Examining the effects of cholesterol on model membranes at high temperatures: Laurdan and Patman see it differently.

At high temperature, the presence of cholesterol in phospholipid membranes alters the influence of membrane dipoles, including water molecules, on naphthalene-based fluorescent probes such as Laurdan and Patman (solvatochromism). Although both of these probes report identical changes to their emission spectra as a function of temperature in pure phosphatidylcholine bilayers, they differ in their response to cholesterol. Computer simulations of the spectra based on a simple model of solvatochromism indicated that the presence of cholesterol reduces the probability of bilayer dipole relaxation and also blunts the tendency of heat to enhance that probability.

Promoting mastery of complex biological mechanisms.

This article describes efforts aimed at improving comprehension and retention of complex molecular mechanisms commonly studied in undergraduate biology and biochemistry courses. The focus is on the design of appropriate assessments, an active classroom emphasizing formative practice, and more effective out-of-class study habits. Assessments that require students to articulate their understanding through writing are the most effective.

Ultraslow dynamics of a complex amphiphile within the phospholipid bilayer: Effect of the lipid pre-transition.

The shape and intensity of fluorescence emission spectra of Merocyanine 540 embedded in dipalmitoylphosphatidylcholine bilayers differ depending on the thermal history of the sample. This apparent hysteresis in fluorescence emission was most prominent in the temperature range of 20 to 35°C. Analysis of kinetic and temperature cycling experiments suggested that Merocyanine 540 slowly (half time of about 30min) assumes a metastable configuration as temperature is raised above the phospholipid pre-transition point.

Promoting the Multidimensional Character of Scientific Reasoning.

This study reports part of a long-term program to help students improve scientific reasoning using higher-order cognitive tasks set in the discipline of cell biology. This skill was assessed using problems requiring the construction of valid conclusions drawn from authentic research data. We report here efforts to confirm the hypothesis that data interpretation is a complex, multifaceted exercise.

Relationships between membrane water molecules and Patman equilibration kinetics at temperatures far above the phosphatidylcholine melting point.

The naphthalene-based fluorescent probes Patman and Laurdan detect bilayer polarity at the level of the phospholipid glycerol backbone. This polarity increases with temperature in the liquid-crystalline phase of phosphatidylcholines and was observed even 90°C above the melting temperature. This study explores mechanisms associated with this phenomenon.

Ionomycin causes susceptibility to phospholipase A2 while temperature-induced increases in membrane fluidity fail: possible involvement of actin fragmentation.

A diminution in the order of membrane lipids, which occurs during apoptosis, has been shown to correlate with increased membrane susceptibility to hydrolysis by secretory phospholipase A2. Studies with artificial membranes, however, have demonstrated that the relationship between membrane order and hydrolysis is more complex than suggested thus far by cell studies. To better resolve this relationship, this study focused on comparisons between increasing temperature and calcium ionophore as means of decreasing membrane order in S49 cells.

Membrane properties involved in calcium-stimulated microparticle release from the plasma membranes of S49 lymphoma cells.

This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current.

Synergistic effects of secretory phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus with cancer chemotherapeutic agents.

Healthy cells typically resist hydrolysis catalyzed by snake venom secretory phospholipase A2. However, during various forms of programmed cell death, they become vulnerable to attack by the enzyme. This observation raises the question of whether the specificity of the enzyme for dying cells could be used as a strategy to eliminate tumor cells that have been intoxicated but not directly killed by chemotherapeutic agents.

Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells.

The membranes of healthy lymphocytes normally resist hydrolysis by secretory phospholipase A(2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface.

Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A(2) activity.

Secretory phospholipase A(2) exhibits much greater activity toward apoptotic versus healthy cells. Various plasma membrane changes responsible for this phenomenon have been proposed, including biophysical alterations described as "membrane fluidity" and "order." Understanding of these membrane perturbations was refined by applying studies with model membranes to fluorescence measurements during thapsigargin-induced apoptosis of S49 cells using probes specific for the plasma membrane: Patman and trimethylammonium-diphenylhexatriene.

Wavelength dependence of patman equilibration dynamics in phosphatidylcholine bilayers.

Assessment of the equilibration kinetics of Patman at the edges of its emission spectra provided additional insights about membrane properties beyond those obtained from end-point fluorescence measurements. Upon introduction of the probe to aqueous suspensions of liposomes, the emission intensity slowly increased about 10-fold (t(½)=~100 s). The rate of equilibration depended on emission wavelength, and was usually faster at 500 than at 435 nm.

Fluorescence anisotropy of diphenylhexatriene and its cationic Trimethylamino derivative in liquid dipalmitoylphosphatidylcholine liposomes: opposing responses to isoflurane.

Background: The mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature.

Development of an instrument for measuring self-efficacy in cell biology.

This article describes the development of a ten-item scale to assess biology majors' self-efficacy towards the critical thinking and data analysis skills taught in an upper-division cell biology course. The original seven-item scale was expanded to include three additional items based on the results of item analysis. Evidence of reliability and validity was collected and reported for the revised scale.

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin.

Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity.

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase.

Relationship between membrane permeability and specificity of human secretory phospholipase A(2) isoforms during cell death.

During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A(2). The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death.

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.

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.

Cloning the professor, an alternative to ineffective teaching in a large course.

Pedagogical strategies have been experimentally applied in large-enrollment biology courses in an attempt to amplify what teachers do best in effecting deep learning, thus more closely approximating a one-on-one interaction with students. Carefully orchestrated in-class formative assessments were conducted to provide frequent, high-quality feedback that allows students to accurately diagnose the current state of their understanding of fundamental biological concepts and make specific plans to remedy any deficiencies. Teachers can also assume responsibility to guide out-of-class study among classmates by promoting Elaborative Questioning, an inquiry exchange that permits misconceptions to be identified and corrected and that promotes long-lasting metacognitive and analytical thinking skills.

The influence of membrane physical properties on microvesicle release in human erythrocytes.

Exposure of human erythrocytes to elevated intracellular calcium causes fragments of the cell membrane to be shed as microvesicles. This study tested the hypothesis that microvesicle release depends on microscopic membrane physical properties such as lipid order, fluidity, and composition. Membrane properties were manipulated by varying the experimental temperature, membrane cholesterol content, and the activity of the trans-membrane phospholipid transporter, scramblase.

Sequence of physical changes to the cell membrane during glucocorticoid-induced apoptosis in S49 lymphoma cells.

During apoptosis, physical changes in the plasma membrane prepare the cell for clearance by phagocytes and hydrolysis by secretory phospholipase A(2) (sPLA(2)). The relationships among these changes have not been adequately established, especially for hormone-stimulated apoptosis. This study addresses these issues for glucocorticoid-induced apoptosis in S49 lymphoma cells.

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.

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2.

The ability of secretory phospholipase A(2) (sPLA(2)) to hydrolyze cell membranes is highly dependent on the physical properties of the membrane. The effects of cholesterol on these properties have been characterized in artificial bilayers and found to alter sPLA(2) activity significantly. It is hypothesized that the natural difference in cholesterol content between erythrocytes and leukocytes is in part responsible for their differing susceptibility to hydrolysis by sPLA(2).

Quantification of DeQi sensation by visual analog scales in healthy humans after immunostimulating acupuncture treatment.

Acupuncture is the most popular component of traditional Chinese medicine in Western countries. However, the mechanisms of its effects remain unclear. The therapeutic effect of acupuncture appears when a sensation of DeQi is achieved.

Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis.

During apoptosis, changes occur in lymphocyte membranes that render them susceptible to hydrolysis by secretory phospholipase A(2) (sPLA(2)). To study the relevant mechanisms, a simplified model of apoptosis using a calcium ionophore was applied. Kinetic and flow cytometry experiments provided key observations regarding ionophore treatment: the initial rate of hydrolysis was elevated at all enzyme concentrations, the total amount of reaction product was increased fourfold, and adsorption of the enzyme to the membrane surface was unaltered.