Assessing mitochondrial outer membrane permeabilization during apoptosis

Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.     

Stability of division-related protein and nucleic acid fractions in synchronized Tetrahymena

The kinetics of RNA synthesis have been studied under various growth conditions using synchronized Tetrahymena pyriformis GL. The curves obtained are approximately linear in growth-supporting media but vary under other circumstances. The presence of amino acids in the medium stimulates RNA synthesis in Tetrahymena as it does in bacteria. Since these induced variations do not influence the essential features of the pre-cytokinetic period it is inferred that fluctuations in the rate of RNA synthesis described by others are probably not essential features of temperature-induced synchrony. In addition, the translational stability of division-related templates at synchronizing temperatures has been investigated. The synthesis of proteins necessary for cytokinesis is shown to be greatly reduced under conditions simulating a heat-shock. Inhibitor studies using cycloheximide indicate that protein synthesis is required longer into the cytokinetic phase than had previously been thought. Collectively the data are all compatible with the hypothesis that synthesis of division-related proteins is drastically reduced in heat-shocked Tetrahymena and that the basis for this reduction is hydrolysis of template RNA without concurrent translation.     

Understanding the High Prevalence of HIV and Other Sexually Transmitted Infections among Socio-Economically Vulnerable Men Who Have Sex with Men in Jamaica

ObjectivesThis study estimates HIV prevalence among men who have sex with men (MSM) in Jamaica and explores social determinants of HIV infection among MSM.DesignAn island-wide cross-sectional survey of MSM recruited by peer referral and outreach was conducted in 2011. A structured questionnaire was administered and HIV/STI tests done. We compared three groups: MSM who accepted cash for sex within the past 3 months (MSM SW), MSM who did not accept cash for sex (MSM non-SW), and MSM with adverse life events (ever raped, jailed, homeless, victim of violence or low literacy).ResultsHIV prevalence among 449 MSM was 31.4%, MSM SW 41.1%, MSM with adverse life events 38.5%, 17 transgender MSM (52.9%), and MSM non-SW without adverse events 21.0%. HIV prevalence increased with age and number of adverse life events (test for trend P < 0.001), as did STI prevalence (P = 0.03). HIV incidence was 6.7 cases/100 person-years (95% CI: 3.74, 12.19). HIV prevalence was highest among MSM reporting high-risk sex; MSM SW who had been raped (65.0%), had a STI (61.2%) and who self identified as female (55.6%). Significant risk factors for HIV infection common to all 3 subgroups were participation in both receptive and insertive anal intercourse, high-risk sex, and history of a STI. Perception of no or little risk, always using a condom, and being bisexual were protective.ConclusionHIV prevalence was high among MSM SW and MSM with adverse life events. Given the characteristics of the sample, HIV prevalence among MSM in Jamaica is probably in the range of 20%. The study illustrates the importance of social vulnerability in driving the HIV epidemic. Programs to empower young MSM, reduce social vulnerability and other structural barriers including stigma and discrimination against MSM are critical to reduce HIV transmission.     

Contact‐induced apical asymmetry drives the thigmotropic responses of Candida albicans hyphae

Filamentous hyphae of the human pathogen, Candida albicans, invade mucosal layers and medical silicones. In vitro, hyphal tips reorient thigmotropically on contact with small obstacles. It is not known how surface topography is sensed but hyphae lacking the cortical marker, Rsr1/Bud1, are unresponsive. We show that, on surfaces, the morphology of hyphal tips and the position of internal polarity protein complexes are asymmetrically skewed towards the substratum and biased towards the softer of two surfaces. In nano‐fabricated chambers, the Spitzenkörper (Spk) responded to touch by translocating across the apex towards the point of contact, where its stable maintenance correlated with contour‐following growth. In the rsr1Δ mutant, the position of the Spk meandered and these responses were attenuated. Perpendicular collision caused lateral Spk oscillation within the tip until after establishment of a new growth axis, suggesting Spk position does not predict the direction of growth in C. albicans. Acute tip reorientation occurred only in cells where forward growth was countered by hyphal friction sufficient to generate a tip force of ～ 8.7 μN (1.2 MPa), more than that required to penetrate host cell membranes. These findings suggest mechanisms through which the organization of hyphal tip growth in C. albicans facilitates the probing, penetration and invasion of host tissue.     

Biophysical properties of normal and diseased renal glomeruli

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.     

Relative resistance of early memory cells in mice primed with sheep erythrocytes to cytotoxic effects of anti-theta serum

A series of human fetal and adult thymuses was fractionated on discontinuous gradients of bovine serum albumin. Fetal thymocytes were 10 times as mitotically active as equivalent adult cells. The predominating cell in the youngest fetus (11.0 cm CR length) was found in the less dense layers of the gradient while the predominating adult cell type was in the more dense layers. As the fetus and infant mature, there is a gradual shift from the fetal to the adult gradient profile.     

oxLDL facilitates flow-induced realignment of aortic endothelial cells

Alignment of vascular endothelial cells (ECs) in the direction of the flow is considered a key factor in maintaining endothelial integrity in an active hemodynamic environment. Our recent studies showed that exposure to oxidized LDL (oxLDL), one of the major proatherogenic lipoproteins, significantly increases the stiffness of human aortic ECs, suggesting that oxLDL may have a significant impact on the sensitivity of ECs to mechanical stimuli. In this study, we show that oxLDL strongly enhances the ability of ECs to realign in the direction of the flow and facilitates the formation of F-actin stress fibers under static and flow conditions. The impact of oxLDL on the flow-induced realignment is observed on whole cell and single-fiber levels. We also show that, similar to the effect of oxLDL on endothelial stiffness, the impact of oxLDL on flow-induced realignment can be simulated by methyl-beta-cyclodextrin-induced cholesterol depletion, supporting the hypothesis that oxLDL acts as cholesterol acceptor, rather than cholesterol donor, for ECs. Finally, we propose that oxLDL/cholesterol depletion-induced sensitization of ECs to flow may be a result of an increase in cellular stiffness and a respective increase in membrane-cytoskeleton tension.