Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c-/- mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c-/- cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c-/- cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.     

Therapeutic potential of low-intensity ultrasound (part 2): biomolecular effects, sonotransfection, and sonopermeabilization

Part one of this review focused on the thermal and mechanical effects of low-intensity ultrasound (US). In this second and final part of the review, we will focus on and discuss various aspects of low-intensity US, with emphasis on the biomolecular effects, US-mediated gene transfection (sonotransfection), and US-mediated permeabilization (sonopermeabilization). Sonotransfection of different cell lines in vitro and target tissues in vivo have been reported. Optimization experiments have been done and different mechanisms investigated. It has also been found that several genes can be up-regulated or down-regulated by sonication. As to the potential therapeutic applications, systemic or local sonotransfection might also be a safe and effective gene therapy method in effecting the cure of local and systemic disorders. Gene regulation of target cells may be utilized in modifying cellular response to a treatment, such as increasing the sensitivity of diseased cells while making normal cells resistant to the side effects of a treatment. Advances in sonodynamic therapy and drug sonopermeabilization also offer an ever-increasing array of therapeutic options for low-intensity US.     

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.     

Interaction of Phosphatidylserine-Phosphatidylcholine Liposomes with Sickle Erythrocytes: EVIDENCE FOR ALTERED MEMBRANE SURFACE PROPERTIES

The sickle erythrocyte (RBC) is a pathologic RBC that contains multiple membrane abnormalities. Some of these abnormalities have been implicated in the pathophysiology of vasoocclusive crises characteristic of sickle cell disease; others have yet to be defined in terms of their clinical significance. Recent information has shown that sickle RBC adhere abnormally to cultured endothelial cells yet little is known about the ways in which sickle cells interact with model membranes of defined size and lipid composition. We investigated this phenomenon by interacting sickle RBC with artificial lipid vesicles (liposomes) containing acidic phospholipids. Our results demonstrate that sickle disease (hemoglobin SS) RBC bind more of these liposomes than do normal or sickle trait (hemoglobin AS) RBC and that these differences are accentuated by hypoxia-induced sickling. Binding of liposome phospholipid to sickled RBC was not attributable to phospholipid exchange between liposomes and RBC and was consistent with a mechanism involving both membrane fusion and a stable reversible adhesion of liposomes to the RBC membrane.Investigations into the mechanism(s) underlying increased liposome binding to sickled RBC suggested that the known reversible translocation of aminophospholipids, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE), from the inner to the outer leaflet of the reversibly sickled RBC (RSC) plasma membrane during sickling may be a component of increased liposome binding to RSC. This idea was supported from results of experiments in which normal RBC were treated with diamide resulting in the expression of outer leaflet PE and PS and a stimulation of liposome binding to these cells. However, sickle RBC separated according to cell density on stractan gradients showed that irreversibly sickled RBC (ISC) were less capable of liposome binding than were discoid RSC. Since ISC are known to contain elevated levels of outer leaflet aminophospholipids, such a result suggests that other changes in the plasma membrane of sickle cells, in addition to phospholipid reorganization, are probably involved in enhanced liposome binding to these cells. In other experiments, we showed that liposomes containing l-phenylalanine were capable of delivering this antisickling agent into intact sickle RBC as demonstrated by the partial inhibition of hypoxia-induced sickling in vitro. Our results suggest that liposomes can be used as sensitive probes for investigating changes in RBC membrane properties, especially those that affect intermembrane interactions, and that liposomal transport systems may have significant implications in the therapy of sickle cell disease.     

Over-Pressure Suppresses Ultrasonic-Induced Drug Uptake

Ultrasound (US) is used to enhance and target delivery of drugs and genes to cancer tissues. The present study further examines the role of acoustic cavitation in US-induced permeabilization of cell membranes and subsequent drug or gene uptake by the cell. Rat colon cancer cells were exposed to ultrasound at various static pressures to examine the hypothesis that oscillating bubbles, also known as cavitating bubbles, permeabilize cells. Increasing pressure suppresses bubble cavitation activity; thus, if applied pressure were to reduce drug uptake, cell permeabilization would be strongly linked to bubble cavitation activity. Cells were exposed to 476 kHz pulsed ultrasound at average intensities of 2.75 W/cm² and 5.5 W/cm² at various pressures and times in an isothermal chamber. Cell fractions with reversible membrane damage (calcein uptake) and irreversible damage (propidium iodide uptake) were analyzed by flow cytometry. Pressurization to 3 atm nearly eliminated the biological effect of US in promoting calcein uptake. Data also showed a linear increase in membrane permeability with respect to insonation time and intensity. This research shows that US-mediated cell membrane permeability is likely linked to cavitation bubble activity. (E-mail: pitt@byu.edu)     

In vivo externalization of phosphatidylserine and phosphatidylethanolamine in the membrane bilayer and hypercoagulability by the lipid peroxidation of erythrocytes in rats.

Phospholipid distribution across erythrocyte membrane bilayer is asymmetrical. In normal erythrocytes, entire phosphatidylserine (PS) and most of the phosphatidylethanolamine (PE) is present on the cytoplasmic side of membrane bilayer, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are predominantly present at the outer side of membrane bilayer. The present study was undertaken to determine whether membrane lipid peroxidation has any effect on the distribution of PS, PE, and PC across erythrocyte membrane bilayer in vivo in an animal model. Erythrocyte membrane lipid peroxidation was induced in rats by administering phenylhydrazine, an oxidant drug. Membrane phospholipid organization was determined by using bee venom phospholipase-A2 and indirectly by measuring clotting time on recalcification of normal human platelet-poor plasma in the presence of Russell's viper venom. Phenylhydrazine administration to rats caused significant membrane lipid peroxidation as measured by the accumulation of malonyldialdehyde (MDA), an end product of fatty acid peroxidation, as well as externalization of a significant portion of PS and PE from the inner to the outer side of membrane bilayer in erythrocytes. There was a significant positive correlation (r) between the amount of MDA accumulated in the erythrocytes and the movement of PS (r = 0.92) and PE (r = 0.96) from inner to the outer membrane bilayer and PC (r = 0.81) from outer to the inner membrane bilayer. Erythrocytes of phenylhydrazine-treated rats also showed significantly reduced clotting time. This reduction in clotting time had a significant positive correlation with MDA accumulation (r = 0.92) and PS externalization (r = 0.90). Both the effect of phenylhydrazine on erythrocyte membrane lipid peroxidation and alterations in phospholipid organization and coagulability were blocked when rats were simultaneously administered with vitamin E or C antioxidants.     

Effect of lipoprotein (a) on annexin A5 binding to cell membrane

BackgroundHigh blood lipoprotein (a) [Lp(a)] concentration is a risk factor for a thrombotic event. Annexin A5 is involved in anticoagulation on the endothelial surface. How Lp(a) affects the annexin A5 function is not clear. This study investigates annexin A5 binding on the cell membrane in the presence of Lp(a).MethodsLp(a) was isolated from human blood plasma by ultracentrifugation and annexin A5 protein was purchased commercially. The cell membrane was prepared from primary human umbilical vein endothelial cells (HUVEC) and cultured cell line HepG2 by sucrose density gradient centrifugation. Enzyme-linked immunosorbent assays (ELISA) were used to examine annexin A5 binding to the cell membrane in the presence of Lp(a). Flow cytometry was used to analyze the binding of fluorescence-labeled annexin A5 to phosphatidylserine (PS)-translocated intact cells in the presence of Lp(a).ResultsAnnexin A5 binding to the cell membrane was attenuated by a high concentration of Lp(a) in both HUVEC and HepG2 membrane surfaces. The phenomenon was also observed with annexin A5 surface labeling of HepG2 cells and flow cytometry analysis.ConclusionsThe results imply that Lp(a) interferes with annexin A5 binding to the procoagulant PS which translocates to the membrane surface under stress condition and therefore may increase the risk for thrombosis.     

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor,Histone H2B,to the macrophage surface

Summary. Background: Plasminogen (Plg) binding to cell surface Plg receptors (Plg‐Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg‐R and its cell surface expression is up‐regulated when monocytes are differentiated to macrophages via a pathway dependent on L‐type Ca²⁺ channels and intracellular Ca²⁺. Objectives: We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on the macrophage surface. Methods: THP‐1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outer membrane exposure of phosphatidylserine (PS). Results: H2B interacted directly with PS via an electrostatic interaction. Anti‐PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L‐type Ca²⁺ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions: These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L‐type Ca²⁺ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to.     

Increased platelet phosphatidylserine exposure and caspase activation in chronic uremia.

Platelet activation is associated with exposure of the aminophospholipid phosphatidylserine (PS) to the outer hemi-leaflet of the plasma membrane bilayer, which seems to be involved in the coagulation process. Because platelet activation may occur in patients suffering from chronic uremia, which is frequently associated with a thrombophilic tendency, we studied whether uremic platelets show an increased propensity to expose PS on the outer membrane leaflet and whether this process is linked with important functional and molecular changes. Flow cytometric percentage of annexin V-positive platelets, a measure of PS externalization, was significantly elevated (P < 0.001) in uremic patients when compared to normal controls under both unstimulated and agonist-stimulated conditions. Uremic platelet procoagulant activity, as measured by thrombin generation, was more than twice as high (4.13 +/- 0.3 micro mL(-1)) as that found in normal controls (1.86 +/- 0.2 micro mL(-1)). Two independent assays showed that the enzymatic activity of caspase-3, a protease involved in the loss of membrane PS asymmetry, was significantly greater in the platelets of uremic subjects than in those of healthy controls. PS exposure in agonist-stimulated platelets was markedly reduced by inhibition of caspase-3 activity but was not affected by inhibition of calpain activity. These results support the view that the thrombophilic susceptibility of uremic patients may be partly ascribed to increased PS exposure to the outer membrane leaflet of platelets. This process seems to be causally linked to an increase in caspase-3 activity, particularly during platelet activation.     

The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis.

The pathogenic role of antiendothelial cell antibodies (AECA) remains unclear. They are frequently associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS), which is difficult to reconcile with the distribution of PL molecular species within the plasma membrane. Since it is already known that PS is transferred to the outer face of the membrane as a preclude to apoptosis, the possibility exists that apoptosis is initiated by AECA. AECA-positive/anti-PL antibody-negative sera from eight patients with systemic sclerosis (SS) and 21 control patients were evaluated. Endothelial cells (EC) were incubated with AECA and the exposure of PS was established through the binding of annexin V. Hypoploid cell enumeration, DNA fragmentation, and optical and ultrastructural analyses of EC were used to confirm apoptosis. Incubation of EC with AECA derived from six of eight patients with SS led to the expression of PS on the surface of the cells. This phenomenon was significantly more frequent in SS (P < 0.04) than in control diseases. The redistribution of plasma membrane PS preceded other events associated with apoptosis: hypoploidy, DNA fragmentation, and morphology characteristic for apoptosis. Apoptosis-inducing AECA did not recognize the Fas receptor. We conclude that AECA may be pathogenic by inducing apoptosis.     

Agp2p,the Plasma Membrane Transregulator of Polyamine Uptake,Regulates the Antifungal Activities of the Plant Defensin NaD1 and Other Cationic Peptides

Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata, has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum. The resistance of agp2Δ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2Δ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.     

Purinergic Modulation of Interleukin-1β Release from Microglial Cells Stimulated with Bacterial Endotoxin

Microglial cells express a peculiar plasma membrane receptor for extracellular ATP, named P2Z/P2X₇ purinergic receptor, that triggers massive transmembrane ion fluxes and a reversible permeabilization of the plasma membrane to hydrophylic molecules of up to 900 dalton molecule weight and eventual cell death (Di Virgilio, F. 1995. Immunol. Today. 16:524–528). The physiological role of this newly cloned (Surprenant, A., F. Rassendren, E. Kawashima, R.A. North and G. Buell. 1996. Science (Wash. DC). 272:735–737) cytolytic receptor is unknown. In vitro and in vivo activation of the macrophage and microglial cell P2Z/P2X₇ receptor by exogenous ATP causes a large and rapid release of mature IL-1β. In the present report we investigated the role of microglial P2Z/P2X₇ receptor in IL-1β release triggered by LPS. Our data suggest that LPS-dependent IL-1β release involves activation of this purinergic receptor as it is inhibited by the selective P2Z/P2X₇ blocker oxidized ATP and modulated by ATP-hydrolyzing enzymes such as apyrase or hexokinase. Furthermore, microglial cells release ATP when stimulated with LPS. LPS-dependent release of ATP is also observed in monocyte-derived human macrophages. It is suggested that bacterial endotoxin activates an autocrine/paracrine loop that drives ATP-dependent IL-1β secretion.     

Characterization of a Novel Small Molecule That Potentiates β-Lactam Activity against Gram-Positive and Gram-Negative Pathogens

In a loss-of-viability screen using small molecules against methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 with a sub-MIC of a β-lactam, we found a small molecule, designated DNAC-1, which potentiated the effect of oxacillin (i.e., the MIC of oxacillin decreased from 64 to 0.25 μg/ml). Fluorescence microscopy indicated a disruption in the membrane structures within 15 min of exposure to DNAC-1 at 2× MIC. This permeabilization was accompanied by a rapid loss of membrane potential, as monitored by use of the DiOC₂ (3,3′-diethyloxacarbocyanine iodide) dye. Macromolecular analysis showed the inhibition of staphylococcal cell wall synthesis by DNAC-1. Transmission electron microscopy of treated MRSA USA300 cells revealed a slightly thicker cell wall, together with mesosome-like projections into the cytosol. The exposure of USA300 cells to DNAC-1 was associated with the mislocalization of FtsZ accompanied by the localization of penicillin-binding protein 2 (PBP2) and PBP4 away from the septum, as well as mild activation of the vraRS-mediated cell wall stress response. However, DNAC-1 does not have any generalized toxicity toward mammalian host cells. DNAC-1 in combination with ceftriaxone is also effective against an assortment of Gram-negative pathogens. Using a murine subcutaneous coinjection model with 10⁸ CFU of USA300 as a challenge inoculum, DNAC-1 alone or DNAC-1 with a sub-MIC of oxacillin resulted in a 6-log reduction in bacterial load and decreased abscess formation compared to the untreated control. We propose that DNAC-1, by exerting a bimodal effect on the cell membrane and cell wall, is a viable candidate in the development of combination therapy against many common bacterial pathogens.     

Bioeffects of Low‐Intensity Ultrasound In Vitro

Objective. The purpose of this study was to evaluate the potential molecular mechanism of low‐intensity ultrasound‐induced apoptosis by analyzing protein profile alteration in response to ultrasound exposure. Methods. Human hepatocarcinoma SMMC‐7721 cells were used in this study. Cell viability was measured by a trypan blue dye exclusion test. Morphologic changes were examined by light microscopy. Apoptosis was assessed by phosphatidylserine externalization and DNA fragmentation. The pattern of the mitochondrial membrane potential decrease was determined by flow cytometry. Protein profile alteration was analyzed by comparative proteomics based on 2‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Results. Low‐intensity ultrasound (3.0 W/cm², 1 minute, cells incubated for 6 hours after ultrasound exposure) induced early apoptosis (mean ± SD, 26.5% ± 6.2%) significantly (P < .05) with minimal lysis in human hepatocarcinoma cells in vitro. On a molecular level, several proteins, eg, cellular tumor antigen protein 53, BH3‐interacting domain death agonist, apoptosis regulator Bcl‐2, and heme oxygenase 1 were identified as responding to ultrasound irradiation, suggesting that mitochondrial dysfunction and oxidative stresses were involved in ultrasound‐induced apoptosis. It was also assumed that mitofilin‐regulated crista remodeling may be a potential channel of mitochondrial membrane permeabilization pore formation involved in low‐intensity ultrasound‐induced apoptosis. Conclusions. This study suggests that 2 potential molecular signaling pathways are involved in ultrasound‐induced apoptosis. It is a first step toward low‐intensity ultrasound‐induced apoptotic cancer therapy via understanding its relevant molecular signaling and key proteins.     

Translocation of silica nanospheres through giant unilamellar vesicles (GUVs) induced by a high frequency electromagnetic field

Membrane model systems capable of mimicking live cell membranes were used for the first time in studying the effects arising from electromagnetic fields (EMFs) of 18 GHz where membrane permeability was observed following exposure. A present lack of understanding of the mechanisms that drive such a rapid change in membrane permeabilization as well as any structural or dynamic changes imparted on biomolecules affected by high-frequency electromagnetic irradiation limits the use of 18 GHz EMFs in biomedical applications. A phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) labelled with a fluorescent marker 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DOPE) was used in constructing the giant unilamellar vesicles (GUVs). After three cycles of exposure, enhanced membrane permeability was observed by the internalisation of hydrophilic silica nanospheres of 23.5 nm and their clusters. All-atom molecular dynamics simulations of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes exposed to high frequency electric fields of different field strengths showed that within the simulation timeframe only extremely high strength fields were able to cause an increase in the interfacial water dynamics characterized by water dipole realignments. However, a lower strength, high frequency EMF induced changes of the water hydrogen bond network, which may contribute to the mechanisms that facilitate membrane permeabilization in a longer timeframe.

Membrane model systems capable of mimicking live cell membranes were used for the first time in studying the effects arising from electromagnetic fields (EMFs) of 18 GHz where membrane permeability was observed following exposure.     

Effects of griseofulvin on apoptosis through caspase-3- and caspase-9-dependent pathways in K562 leukemia cells: An in vitro study

Background: Griseofulvin, an oral nontoxic antifungal drug, has been reported to possess anticancer effect in human cancer cells, while the mechanisms are not completely understood.Objective: The aim of this study was to investigate the cytotoxic effect of griseofulvin on K562 cells and to understand its underlying molecular pathways.Methods: K562 cells were treated with griseofulvin at different concentrations for 24 hours, and the inhibition effect of griseofulvin on K562 cell proliferation was assessed by tetrazolium salt colorimetric assay. Apoptosis was assessed by examining nuclear morphology and quantifying phosphatidylserine externalization, and alterations in cellular morphology were analyzed by laser scanning confocal microscopy for fluorescent analysis. Flow cytometry was used in the analysis of cell cycle, mitochondrial membrane potential, and caspase pathways.Results: Griseofulvin could inhibit the growth of K562 cells in a dose-dependent manner with a mean (SD) inhibitory concentration of 50% value of 15.38 (1.35) μg/mL compared with untreated controls. Apoptosis was induced in K562 cells (38.35% [2.73%]; P < 0.01) by griseofulvin with the observation of both an increase in phosphatidylserine level and accumulation of chromatin nucleation in griseofulvintreated cells. In addition, cell-cycle analysis using propidium iodide staining suggested a significant G2/M accumulation (increase from mean 17.64% [4.49%] to 48.29 [1.89%]; P < 0.01) as a result of griseofulvin treatment. Flow cytometry analysis found that griseofulvin treatment was associated with the depolarization of the mitochondrial membrane in K562 cells. Furthermore, increased activities of caspase-3 by 22.15-fold (P < 0.01) and caspase-9 by 16.73-fold (P < 0.01) were observed in K562 cells after griseofulvin treatment compared with the untreated control; a decrease of caspase-8 activity was also observed, but the change was not statistically significant.Conclusions: These findings suggest that griseofulvin inhibited growth of K562 cells and induced cell apoptosis through cell-cycle arrest and mitochondrial membrane potential decrease as well as caspase-3 and -9 activation. Further testing is needed to evaluate the potential of griseofulvin as a candidate in the chemotherapy of hematologic malignancies.Key words: griseofulvin, caspase-3, caspase-9, K562 cells, apoptosis     

Transient Focal Membrane Deformation Induced by Arginine-rich Peptides Leads to Their Direct Penetration into Cells

Endocytosis has been implicated in the cellular uptake of arginine-rich, cell-penetrating peptides (CPPs). However, accumulating evidence suggests that certain conditions allow the direct, non-endocytic penetration of arginine-rich peptides through the plasma membrane. We previously showed that Alexa Fluor 488-labeled dodeca-arginine (R12-Alexa488) directly enters cells at specific sites on the plasma membrane and subsequently diffuses throughout cells. In this study, we found that the peptide influx was accompanied by the formation of unique, “particle-like” multivesicular structures on the plasma membrane, together with topical inversion of the plasma membrane. Importantly, the conjugation of dodeca-arginine (R12) to Alexa Fluor 488 or a peptide tag derived from hemagglutinin (HAtag) significantly accelerated particle formation, suggesting that the chemical properties of the attached molecules (cargo molecules) may contribute to translocation of the R12 peptide. Coincubation with R12-HAtag allowed the membrane-impermeable R4-Alexa488 to permeate cells. These results suggest that R12 peptides attached to hydrophobic cargo molecules stimulate dynamic morphological alterations in the plasma membrane, and that these structural changes allow the peptides to permeate the plasma membrane. These findings may provide a novel mode of cell permeabilization by arginine-rich peptides as a means of drug delivery.     

Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells

The phagocytosis of apoptotic cells within an organism is a critical terminal physiological process in programmed cell death. Evidence suggests that apoptotic cell engulfment and removal by macrophages is facilitated by phosphatidylserine (PS) displayed at the exofacial surface of the plasma membrane; however, neither the macrophage receptors responsible for PS recognition, nor characterization of the PS molecular species potentially involved, have been clearly defined. We show that the class B scavenger receptor CD36 plays an essential role in macrophage clearance of apoptotic cells in vivo. Further, macrophage recognition of apoptotic cells via CD36 is shown to occur via interactions with membrane-associated oxidized PS (oxPS) and, to a lesser extent, oxidized phosphatidylcholine, but not nonoxidized PS molecular species. Mass spectrometry analyses of oxPS species identify structures of candidate ligands for CD36 in apoptotic membranes that may facilitate macrophage recognition. Collectively, these results identify oxPS-CD36 interactions on macrophages as potential participants in a broad range of physiologic processes where macrophage-mediated engulfment of apoptotic cells is involved.     

Mechanisms of Action of Substituted β-Amino Alkanols on Leishmania donovani

Leishmaniasis is the protozoan disease second in importance for human health, superseded only by malaria; however, the options for chemotherapeutic treatment are increasingly limited due to drug resistance and toxicity. Under this perspective, a quest for new chemical compounds is urgently needed. An N-substituted 2-aminoalkan-1-ol scaffold has been shown to be a versatile scaffold for antiparasitic activity. Knowledge about its mechanism of action is still rather limited. In this work, we endeavored to define the leishmanicidal profile of such β-amino alkanol derivatives using a set of 15 N-mono- and disubstituted surrogates, tested on Leishmania donovani promastigotes and intracellular amastigotes. The best compound (compound 5), 2-ethylaminododecan-1-ol, had a 50% effective concentration (EC₅₀) of 0.3 μM and a selectivity index of 72 for infected THP-1 cells and was selected for further elucidation of its leishmanicidal mechanism. It induced fast depletion of intracellular ATP content in promastigotes in the absence of vital dye intracellular entry, ruling out plasma membrane permeabilization as its origin. Confocal and transmission electron microscopy analyses showed that compound 5 induced severe mitochondrial swelling and vesiculation. Polarographic analysis using an oxygen electrode demonstrated that complex II of the respiratory chain (succinate reductase) was strongly inhibited by compound 5, identifying this complex as one of the primary targets. Furthermore, for other β-amino alkanols whose structures differed subtly from that of compound 5, plasma membrane permeabilization or interference with membrane traffic was also observed. In all, N-substituted β-amino alkanols were shown as appealing leishmanicidal candidates deserving further exploration.     

Platelet‐ and erythrocyte‐derived microparticles trigger thrombin generation via factor XIIa

See also Shapiro S, Laffan M. Making contact with microparticles. This issue, pp 1352–4. Summary. Background: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. Objectives: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. Methods: Microparticles were isolated from calcium ionophore‐stimulated platelets, erythrocytes and monocytic THP‐1 cells. MPs were quantified, characterized for cell‐specific antigens and analyzed for TF, PS exposure and their thrombin‐generating potential. Results: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet‐ and erythrocyte‐derived MPs (< 1 fm nm ^?1 PS), whereas monocyte‐derived MPs exposed TF (32 fm nm ^?1 PS). Platelet‐, erythrocyte‐ and monocyte‐derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet‐ and erythrocyte‐derived MPs, but interfered with monocyte MP‐triggered coagulation. Platelet‐ and erythrocyte‐derived MPs completely failed to induce thrombin generation in FXII‐deficient plasma. In contrast, monocyte‐derived MPs induced similar thrombin generation in normal vs. FXII‐deficient plasma. Conclusion: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII‐dependent manner. In contrast, monocyte‐derived MPs trigger coagulation predominantly via TF.