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In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

High‐field (11.7 and 14 T) proton high‐resolution NMR spectra of apple juices obtained from a variety of cultivars are reported and partial spectral assignments are made. There are significant spectral differences between cultivars, which may be of value in identifying the origins of apple juices. The results also indicate that the method is likely to be of value in the authentication of fruit juices. Careful spectral analysis shows that some differences arise simply as a result of the differences in the pH of the juices and that microbiological and oxidative effects must be taken into account. Care must therefore be exercised in the application of multivariate methods to the data as spurious or trivial correlations may be obtained. It is concluded that the richness of the spectra and the ease with which they may be obtained indicate that high‐field proton NMR will prove valuable not only in speciation and authentication studies, but also in the analysis of biochemical changes occurring in fruits and their juices. © 1997 John Wiley & Sons, Ltd.

BackgroundPlasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated.MethodsConcentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group.ResultsAnaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance.ConclusionsThese findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

We present Defocusing Microscopy (DM), a bright-field optical microscopy technique able to perform total 3D imaging of transparent objects. By total 3D imaging we mean the determination of the actual shapes of the upper and lower surfaces of a phase object. We propose a new methodology using DM and apply it to red blood cells subject to different osmolality conditions: hypotonic, isotonic and hypertonic solutions. For each situation the shape of the upper and lower cell surface-membranes (lipid bilayer/cytoskeleton) are completely recovered, displaying the deformation of RBCs surfaces due to adhesion on the glass-substrate. The axial resolution of our technique allowed us to image surface-membranes separated by distances as small as 300 nm. Finally, we determine volume, superficial area, sphericity index and RBCs refractive index for each osmotic condition.Standard optical microscopy imaging of phase objects is generally obtained with microscopes operating in Phase Contrast or Nomarsky configurations 1,2 . However, even for objects with uniform refractive index these techniques present difficulties for obtaining accurate full-field thickness profiles. Recently, new approaches known as quantitative phase microscopy techniques 3-7 , have adequately obtained full-field height profile of phase objects with successful application in living cells. Despite that, in the case of red blood cells (RBCs) these techniques are only able to provide the thickness profile and thickness amplitude fluctuations, such that height profile information of each particular cell surface-membrane (lipid bilayer/cytoskeleton) is not accessed.Using Defocusing Microscopy (DM), we present a new method able to perform total 3D imaging of RBC, and thus, capable of determining the actual shape of the cell's upper and lower surface-membranes, separately. New procedures to retrieve RBC volume, superficial area, sphericity index and refractive index using DM are also exposed. All developed methods are applied to data of 25 RBCs immersed in three different solutions: hypotonic (200 mOsm/kg), isotonic (300 mOsm/kg) and hypertonic (400 mOsm/kg), showing the differences in the lower membrane adhesion to the glass substrate. For assessment of bio-mechanical properties along the RBCs surfaces, nanometer height fluctuations for each interface can be obtained separately, such that the effect of adhesion to the substrate can also be evaluated 8,9 . A defocusing technique has been recently applied for 3D imaging of cells using a phase contrast microscope under white-light illumination, with transverse resolution of 350 nm and axial resolution of 900 nm 10 . This technique cannot resolve surface-membranes separated by an axial distance smaller than 900 nm, which is the case of most RBCs. Strikingly, our Defocusing Microscopy technique, using a bright-field defocused microscope and our a) E-mail me at: mesquita@fisica.ufmg.br theoretical framework, can resolve surface-membranes of RBCs separated by axial distances down to 300 nm.Transparent objects that would be...