scholarly journals Organization on the plasma membrane of the retinitis pigmentosa protein RP2: investigation of association with detergent-resistant membranes and polarized sorting

2003 ◽  
Vol 372 (2) ◽  
pp. 427-433 ◽  
Author(s):  
J. Paul CHAPPLE ◽  
Celene GRAYSON ◽  
Alison J. HARDCASTLE ◽  
Tracey A. BAILEY ◽  
Karl MATTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Retinitis Pigmentosa ◽  
Cell Line ◽  
Lipid Rafts ◽  
Retinal Pigment ◽  
Significant Proportion ◽  
Wide Range ◽  
Detergent Resistant Membranes ◽  
Acylated Proteins

Mutations in the retinitis pigmentosa protein gene RP2 account for up to 15% of X-linked retinitis pigmentosa. RP2 is a novel protein of unknown function, which is targeted to the plasma membrane by dual N-terminal acyl-modification. Dual-acylated proteins are targeted to lipid rafts, and some are subject to polarized sorting. Therefore we investigated the organization of RP2 on the plasma membrane. Endogenous RP2 protein was predominantly localized at the plasma membrane, and exogenously expressed green-fluorescent-protein-tagged protein was also targeted to the membrane in a wide range of cultured cells. High levels of endogenous RP2 protein were present in HeLa cells and in the retinal pigment epithelium-derived cell line ARPE19. A significant proportion of RP2 in cultured neuroblastoma cells was associated with detergent-resistant membranes (DRMs), but much less than other dually acylated proteins (e.g. Lyn and Fyn). In contrast, the RP2-interacting protein Arl3 (ADP-ribosylation factor-like 3) was not found to be associated with DRMs. The association of RP2 with DRMs was cholesterol-dependent. In polarized epithelial cells in culture and in vivo, RP2 was present in both the apical and basolateral domains of the plasma membrane. These data show that RP2 is not specific to either domain, unlike some other dually acylated proteins. Interestingly, the level of RP2 protein increased in the epithelial cell line Caco-2 with differentiation and polarization. These data show that RP2 is present on the membrane of all cell types examined both in vitro and in vivo, and that RP2 associates with lipid rafts, suggesting a potential role for the protein in signal transduction.

scholarly journals Cell-Fusing Agent Virus Reduces Arbovirus Dissemination in Aedes aegypti Mosquitoes In Vivo

2019 ◽  
Vol 93 (18) ◽  
Author(s):  
Artem Baidaliuk ◽  
Elliott F. Miot ◽  
Sebastian Lequime ◽  
Isabelle Moltini-Conclois ◽  
Fanny Delaigue ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Dengue Virus ◽  
Cell Line ◽  
Content Type ◽  
Cells In Culture ◽  
Mosquito Cells ◽  
Wide Range ◽  
Natural Hosts

ABSTRACT Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance, such as the flaviviruses dengue virus (DENV) and Zika virus (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an A. aegypti cell line and subsequently detected in natural A. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the A. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai A. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that the interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce the transmission of human-pathogenic flaviviruses. IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans, including dengue and Zika viruses. Interestingly, A. aegypti is also naturally infected with insect-only viruses, such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in A. aegypti mosquitoes. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance and that they could possibly be manipulated to reduce arbovirus transmission.

scholarly journals The Vesicle- and Target-SNARE Proteins That Mediate Glut4 Vesicle Fusion Are Localized in Detergent-insoluble Lipid Rafts Present on Distinct Intracellular Membranes

2002 ◽  
Vol 277 (51) ◽  
pp. 49750-49754 ◽  
Author(s):  
Luke H. Chamberlain ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Lipid Rafts ◽  
Significant Proportion ◽  
Vesicle Fusion ◽  
Spatial Integration ◽  
Attachment Protein ◽  
Intracellular Membranes ◽  
Syntaxin 4 ◽  
Protein Attachment

Insulin stimulates the fusion of intracellular vesicles containing the glucose transporter Glut4 with the plasma membrane in adipocytes and muscle cells. Glut4 vesicle fusion is thought to be catalyzed by the interaction of the vesicle solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptor VAMP2 with the target solubleN-ethyl-maleimide-sensitive fusion protein attachment protein receptors SNAP-23 and syntaxin 4. Here, we use combined membrane fractionation, detergent solubility, and sucrose gradient flotation to demonstrate that the large majority (>70%) of SNAP-23 and a significant proportion of syntaxin 4 (∼35%) are associated with plasma membrane lipid rafts in 3T3-L1 adipocytes. Furthermore, VAMP2 is shown to be concentrated in lipid rafts isolated from intracellular membranes. Insulin stimulation had no effect on the plasma membrane raft association of SNAP-23 or syntaxin 4 but promoted VAMP2 insertion into plasma membrane rafts. Immunofluorescence analysis revealed that SNAP-23 was clustered at the plasma membrane and almost completely segregated from the transferrin receptor. SNAP-23 distribution seemed to be distinct from caveolin-1, and clusters of SNAP-23 were dispersed after cholesterol extraction with methyl-β-cyclodextrin, suggesting that the majority of SNAP-23 is associated with non-caveolar, cholesterol-rich lipid rafts. The results described implicate lipid rafts as important platforms for Glut4 vesicle fusion and suggest the hypothesis that such rafts may represent a spatial integration point of insulin signaling and membrane traffic.

scholarly journals Phospholipase C-β1 Signaling Affects Reproductive Behavior, Ovulation, and Implantation

Endocrinology ◽  
2009 ◽  
Vol 150 (7) ◽  
pp. 3259-3266 ◽  
Author(s):  
Panayiotis Filis ◽  
Tamsin Lannagan ◽  
Ashley Thomson ◽  
Alison A. Murray ◽  
Peter C. Kind ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Reproductive Behavior ◽  
Gonadal Development ◽  
G Protein Coupled Receptors ◽  
Male And Female ◽  
Wide Range ◽  
Reproductive Processes ◽  
G Protein Coupled

Infertility can result from a wide range of defects, from behavioral, through germ cell development and maturation, to fertilization or embryo development. Many of the hormones regulating these processes signal via G protein-coupled receptors, which in turn activate a range of plasma membrane enzymes including phospholipase C (PLC)-β isoforms. Transgenic mice lacking functional Plc-β1 (Plc-β1 KO mice) have been noted to have severely impaired fertility, but there has been little study of the reproductive processes affected by lack of this enzyme. This study examined reproductive behavior, gonadal development, fertilization, and implantation in Plc-β1 KO mice. Male and female Plc-β1 KO mice exhibited impaired reproductive behavior. No other defect in reproduction was noted in males, raising the possibility that the reduced fertility of Plc-β1 KO males could be due solely to impaired behavior. In contrast, female Plc-β1 KO mice exhibited both behavioral and nonbehavioral defects. Plc-β1 KO females ovulated only in response to exogenous hormones, with a large proportion of in vivo embryos recovered on embryonic d 4.5 exhibiting abnormal morphology. In addition, uteri of pregnant Plc-β1 KO females exhibited an implantation defect, with poor embryo attachment and a failure to up-regulate cyclooxygenase-2 mRNA.

scholarly journals Analysis of Detergent-Resistant Membranes in Arabidopsis. Evidence for Plasma Membrane Lipid Rafts

2004 ◽  
Vol 137 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Georg H.H. Borner ◽  
D. Janine Sherrier ◽  
Thilo Weimar ◽  
Louise V. Michaelson ◽  
Nathan D. Hawkins ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Plasma Membrane Lipid ◽  
Detergent Resistant Membranes

An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo

1994 ◽  
Vol 107 (4) ◽  
pp. 813-825 ◽  
Author(s):  
M.R. Shanks ◽  
D. Cassio ◽  
O. Lecoq ◽  
A.L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Membrane Proteins ◽  
Cell Line ◽  
Hybrid Cell ◽  
The Other ◽  
Hepatocyte Polarity ◽  
Rat Hepatoma

Studies of hepatocyte polarity, an important property of liver epithelial cells, have been hampered by the lack of valid in vitro models. We report here that a new polarized hepatoma-derived hybrid cell line, called WIF-B, has improved characteristics to those of its parent, WIF12-1. This latter line originated from the fusion of non-polarized rat hepatoma Fao cells with human fibroblasts (WI-38) and selection for a polarized phenotype. We generated the WIF-B line by growing WIF12-1 cells as unattached aggregates for three weeks and selecting for survivors. Karyotype analysis showed a broad chromosome pattern in the initial WIF-B population, but this pattern stabilized after a few passages. The growth and phenotypic properties of these cells were quite different from those of their polarized WIF12-1 parent. WIF-B cells attained a 4-fold higher maximal density in monolayer culture, survived at this density for > 5 days rather than 1 day, and exhibited two to three times more apical structures during this period (80 to 95%). We compared several parameters of liver differentiation in the WIF-B cells with those of a related hybrid clone, WIF12-E, which is extinguished for most liver-specific functions, and with the common hepatoma parent, Fao. By immunoblot analysis, the levels of expression of eight plasma membrane proteins were higher in the WIF-B cells than in either of the other two cell lines and ranged from 10 to 200% of those in vivo. Two plasma membrane proteins were not detected in WIF12-E cells. By immunofluorescence, the apical membrane proteins in WIF-B displayed different cellular localizations than in either of the other two cell lines. In WIF-B cells, apical proteins were confined to a plasma membrane region that we have identified as the apical domain by several criteria (Ihrke, G., Neufeld, E.D., Meads, T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R. E. and Hubbard, A. L. J. Cell Biol., 123, 1761–1765). The same molecules were distributed over the entire plasma membrane of Fao and WIF12-E cells and also (for Fao cells) in intracellular punctate structures that did not colocalize with the majority of structures containing a secretory protein, albumin. Our results indicate that the WIF-B cells are more highly differentiated than any of their ancestors (Fao or WIF12-1 cells) and thus, are promising candidates for in vitro studies of hepatocyte polarity.

The Analysis of Malignancy by Cell Fusion

1971 ◽  
Vol 8 (3) ◽  
pp. 659-672
Author(s):  
G. KLEIN ◽  
U. BREGULA ◽  
F. WIENER ◽  
H. HARRIS
Keyword(s):  
Cell Line ◽  
Tumour Cell ◽  
Malignant Cell ◽  
Hybrid Cells ◽  
Metabolic Defects ◽  
L Cell ◽  
Wide Range ◽  
Derivatives Of ◽  
Selection Of

A wide range of different kinds of malignant cell were fused with certain derivatives of the L cell line and the ability of the resulting hybrid cells to grow progressively in vivo was examined. In all cases the highly malignant character of the tumour cells was suppressed by fusion with the L cell derivatives, whether or not these had metabolic defects that facilitated selection of the hybrid cells. So long as the hybrid cells retained the complete chromosome complements of the two parent cells, their ability to grow progressively in vivo was very limited, for tumours composed of such unreduced hybrids were not found. However, when they lost certain specific, but as yet unidentified, chromosomes, the hybrid cells regained the ability to grow progressively in vivo and gave rise to a tumour. These findings thus indicated that the L cell derivatives contributed something to the hybrid that suppressed the malignancy of the tumour cell, and that this contribution was lost when certain specific chromosomes were eliminated.

Pharmacokinetics- and Pharmacodynamics-Guided Optimization of the Dose and Treatment Schedule for the Dual SRC/ABL Inhibitor BMS-354825.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1987-1987 ◽  
Author(s):  
Feng Roger Luo ◽  
Zheng Yang ◽  
Amy Camuso ◽  
Richard Smykla ◽  
Stephen Castenada ◽  
...  
Keyword(s):  
Phase I ◽  
Philadelphia Chromosome ◽  
Significant Proportion ◽  
Chronic Phase ◽  
Treatment Schedule ◽  
Antileukemic Activity ◽  
Phase I Clinical Trials ◽  
Post Dose ◽  
Wide Range

Abstract Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) originates from multipotential stem cells and is caused by a reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the fusion protein BCR-ABL, a constitutively activated tyrosine kinase. The discovery of imatinib, which selectively targets BCR-ABL, represents a breakthrough treatment for this disorder. However, emerging evidence indicates that a significant proportion of patients in the early chronic phase of the disease fail to achieve optimal response to imatinib due to innate or acquired drug resistance. Moreover, patients in the accelerated or blast crisis phases of the disease in general respond far less favorably to imatinib therapy. There is an urgent need for improved treatment options for these patient populations. BMS-354825, a dual-selective inhibitor of SRC and ABL kinases has demonstrated promising antileukemic activity in vitro and in vivo against preclinical models of human CML, including several that were resistant to imatinib through a variety of mechanisms (Lee et al., Proceedings of the AACR, 2004; Donato et al., Proceedings of the AACR, 2004; Shah et al., Science, 16:399–401, 2004). BMS-354825 is currently being evaluated in a Phase I clinical trial. We performed studies to gain better understanding of the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD) of BMS-354825 and its antileukemic activity. Efficacy was determined in a human CML model (K562) grown subcutaneously in mice. Mouse plasma PK was determined by liquid chromatography/mass spectrometry. Inhibitions of the phosphorylation of tumoral BCR-ABL and its down-stream substrate CrkL, were used as PD markers and were measured by Western blot analysis. BMS-354825 administered orally was efficacious (curative) over a wide range of doses (1.25–50 mg/kg/dose) and exhibited predictable and dose-dependent pharmacokinetics. The time-course of tumoral BCR-ABL and CrkL inhibition and recovery directly correlated with plasma level of BMS-354825. At the minimum effective dose of 1.25 mg/kg/dose, maximum inhibition of BCR-ABL was observed at ~3 hr post-dose, and the inhibition was partially reversed at 7 hr and completely recovered between 7–17 hr post-dose. Based on these data, a PK/PD model was established which predicted that the plasma concentration of BMS-354825 required to effectively inhibit BCR-ABL in K562 cells was ~20 nM. In addition, the model predicted that at a given total dose, twice-a-day split-dose regimens should be more efficacious than once-a-day regimens. This was supported by in vivo efficacy studies in K562 xenografts where BMS-354825 was curative at 1.25 mg/kg/dose when administered twice-a-day, whereas once-a-day dosing required 5 mg/kg/dose to achieve the same response. In summary, BCR-ABL and/or CrkL phosphorylation in CML cells can serve as in vivo PD markers for BMS-354825. The safety/efficacy of BMS-354825, administered either once daily or twice-a-day, is currently being evaluated in Phase I clinical trials in CML patients.

scholarly journals Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells

2004 ◽  
Vol 380 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Galina RADEVA ◽  
Frances J. SHAROM
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Lipid Rafts ◽  
Receptor Protein ◽  
Membrane Microdomains ◽  
Plasma Membrane Vesicles ◽  
Leukaemia Cell ◽  
Leukaemia Cell Line ◽  
Isolation And Characterization ◽  
Triton X

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135–170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.

scholarly journals Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1226
Author(s):  
Adriano Nunes-Nesi ◽  
João Henrique F. Cavalcanti ◽  
Alisdair R. Fernie
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Genetic Variants ◽  
Mitochondrial Carrier ◽  
Wide Range ◽  
Mitochondrial Carrier Family ◽  
Insight Into ◽  
Green Lineage

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

scholarly journals The influence of chromone based hydrazones on lipid peroxidation and bFGF concentration in the HL-60 cell line.

2013 ◽  
Vol 60 (2) ◽  
Author(s):  
Andrzej Łazarenkow ◽  
Marta Michalska ◽  
Anna Gorąca ◽  
Marek Mirowski ◽  
Jolanta Nawrot-Modranka ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Line ◽  
Significant Influence ◽  
Biological Properties ◽  
Synthetic Analogues ◽  
Wide Range ◽  
Synthetic Derivatives ◽  
Derivatives Of

Natural and synthetic derivatives of benzo-γ-pyrones (i.e. flavones, chromones, and coumarins) and their synthetic analogues possess a wide range of biological properties in vitro and in vivo. In this paper we investigated the influence of two hydrazone compounds of chromones, 3-{[(2-dimethoxytiophosphoryl)-2-methylhydrazono]-methyl}-chromen-4-one (CH-3) and 2-amino-6-chloro-3-[(2-hydroxyethyl)-hydrazonomethyl]-chromen-4-one (A-12), on lipid peroxidation and bFGF concentration in the HL-60 cells. Both of the studied compounds had a significant influence on bFGF and TBARS in ranges -137.20 ~ 380.26% and -81.66 ~ -28.68%, respectively, in comparison with the control (counted as 0%).

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