Cell Ghosts Research Articles

Melanin particles isolated from the fungus Fonsecaea pedrosoi activates the human complement system.

BACKGROUNDMelanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America.OBJECTIVEThe aim of this study was to evaluate the effects of acid-basic extracted F. pedrosoi melanin particles and fungal cell ghosts obtained by Novozym 234 treatment on their ability to activate the human complement system.METHODSThe ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA).FINDINGSUnsensitised melanin particles and melanin ghosts presented complement consumption of 82.67 ± 2.08% and 96.04 ± 1.13%, respectively. Immunofluorescence assays revealed intense deposition of the C3 and C4 fragments on the surface of melanin particles and ghosts extracted from F. pedrosoi. Deposition of the C3, C4, and C5 fragments onto melanin samples and zymosan was confirmed by ELISA. Deposition of small amounts of C1q and C9 onto melanin samples and zymosan was detected by ELISA.CONCLUSION Fonsecaea pedrosoi melanin particles and fungal cell ghosts activated the complement system mainly through an alternative pathway.

Native Gel Electrophoresis Reveals Partial Physical Association of band 3 and aquaporins on the Erythrocyte Membrane

Band 3 is the most abundant membrane protein in human red cells. Band 3 primarily functions as a bi‐directional anion transporter for Cl− and HCO3− to support blood CO2 exchange. Because CO2 mainly exists in the form of HCO3− (aq) in the human body, the rate of CO2 blood respiration is dependent on the intraerythrocytic reaction CO2(g) + H2O □ HCO3− (aq) + H+ (aq). By FLIM‐FRET, we recently uncovered the existence of mobile interaction between band 3 and water channel AQP1. Their interaction could be disrupted when red cell vesicles were put in a hypotonic milieu, suggesting AQP1 bearing a functional role in the band 3‐central CO2 transport metabolon.ObjectiveAs there are at least two types of band 3 protein complexes on the erythrocyte membrane, the goal of this study was to probe the physical associations between aquaporins and different band 3 complexes.MethodWe utilized semi‐native and native gel electrophoresis to identify aquaporin complexes and to differentiate their associations with the different band 3 complexes.ResultsWater channel AQP1 and aquaglyceroporin AQP3 complexes were both identified in red cell ghosts by semi‐native gel electrophoresis. Some of the aquaporin complexes were found to be structurally linked to band 3 protein complexes.ConclusionThe physical association between AQP1 and band 3 and that between AQP3 and band 3 can be differentiated by semi‐native gel electrophoresis. Erythrocyte aquaporins also appear physically linked to other membrane‐associated proteins in the human red cells.Support or Funding InformationMOST 106‐2320‐B‐195‐002 from Taiwan Ministry of Science & TechnologyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of hydroxycarbamide and interferon-α on red cell adhesion and membrane protein expression in polycythemia vera.

Polycythemia vera is a chronic myeloproliferative neoplasm characterized by the JAK2V617F mutation, elevated blood cell counts and a high risk of thrombosis. Although the red cell lineage is primarily affected by JAK2V617F, the impact of mutated JAK2 on circulating red blood cells is poorly documented. Recently, we showed that in polycythemia vera, erythrocytes had abnormal expression of several proteins including Lu/BCAM adhesion molecule and proteins from the endoplasmic reticulum, mainly calreticulin and calnexin. Here we investigated the effects of hydroxycarbamide and interferon-α treatments on the expression of erythroid membrane proteins in a cohort of 53 patients. Surprisingly, while both drugs tended to normalize calreticulin expression, proteomics analysis showed that hydroxycarbamide deregulated the expression of 53 proteins in red cell ghosts, with overexpression and downregulation of 37 and 16 proteins, respectively. Within over-expressed proteins, hydroxycarbamide was found to enhance the expression of adhesion molecules such as Lu/BCAM and CD147, while interferon-α did not. In addition, we found that hydroxycarbamide increased Lu/BCAM phosphorylation and exacerbated red cell adhesion to its ligand laminin. Our study reveals unexpected adverse effects of hydroxycarbamide on red cell physiology in polycythemia vera and provides new insights into the effects of this molecule on gene regulation and protein recycling or maturation during erythroid differentiation. Furthermore, our study shows deregulation of Lu/BCAM and CD147 that are two ubiquitously expressed proteins linked to progression of solid tumors, paving the way for future studies to address the role of hydroxycarbamide in tissues other than blood cells in myeloproliferative neoplasms.

The Molecular Structure of Human Red Blood Cell Membranes From Highly Oriented, Solid Supported Multi-Lamellar Membranes

The preparation of Red Blood Cell (RBC) Ghosts is a well-known protocol in biological and medical research and describes the extraction of the membrane from RBCs. Another well-known protocol is the preparation of highly ordered stacks of artificial lipid bilayers on silicon wafers. There are various attempts to adapt this protocol to a native cell membrane. For the first time we were able to combine both described techniques and to prepare highly ordered stacks of RBC membranes on silicon wafers [1]. These systems can now be used as inexpensive and safe platforms for testing the effect of drugs and bacteria on RBC membranes in-vitro using biophysical techniques, such as X-ray and neutron diffraction, optical spectroscopy and atomic force microscopy. We present direct experimental evidence that these RBC membranes consist of nanometer sized domains of integral coiled-coil peptides, as well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, membrane and hydration water layer thicknesses, areas per lipid tail and domain sizes were determined. The common drug aspirin was added to the RBC membranes and found to interact with RBC membranes and preferably partition in the head group region of the lo domain leading to a fluidification of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail spacing. [1] Himbert S. et al., The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes, Scientific Reports 7, Article number: 39661 (2017).

Evidence that asymmetry of the membrane/cytoskeletal complex in human red blood cell ghosts is responsible for their biconcave shape

The main conclusion of the results reported in this article is that during centrifugation, sphered red blood cell ghosts become oriented in their attachment to a coverslip such that a dense band within the ghosts lies parallel to the centrifugal field. The result of the orientation of this dense band is that when the attached spherical ghosts are shrunken to become biconcave discs, they do so by directly collapsing on themselves without any lateral motion. This result is interpreted to suggest that a dense band, relative to the dimple, resides in the rim of the ghost and is responsible for its biconcave shape. These results confirm the conclusions reached in a previous publication in which there was the uncertainty that the shape change of the spherical ghosts to discs could not be directly imaged. The present work corrects this limitation by use of a chamber in which the tonicity of the solutions in the ghosts' surround could be altered by perfusion coupled with constant microscopic imaging. The identity of the components that are responsible for the differences in the density (mass) between the rim and the dimple regions of the cytoskeletal/membrane complex in the biconcave disk are unknown. It is also unknown what forces apply or what the explanation is for the unique orientation of the dense band during the ghosts' centrifugation, as described in this article. Nevertheless, the results reported in this article indicate the membrane's underlying cytoskeletal complex is asymmetrically distributed.

Time-lapse imaging of cell death in cell culture and whole living organisms using turn-on deep-red fluorescent probes.

Cell death is a central process in developmental biology and also an important indicator of disease status and treatment efficacy. Two related fluorescent probes are described that are molecular conjugates of one or two zinc dipicolylamine (ZnDPA) coordination complexes with an appended solvatochromic benzothiazolium squaraine dye. The probes were designed to target the anionic phospholipid, phosphatidylserine (PS), that is exposed on the surface of dead and dying cells. A series of spectrometric and microscopy studies using liposomes and red blood cell ghosts as models showed that the probe with two ZnDPA targeting units produced higher affinity, stronger fluorescence "turn-on" effect, and better image contrast than the probe with one ZnDPA. Both fluorescent probes enabled "no-wash" time-lapse microscopic imaging of mammalian cell death within a culture. The probe with two ZnDPA units was used for non-invasive time-lapse imaging of cell death during the development of Xenopus laevis (frog) embryos. In vivo fluorescence micrographs revealed probe accumulation within the embryo tail, head and spine regions that were undergoing regression and apoptosis during growth and maturation. These new fluorescent probes are likely to be useful for time-resolved, non-invasive in vivo imaging of cell death process in range of living organisms. From a broader perspective, it should be possible to utilize the negative solvatochromism exhibited by benzothiazolium squaraine dyes for development of various "turn-on" deep-red fluorescent probes and materials that target cell surface biomarkers for in vitro and in vivo imaging.

High-speed polarization-resolved coherent Raman scattering imaging

Polarization-resolved coherent Raman scattering (polar-CRS) provides rich information on molecular orientational organization, with the strong advantages of being a label-free and chemically specific imaging method. Its implementation, however, strongly reduces the imaging acquisition rate, due to limits imposed by polarization tuning. Here we demonstrate fast-polar-CRS imaging based on combined electro-optic polarization and acousto-optic amplitude modulations, applicable to both stimulated Raman scattering and coherent anti-Stokes Raman scattering imaging. The proposed scheme adds polarization information without compromising the capacities of regular CRS intensity imaging; increases the speed of orientational imaging by two orders of magnitude as compared with previous approaches; and does not require post-processing analyses. We show that this method permits sub-second time-scale imaging of lipid order packing and local lipid membrane deformations in artificial lipid multilayers, but also in red blood cell ghosts, demonstrating its high sensitivity down to a single lipid bilayer membrane.

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction.

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.

USE OF VIBRIO CHOLERAE SURFACE STRUCTURES FOR SPECIFIC PROPHYLAXIS AND DIAGNOSTICS OF CHOLERA

The need for efficient and cost-effective cholera vaccine hasn't lost its actuality in view of the emergence of new strains leading to severe clinical forms of cholera and capable to replace strains of the seventh.cholera pandemic, and in connection with the threat of cholera spreading beyond the borders of endemic countries. In this review data from literature sources are presented about the use of outer membrane proteins, vesicles, cell ghosts of the cholera causative agent in specific prophylaxis and diagnostics of the disease.

IUGR, lipid peroxidation, and calcium ATPase activity of maternal red blood cell ghosts

IUGR, lipid peroxidation, and calcium ATPase activity of maternal red blood cell ghosts

Red blood cell ghosts as promising drug carriers to target wound infections

Autologous red blood cell ghosts (RBC ghosts) can carry cytokines to the sites of inflammation. The targeting moiety of the RBC ghosts is associated with the nature of purulent inflammation, where the erythrocytes are phagocyted and encapsulated drugs are released. In the present study we have investigated the healing potential of RBC ghosts loaded with cytokine IL-1β and antibiotic. Additionally, the pharmacokinetic properties of RBC ghosts loaded with IL-1β were studied. 35 Male Wistar rats (250–300g) were used in the pharmacokinetic study and in a wound infection model where a suspension of Staphylococcus aureus was placed into a surgical cut of the skin and subcutaneous tissue in the femoral region. In order to monitor progression of the wound repair processes, wound swabs or aspiration biopsies were taken for analyses on the 1st–6th days. Wound repair dynamics assessment was based on suppression of S. aureus growth, signs of pain, time of disappearance of pus and infiltration around the wound. Visual observations, as well as microbiological and cytological analysis of wound exudates demonstrated a significant acceleration of healing processes in a group of animals treated with a local injection of IL-1β and ceftriaxone encapsulated into RBC ghosts when compared to the animals treated either with a local or IM injection of free drugs. For the pharmacokinetic study, single IV injections of either free or encapsulated IL-1β were made and the concentration of IL-1β in serum samples and tissue homogenates were determined. Encapsulation in RBC ghosts improved pharmacokinetic profiles of IL-1β by increasing the half-life, reducing its clearance, and increasing the deposition of the drug in the liver, spleen and lungs. These data suggest that RBC ghosts are effective drug carriers for targeted delivery of cytokines to the sites of inflammation, and have a potential for improving the treatment outcomes of purulent diseases.

Interactions of dendritic glycopolymer with erythrocytes, red blood cell ghosts and membrane enzymes

Interactions between maltose functionalized hyperbranched poly(ethylene imine)s (95% maltose decoration denoted as Mal-PEI A; 33% maltose decoration denoted as Mal-PEI B) and red blood cells (RBCs) and between red blood cell membranes were investigated. We monitored the degree of hemolysis, the change in cell shape, the influence of polymers on the fluidity of the cell membrane and some cell membrane enzymes to determine their possible cytotoxic impact on them. To observe the extent of hemolysis, the RBCs were incubated with different concentrations of Mal-PEIs. The first significant lysis of RBCs was observed after 6h of incubation. Prolongation of the incubation time increased the number of ruptured cells. Moreover, we observed that Mal-PEI B was more hemolytic than Mal-PEI A in buffer solution. In contrast, an incubation of RBCs with Mal-PEIs in human plasma significantly decreased the hemolytic process and showed higher hemolytic property of Mal-PEI A compared to Mal-PEI B. Also several changes in the shape of the RBCs occurred after incubation with Mal-PEIs. Some of the erythrocytes shrank (echinocytes), but their morphology generally remained unchanged during the incubation. As shown by fluorescence experiments, both polymers induced the increase of fluidity of RBCs membranes. In summary, both types of hyperbranched poly(ethylene imine)s were practically non-hemolytic even at high polymer concentrations. Mal-PEI B was slightly more noxious than the Mal-PEI A in a buffer solution, while in blood plasma, the situation was opposite. Decrease of Na+/K+ ATPase and total ATPase enzymes activity was related with molecule size and number of maltose groups on the surface of molecule. The low hemolytic properties only observed at higher concentration (100μM and 400μM) indicated that Mal-PEIs are promising macromolecules in the area of drug delivery systems.

4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Biologically Active Precursor for the Generation of 2-(ω-Carboxyethyl)pyrrole (CEP) Derivatives of Proteins and Ethanolamine Phospholipids.

2-(ω-Carboxyethyl)pyrrole (CEP) derivatives of proteins were previously shown to have significant pathological and physiological relevance to age-related macular degeneration, cancer and wound healing. Previously, we showed that CEPs are generated in the reaction of ε-amino groups of protein lysyl residues with 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphatidylcholine (HOHA-PC), a lipid oxidation product uniquely generated by oxidative truncation of docosahexanenate-containing phosphatidylcholine. More recently, we found that HOHA-PC rapidly releases HOHA-lactone and 2-lyso-PC (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation. Now we report that HOHA-lactone reacts with Ac-Gly-Lys-OMe or human serum albumin to form CEP derivatives in vitro. Incubation of human red blood cell ghosts with HOHA-lactone generates CEP derivatives of membrane proteins and ethanolamine phospholipids. Quantitative analysis of the products generated in the reaction HOHA-PC with Ac-Gly-Lys-OMe showed that HOHA-PC mainly forms CEP-dipeptide that is not esterified to 2-lysophosphatidycholine. Thus, the HOHA-lactone pathway predominates over the direct reaction of HOHA-PC to produce the CEP-PC-dipeptide derivative. Myleoperoxidase/H2O2/NO2(-) promoted in vitro oxidation of either 1-palmityl-2-docosahexaneoyl-sn-glycero-3-phosphatidylcholine (DHA-PC) or docosahexaenoic acid (DHA) generates HOHA-lactone in yields of 0.45% and 0.78%, respectively. Lipid oxidation in human red blood cell ghosts also releases HOHA-lactone. Oxidative injury of ARPE-19 human retinal pigmented epithelial cells by exposure to H2O2 generated CEP derivatives. Treatment of ARPE-19 cells with HOHA-lactone generated CEP-modified proteins. Low (submicromolar), but not high, concentrations of HOHA-lactone promote increased vascular endothelial growth factor (VEGF) secretion by ARPE-19 cells. Therefore, HOHA-lactone not only serves as an intermediate for the generation of CEPs but also is a biologically active oxidative truncation product from docosahexaenoate lipids.

Effect of magnesium gluconate on the level of lipid peroxidation in placental homogenates and red cells ghosts in salt-loaded pregnant rats

Effect of magnesium gluconate on the level of lipid peroxidation in placental homogenates and red cells ghosts in salt-loaded pregnant rats

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

Human amniotic epithelial cells: Proliferation and apoptosis during their hepatic differentiation

Effect of magnesium gluconate on Ca-ATPase activity and lipid peroxidation levels in red blood cell ghosts from preeclamptic pregnant women

Effect of magnesium gluconate on Ca-ATPase activity and lipid peroxidation levels in red blood cell ghosts from preeclamptic pregnant women

Osmotic fragility of red blood cells, lipid peroxidation and Ca2+-ATPase activity of placental homogenates and red blood cell ghosts in salt-loaded pregnant rats

Objective: To evaluate the osmotic fragility of red blood cells and the level of lipid peroxidation, the Ca2+-ATPase activity of red cell ghosts and placental homogenates from salt-loaded pregnant rats.Methods: Salt-loaded pregnant rats received 1.8% NaCl solution ad libitum as a beverage for seven days, starting on 15th day of pregnancy. Then, it was evaluated the level of lipid peroxidation and the Ca2+-ATPase activity of placental homogenates and red blood cell ghosts from control and experimental rats. Furthermore, the osmotic fragility of the red blood cells was evaluated by measuring the lysis of these cells when incubated with a NaCl solution with different osmolarities.Results: It was found that placental homogenates and red blood cell ghosts from experimental pregnant rats showed an increased level of lipid peroxidation and a lowered Ca2+-ATPase activity, as compared to control pregnant rats. They also presented an increased osmotic fragility of their red blood cells.Conclusions: Salt-loaded pregnant rats showed, similar to preeclamptic women, an increased level of lipid peroxidation and a lowered Ca2+-ATPase activity in placental and red blood cells membranes, as well as an increased osmotic fragility of the red blood cells.

Increased water flux induced by an aquaporin-1/carbonic anhydrase II interaction

Aquaporin-1 (AQP1) enables greatly enhanced water flux across plasma membranes. The cytosolic carboxy terminus of AQP1 has two acidic motifs homologous to known carbonic anhydrase II (CAII) binding sequences. CAII colocalizes with AQP1 in the renal proximal tubule. Expression of AQP1 with CAII in Xenopus oocytes or mammalian cells increased water flux relative to AQP1 expression alone. This required the amino-terminal sequence of CAII, a region that binds other transport proteins. Expression of catalytically inactive CAII failed to increase water flux through AQP1. Proximity ligation assays revealed close association of CAII and AQP1, an effect requiring the second acidic cluster of AQP1. This motif was also necessary for CAII to increase AQP1-mediated water flux. Red blood cell ghosts resealed with CAII demonstrated increased osmotic water permeability compared with ghosts resealed with albumin. Water flux across renal cortical membrane vesicles, measured by stopped-flow light scattering, was reduced in CAII-deficient mice compared with wild-type mice. These data are consistent with CAII increasing water conductance through AQP1 by a physical interaction between the two proteins.

Magnesium sulfate affords protection against oxidative damage during severe preeclampsia

IntroductionMgSO4 is the drug of choice to prevent seizures in preeclamptic pregnant women, but its mechanism of action at the molecular level remains an enigma. In previous works, we found that treating preeclamptic women with MgSO4 reduces the lipid peroxidation of their red blood cell membranes to normal levels and leads to a significant reduction in the osmotic fragility of the red blood cells that is increased during preeclampsia. In addition, the increase in lipid peroxidation of red cell membranes induced by the Fenton reaction does not occur when MgSO4 is present. MethodsThe antioxidant protection of MgSO4 was evaluated in UV-C-treated red blood cell ghosts and syncytiotrophoblast plasma membranes by measuring their level of lipid peroxidation. The interaction of MgSO4 with free radicals was assessed for its association with the galvinoxyl radical, the quenching of H2O2-induced chemiluminescence and its effect on sensitized peroxidation of linoleic acid. Resultsa) MgSO4 protected red blood cell ghosts and the syncytiotrophoblast plasma membranes of normotensive pregnant women against lipid peroxidation induced by UV-C irradiation. b) MgSO4 does not seem to scavenge the galvinoxyl free radical. c) The quenching of the H2O2-enhanced luminol chemiluminescence is increased by the presence of MgSO4. d) The peroxidation of linoleic acid is significantly blocked by MgSO4. DiscussionMgSO4 may provide protection against oxidative damage of plasma membranes through interactions with alkyl radicals.

Nitric oxide levels in the aqueous humor vary in different ocular hypertension experimental models

This study investigated the relationships among intraocular pressure (IOP), nitric oxide (NO) levels, and aqueous flow rates in experimental ocular hypertension models. A total of 75 rabbits were used. One of four different materials [i.e., α-chymotrypsin, latex microspheres (Polybead), red blood cell ghosts, or sodium hyaluronate (Healon GV)] was injected into the eyes of the 15 animals in each experimental group; the remaining 15 rabbits were reserved for a control group. The IOP changes in the five groups were recorded on postinduction Days 1–3, Day 7, Day 14, Day 30, Day 60, Day 90, and Day 120. On postinduction Day 7, the dynamics and NO levels in the aqueous humor were recorded. Significant IOP elevations were induced by α-chymotrypsin (p < 0.01) and Polybead (p < 0.01) on each postinduction day. In the red blood cell ghosts model, significant elevations (p < 0.01) were found on postinduction Days 1–3; Healon GV significantly elevated IOP (p < 0.01) on postinduction Day 1 and Day 2. On postinduction Day 7, the aqueous humor NO levels increased significantly in the models of α-chymotrypsin, Polybead, and red blood cell ghosts (all p < 0.01), while the aqueous flow rates were significantly reduced in the models of α-chymotrypsin and Polybead (p < 0.005). Persistent ocular hypertension models were induced with α-chymotrypsin and Polybead in the rabbits. The Polybead model exhibited the characteristic of an increased aqueous humor NO level, similar to human eyes with acute angle-closure glaucoma and neovascular glaucoma.