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1.  Molecular Details of Membrane Fluidity Changes during Apoptosis and Relationship to Phospholipase A2 Activity 
Biochimica et biophysica acta  2012;1828(2):887-895.
Summary
Secretory phospholipase A2 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. Alterations in emission properties of these probes corresponded with enhanced susceptibility of the cells to hydrolysis by secretory phospholipase A2. By applying a quantitative model, additional information was extracted from the kinetics of Patman equilibration with the membrane. Taken together, these data suggested that the phospholipids of apoptotic membranes display greater spacing between adjacent headgroups, reduced interactions between neighboring lipid tails, and increased penetration of water among the heads. The phase transition of artificial bilayers was used to calibrate quantitatively the relationship between probe fluorescence and the energy of interlipid interactions. This analysis was applied to results from apoptotic cells to estimate the frequency with which phospholipids protrude sufficiently at the membrane surface to enter the enzyme’s active site. The data suggested that this frequency increases 50–100-fold as membranes become susceptible to hydrolysis during apoptosis.
doi:10.1016/j.bbamem.2012.08.024
PMCID: PMC3529823  PMID: 22967861
two-photon microscopy; merocyanine 540; Laurdan; hydrolysis kinetics; Patman; diphenylhexatriene
2.  Membrane Properties Involved in Calcium-Stimulated Microparticle Release from the Plasma Membranes of S49 Lymphoma Cells 
The Scientific World Journal  2014;2014:537192.
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. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32–42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes.
doi:10.1155/2014/537192
PMCID: PMC3918691  PMID: 24578641
3.  Investigation into the Role of Phosphatidylserine in Modifying the Susceptibility of Human Lymphocytes to Secretory Phospholipase A2 using Cells Deficient in the Expression of Scramblase 
Biochimica et Biophysica Acta  2012;1818(5):1196-1204.
Summary
Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 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. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.
doi:10.1016/j.bbamem.2012.01.005
PMCID: PMC3319224  PMID: 22266334
4.  Synergistic Effects of Secretory Phospholipase A2 from the Venom of Agkistrodon piscivorus piscivorus with Cancer Chemotherapeutic Agents 
BioMed Research International  2012;2013:565287.
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. This idea was tested with S49 lymphoma cells and a broad range of antineoplastic drugs: methotrexate, daunorubicin, actinomycin D, and paclitaxel. In each case, a substantial population of treated cells was still alive yet vulnerable to attack by the enzyme. Induction of cell death by these agents also perturbed the biophysical properties of the membrane as detected by merocyanine 540 and trimethylammonium-diphenylhexatriene. These results suggest that exposure of lymphoma cells to these drugs universally causes changes to the cell membrane that render it susceptible to enzymatic attack. The data also argue that the snake venom enzyme is not only capable of clearing cell corpses but can aid in the demise of tumor cells that have initiated but not yet completed the death process.
doi:10.1155/2013/565287
PMCID: PMC3591165  PMID: 23509743
5.  Relationship between Membrane Permeability and Specificity of Human Secretory Phospholipase A2 Isoforms during Cell Death 
Biochimica et biophysica acta  2011;1808(7):1913-1920.
Summary
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 A2 (sPLA2). 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. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to sPLA2. The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various sPLA2 isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group × enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for sPLA2 and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells.
doi:10.1016/j.bbamem.2011.04.003
PMCID: PMC3102113  PMID: 21510917
6.  Lipid-mediated unfolding of 3-beta hydroxysteroid dehydrogenase2 is essential for steroidogenic activity 
Biochemistry  2011;50(51):11015-11024.
For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, their activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3-beta 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 (DHEA) to androstenedione. Conversion requires that 3βHSD2 serves as both a dehydrogenase and isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using Fluorescence Resonance Energy Transfer (FRET), we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (Tm), from 42° C to 37° C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without affecting the β-sheet conformation. With the exception of the N-terminal 20 amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.
doi:10.1021/bi2016102
PMCID: PMC3399592  PMID: 22106846
7.  Fluorescence anisotropy of diphenylhexatriene and its cationic Trimethylamino derivative in liquid dipalmitoylphosphatidylcholine liposomes: opposing responses to isoflurane 
BMC Biophysics  2012;5:5.
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.
Results
In liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount.
Conclusions
The anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.
doi:10.1186/2046-1682-5-5
PMCID: PMC3359235  PMID: 22444827
8.  Rethinking Exams and Letter Grades: How Much Can Teachers Delegate to Students? 
CBE— Life Sciences Education  2006;5(3):270-280.
In this article we report a 3-yr study of a large-enrollment Cell Biology course focused on developing student skill in scientific reasoning and data interpretation. Specifically, the study tested the hypothesis that converting the role of exams from summative grading devices to formative tools would increase student success in acquiring those skills. Traditional midterm examinations were replaced by weekly assessments administered under test-like conditions and followed immediately by extensive self, peer, and instructor feedback. Course grades were criterion based and derived using data from the final exam. To alleviate anxiety associated with a single grading instrument, students were given the option of informing the grading process with evidence from weekly assessments. A comparative analysis was conducted to determine the impact of these design changes on both performance and measures of student affect. Results at the end of each year were used to inform modifications to the course in subsequent years. Significant improvements in student performance and attitudes were observed as refinements were implemented. The findings from this study emphasized the importance of prolonging student opportunity and motivation to improve by delaying grade decisions, providing frequent and immediate performance feedback, and designing that feedback to be maximally formative and minimally punitive.
doi:10.1187/cbe.05-11-0123
PMCID: PMC1618686  PMID: 17012219
9.  Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers 
PMC Biophysics  2010;3:14.
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 (Lβ'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pβ') 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
doi:10.1186/1757-5036-3-14
PMCID: PMC2993649  PMID: 21054864
10.  Cloning the Professor, an Alternative to Ineffective Teaching in a Large Course 
CBE Life Sciences Education  2009;8(3):252-263.
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. Data are presented that demonstrate the positive impact of these innovations on student performance and affect.
doi:10.1187/cbe.09-01-0006
PMCID: PMC2736028  PMID: 19723819
11.  Teaching Cell Biology in the Large-Enrollment Classroom: Methods to Promote Analytical Thinking and Assessment of Their Effectiveness 
Cell Biology Education  2003;2:180-194.
A large-enrollment, undergraduate cellular biology lecture course is described whose primary goal is to help students acquire skill in the interpretation of experimental data. The premise is that this kind of analytical reasoning is not intuitive for most people and, in the absence of hands-on laboratory experience, will not readily develop unless instructional methods and examinations specifically designed to foster it are employed. Promoting scientific thinking forces changes in the roles of both teacher and student. We describe didactic strategies that include directed practice of data analysis in a workshop format, active learning through verbal and written communication, visualization of abstractions diagrammatically, and the use of ancillary small-group mentoring sessions with faculty. The implications for a teacher in reducing the breadth and depth of coverage, becoming coach instead of lecturer, and helping students to diagnose cognitive weaknesses are discussed. In order to determine the efficacy of these strategies, we have carefully monitored student performance and have demonstrated a large gain in a pre- and posttest comparison of scores on identical problems, improved test scores on several successive midterm examinations when the statistical analysis accounts for the relative difficulty of the problems, and higher scores in comparison to students in a control course whose objective was information transfer, not acquisition of reasoning skills. A novel analytical index (student mobility profile) is described that demonstrates that this improvement was not random, but a systematic outcome of the teaching/learning strategies employed. An assessment of attitudes showed that, in spite of finding it difficult, students endorse this approach to learning, but also favor curricular changes that would introduce an analytical emphasis earlier in their training.
doi:10.1187/cbe.02-11-0055
PMCID: PMC192442  PMID: 14506506
Rasch analysis; item response theory; student outcome; student attitude; student confidence
12.  The influence of membrane physical properties on microvesicle release in human erythrocytes 
PMC Biophysics  2009;2:7.
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. Microvesicle release was enhanced by increasing the experimental temperature. Reduction in membrane cholesterol content by treatment with methyl-β-cyclodextrin also facilitated vesicle shedding. Inhibition of scramblase with R5421 impaired vesicle release. These data were interpreted in the context of membrane characteristics assessed previously by fluorescence spectroscopy with environment-sensitive probes such as laurdan, diphenylhexatriene, and merocyanine 540. The observations supported the following conclusions: 1) calcium-induced microvesicle shedding in erythrocytes relates more to membrane properties detected by diphenylhexatriene than by the other probes; 2) loss of trans-membrane phospholipid asymmetry is required for microvesicle release.
PACS Codes: 87.16.dj, 87.16.dt
doi:10.1186/1757-5036-2-7
PMCID: PMC2739839  PMID: 19703298

Results 1-12 (12)