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1
Ali, Randa H., Mohamed E. Ali e Reham Samir. "Production and Characterization of Bacterial Ghost Vaccine against Neisseria meningitidis". Vaccines 11, n. 1 (23 dicembre 2022): 37. http://dx.doi.org/10.3390/vaccines11010037.
Abstract (sommario):
Bacterial ghosts (BGS) are empty non-living envelopes produced either genetically or chemically. This study investigated a novel chemical protocol for the production of Neisseria meningitidis ghost vaccine using tween 80 followed by a pH reduction with lactic acid. For our vaccine candidate, both safety and immunogenicity aspects were evaluated. The ghost pellets showed no sign of growth upon cultivation. BGS were visualized by scanning electron microscopy, illustrating the formation of trans-membrane tunnels with maintained cell morphology. Gel electrophoresis showed no distinctive bands of the cytoplasmic proteins and DNA, assuring the formation of ghost cells. In animal model, humoral immune response significantly increased when compared to commercial vaccine (p < 0.01). Moreover, serum bactericidal assay (SBA) recorded 94.67% inhibition compared to 64% only for the commercial vaccine after three vaccination doses. In conclusion, this is the first N. meningitidis ghost vaccine candidate, proven to be effective, economic, and with significant humoral response and efficient SBA values; however, clinical studies should be performed.
2
Golan, D. E., C. S. Brown, C. M. Cianci, S. T. Furlong e J. P. Caulfield. "Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components." Journal of Cell Biology 103, n. 3 (1 settembre 1986): 819–28. http://dx.doi.org/10.1083/jcb.103.3.819.
Abstract (sommario):
Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.
3
Jawale, Chetan V., Nithiphonh Somsanith, Seong Kug Eo, Sang-Youel Park e John Hwa Lee. "Evaluation of Salmonella Gallinarum ghost formulated with Montanide™ ISA 70 VG adjuvant as a vaccine against fowl typhoid". Acta Veterinaria Hungarica 63, n. 4 (dicembre 2015): 401–12. http://dx.doi.org/10.1556/004.2015.038.
Abstract (sommario):
Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent adjuvant. Salmonella Gallinarum ghosts carrying LTB (S. Gallinarum-LTB ghosts) were genetically constructed using a plasmid, pJHL187-LTB, designed for the co-expression of the LTB and E lysis proteins. This study evaluates the immunopotentiating effects of Montanide™ ISA 70 VG on S. Gallinarum-LTB ghost vaccination against fowl typhoid. Five-week-old layer chickens were injected intramuscularly with sterile PBS (non-immunised control, Group A), S. Gallinarum-LTB ghost (Group B) or S. Gallinarum-LTB ghost emulsified with Montanide™ ISA 70 VG adjuvant (Group C). Chickens from both Groups B and C showed significant induction of antigen-specific systemic IgG response compared to controls; in addition, Group C showed enhanced induction of systemic IgG response compared to Group B. We observed significant induction of antigen-specific lymphocyte proliferative response and increased mRNA levels of Th1 cytokines (IFN-γ and IL2) in both Groups B and C. Furthermore, in the challenge experiment with a virulent strain of S. Gallinarum, Group C showed higher survival rates compared with other groups. These results indicate that vaccination with the S. Gallinarum-LTB ghost in combination with Montanide™ ISA 70 VG may enhance the protective immunity against fowl typhoid.
4
Wooden, Jason M., Greg Finney, Michael MacCoss e Diana M. Gilligan. "Comprehensive Analysis of Murine and Human RBC Ghost Proteomes." Blood 108, n. 11 (16 novembre 2006): 1569. http://dx.doi.org/10.1182/blood.v108.11.1569.1569.
Abstract (sommario):
Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. Mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton. Genetic knock-out of various components of this apparatus has led to the creation of mouse models which have contributed significantly to our understanding of these disorders in humans. However, the mouse and human RBC protein complements have not been comprehensively compared. Using newly developed proteomic methodology, we conducted a peptide level ‘bottom-up’ analysis of the normal mouse and human RBC ghost (i.e., RBC membrane skeleton and associated proteins). RBCs were purified using cellulose acetate chromatography from whole blood taken from three genetically identical mice and two hematologically normal yet genetically diverse humans. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin and analyzed by shotgun proteomics using microcapillary liquid chromatography coupled with tandem mass spectrometry. In total, 400 and 491 unique proteins were identified in human samples A and B respectively while 469 proteins were found in common across the three mouse samples. All previously identified membrane skeleton proteins were found in the human and mouse samples. Likewise, well-known RBC membrane proteins were represented. Of interest, a surprising number of proteins were found associated with the RBC ghost involved in processes such as protein repair (15–20), protein degradation (30–43), oxidative stress response (4–6), Ras oncogene biology (28–30), and glycolysis (3–6). Collectively, the two human samples represented 544 unique proteins. These results affirm the usefulness of inherited anemia mouse models given the observed conservation of membrane skeleton components and the inherent challenges with doing normal versus diseased RBC analysis in humans due to genetic variation.
5
Nunes-Correia, Isabel, João Ramalho-Santos e Maria C. Pedroso de Lima. "Sendai Virus Fusion Activity as Modulated by Target Membrane Components". Bioscience Reports 18, n. 2 (1 aprile 1998): 59–68. http://dx.doi.org/10.1023/a:1020180109275.
Abstract (sommario):
We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.
6
Hopgood, M. F., S. E. Knowles e F. J. Ballard. "Proteolysis of N-ethylmaleimide-modified aldolase loaded into erythrocyte ghosts: prevention by inhibitors of calpain". Biochemical Journal 259, n. 1 (1 aprile 1989): 237–42. http://dx.doi.org/10.1042/bj2590237.
Abstract (sommario):
1. When rabbit muscle aldolase labelled with tritium and inactivated by N-ethylmaleimide (NEM) was loaded into erythrocyte ghosts, significant proteolysis of the loaded protein occurred. The major product of this proteolysis, separated by electrophoresis under dissociating conditions, was found to be approx. 2 kDa smaller than the parent protein. 2. Proteolysis was detectable during erythrocyte ghost loading at 0 degrees C, reaching a plateau after approx. 12 min. Subsequent incubation at 37 degrees C to allow resealing of the ghosts resulted in additional proteolysis, and up to 20% of the loaded protein was converted to the smaller 38 kDa derivative. 3. EDTA, EGTA, leupeptin and chymostatin, each inhibitors of calcium-activated neutral proteinases (calpains), were the most effective inhibitors of the proteolysis of NEM-inactivated aldolase in ghosts. Other proteinase inhibitors were ineffective, while phenylmethanesulphonyl fluoride was only partially effective. 4. Inhibition of the proteolysis by EGTA was prevented by CaCl2, supporting the involvement of erythrocyte calpain. 5. Pretreatment of ghosts with EGTA prior to loading of NEM-modified aldolase followed by microinjection of the protein into HeLa cells did not result in a different rate of its overall breakdown to acid-soluble products. EGTA is suggested as a useful agent for the erythrocyte ghost-mediated microinjection of calpain-sensitive proteins.
7
Yang, Chinglai, Qingyuan Yang e Richard W. Compans. "Coreceptor-Dependent Inhibition of the Cell Fusion Activity of Simian Immunodeficiency Virus Env Proteins". Journal of Virology 74, n. 13 (1 luglio 2000): 6217–22. http://dx.doi.org/10.1128/jvi.74.13.6217-6222.2000.
Abstract (sommario):
ABSTRACT The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.
8
Turrini, F., A. Naitana, L. Mannuzzu, G. Pescarmona e P. Arese. "Increased red cell calcium, decreased calcium adenosine triphosphatase, and altered membrane proteins during fava bean hemolysis in glucose-6- phosphate dehydrogenase-deficient (Mediterranean variant) individuals". Blood 66, n. 2 (1 agosto 1985): 302–5. http://dx.doi.org/10.1182/blood.v66.2.302.302.
Abstract (sommario):
Abstract RBCs from four glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) subjects were studied during fava bean hemolysis. In the density-fractionated RBC calcium level, Ca2+-ATPase activity, reduced glutathione level, and ghost protein pattern were studied. In the bottom fraction, containing most heavily damaged RBCs, calcium level ranged from 143 to 244 mumol/L RBCs (healthy G6PD- deficient controls: 17 +/- 5 mumol/L RBCs). The Ca2+-ATPase activity ranged from 0.87 to 1.84 mumol ATP consumed/g Hb/min (healthy G6PD- deficient controls: 2.27 +/- 0.4). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts showed: (1) the presence of high mol wt aggregates (in three cases they were reduced by dithioerythritol; in one case, only partial reduction was possible); (2) the presence of multiple, scattered new bands; and (3) the reduction of band 3. Oxidant-mediated damage to active calcium extrusion, hypothetically associated with increased calcium permeability, may explain the large increase in calcium levels. They, in turn, could activate calcium-dependent protease activity, giving rise to the profound changes in the ghost protein pattern.
9
Turrini, F., A. Naitana, L. Mannuzzu, G. Pescarmona e P. Arese. "Increased red cell calcium, decreased calcium adenosine triphosphatase, and altered membrane proteins during fava bean hemolysis in glucose-6- phosphate dehydrogenase-deficient (Mediterranean variant) individuals". Blood 66, n. 2 (1 agosto 1985): 302–5. http://dx.doi.org/10.1182/blood.v66.2.302.bloodjournal662302.
Abstract (sommario):
RBCs from four glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) subjects were studied during fava bean hemolysis. In the density-fractionated RBC calcium level, Ca2+-ATPase activity, reduced glutathione level, and ghost protein pattern were studied. In the bottom fraction, containing most heavily damaged RBCs, calcium level ranged from 143 to 244 mumol/L RBCs (healthy G6PD- deficient controls: 17 +/- 5 mumol/L RBCs). The Ca2+-ATPase activity ranged from 0.87 to 1.84 mumol ATP consumed/g Hb/min (healthy G6PD- deficient controls: 2.27 +/- 0.4). Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts showed: (1) the presence of high mol wt aggregates (in three cases they were reduced by dithioerythritol; in one case, only partial reduction was possible); (2) the presence of multiple, scattered new bands; and (3) the reduction of band 3. Oxidant-mediated damage to active calcium extrusion, hypothetically associated with increased calcium permeability, may explain the large increase in calcium levels. They, in turn, could activate calcium-dependent protease activity, giving rise to the profound changes in the ghost protein pattern.
10
Cardon, Tristan, Isabelle Fournier e Michel Salzet. "SARS-Cov-2 Interactome with Human Ghost Proteome: A Neglected World Encompassing a Wealth of Biological Data". Microorganisms 8, n. 12 (19 dicembre 2020): 2036. http://dx.doi.org/10.3390/microorganisms8122036.
Abstract (sommario):
Conventionally, eukaryotic mRNAs were thought to be monocistronic, leading to the translation of a single protein. However, large-scale proteomics have led to a massive identification of proteins translated from mRNAs of alternative ORF (AltORFs), in addition to the predicted proteins issued from the reference ORF or from ncRNAs. These alternative proteins (AltProts) are not represented in the conventional protein databases and this “ghost proteome” was not considered until recently. Some of these proteins are functional and there is growing evidence that they are involved in central functions in physiological and physiopathological context. Based on our experience with AltProts, we were interested in finding out their interaction with the viral protein coming from the SARS-CoV-2 virus, responsible for the 2020 COVID-19 outbreak. Thus, we have scrutinized the recently published data by Krogan and coworkers (2020) on the SARS-CoV-2 interactome with host cells by affinity purification in co-immunoprecipitation (co-IP) in the perspective of drug repurposing. The initial work revealed the interaction between 332 human cellular reference proteins (RefProts) with the 27 viral proteins. Re-interrogation of this data using 23 viral targets and including AltProts, followed by enrichment of the interaction networks, leads to identify 218 RefProts (in common to initial study), plus 56 AltProts involved in 93 interactions. This demonstrates the necessity to take into account the ghost proteome for discovering new therapeutic targets, and establish new therapeutic strategies. Missing the ghost proteome in the drug metabolism and pharmacokinetic (DMPK) drug development pipeline will certainly be a major limitation to the establishment of efficient therapies.
11
Ito, Shota, Hideki Kandori e Victor A. Lorenz-Fonfria. "Potential Second-Harmonic Ghost Bands in Fourier Transform Infrared (FT-IR) Difference Spectroscopy of Proteins". Applied Spectroscopy 72, n. 6 (13 febbraio 2018): 956–63. http://dx.doi.org/10.1177/0003702818757521.
Abstract (sommario):
Fourier transform infrared (FT-IR) difference absorption spectroscopy is a common method for studying the structural and dynamical aspects behind protein function. In particular, the 2800–1800 cm−1 spectral range has been used to obtain information about internal (deuterated) water molecules, as well as site-specific details about cysteine residues and chemically modified and artificial amino acids. Here, we report on the presence of ghost bands in cryogenic light-induced FT-IR difference spectra of the protein bacteriorhodopsin. The presence of these ghost bands can be particularly problematic in the 2800–1900 cm−1 region, showing intensities similar to O–D vibrations from water molecules. We demonstrate that they arise from second harmonics from genuine chromophore bands located in the 1400–850 cm−1 region, generated by double-modulation artifacts caused from reflections of the IR beam at the sample and at the cryostat windows back to the interferometer (inter-reflections). The second-harmonic ghost bands can be physically removed by placing an optical filter of suitable cutoff in the beam path, but at the cost of losing part of the multiplexing advantage of FT-IR spectroscopy. We explored alternatives to the use of optical filters. Tilting the cryostat windows was effective in reducing the intensity of the second harmonic artifacts but tilting the sample windows was not, presumably by their close proximity to the focal point of the IR beam. We also introduce a simple numerical post-processing approach that can partially, but not fully, correct for second-harmonic ghost bands in FT-IR difference spectra.
12
Plundrich, Nathalie, Mary Ann Lila, Edward Foegeding e Scott Laster. "Protein-bound polyphenols create “ghost” band artifacts during chemiluminescence-based antigen detection". F1000Research 6 (13 marzo 2017): 254. http://dx.doi.org/10.12688/f1000research.10622.1.
Abstract (sommario):
Antigen detection during Western blotting commonly utilizes a horseradish peroxidase-coupled secondary antibody and enhanced chemiluminescent substrate. We utilized this technique to examine the impact of green tea-derived polyphenols on the binding of egg white protein-specific IgE antibodies from allergic human plasma to their cognate antigens. Our experiments unexpectedly showed that green tea-derived polyphenols, when stably complexed with egg white proteins, caused hyperactivation of horseradish peroxidase resulting in the appearance of white “ghost” bands. This study suggests that caution should be taken when evaluating polyphenol-bound proteins by enhanced chemiluminescence Western blotting using horseradish peroxidase and demonstrates that protein-bound polyphenols can be a source of “ghost” band artifacts on Western blots.
13
Plundrich, Nathalie, Mary Ann Lila, Edward Foegeding e Scott Laster. "Protein-bound polyphenols create “ghost” band artifacts during chemiluminescence-based antigen detection". F1000Research 6 (26 maggio 2017): 254. http://dx.doi.org/10.12688/f1000research.10622.2.
Abstract (sommario):
Antigen detection during Western blotting commonly utilizes a horseradish peroxidase-coupled secondary antibody and enhanced chemiluminescent substrate. We utilized this technique to examine the impact of green tea-derived polyphenols on the binding of egg white protein-specific IgE antibodies from allergic human plasma to their cognate antigens. Our experiments unexpectedly showed that green tea-derived polyphenols, when stably complexed with egg white proteins, caused “ghost” band formation in the presence of horseradish peroxide. This study suggests that caution should be taken when evaluating polyphenol-bound proteins by enhanced chemiluminescence Western blotting using horseradish peroxidase and demonstrates that protein-bound polyphenols can be a source of “ghost” band artifacts on Western blots.
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ørntoft, T. F., e N. Clausen. "Hereditary spherocytosis: Diagnostic and anaemia-associated aberrations of ghost proteins". Scandinavian Journal of Clinical and Laboratory Investigation 54, n. 2 (gennaio 1994): 95–103. http://dx.doi.org/10.3109/00365519409086515.
15
Cardon, Tristan, Flore Hervé, Vivian Delcourt, Xavier Roucou, Michel Salzet, Julien Franck e Isabelle Fournier. "Optimized Sample Preparation Workflow for Improved Identification of Ghost Proteins". Analytical Chemistry 92, n. 1 (12 dicembre 2019): 1122–29. http://dx.doi.org/10.1021/acs.analchem.9b04188.
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SCHWARTZ, S. Robert, C. Anne RYBICKI e L. Ronald NAGEL. "Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin". Biochemical Journal 327, n. 2 (15 ottobre 1997): 609–16. http://dx.doi.org/10.1042/bj3270609.
Abstract (sommario):
We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293 Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and β-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.
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Cardon, Tristan, Michel Salzet, Julien Franck e Isabelle Fournier. "Nuclei of HeLa cells interactomes unravel a network of ghost proteins involved in proteins translation". Biochimica et Biophysica Acta (BBA) - General Subjects 1863, n. 10 (ottobre 2019): 1458–70. http://dx.doi.org/10.1016/j.bbagen.2019.05.009.
18
Barel, Gilli, Alexandra Sirota, Hanne Volpin e Edouard Jurkevitch. "Fate of Predator and Prey Proteins during Growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae Prey". Journal of Bacteriology 187, n. 1 (1 gennaio 2005): 329–35. http://dx.doi.org/10.1128/jb.187.1.329-335.2005.
Abstract (sommario):
ABSTRACT A two-dimensional electrophoretic analysis of protein distribution followed by identification of selected proteins by mass spectrometry was performed on fresh bdellovibrio cultures containing attack phase cells of the predatory bacterium Bdellovibrio bacteriovorus strain 109J-1 and the remains of an Escherichia coli or a Pseudomonas syringae pv. tomato prey. Cleavage of the peptidoglycan-associated outer membrane proteins (OMPs) OmpA in E. coli and OprF in P. syringae occurred in both prey. The tryptic peptides obtained from the cleavage products of OmpA and OprF were all located within the 19-kDa pronase-resistant N-terminal parts of the corresponding proteins. The predator cell fraction was separated from the prey ghosts in fresh bdellovibrio cultures by centrifugation on a Percoll-sucrose cushion. Proteins from each fraction were separated by two-dimensional electrophoresis and identified by mass spectrometric analysis. As no prey OMP could be detected in the predator cell fraction, it was concluded that prey OMPs are not transferred to the predator, as had been suggested previously. However, a protein from the predator was found bound to ghost cell envelopes. This protein may correspond to a protein earlier suggested to be associated with the prey outer or cytoplasmic membranes. Along with recently described polypeptides from B. bacteriovorus strains 100 and 114, it forms a new family of putative outer membrane proteins.
19
Murgoci, Adriana-Natalia, Tristan Cardon, Soulaimane Aboulouard, Marie Duhamel, Isabelle Fournier, Dasa Cizkova e Michel Salzet. "Reference and Ghost Proteins Identification in Rat C6 Glioma Extracellular Vesicles". iScience 23, n. 5 (maggio 2020): 101045. http://dx.doi.org/10.1016/j.isci.2020.101045.
20
Xiao, Guipeng, Jintao Lu, Zhende Yang, Hengfei Fu e Ping Hu. "A Study of Adult Olfactory Proteins of Primitive Ghost Moth, Endoclita signifer (Lepidoptera, Hepialidae)". Life 13, n. 12 (27 novembre 2023): 2264. http://dx.doi.org/10.3390/life13122264.
Abstract (sommario):
Endoclita signifer is a prominent wood-boring insect species in eucalyptus plantations in Guangxi, China, causing significant ecological and economic damage. A novel approach to controlling the challenging wood-boring pest involves disrupting the olfactory communication between insects and the volatile compounds emitted by plants. To identify the olfactory proteins contributing to host selection based on 11 GC-EAD-active volatiles from eucalyptus leaves and to discover the highly expressed olfactory proteins, we conducted a study on the antennal transcriptomes of adult E. signifer and screened key olfactory proteins in the antennae. We identified a total of 69 olfactory proteins. When compared to the larval transcriptomes, the antennal transcriptome of adult E. signifer revealed the presence of 17 new odorant-binding proteins (OBPs), including 2 pheromone-binding proteins (PBPs), 7 previously unreported chemosensory proteins (CSPs), 17 new odorant receptors (ORs), 4 new gustatory receptors (GRs), 11 novel ionotropic receptors (IRs), and 2 sensory neuron membrane proteins (SNMPs). Through the phylogenetic tree of OBPs and ORs, we identified EsigPBP2 and EsigPBP3 as two of the three PBPs, designated EsigOR13 as EsigOrco, and recognized EsigOR10 and EsigOR22 as the newly discovered EsigPRs in E. signifer. In the adult antennae, the expression levels of EsigGOBP14, EsigGOBP13, EsigOBP14, EsigOBP17, EsigCSP14, and EsigOR16 were notably high, indicating that these proteins could be pivotal in binding to plant volatiles.
21
Bradford, Emily, Gary Shull e Marian Miller. "The Ghost of the IEL: A Halloween Photoshop Exercise". Microscopy Today 16, n. 6 (novembre 2008): 75. http://dx.doi.org/10.1017/s1551929500062465.
Abstract (sommario):
Image of an intraepithelial lymphocyte (IEL) from a CLIC5 mutant mouse small intestine. The CLIC (Chloride Intracellular Channel) family of proteins is expressed in a wide variety of cell types, and several isoforms are known to cycle between soluble and membranebound forms. As well as being widely expressed, the CLICs are involved in diverse functions, including tubulogenesis, immune cell activation, apoptosis and calcium handling. CLIC5 has been shown to associate with cytoskeletal proteins in placental microvilli and inner ear cells, and is required for proper maintenence of hair cell steriocilia. It has also been localized to the cytosol of human intestinal epithelial cells, though its function there remains unclear. The study in which this particular “IEL” was found, involved a search to see what function CLIC5 played in the modulation of tubulovesicles and microvillar apical membranes in the process of acid secretion. In particular, the relative amounts and structural characteristics of these two membrane types was quantified in parietal cells.
22
Wooden, Jason M., Greg L. Finney, Michael J. MacCoss, Luanne L. Peters e Diana M. Gilligan. "Proteomic Analysis of RBC Ghosts from the Beta-Adducin and Protein 4.2 Knock-Out Mouse Models of Inherited Hemolytic Anemia." Blood 110, n. 11 (16 novembre 2007): 1728. http://dx.doi.org/10.1182/blood.v110.11.1728.1728.
Abstract (sommario):
Abstract Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases with an incidence of 1:2500 to 1:5000 in populations of Northern European descent. While it is known that mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability and non-erythroid effects of known RBC membrane skeleton mutations. To identify proteins that may be involved in disease severity and secondary effects, we used shotgun proteomics to globally profile proteins in RBC ghosts (i.e., RBC membrane skeleton and associated proteins) from well-defined mouse models of inherited hemolytic anemia. A peptide level ‘bottom-up’ analysis was performed on RBCs from normal mice, beta-adducin knock-out mice (Add2-KO, compensated anemia), and protein 4.2 knock-out mice (4.2-KO, mild anemia). For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, five replicate runs utilizing 0.1 ug digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Normal versus diseased comparisons were made using a protein profile found consistently across all independent samples for each genotype. In total, 435 unique proteins were identified for the normal mouse RBC ghost. In contrast, 731 and 848 unique proteins were identified for the Add2-KO and 4.2-KO mice RBC ghosts respectively. Previously identified membrane skeleton proteins were found for all three genotypes with the predicted absence of the knock-out proteins. In addition to well-known membrane proteins, a surprising number of proteins were found involved in processes such as protein repair, protein degradation, Ras oncogene biology, and glycolysis. For both knock-out mice, a large number of proteins involved in translation were identified most likely reflecting their elevated reticulocytosis status. Comparison of the normal and Add2-KO RBC profiles revealed 5 proteins present only in normal the RBC while 53 proteins were present only in the diseased RBC. Likewise, normal vs 4.2 KO comparison revealed 6 proteins present only in the normal RBC while 111 were only in the diseased RBC. Comparison between the two KO mice revealed 34 proteins present only in the Add2-KO and 88 proteins present only in the 4.2 KO. Some of the identified differences are proteins with unknown functions (example, SH3-binding domain glutamic acid-rich protein like). Other differences involve proteins associated with diverse processes such as protein folding (Bcl2-associated athanogene 2), protein modification (magnesium-dependent phosphatase-1), protein transport (RAB35), metabolism (N-acetylneuraminic acid phosphatase), signal transduction (Prohibitin 2), and apoptosis (Rho GTPase activating protein 1). We report that tandem mass spec analysis of disease model RBC ghosts have demonstrated differences in their proteomes and that these identified differences potentially represent candidate proteins involved in disease severity and secondary effects.
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Vaziri, C., e C. P. Downes. "G-protein-mediated activation of turkey erythrocyte phospholipase C by β-adrenergic and P2y-purinergic receptors". Biochemical Journal 284, n. 3 (15 giugno 1992): 917–22. http://dx.doi.org/10.1042/bj2840917.
Abstract (sommario):
Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5′-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5′-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.
24
Hjelm, Anna, Bill Söderström, David Vikström, Wouter S. P. Jong, Joen Luirink e Jan-Willem de Gier. "Autotransporter-Based Antigen Display in Bacterial Ghosts". Applied and Environmental Microbiology 81, n. 2 (14 novembre 2014): 726–35. http://dx.doi.org/10.1128/aem.02733-14.
Abstract (sommario):
ABSTRACTBacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered theEscherichia coliautotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using theMycobacterium tuberculosisvaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorateE. coliandSalmonellaghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E inE. coliandSalmonellacan be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.
25
Collin, Matthew A., Kazuei Mita, Frantisek Sehnal e Cheryl Y. Hayashi. "Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus". Journal of Molecular Evolution 70, n. 5 (maggio 2010): 519–29. http://dx.doi.org/10.1007/s00239-010-9349-8.
26
Plattner, H., R. Pape, B. Haacke, K. Olbricht, C. Westphal e H. Kersken. "Synchronous exocytosis in Paramecium cells. VI. Ultrastructural analysis of membrane resealing and retrieval". Journal of Cell Science 77, n. 1 (1 agosto 1985): 1–17. http://dx.doi.org/10.1242/jcs.77.1.1.
Abstract (sommario):
After the synchronous induction of exocytosis of secretory organelles (trichocysts) in Paramecium tetraurelia cells the process of membrane resealing and retrieval could be followed under synchronous conditions. The characteristic aggregates of membrane intercalated particles (MIPs) contained within the freeze-fractured cell membrane (rings and rosettes) and trichocyst membranes (annulus MIPs), in addition to collar striations on the top of trichocyst membranes, served as endogenous ultrastructural markers. This allowed us to follow the re-arrangement of membrane constituents during and after exocytosis with high temporal and spatial precision. Membrane specificity is maintained to a considerable extent (approximately 99.5%), as judged from the rare occurrence of aberrant resealing (according to freeze-fracture data) and from the rather minute shift of glycocalyx components (according to electron staining experiments) during normal membrane resealing. Coated pits are not involved in membrane retrieval (155 ghosts analysed); since the membrane regions involved in exocytotic fusion are backed by apposed materials, probably proteins, this may restrain membrane constituents from intermixing. Another factor for maintaining membrane specificity is the fact that resealing of the exocytotic opening occurs much more rapidly than in most other systems. The retrieval operates with a half-life of 3 (strain 7S) to 9 min (K401); the involvement of cortical microtubules in the retrieval can be largely excluded, since only two microtubules (of unidentified origin) were seen to approach ghost structures in 4074 cases analysed during this period of intense ghost retrieval. Phalloidin microinjected at a dose that blocked all cytoplasmic streaming (before synchronous exocytosis was induced) did not abolish membrane resealing and retrieval, which, therefore, may be passive processes.
27
Żyłka, Romuald, Justyna Kupiec e Stanislaw Przestalski. "Peptides conformational changes of the erythrocyte membrane induced by organometallic tin compounds". Current Topics in Biophysics 34, n. 1 (1 gennaio 2011): 31–35. http://dx.doi.org/10.2478/v10214-011-0005-2.
Abstract (sommario):
Peptides conformational changes of the erythrocyte membrane induced by organometallic tin compoundsThe paper presents the results of a study on the effect of selected organic chlorides of tin on peptide conformations of erythrocyte ghosts from pig blood. The following compounds were used: dibutyltin dichloride (DBT), tributyltin chloride (TBT), diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT). Peptide conformation changes were determined on the basis of measurements done with the ATR FTIR technique. This method made it possible to measure the percent share of a peptide with specified conformation in the whole amount of the peptides in the membranes studied. The investigation showed that all the tin organic compounds studied cause a several-percent decrease in the quantities of both the peptides with the α-helix and turn conformation, and about a 20% increase in ghost peptides with β-sheet conformation. It seems that the changes observed can cause disturbances in the function of proteins and, consequently, the activity of the membrane; and this may be one of the aspects of the toxic properties of organotins.
28
Rybicki, AC, RS Schwartz, EJ Hustedt e CE Cobb. "Increased rotational mobility and extractability of band 3 from protein 4.2-deficient erythrocyte membranes: evidence of a role for protein 4.2 in strengthening the band 3-cytoskeleton linkage". Blood 88, n. 7 (1 ottobre 1996): 2745–53. http://dx.doi.org/10.1182/blood.v88.7.2745.bloodjournal8872745.
Abstract (sommario):
Band 3 (anion-exchange protein 1-[AE1]) is the major integral membrane protein of human erythrocytes and links the membrane to the underlying cytoskeleton via high-affinity binding to ankyrin. It is unclear whether other cytoskeletal proteins participate in strengthening the ankyrin-band 3 linkage, but a putative role for protein 4.2 (P4.2) has been proposed based on the increased osmotic fragility and spherocytic morphology of P4.2-deficient red blood cells (RBCs). The present study was designed to investigate the hypothesis that P4.2 has a direct role in strengthening the band 3-cytoskeleton linkage in human RBCs, by measuring independent features of this interaction in normal and P4.2-deficient RBCs. The features examined were the rotational mobility of band 3 assayed by time-resolved phosphorescence emission anisotropy (TPA), and the extractability of band 3 by octyl-beta-glucoside, the latter being a nonionic detergent that selectively extracts only band 3 that is not anchored to the cytoskeleton. We find that the amplitude of the most rapidly rotating population of band 3 (correlation time, approximately 30 to 60 microseconds) is increased 81% and 67% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts. The amplitude of the intermediate speed rotating population of band 3 (correlation time, approximately 200 to 500 microseconds) is increased 23% and 8% in P4.2-deficient ghosts (P4.2NIPPON and band 3MONTEFIORE, respectively) compared with control ghosts, at the expense of the slowly rotating component (correlation time, approximately 2,000 to 3,000 microseconds, amplitude decreased 43% and 39% in P4.2NIPPON and band 3MONTEFIORE, respectively) and immobile component (immobile on this experimental time scale; amplitude decreased 26% and 10% in P4.2NIPPON and band 3MONTEFIORE, respectively) of band 3. These results demonstrate that P4.2 deficiency partially removes band 3 rotational constraints, ie, it increases band 3 rotational mobility. The nonionic detergent octyl-beta-glucoside, which does not disturb band 3-cytoskeleton associations, ie, it extracts only band 3 that is not attached to the cytoskeleton, extracted 30% and 61% more band 3 from P4.2NIPPON and band 3MONTEFIORE ghost membranes, respectively, compared with control ghosts. The octyl-beta-glucoside ghost extracts from both P4.2-deficient phenotypes were enriched in band 3 oligomeric species (tetramers, higher-order oligomers, and aggregates) compared with controls. Since band 3 oligomers selectively associate with the cytoskeleton, these results are consistent with a weakened band 3-cytoskeleton linkage in P4.2-deficient RBC membranes. P4.2 deficiency does not affect band 3 anion transport activity, since uptake of radiolabeled sulfate was similar for control and P4.2-deficient RBCs. Thus, we propose that P4.2 directly participates in strengthening the band 3-cytoskeleton linkage.
29
Glowacki, David R., Jeremy N. Harvey e Adrian J. Mulholland. "Protein dynamics and enzyme catalysis: the ghost in the machine?" Biochemical Society Transactions 40, n. 3 (22 maggio 2012): 515–21. http://dx.doi.org/10.1042/bst20120047.
Abstract (sommario):
One of the most controversial questions in enzymology today is whether protein dynamics are significant in enzyme catalysis. A particular issue in these debates is the unusual temperature-dependence of some kinetic isotope effects for enzyme-catalysed reactions. In the present paper, we review our recent model [Glowacki, Harvey and Mulholland (2012) Nat. Chem. 4, 169–176] that is capable of reproducing intriguing temperature-dependences of enzyme reactions involving significant quantum tunnelling. This model relies on treating multiple conformations of the enzyme–substrate complex. The results show that direct ‘driving’ motions of proteins are not necessary to explain experimental observations, and show that enzyme reactivity can be understood and accounted for in the framework of transition state theory.
30
Hopgood, M. F., S. E. Knowles, J. S. Bond e F. J. Ballard. "Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells". Biochemical Journal 256, n. 1 (15 novembre 1988): 81–88. http://dx.doi.org/10.1042/bj2560081.
Abstract (sommario):
The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.
31
Willett, Brian J., Celia A. Cannon e Margaret J. Hosie. "Upregulation of Surface Feline CXCR4 Expression following Ectopic Expression of CCR5: Implications for Studies of the Cell Tropism of Feline Immunodeficiency Virus". Journal of Virology 76, n. 18 (15 settembre 2002): 9242–52. http://dx.doi.org/10.1128/jvi.76.18.9242-9252.2002.
Abstract (sommario):
ABSTRACT Feline CXCR4 and CCR5 were expressed in feline cells as fusion proteins with enhanced green fluorescent protein (EGFP). Expression of the EGFP fusion proteins was localized to the cell membrane, and surface expression of CXCR4 was confirmed by using a cross-species-reactive anti-CXCR4 monoclonal antibody. Ectopic expression of feline CCR5 enhanced expression of either endogenous feline CXCR4 or exogenous feline or human CXCR4 expressed from a retrovirus vector, indicating that experiments investigating the effect of CCR5 expression on feline immunodeficiency virus (FIV) infection must be interpreted with caution. Susceptibility to infection with cell culture-adapted strains of FIV or to syncytium formation following transfection with a eukaryotic vector expressing an env gene from a cell culture-adapted strain of virus correlated with expression of either human or feline CXCR4, whereas feline CCR5 had no effect. In contrast, neither CXCR4 nor CCR5 rendered cells permissive to either productive infection with primary strains of FIV or syncytium formation following transfection with primary env gene expression vectors. Screening a panel of Ghost cell lines expressing diverse human chemokine receptors confirmed that CXCR4 alone supported fusion mediated by the FIV Env from cell culture-adapted viruses. CXCR4 expression was upregulated in Ghost cells coexpressing CXCR4 and CCR5 or CXCR4, CCR5, and CCR3, and susceptibility to FIV infection could be correlated with the level of CXCR4 expression. The data suggest that β-chemokine receptors may influence FIV infection by modulating the expression of CXCR4.
32
Trindade, I. B., G. Hernandez, E. Lebègue, F. Barrière, T. Cordeiro, M. Piccioli e R. O. Louro. "Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli". JBIC Journal of Biological Inorganic Chemistry 26, n. 2-3 (9 febbraio 2021): 313–26. http://dx.doi.org/10.1007/s00775-021-01854-y.
Abstract (sommario):
AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract
33
MAYNARD, D. M., H. F. G. HEIJNEN, W. A. GAHL e M. GUNAY-AYGUN. "The α-granule proteome: novel proteins in normal and ghost granules in gray platelet syndrome". Journal of Thrombosis and Haemostasis 8, n. 8 (27 maggio 2010): 1786–96. http://dx.doi.org/10.1111/j.1538-7836.2010.03932.x.
34
Kuross, SA, BH Rank e RP Hebbel. "Excess heme in sickle erythrocyte inside-out membranes: possible role in thiol oxidation". Blood 71, n. 4 (1 aprile 1988): 876–82. http://dx.doi.org/10.1182/blood.v71.4.876.876.
Abstract (sommario):
Abstract It has been suggested that the development of sickle RBC membrane defects might be related to abnormal amounts of membrane-associated heme (a term we use in its generic sense to include hemoglobins, hemichromes, and free heme). Techniques previously used to measure membrane heme, however, would not distinguish between what is truly membrane-associated and what is merely trapped in RBC ghost preparations. Consequently, we have examined extensively washed inside- out membranes (IOM) prepared from normal and sickle RBC. Approximately 25% of the sickle ghost heme is lost upon conversion to IOM, but sickle IOM still have a significant excess (1.6 +/- 0.3 nmol heme/mg membrane protein compared with 0.7 +/- 0.2 nmol/mg for normal IOM, P less than .001). Amounts of ghost heme are only poorly predictive of amounts of IOM heme (r = .664). Preparation of IOM by using isotonic lysis with saponin yields virtually identical amounts of IOM heme. Small amounts of heme (less than 15%) can be displaced from IOM by using manipulations that elute spectrin, displace electrostatically bound proteins, or cleave the cytoplasmic portion of band 3. Treatment of IOM with dithiothreitol (DTT), however, displaces the most heme (35%), and this is almost reproduced (25% displacement) by the treatment of intact RBC with DTT before IOM preparation. Sequential treatment with all manipulations still leaves about 40% of the heme in sickle IOM, which indicates a compartment more intimately associated with the membrane. At least part of this is free heme without globin, as evidenced by abnormal binding of radiochloroquine to sickle IOM. Conversely, some IOM-associated globin is globin without heme because the measurement of globin per se markedly overpredicts amount of IOM heme. There is a strong correlation between RBC density and amounts of either ghost or IOM heme. Finally, the amount of membrane thiol oxidation (as measured by thiol-disulfide-exchange chromatography) does not correlate at all with ghost heme (r = .105), but it correlates well with IOM heme (r = .877, P less than .001). These data demonstrate that there are abnormal amounts of heme truly associated with sickle RBC membranes, and they are consistent with the hypothesis that this membrane-associated heme participates in the pathobiology of the sickle RBC membrane, particularly those aspects perhaps related to thiol oxidation.
35
Kuross, SA, BH Rank e RP Hebbel. "Excess heme in sickle erythrocyte inside-out membranes: possible role in thiol oxidation". Blood 71, n. 4 (1 aprile 1988): 876–82. http://dx.doi.org/10.1182/blood.v71.4.876.bloodjournal714876.
Abstract (sommario):
It has been suggested that the development of sickle RBC membrane defects might be related to abnormal amounts of membrane-associated heme (a term we use in its generic sense to include hemoglobins, hemichromes, and free heme). Techniques previously used to measure membrane heme, however, would not distinguish between what is truly membrane-associated and what is merely trapped in RBC ghost preparations. Consequently, we have examined extensively washed inside- out membranes (IOM) prepared from normal and sickle RBC. Approximately 25% of the sickle ghost heme is lost upon conversion to IOM, but sickle IOM still have a significant excess (1.6 +/- 0.3 nmol heme/mg membrane protein compared with 0.7 +/- 0.2 nmol/mg for normal IOM, P less than .001). Amounts of ghost heme are only poorly predictive of amounts of IOM heme (r = .664). Preparation of IOM by using isotonic lysis with saponin yields virtually identical amounts of IOM heme. Small amounts of heme (less than 15%) can be displaced from IOM by using manipulations that elute spectrin, displace electrostatically bound proteins, or cleave the cytoplasmic portion of band 3. Treatment of IOM with dithiothreitol (DTT), however, displaces the most heme (35%), and this is almost reproduced (25% displacement) by the treatment of intact RBC with DTT before IOM preparation. Sequential treatment with all manipulations still leaves about 40% of the heme in sickle IOM, which indicates a compartment more intimately associated with the membrane. At least part of this is free heme without globin, as evidenced by abnormal binding of radiochloroquine to sickle IOM. Conversely, some IOM-associated globin is globin without heme because the measurement of globin per se markedly overpredicts amount of IOM heme. There is a strong correlation between RBC density and amounts of either ghost or IOM heme. Finally, the amount of membrane thiol oxidation (as measured by thiol-disulfide-exchange chromatography) does not correlate at all with ghost heme (r = .105), but it correlates well with IOM heme (r = .877, P less than .001). These data demonstrate that there are abnormal amounts of heme truly associated with sickle RBC membranes, and they are consistent with the hypothesis that this membrane-associated heme participates in the pathobiology of the sickle RBC membrane, particularly those aspects perhaps related to thiol oxidation.
36
Jain, Amisha, Himanshu Dhanodkar, Anjali Shujalpurkar e Gauri Motiwale. "Calcifying Odontogenic Cyst: An Enigma". International Journal of Orofacial Research 7, n. 2 (5 novembre 2023): 37–41. http://dx.doi.org/10.56501/intjorofacres.v7i2.895.
Abstract (sommario):
The calcifying odontogenic cyst (COC), discovered in 1962, is a rare developmental odontogenic cyst clinically present as slow-growing swelling mainly in the anterior portion of the jaws, generally present in the second and sixth decades of life. It accounts for 0.3%–0.8% of odontogenic cysts. COC showed variations in clinical and radiographic features that are not pathognomic, whereas histomorphology forms exist in 3 patterns: benign cystic, solid (neoplastic), and aggressive (malignant) forms. The radiograph shows well-defined radiolucency with irregular masses, while the histopathologic features include a cystic lining with characteristic “Ghost” cells and immunohistochemical reactions positive for various enamel proteins.
37
Angelescu, Iulia-Roxana, Medana Zamfir, Emanuela-Cătălina Ionetic e Silvia-Simona Grosu-Tudor. "The Biological Role of the S-Layer Produced by Lactobacillus helveticus 34.9 in Cell Protection and Its Probiotic Properties". Fermentation 10, n. 3 (6 marzo 2024): 150. http://dx.doi.org/10.3390/fermentation10030150.
Abstract (sommario):
Lactobacillus helveticus 34.9 was isolated from a sample of Romanian home-made fermented milk, producing both surface layer proteins and a class III bacteriocin. The present study aimed to investigate the biological and functional role of the S-layer in correlation with its probiotic properties. The presence of S-layer proteins resulted in various degrees of co-aggregation of L. helveticus 34.9 with pathogens and with other lactic acid bacteria, but the removal of these proteins reduced the co-aggregation with all the tested strains. Moreover, the S-layer proved to be involved in cell wall hydrophobicity and cellular protection during freeze-drying. In the simulated passage through the gastrointestinal tract, S-layer depleted cells exhibited increased vulnerability, with greater viability loss in low pH and pepsin treatment compared to control cells. Subsequently, in the small intestine simulation, these cells lost all viability, underscoring the vital role of extracellular proteins for cell protection. The morphological effects of these treatments were observed by scanning electron microscopy. Severe structural damage was noticed when the S-layer was absent, including loss of cell shape and integrity as well as many ghost cells emptied of their content. Finally, the elimination of surface proteins reduced the interaction between L. helveticus 34.9 and mammalian cells.
38
May, James M., Zhi-chao Qu e Charles E. Cobb. "Reduction and uptake of methylene blue by human erythrocytes". American Journal of Physiology-Cell Physiology 286, n. 6 (giugno 2004): C1390—C1398. http://dx.doi.org/10.1152/ajpcell.00512.2003.
Abstract (sommario):
A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB+) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB+. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB+ <5 μM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB+-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB+-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB+, which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB+, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB+ reduction may correspond to MB+-dependent NAD(P)H reductase activity in erythrocyte ghosts.
39
Homer, Bruce L., Kenneth R. Pierce, Charles H. Bridges, James E. Womack, Blair A. Sowa e Ramon C. Littell. "Inhibition of Copper-Associated Erythrocyte Ghost Membrane Lipid Peroxidation by Hepatic Cytosolic Low Molecular Weight Proteins". Toxicologic Pathology 19, n. 3 (aprile 1991): 206–13. http://dx.doi.org/10.1177/019262339101900302.
40
Meng, Qian, Ji‐Hong Zhang, Huan Zhang, Gui‐Ling Zhou, Ruo‐Yao Ni, Yan‐Ni Zhao, Qi‐Lian Qin e Zhen Zou. "Comparative analysis of C‐type lectin domain proteins in the ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae)". Insect Science 26, n. 3 (22 febbraio 2018): 453–65. http://dx.doi.org/10.1111/1744-7917.12564.
41
Beck, K. A., J. A. Buchanan e W. J. Nelson. "Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex". Journal of Cell Science 110, n. 10 (15 maggio 1997): 1239–49. http://dx.doi.org/10.1242/jcs.110.10.1239.
Abstract (sommario):
To extend our finding of a Golgi-localized form of the membrane skeleton protein spectrin, we have identified an isoform of ankyrin that associates at steady state with the Golgi complex. Immuno-light and -electron microscopy show that this ankyrin isoform localizes to the perinuclear cytoplasm on tubular vesicular structures that co-stain with Golgi marker proteins. An antiserum raised against erythrocyte ankyrin, which was used to identify the Golgi ankyrin, recognized three prominent polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purification of this antiserum against each of these MDCK cell ankyrins revealed that only an antibody specific for the 195 kDa form retained the ability to stain the Golgi complex; affinity purified antibody preparations specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Antibody specific for the 195 kDa ankyrin did not recognize a recently identified 119 kDa ankyrin that is also localized to the Golgi. The 195 kDa Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-sediments with Golgi beta-spectrin during brief, low speed centrifugation of Triton X-100 extracts of MDCK cells. Golgi ankyrin and beta-spectrin are retained on tubular vesicular ‘Golgi ghosts’ following extraction of cultured cells with Triton X-100. Significantly, Golgi ghost tubules containing ankyrin/spectrin are co-linear with individual microtubules, suggesting a role for both Golgi membrane skeleton and microtubules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldin A, indicating that Golgi ankyrin has a dynamic assembly state similar to that of Golgi spectrin and other Golgi membrane coat proteins.
42
Klei, Thomas, Robin Van Bruggen, Jill Dalimot, Martijn Veldthuis, Erik Mul, Timo Rademakers, Mark Hoogenboezem et al. "Hemolysis in the Spleen Drives Erythrocyte Turnover". Blood 134, Supplement_1 (13 novembre 2019): 946. http://dx.doi.org/10.1182/blood-2019-124342.
Abstract (sommario):
Erythrocytes circulate for an average of 120 days before they are removed from the circulation. Various processes and factors have been identified that may contribute to degradation of senescent erythrocytes, but this complex process is still not completely understood. Accumulation of removal signals such as phosphatidylserine exposure, changes in CD47 expression and oxidation of proteins and lipids that render them susceptible to complement deposition, may contribute to recognition and degradation by red pulp macrophages (RPM) of the spleen. However, many questions remain on the exact mechanisms that determine the fate of aged erythrocytes. This is well exemplified in a mouse study in which physiologically aged erythrocytes were found to undergo phagocytosis by RPM in vivo but not in vitro. This finding suggested that the splenic architecture may play an important role in facilitating erythrocyte turnover. Loss of membrane deformability may lead to the initial trapping of aged or damaged erythrocytes in the spleen, an event that precedes their degradation by macrophages. Loss of deformability can explain why certain genetic diseases that affect erythrocyte membrane deformability, such as is the case in sickle cell disease and spherocytosis, result in trapping in the spleen, giving rise to anaemia. Next to loss of deformability, activation of adhesion molecules, such as Lu/BCAM and CD44, specifically on aged erythrocytes has been proposed to contribute to retention of erythrocytes within the spleen, leading to their turnover. In this study we provide evidence that the splenic environment is of key importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPM we noted that only a small proportion of the macrophages were in the process of phagocytosing intact erythrocytes. Based on a range of variables, including the number of erythrocytes that are cleared daily, the number of RPM present in the spleen, the degradation rate of erythrocytes as well as differential contribution of spleen and liver to erythrocyte turnover, conservative estimates approximate that at least a 30-fold fewer erythrophagocytic events are observed in RPM than anticipated. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of a large population of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By in vivo imaging of the spleen and transfusion experiments we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis, thereby forming erythrocyte ghosts. Of note, we found that the levels of haptoglobin and hemopexin, two plasma proteins that are involved in scavenging of haemoglobin and heme, respectively, correlate well with the rate of hemolysis that was observed in the spleen. Additionally, we show that the erythrocyte adhesion molecules which are specifically activated on aged erythrocytes, Lu/BCAM and CD44, cause senescent erythrocytes to interact with the extracellular matrix of the spleen. This adhesion molecule-driven retention of senescent erythrocytes, under low shear conditions, was found to result in steady shrinkage of the erythrocytes and ultimately resulted in hemolysis and ghost formation. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghosts were found to be immediately recognized and rapidly degraded (1-3 hours) by RPM, thereby explaining the lack of phagocytosis of intact erythrocytes in the spleen. Together, these data identify hemolysis and ghost formation as key events in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated. Disclosures No relevant conflicts of interest to declare.
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Yuan, J., A. Bunyaratvej, S. Fucharoen, C. Fung, E. Shinar e SL Schrier. "The instability of the membrane skeleton in thalassemic red blood cells". Blood 86, n. 10 (15 novembre 1995): 3945–50. http://dx.doi.org/10.1182/blood.v86.10.3945.bloodjournal86103945.
Abstract (sommario):
The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta- thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha- globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.
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Mhatre, Radhika D., e Sucheta P. Dandekar. "Evaluation of erythrocyte membrane lipids and proteins in renal disorders". International Journal of Research in Medical Sciences 10, n. 1 (28 dicembre 2021): 70. http://dx.doi.org/10.18203/2320-6012.ijrms20214870.
Abstract (sommario):
Background: Membrane lipids and proteins play a significant part in imparting membrane its rheological properties. These parameters are altered in diseased states. Exploring the conformational changes in renal disorders can widen our understanding of its impact on the circulatory system. This could lead to a new diagnostic parameter to study the progress of a disease.Methods: 120 blood samples collected from 30 kidney donors, 30 stage 3-4 Chronic kidney disease (CKD) patients (group 1) and 30 stage 5 CKD patients on dialysis (pre and post dialysis) (group 2) were lysed and washed to obtain erythrocyte ghost membranes. The proteins extracted from these membranes were estimated colorimetrically using Micro BCA kit. Phospholipids were separated and quantified using HPTLC. Fatty acids and cholesterol were analysed using GCMS.Results: The erythrocyte membrane protein profile showed lower values in group 2 participants than group 1 participants, but this difference was not significant. Distinct decreases in percentages of palmitic acid, myristic acid, stearic acid, dodecanoic acid, cholesterol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine were observed in both groups, with the lowest values in patients undergoing dialysis. Sphingomyelin and linoleic acid did not show any such trend across groups.Conclusions: The data is suggestive of an altered membrane structure in participants undergoing dialysis patients than the control group. This could be because of uremic toxins in the circulatory system affecting the membrane lipids. Decreased levels of essential phospholipids can impact the functions and lifespan of the erythrocytes. This could be a reason behind anaemia seen in most patients with CKD.
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Dubreuil, R. R., e G. B. Bouck. "The membrane skeleton of a unicellular organism consists of bridged, articulating strips." Journal of Cell Biology 101, n. 5 (1 novembre 1985): 1884–96. http://dx.doi.org/10.1083/jcb.101.5.1884.
Abstract (sommario):
In this paper we show that a membrane skeleton associated with the plasma membrane of the unicellular organism Euglena consists of approximately 40 individual S-shaped strips that overlap along their lateral margins. The region of strip overlap is occupied by a set of microtubule-associated bridges and microtubule-independent bridges. Both cell form and plasma membrane organization are dependent on the integrity of this membrane skeleton. Removal of the membrane skeleton with a low-molar base results in loss of membrane form and randomization of the paracrystalline membrane interior characteristic of untreated cells. Conversely, removal of the plasma membrane and residual cytoplasm with lithium 3,5-diiodosalicylate/Nonidet P-40 yields cell ghosts that retain the form of the original cell but consist only of the membrane skeleton. Two major polypeptides of 86 and 80 KD persist in the skeleton and two other major proteins of 68 and 39 kD are associated with the plasma membrane fraction. None of these components appears to be the same as the major polypeptides (spectrins, band 3) of the erythrocyte ghost, the other cell system in which a well-defined peripheral membrane skeleton has been identified. We suggest that the articulating strips of euglenoids are not only the basic unit of cell and surface form, but that they are also positioned to mediate or accommodate surface movements by sliding, and to permit surface replication by intussusception.
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Fye, Haddy KS, Paul Mrosso, Frédéric B. Piel, Simon Davis, Roman Fischer, Gration Rwegasira, Benedikt Kessler e Julie Makani. "Proteomics Pathways of Sickle Cell Anemia (P2SCA): A Comprehensive Analysis By Liquid Chromatography Mass Spectrometry of Erythrocyte Membrane Proteins Characterized from the Muhimbili Sickle Cell Programme, Tanzania". Blood 132, Supplement 1 (29 novembre 2018): 3653. http://dx.doi.org/10.1182/blood-2018-99-114650.
Abstract (sommario):
Abstract Annually, there are 312 000 births with Sickle Cell Anemia (SCA), which has been recognized as one of the most common inherited conditions in Africa and is currently emerging as a condition of prominence in much of the developed world due to migration patterns. Despite its growing importance, there has been a significant lag in the application of emerging methodologies to its research, in particular to support the discovery of disease-associated markers of potential implication in therapeutics and informing a more comprehensive understanding of the condition. To bring SCA in line with cutting-edge 'omics research, we conducted a study applying advanced mass spectrometry methods for the comprehensive characterization of the protein profiles of erythrocyte membranes. One hundred and twenty participants of confirmed Hemoglobin (Hb) phenotypes HbAA (18), HbAS (21) and HbSS (81) were enrolled from the Muhimbili Sickle Cell Programme in Tanzania. All consented individuals were confirmed to not be on hydroxyurea treatment and not having received a blood transfusion in the preceding 3 months. Whole blood was collected and mixed in 10 ml EDTA vials sent within 72 hours to the Target Discovery Institute (TDI), University of Oxford, UK. Once received, packed erythrocyte fractions were isolated and underwent a series of wash steps followed by erythrocyte lysis and the isolation of the ghost membranes by ultracentrifugation. The ghost cells were then prepared for liquid chromatography mass spectrometry (LCMS) analysis using a novel published method. Proteomics analysis on a Thermo Scientific Q-Exactive High Field MS identified 2,288 membrane or membrane associated proteins of which 1,605 showed significant difference between at least two of the Hb phenotypes with fold changes of up to 22. Specific comparisons identified 1,286 proteins showing significant (p value cut-off, 0.05) changes in expression between the HbAA and HbSS groups; 1,248 between the HbAS and HbSS groups and 278 between the HbAA and HbAS groups. A summary of 30 markers have been presented including a range of proteins of potential therapeutic impact. These were selected based on their ranked significance following statistical analysis, the overall distinction seen between the controls and the HbSS group and the robustness of their identification. The results obtained show significant changes in the presence or absence as well as levels of numerous proteins in the HbSS population compared to controls. This research is a significant contribution to the field of SCA research and forms the foundation to direct further study on the sickle erythrocyte membrane. The large number of proteins identified to be of association to individuals' Hb genotypes represents a significant breakthrough and could contribute to a better understanding of the pathophysiological mechanisms involved in the disease. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Iwaki-Egawa, Sachiko, e Garret M. Ihler. "Comparison of the abilities of proteins from Bartonella bacilliformis and Bartonella henselae to deform red cell membranes and to bind to red cell ghost proteins". FEMS Microbiology Letters 157, n. 1 (17 gennaio 2006): 207–17. http://dx.doi.org/10.1111/j.1574-6968.1997.tb12775.x.
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Sun, Zixuan, Wenjing Wu e Guren Zhang. "Characterization of novel β-1,3-glucan recognition proteins from a Tibetan Plateau ghost moth Thitarodes pui (Lepidoptera, Hepialidae)". Physiological Entomology 44, n. 1 (23 novembre 2018): 20–32. http://dx.doi.org/10.1111/phen.12272.
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Ohtani, Naoto, e Makoto Miyata. "Identification of a novel nucleoside triphosphatase from Mycoplasma mobile: a prime candidate motor for gliding motility". Biochemical Journal 403, n. 1 (13 marzo 2007): 71–77. http://dx.doi.org/10.1042/bj20061439.
Abstract (sommario):
A protein with a molecular mass of 42 kDa (P42) from Mycoplasma mobile, one of several mycoplasmas that exhibit gliding motility, was shown to be a novel NTPase (nucleoside triphosphatase). Although the P42 protein lacks a common ATP-binding sequence motif (Walker A), the recombinant proteins expressed in Escherichia coli certainly hydrolysed some nucleoside triphosphates, including ATP. The results of photoaffinity labelling by an ATP analogue supported that the P42 protein contains a specific binding site for ATP (or another nucleoside triphosphate). In the M. mobile genome, the P42 gene is located downstream of gli123, gli349 and gli521 genes, and they have been reported to be polycis-tronically transcribed. As the huge proteins encoded by gli123, gli349 and gli521 play a role in gliding motility of M. mobile, P42 might also have some kind of function in the gliding motility. The gliding motility of M. mobile is driven directly by ATP hydrolysis, but the key ATPase has not been identified. Our results showed that, among these four proteins, only P42 exhibited ATPase activity. Biochemical characteristics – optimal conditions for activity, substrate specificities, and inhibiting effects by ATP analogues – of the recombinant P42 proteins were very similar to those of a putative ATPase speculated from a previous analysis with a gliding ‘ghost’ whose cell membrane was permeabilized by Triton X-100. These results support the hypothesis that the P42 protein is the key ATPase in the gliding motility of M. mobile.
50
Vasievich, Matthew, Bin Zhang, Jesse Rinehart, Morgan Jones, Ivan Maillard e David Ginsburg. "Sec23b deficiency In Mice Results In Pancreatic Destruction and Defective long Term Hematopoietic Stem Cell Function". Blood 116, n. 21 (19 novembre 2010): 2038. http://dx.doi.org/10.1182/blood.v116.21.2038.2038.
Abstract (sommario):
Abstract Abstract 2038 Congenital Dyserythropoietic Anemia type II (CDAII) is an autosomal recessive disorder caused by mutations in the gene SEC23B. SEC23B is a component of the COPII coat complex that transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. CDAII is characterized by mild to moderate anemia and >10% bi- or multi- nucleate erythroblasts in the bone marrow. We generated Sec23b deficient mice (Sec23b gt/gt) from ES cells with a genetrap cassette inserted into the last intron of Sec23b. Sec23b gt/gt mice die at birth with destruction of the exocrine pancreas. Eight to 12 week old lethally irradiated C57BL/6J recipients were transplanted with 106 E17.5 fetal liver cells from either wildtype or Sec23b gt/gt mice. Mice were bled at 6, 8 and 12 weeks post transplant and no significant difference was observed in either hematocrit or hemoglobin (N=3-7 mice per group). Red blood cell (rbc) ghosts prepared from peripheral blood of transplanted mice showed no shift of Band 3 on SDS-PAGE analysis. Ghost fractions from Sec23b gt/gt recipients analyzed by western blot for the presence of residual ER proteins showed no difference between wildtype and Sec23b gt/gt fetal liver cell recipients. No significant difference was found in bi-nucleate erythroblasts in the bone marrow, myeloid:erythroid ratio or spleen mass. We quantitatively analyzed the proteome of mature rbc ghosts from Sec23b gt/gt recipients by Stabile Isotope Labeling of Amino acids in Cells (SILAC) proteomics using rbc ghosts from mice fed lysine labeled with 13C (SILAC chow). When SILAC-labeled rbc ghosts were compared by mass spectrometry analysis with ghosts from Sec23b gt/gt fetal liver cell recipients, no significant differences in abundance of rbc membrane proteins were observed. To more stringently test the ability of Sec23b gt/gt fetal liver cells to engraft, 5×105 wildtype or Sec23b gt/gt fetal liver cells were co-transplanted with 5×105 wildtype fetal liver cells expressing a GFP transgene. FACS analysis of peripheral blood obtained at 6 and 8 weeks post transplant showed significant out-competition of Sec23b gt/gt cells by GFP+ progenitors in Ter119+ rbcs (p<0.01, at all time points), CD3+ T cells (p<0.01 at 8 weeks), B220+ B cells (p<0.01, at all time points) and Mac1+Gr1+ myeloid cells (p<0.01, at all time points) compared to wildtype controls (N=5-7 mice per group). Fetal livers from wildtype and Sec23b gt/gt mice showed no difference in the total number of CD150+CD48-Sca1+cKit+(lineage-) long term hematopoietic stem cells (LT-HSCs) per fetal liver (p=0.45). In conclusion, Sec23b deficient humans and mice exhibit disparate phenotypes, apparently restricted to CDAII in humans and a prominent neonatal pancreatic insufficiency in mice. These differences could be due to evolutionary changes in relative expression and/or function of the Sec23a and b paralogs in humans and mice. However, we cannot exclude an allele-specific defect due to residual or aberrant function of the Sec23b gt allele. Analyses of a deleted and a conditional floxed Sec23b null allele are currently in progress. Finally our transplant data suggest that Sec23b traffics cargoes important for the function of murine LT-HSCs. This finding contrasts with the apparently erythroid-specific human phenotype, raising the possibility of a requirement for Sec23b function in the marrow microenvironment not tested by these transplant studies. Tissue specific deletion of Sec23b is currently in progress to address this question. Disclosures: No relevant conflicts of interest to declare.