicles containing cholesterol allowed creation of a phase map. Superimposition of data from the three simultaneous measurements provided details about the various phase regions in the map not discernible from any one of the three alone. The results were applied to assessment of calcium-induced membrane changes in living cells.PACS Codes: 87.16.dt.

Use of fluorescence to determine the effects of cholesterol on lipid behavior in sphingomyelin liposomes and erythrocyte membranes.

The purpose of this study was to generate the equivalent of a cholesterol/temperature phase map for a biological membrane using fluorescence spectroscopy. The pseudo-phase map was created using human erythrocytes treated with various concentrations of methyl-beta-cyclodextrin to remove defined amounts of cholesterol and a trio of fluorescent probes that assess different membrane properties (laurdan, diphenylhexatriene, and merocyanine 540). Parallel experiments with two-photon microscopy suggested that changes in cellular cholesterol content affected the entire membrane rather than being localized to specific macroscopic domains. The various regions of the composite erythrocyte pseudo-phase map were interpreted using analogous data acquired from multilamellar vesicles that served as simplified models of cholesterol-dependent phases. The vesicles consisted of various concentrations of cholesterol (0 to 50 mol%) with either palmitoyl sphingomyelin, 1:1 dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine, or phospholipid mixtures intended to simulate either the inner or outer leaflet of erythrocyte membranes. Four distinguishable regions were observed in sphingomyelin phase maps corresponding to the traditional solid-ordered and liquid-disordered phases and two types of liquid-ordered behavior. Physical properties were less diverse in the mixed phospholipid vesicles, as expected, based on previous studies. Erythrocytes displayed five regions of different combinations of membrane properties along the phase map. Some of the observations identified similarities between the cells and liquid-ordered behavior observed in the various types of liposomes as well as some interesting differences.

Differential detection of phospholipid fluidity, order, and spacing by fluorescence spectroscopy of bis-pyrene, prodan, nystatin, and merocyanine 540.

The properties of liquid-ordered, solid-ordered, and liquid-disordered phases were investigated by steady-state fluorescence spectroscopy in liposomes composed of mixtures of dipalmitoylphosphatidylcholine and cholesterol (0-40 mol %) as a function of temperature (24-51 degrees C). The fluorescent probes used (bis-pyrene, nystatin, prodan, and merocyanine) were chosen because they differ in the location they occupy in the membrane and in the types of properties they sense. Comparison of phase diagrams with contour plots of the fluorescence data suggested that bis-pyrene is sensitive primarily to lipid order. In contrast, nystatin fluorescence intensity responded to changes in lipid fluidity. The shape of the prodan emission spectrum detected both liquid-solid and order-disorder transitions in the phase diagram. Merocyanine's behavior was more complex. First, it was more sensitive than any of the other probes to the membrane pretransition that occurs in the absence of cholesterol. Second, regardless of whether emission intensity, anisotropy, or spectral shape was observed, the probe appeared to distinguish two types of liquid-ordered phases, one with tightly packed lipids and one in which the apparent spacing among lipids was increased. The prodan data supported these results by displaying modest versions of these two observations. Together, the results identify eight regions within the phase diagram of distinguishable combinations of these physical properties. As an example of how this combined analysis can be applied to biological membranes, human erythrocytes were treated similarly. Temperature variation at constant cholesterol content revealed three of the eight combinations identified in our analysis of liposomes.

Mechanisms governing the level of susceptibility of erythrocyte membranes to secretory phospholipase A2.

Although cell membranes normally resist the hydrolytic action of secretory phospholipase A(2) (sPLA(2)), they become susceptible during apoptosis or after cellular trauma. Experimentally, susceptibility to the enzyme can be induced by loading cells with calcium. In human erythrocytes, the ability of the calcium ionophore to cause susceptibility depends on temperature, occurring best above approximately 35 degrees C. Considerable evidence from experiments with artificial bilayers suggests that hydrolysis of membrane lipids requires two steps. First, the enzyme adsorbs to the membrane surface, and second, a phospholipid diffuses from the membrane into the active site of the adsorbed enzyme. Analysis of kinetic experiments suggested that this mechanism can explain the action of sPLA(2) on erythrocyte membranes and that temperature and calcium loading promote the second step. This conclusion was further supported by binding experiments and assessment of membrane lipid packing. The adsorption of fluorescent-labeled sPLA(2) was insensitive to either temperature or ionophore treatment. In contrast, the fluorescence of merocyanine 540, a probe sensitive to lipid packing, was affected by both. Lipid packing decreased modestly as temperature was raised from 20 to 60 degrees C. Calcium loading enhanced packing at temperatures in the low end of this range, but greatly reduced packing at higher temperatures. This result was corroborated by measurements of the rate of extraction of a fluorescent phosphatidylcholine analog from erythrocyte membranes. Furthermore, drugs known to inhibit susceptibility in erythrocytes also prevented the increase in phospholipid extraction rate. These results argue that the two-step model applies to biological as well as artificial membranes and that a limiting step in the hydrolysis of erythrocyte membranes is the ability of phospholipids to migrate into the active site of adsorbed enzyme.