scholarly journals In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1784-1792 ◽  
Author(s):  
Gianluca Civenni ◽  
Samuel T. Test ◽  
Urs Brodbeck ◽  
Peter Bütikofer
Keyword(s):  
Membrane Vesicle ◽  
Human Erythrocytes ◽  
Membrane Extraction ◽  
Membrane Microdomains ◽  
Membrane Insertion ◽  
Vesicle Formation ◽  
Calcium Loading ◽  
Lipid Moiety ◽  
Triton X

Abstract In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

scholarly journals In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1784-1792 ◽  
Author(s):  
Gianluca Civenni ◽  
Samuel T. Test ◽  
Urs Brodbeck ◽  
Peter Bütikofer
Keyword(s):  
Membrane Vesicle ◽  
Human Erythrocytes ◽  
Membrane Extraction ◽  
Membrane Microdomains ◽  
Membrane Insertion ◽  
Vesicle Formation ◽  
Calcium Loading ◽  
Lipid Moiety ◽  
Triton X

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

scholarly journals Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Vivek Kumar Gupta ◽  
Rajnish Pal ◽  
Nikhat Jamal Siddiqi ◽  
Bechan Sharma
Keyword(s):  
Lead Exposure ◽  
Phosphate Buffer ◽  
Human Erythrocytes ◽  
Enzyme Substrate ◽  
The People ◽  
Occupationally Exposed ◽  
Membrane Bound ◽  
Triton X ◽  
Made In

Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte’s AChE exhibited Km for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (Ki value, 3.6 mM) which negatively influenced both the Vmax and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead.

scholarly journals Antibiotics Stimulate Formation of Vesicles inStaphylococcus aureusin both Phage-Dependent and -Independent Fashions and via Different Routes

2018 ◽  
Vol 63 (2) ◽  
pp. e01439-18 ◽  
Author(s):  
Federica Andreoni ◽  
Masanori Toyofuku ◽  
Carmen Menzi ◽  
Ratchara Kalawong ◽  
Srikanth Mairpady Shambat ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Vesicle Formation ◽  
Independent Manner ◽  
Content Type ◽  
Lactam Antibiotic ◽  
Underlying Mechanisms

ABSTRACTBacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogenStaphylococcus aureus. DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains ofS. aureusbut not in their phage-devoid counterparts. The β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by β-lactam antibiotic-induced blebbing. The purified MVs derived fromS. aureusprotected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, bothin vitroandex vivoin whole blood. In addition, the MVs protectedS. aureusfrom killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

scholarly journals ARF1 dimerization is essential for vesicle trafficking and dependent on activation by ARF-GEF dimers in Arabidopsis

2020 ◽  
Author(s):  
Sabine Brumm ◽  
Mads Eggert Nielsen ◽  
Sandra Richter ◽  
Hauke Beckmann ◽  
York-Dieter Stierhof ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Membrane Vesicle ◽  
Biological Significance ◽  
Eukaryotic Cell ◽  
Vesicle Formation ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Sites Of Action

AbstractMembrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs have been shown to form dimers in vivo. This feature is conserved across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 dimerization in vivo and we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in Arabidopsis. Mutations disrupting simultaneous binding of two ARF1•GDPs by the two SEC7 domains of GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells

1993 ◽  
Vol 104 (4) ◽  
pp. 1281-1290 ◽  
Author(s):  
M. Garcia ◽  
C. Mirre ◽  
A. Quaroni ◽  
H. Reggio ◽  
A. Le Bivic
Keyword(s):  
Intracellular Transport ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Membrane Microdomains ◽  
Ph Variation ◽  
Glycosyl Phosphatidylinositol ◽  
Apical Sorting ◽  
Triton X

In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.

IN VITRO UPTAKE OF RADIOACTIVE 131I LABELLED L-TRIIODOTHYRONINE BY HUMAN ERYTHROCYTES AS AN INDEX OF THYROID FUNCTION

1960 ◽  
Vol XXXIV (II) ◽  
pp. 305-311 ◽  
Author(s):  
M. G. Woldring ◽  
A. Bakker ◽  
H. Doorenbos
Keyword(s):  
Thyroid Function ◽  
Incubation Time ◽  
Clinical Results ◽  
Human Erythrocytes ◽  
Red Cell ◽  
Clinical Value ◽  
Blood Samples ◽  
In Vitro Uptake

ABSTRACT The red cell triiodothyronine uptake technique as used in our hospital is described. Incubation time is of almost no importance. The temperature during incubation should be 37° C. Further improvement of the technique is obtained when all blood samples are brought up to 40 % haematocrit prior to incubation. Clinical results are discussed. It is yet too early to give a definite assessment of its clinical value, but it is definitely superior to the measurement of the BMR.

Preparation and In Vitro Evaluation of Resealed Erythrocytes as A New Trend in Treatment of Asthma

2016 ◽  
Vol 6 (3) ◽  
Author(s):  
Mohammed Ibrahim ◽  
Alaa Zaky ◽  
Mohsen Afouna ◽  
Ahmed Samy
Keyword(s):  
In Vitro Release ◽  
Human Erythrocytes ◽  
The Body ◽  
Blood Levels ◽  
Time Interval ◽  
Burst Release ◽  
Prolonged Release ◽  
Nocturnal Asthma ◽  
Carrier Erythrocytes

Carrier erythrocytes are emerging as one of the most promising biological drug delivery systems investigated in recent decades. Beside its biocompatibility, biodegradability and ability to circulate throughout the body, it has the ability to perform extended release system of the drug for a long period. The ultimate goal of this study is to introduce a new carrier system for Salbutamol, maintaining suitable blood levels for a long time, as atrial to resolve the problems of nocturnal asthma medication Therefore in this work we study the effect of time, temperature as well as concentration on the loading of salbutamol in human erythrocytes to be used as systemic sustained release delivery system for this drug. After the loading process is performed the carrier erythrocytes were physically and cellulary characterized. Also, the in vitro release of salbutamol from carrier erythrocytes was studied over time interval. From the results it was found that, human erythrocytes have been successfully loaded with salbutamol using endocytosis method either at 25 Co or at 37 Co . The highest loaded amount was 3.5 mg/ml and 6.5 mg/ml respectively. Moreover, the percent of cells recovery is 90.7± 1.64%. Hematological parameters and osmotic fragility behavior of salbutamol loaded erythrocytes were similar that of native erythrocytes. Scanning electron microscopy demonstrated that the salbutamol loaded cells has moderate change in the morphology. Salbutamol releasing from carrier cell was 43% after 36 hours in phosphate buffer saline. The releasing pattern of the drug from loaded erythrocytes showed initial burst release in the first hour followed by a very slow release, obeying zero order kinetics. It concluded that salbutamol is successfully entrapped into erythrocytes with acceptable loading parameters and moderate morphological changes, this suggesting that erythrocytes can be used as prolonged release carrier for salbutamol.

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

scholarly journals Surface-Engineered Dendrimeric Nanoconjugates for Macrophage-Targeted Delivery of Amphotericin B: Formulation Development andIn VitroandIn VivoEvaluation

2015 ◽  
Vol 59 (5) ◽  
pp. 2479-2487 ◽  
Author(s):  
Keerti Jain ◽  
Ashwni Kumar Verma ◽  
Prabhat Ranjan Mishra ◽  
Narendra Kumar Jain
Keyword(s):  
Drug Release ◽  
Amphotericin B ◽  
Human Erythrocytes ◽  
Antileishmanial Activity ◽  
Cell Uptake ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Drug Localization

ABSTRACTThe present study aimed to develop an optimized dendrimeric delivery system for amphotericin B (AmB). Fifth-generation (5.0G) poly(propylene imine) (PPI) dendrimers were synthesized, conjugated with mannose, and characterized by use of various analytical techniques, including Fourier transform infrared spectroscopy (FTIR),1H nuclear magnetic resonance (1H-NMR) spectroscopic analysis, and atomic force microscopy (AFM). Mannose-conjugated 5.0G PPI (MPPI) dendrimers were loaded with AmB and evaluated for drug loading efficiency,in vitrodrug release profile, stability, hemolytic toxicity to human erythrocytes, cytotoxicity to and cell uptake by J774A.1 macrophage cells, antiparasitic activity against intracellularLeishmania donovaniamastigotes,in vivopharmacokinetic and biodistribution profiles, drug localization index, toxicity, and antileishmanial activity. AFM showed the nanometric size of the MPPI dendrimers, with a nearly globular architecture. The conjugate showed a good entrapment efficiency for AmB, along with pH-sensitive drug release. Highly significant reductions in toxicity toward human erythrocytes and macrophage cells, without compromising the antiparasitic activity of AmB, were observed. The dendrimeric formulation of AmB showed a significant enhancement of the parasiticidal activity of AmB toward intramacrophagicL. donovaniamastigotes. In thein vitrocell uptake studies, the formulation showed selectivity toward macrophages, with significant intracellular uptake. Further pharmacokinetic and organ distribution studies elucidated the controlled delivery behavior of the formulation. The drug localization index was found to increase significantly in macrophage-rich organs.In vivostudies showed a biocompatible behavior of MPPIA, with negligible toxicity even at higher doses, and promising antileishmanial activity. From the results, we concluded that surface-engineered dendrimers may serve as optimized delivery vehicles for AmB with enhanced activity and low or negligible toxicity.

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