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. Flow cytometry, microscopy, and fluorescence spectroscopy were used to assess merocyanine 540 emission, laurdan generalized polarization, phosphatidylserine exposure, caspase activation, and membrane permeability to propidium iodide in the absence and presence of sPLA(2). The earliest event observed was activation of cellular caspases. Results with membrane probes suggest that interlipid spacing also increases early during apoptosis and precedes transbilayer migration of phosphatidylserine, DNA fragmentation, and a general increase in lipid order associated with blebbing and dissolution of the cells. The activity of sPLA(2) appeared to be linked more to lipid spacing than to loss of membrane asymmetry. The early nature of some of these events and their ability to promote activity of a proinflammatory enzyme suggests the possibility of an inflammatory response during T-lymphocyte 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. Analysis of these results suggested that susceptibility during calcium-induced apoptosis is limited by availability of substrate rather than adsorption of enzyme. Fluorescence experiments identified three membrane alterations during apoptosis that might affect substrate access to the sPLA(2) active site. First, intercalation of merocyanine 540 into the membrane was improved, suggesting an increase in lipid spacing. Second, laurdan detected increased solvation of the lower headgroup region of the membrane. Third, the rate at which fluorescent lipids could be removed from the membrane by albumin was enhanced, implying greater vertical mobility of phospholipids. Thus, it is proposed that the membranes of apoptotic cells become susceptible to sPLA(2) through a reduction in lipid-neighbor interactions that facilitates migration of phospholipids into the enzyme active site.

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-beta-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.

Female students are less likely to participate in class than male peers in life sciences courses of all levels, but they are more likely to participate in classrooms with more female peers. Female students earn lower final course grades than males in classes taught by a male instructor and/or when female students are in the minority.

A course format with a creative grading scheme and weekly formative midterms produced large gains in student success with test items requiring higher-order cognitive skills. This format is designed to provide multiple opportunities to attempt demanding problems on exams, immediate feedback, and incentives to improve.

Accumulating evidence implicates the voltage-dependent anion channel (VDAC) as functioning in mitochondria-mediated apoptosis involving cytochrome c release, leading to caspases activation and apoptosis. The mechanisms regulating cytochrome c release and the molecular architecture of the cytochrome c conducting channel remain unknown. Previously, we demonstrated that apoptosis induction was accompanied by VDAC oligomerization, as revealed by cross-linking and directly monitored in living cells using Bioluminescence Resonance Energy Transfer technology. Moreover, apoptosis inhibitors inhibited VDAC oligomerization and a correlation between the levels of VDAC oligomerization and apoptosis was observed. Here, we combined sitedirected mutagenesis with chemical cross-linking to reveal the contact sites between VDAC1 molecules in dimers and higher oligomers. Replacing hydrophobic amino acids with charged amino acids in b-strands 1,2 and 19, but not 14, interfered with VDAC1 oligomerization and apoptosis induction. Cysteine cross-linking results, from introducing cysteine at a defined position in cysteineless VDAC1 and applying the cysteine-specific cross-linker, BMOE, supported the close vicinity of b-strands 1,2 and 19 in VDAC1 dimer. Moreover, the results suggest that VDAC1 exists as a dimer that undergoes conformational changes upon apoptosis induction to assemble into a higher oligomeric state. Additionally we demonstrated that the N-terminal region of VDAC1 lies inside the pore, but could also move and interact with the N-terminus from a second molecule to form a dimer. Our results suggest that the glycine rich sequence 21-GYGFG-25 is involved in the N-terminus translocation from the internal pore to the channel face. These results provide structural insight into cellular VDAC1's oligomeric state and its N-terminal region location and translocation.

The effectiveness of teaching Punnett squares as a calculation aid for Hardy-Weinberg equilibrium with a quasi-experimental design was tested. On its own, this aid may allow for increased calculation success, and it may increase mastery of equation derivation when taught before the equations. Benefits for students with math anxiety are discussed.

Despite extensive scientific research supporting the safety and effectiveness of approved vaccines, debates about their use continue in the public sphere. A paper prominently circulated on social media concluded that countries requiring more infant vaccinations have higher infant mortality rates (IMR), which has serious public health implications. However, inappropriate data exclusion and other statistical flaws in that paper merit a closer examination of this correlation. We re-analyzed the original data used in Miller and Goldman's study to investigate the relationship between vaccine doses and IMR. We show that the sub-sample of 30 countries used in the original paper was not a random sample from the entire dataset, and the correlation coefficient of 0.49 reported in that study is virtually impossible without data manipulation. Next, we show IMR as a function of countries' actual vaccination rates, rather than vaccination schedule, and show a strong negative correlation between vaccination rates and IMR. Finally, we analyze United States IMR data as a function of Hepatitis B vaccination rate to show an example of increased vaccination rates corresponding with reduced infant death over time. From our analyses, it is clear that vaccination does not predict higher IMR as previously reported.