scholarly journals Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line.

1992 ◽  
Vol 12 (4) ◽  
pp. 1864-1871 ◽  
Author(s):  
G Q Daley ◽  
R A Van Etten ◽  
P K Jackson ◽  
A Bernards ◽  
D Baltimore
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

scholarly journals Nonmyristoylated Abl proteins transform a factor-dependent hematopoietic cell line

1992 ◽  
Vol 12 (4) ◽  
pp. 1864-1871
Author(s):  
G Q Daley ◽  
R A Van Etten ◽  
P K Jackson ◽  
A Bernards ◽  
D Baltimore
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Hematopoietic Cell ◽  
Myelogenous Leukemia ◽  
3T3 Fibroblasts ◽  
Nih 3T3

N-terminal myristoylation can promote the association of proteins with the plasma membrane, a property that is required for oncogenic variants of Src and Abl to transform fibroblastic cell types. The P210bcr/abl protein of chronic myelogenous leukemia cells is not myristoylated and does not stably transform NIH 3T3 fibroblasts; however, it will transform lymphoid and myeloid cell types in vitro and in vivo, suggesting that myristoylation is not required for Abl variants to transform hematopoietic cells. To test this hypothesis, we introduced point mutations that disrupt myristoylation into two activated Abl proteins, v-Abl and a deletion mutant of c-Abl (delta XB), and examined their ability to transform an interleukin-3-dependent lymphoblastoid cell line, Ba/F3. Neither of the nonmyristoylated Abl proteins transformed NIH 3T3 fibroblasts, but like P210bcr/abl, both were capable of transforming the Ba/F3 cells to factor independence and tumorigenicity. Nonmyristoylated Abl variants did not associate with the plasma membrane in the transformed Ba/F3 cells. These results demonstrate that Abl proteins can transform hematopoietic cells in the absence of membrane association and suggest that distinct functions of Abl are required for transformation of fibroblast and hematopoietic cell types.

scholarly journals Sphingosine 1-phosphate in inflammation

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Lijuan Li ◽  
Lixia An ◽  
Lifang Li ◽  
Yongjuan Zhao
Keyword(s):  
Plasma Membrane ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Cell Types ◽  
In Vivo Models ◽  
Integral Role ◽  
Sphingosine 1 Phosphate ◽  
And Migration

Sphingolipids are formed via the metabolism of sphingomyelin, aconstituent of the plasma membrane, or by denovosynthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1-phosphate and sphingosine 1-phosphate (S1P), are likely to have an integral role in in?ammation. This can involve, for example, activation of pro-in?ammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-in?ammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high-af?nity G-protein-coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on in?ammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in in?ammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and in?ammatory bowel disease, which involve important in?ammatory components. A signi?cant body of research now indicates that sphingolipids are intimately involved in the in?ammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological in?ammation.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Abstract Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

scholarly journals Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1252-1262 ◽  
Author(s):  
Dale J. Powner ◽  
Matthew N. Hodgkin ◽  
Michael J.O. Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Enzyme Activation ◽  
Dominant Negative ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

Mcl-1 in Transgenic Mice Promotes Survival in a Spectrum of Hematopoietic Cell Types and Immortalization in the Myeloid Lineage

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3226-3239 ◽  
Author(s):  
Ping Zhou ◽  
Liping Qian ◽  
Christine K. Bieszczad ◽  
Randolph Noelle ◽  
Michael Binder ◽  
...  
Keyword(s):  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Types ◽  
Cell Number ◽  
Hematopoietic Cell ◽  
Viable Cell Number ◽  
Wide Range ◽  
Transgenic Cells

Mcl-1 is a member of the Bcl-2 family that is expressed in early monocyte differentiation and that can promote viability on transfection into immature myeloid cells. However, the effects of Mcl-1 are generally short lived compared with those of Bcl-2 and are not obvious in some transfectants. To further explore the effects of this gene, mice were produced that expressed Mcl-1 as a transgene in hematolymphoid tissues. The Mcl-1 transgene was found to cause moderate viability enhancement in a wide range of hematopoietic cell types, including lymphoid (B and T) as well as myeloid cells at both immature and mature stages of differentiation. However, enhanced hematopoietic capacity in transgenic bone marrow and spleen was not reflected in any change in pool sizes in the peripheral blood. In addition, among transgenic cells, mature T cells remained long lived compared with B cells and macrophages could live longer than either of these. Interestingly, when hematopoietic cells were maintained in tissue culture in the presence of interleukin-3, Mcl-1 enhanced the probability of outgrowth of continuously proliferating myeloid cell lines. Thus, Mcl-1 transgenic cells remained subject to normal in vivo homeostatic mechanisms controlling viable cell number, but these constraints could be overridden under specific conditions in vitro. Within the organism, Bcl-2 family members may act at “viability gates” along the differentiation continuum, functioning as part of a system for controlled hematopoietic cell amplification. Enforced expression of even a moderate viability-promoting member of this family such as Mcl-1, within a conducive intra- and extracellular environment in isolation from normal homeostatic constraints, can substantially increase the probability of cell immortalization. © 1998 by The American Society of Hematology.

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.

scholarly journals Preparation and characterization of nano chitosan for hGM-CSF release

2015 ◽  
Vol 18 (2) ◽  
pp. 64-73
Author(s):  
Truong Tat Dang ◽  
Thuan Cong Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Cell Line ◽  
Average Diameter ◽  
Cell Types ◽  
High Concentration ◽  
Dose Requirement ◽  
Chitosan Beads ◽  
Release Capacity ◽  
Antigen Presenting

hGM-CSF (human granulocytemacrophage colony stimulating factor) is a cytokine secreted by many cell types. Its characters are suitable for vaccine adjuvant such as ability to stimulate survival, differentiation and enhancement the functions of antigen-presenting cells. This cytokine is also a chemoattractant for monocytes and neutrophils to the infected sites, stimulates the expression of several cytokines like IL-1, IL-6, TNF, which are essential for B and T lymphocyte differentiation. However, hGM-CSF has some drawbacks for being an adjuvant candidate due to its easy degradation, toxicity at high concentration and low-dose requirement for therapeutic effect. Drugs delivery system using chitosan can overcome these disadvantages of hGM-CSF. In this present study, chitosan particles were prepared and evaluated the absorption and release of human hGM-CSF. Firstly, the activity of hGM CSF was evaluated by proliferation bioassay using TF-1 cell line. Afterward, chitosan particles were prepared by ionic gelation method and were examined for its toxicity on TF‑1 cell line. After protein absorbance onto chitosan particles, the release capacity and in vitro protection of chitosan for hGM-CSF were assessed. The result showed that hGM-CSF had an ED50 value of 106 pg/mL. The synthesized chitosan particles had an average diameter of 24.5 nm and were nontoxic. Based on the results of SDS-PAGE and Bradford, the adsorption efficiency of hGM‑CSF onto chitosan particles reached 99 % and chitosan has the ability to release hGM-CSF and protects it from hydrolysis of trypsin. In conclusion, the synthesized chitosan beads absorbed and released hGMCSF with its activity remained. This result provides the evidence for further in vivo researches.

scholarly journals Galectin–glycan lattices regulate cell-surface glycoprotein organization and signalling

2008 ◽  
Vol 36 (6) ◽  
pp. 1472-1477 ◽  
Author(s):  
Omai B. Garner ◽  
Linda G. Baum
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Targeted Delivery ◽  
Cell Types ◽  
Surface Glycoprotein ◽  
Membrane Domains ◽  
Cellular Functions ◽  
Cell Surface Glycoprotein

The formation of multivalent complexes of soluble galectins with glycoprotein receptors on the plasma membrane helps to organize glycoprotein assemblies on the surface of the cell. In some cell types, this formation of galectin–glycan lattices or scaffolds is critical for organizing plasma membrane domains, such as lipid rafts, or for targeted delivery of glycoproteins to the apical or basolateral surface. Galectin–glycan lattice formation is also involved in regulating the signalling threshold of some cell-surface glycoproteins, including T-cell receptors and growth factor receptors. Finally, galectin–glycan lattices can determine receptor residency time by inhibiting endocytosis of glycoprotein receptors from the cell surface, thus modulating the magnitude or duration of signalling from the cell surface. This paper reviews recent evidence in vitro and in vivo for critical physiological and cellular functions that are regulated by galectin–glycoprotein interactions.

Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice

2005 ◽  
Vol 289 (3) ◽  
pp. C697-C707 ◽  
Author(s):  
Takayuki Ogawa ◽  
Takeshi Nikawa ◽  
Harumi Furochi ◽  
Miki Kosyoji ◽  
Katsuya Hirasaka ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gastrocnemius Muscle ◽  
Binding Motif ◽  
Type I ◽  
Heparin Binding ◽  
3T3 Fibroblasts ◽  
Sciatic Neurectomy ◽  
Nih 3T3

In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse C2C12 myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix.

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