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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0271.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Molecular and Experimental
Medicine and the Department of Vascular Biology, The Scripps Research
Institute, La Jolla, CA.
Phospholipid scramblase 1 (PLSCR1) is an endofacial plasma
membrane protein proposed to participate in transbilayer movement of
phosphatidylserine and other phospholipids. In addition to its putative
role in the reorganization of plasma membrane phospholipids, PLSCR1 is
a substrate of intracellular kinases that imply its possible
participation in diverse signaling pathways underlying proliferation,
differentiation, or apoptosis. Because PLSCR1 is prominently expressed
in a variety of blood cells, we evaluated PLSCR activity in platelets
and erythrocytes, and cytokine-dependent growth of hematopoietic
precursor cells, of PLSCR1 knock-out mice. Adult
PLSCR1 Phospholipid scramblase 1 (PLSCR1) is a member of a
recently identified family of membrane proteins that has been proposed to contribute to the reorganization of plasma membrane phospholipids (PLs) in activated platelets and in injured or apoptotic cells exposed
to elevated intracellular Ca++.1-3 In addition
to a putative role in promoting transbilayer redistribution of plasma
membrane PLs through this Ca++-activated PL scramblase
pathway, PLSCR1 has also been reported to be a substrate of several
kinases that participate in cell proliferative, differentiation, or
apoptotic responses, including protein kinase C Although suggested to play a role in the redistribution of plasma
membrane PLs through the PL scramblase pathway, the actual cellular
function(s) of PLSCR1 and related members of this gene family
remains largely unresolved. The putative role of PLSCR1 in mediating
Ca++-dependent accelerated transbilayer migration of plasma
membrane PLs derived from its capacity to mediate this function in
reconstituted membrane systems and the apparent correlation between
levels of endogenous expression of cellular PLSCR1 and the propensity
of various cells to expose phosphatidylserine (PS) in response to influx of Ca++.1,2,8 Subsequently, it was
reported that the Thr phosphorylation of PLSCR1 by cellular protein
kinase C The biologic significance of the phosphorylation of PLSCR1 by
multiple cellular kinases also remains unclear. De novo expression of a
mutant messenger RNA (mRNA) encoding a truncated form of murine PLSCR1
(deleting the proline-rich segment contained between codons 1-128) was
identified in a monocytic leukemia cell line, and this mutation was
found to correlate with the ability of these cells to proliferate in
vivo. By contrast, expression of full-length PLSCR1 increased upon
induced differentiation of these tumor cells to macrophages, suggesting
that PLSCR1 might normally play some role in regulated cell growth
and/or differentiation.11 Furthermore, ectopic expression
of recombinant human PLSCR1 in the HEY1B carcinoma cell line was found
to markedly inhibit growth of solid tumors from these cells after
subcutaneous transplantation.12 Finally, in yeast there is
evidence that the single apparent PLSCR ortholog (YJR100C) is a
stress-induced gene,13 whereas human PLSCR1 expression is
highly induced by the interferons, suggesting possible roles in
immune/stress responses, cell cycle regulation, or
apoptosis.10,14
To gain additional insight into what role PLSCR1 might play in the PL
scramblase pathway and in cytokine-dependent cell growth and
differentiation in vivo, we have investigated hematopoietic cell growth
and differentiation in mice that are deficient in PLSCR1 due to
homozygous disruption of the PLSCR1 gene locus. Whereas these mice
display no apparent hematologic or hemostatic abnormality at steady
state and their blood cells show normal PL scramblase activity, our
results suggest that PLSCR1 Cytokines, antibodies, and other reagents
PLSCR1 knock-out mice
Southern blotting Purified tail genomic DNA was digested with XbaI, separated on a 0.9% agarose gel, and transferred to a nylon membrane. PLSCR1 genomic DNA was detected by hybridization with a 32P-labeled PLSCR1 DNA probe consisting of a 408-base pair PLSCR1 genomic DNA fragment within the targeted region. Insertion of the targeting vector into the PLSCR1 locus shifts the detected XbaI-derived fragment of genomic DNA from 2.9 kilobases (WT) to 7.5 kilobases (knock-out).Northern blotting Northern blotting was performed as previously described.2,15 In brief, total RNA from mouse kidney was resolved on a 1% denatured agarose gel and transferred to a nylon membrane. PLSCR1 mRNA was detected by hybridization with a 32P-labeled mouse PLSCR1 complementary DNA fragment (nucleotides 1086-1465 of GenBank accession no. AF159593).Western blotting Cell lysates were mixed with sample buffer (50 mM Tris [pH 6.8], 4% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue) and heated at 100°C for 5 minutes. Proteins were resolved on 10% sodium dodecyl sulfate-polyacryamide gel and transferred to nitrocellulose membrane. The membrane was blocked in 5% nonfat milk in 20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20 and incubated sequentially with 2 µg/mL anti-PLSCR1 mab 219.16 and horseradish peroxidase-conjugated goat antimouse IgG (Sigma). Bound antibodies were detected by SuperSignal chemiluminescence substrate (Pierce, Rockford, IL) and exposure to x-ray film.Blood collection and hematologic profiles For cell lineage analysis, red blood cell (RBC) and platelet studies, and apoptosis assay, blood was collected by heart puncture and mixed 9:1 with 500 U/mL heparin. For analysis of G-CSF-induced granulocytosis, blood was collected by eye puncture using heparinized capillaries. Newborn mouse blood for blood cell counts was collected from a tail cut. Platelets and RBCs were counted in a Coulter particle counter, and total white blood cell (WBC) counts were determined by hemacytometer. WBC differential counts were performed on a blood smear stained by Wright-Giemsa (Thermo Shandon, Pittsburgh, PA) or Hema 3 stain set (Biochemical Science, Swedesboro, NJ).Tail bleeding times Mouse tail bleeding time was performed as described.16 In brief, the tail was cut 2 mm from the end and immediately immersed into saline at 37°C, and time to cessation of bleeding was recorded.Surface exposure of PS in RBCs Blood was collected by heart puncture, buffy coat was removed by centrifugation, and the cells were washed and suspended at 108/mL in Hanks balanced salt solution, 0.1% bovine serum albumin (BSA), 1 mM Ca++ at 37°C. Ca++ ionophore A23187 (1 µM) was added, and at times indicated the reaction was stopped by addition of 10 mM ethyleneglycotetraacetic acid. Cell surface-exposed PS was detected by the specific binding of factor Va.17 At each time point, 10 µg/mL bovine factor Va (Haematologic Technologies, Essex Junction, VT) was added to a 50-µL aliquot of the cell suspension, followed by incubation with 10 µg/mL FITC-labeled mab V237 specific for the light chain of factor Va (Dr Charles Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK). All samples were additionally stained with 0.1 µM DiIC16(3) (Molecular Probes, Eugene, OR). Cell-associated factor Va was quantified by flow cytometry on a FACSCalibur cytometer (Becton Dickinson, San Jose, CA), with acquisition gated on DiIC16(3)-positive particles.Platelet activation Blood was collected by heart puncture. Platelets were isolated by centrifugation as described18 and suspended to 2 × 107/mL in HEPES buffer (137 mM NaCl, 4 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 20 mM HEPES, pH 7.4) at 37°C. For measurement of cell surface-exposed PS or P-selectin, platelets (100 µL per tube) were incubated for 5 minutes in the presence of ADP (10 µM) plus epinephrine (10 µM), collagen (10 µg/mL), -thrombin (0.5 U/mL), -thrombin (0.5 U/mL) plus
collagen (10 µg/mL), or A23187 (1 µM). PS exposure was detected by
the binding of factor Va as described for RBCs above and cell surface
P-selectin with a FITC-labeled antibody specific for CD62p. For
quantification of fibrinogen binding to platelets, FITC-labeled
fibrinogen (20 µg/mL) was added prior to the addition of the
agonists, except in the case of thrombin, which was first reacted with
platelets followed by inhibition of the enzyme with hirudin (2.5 U/mL).
Fibrinogen-containing samples were fixed with 1% formaldehyde. All
samples were additionally stained with biotinylated antimouse CD41 as a
platelet-specific marker, detected with phycoerythrin-labeled
streptavidin (2.5 µg/mL). Single-cell fluorescence was analyzed on a
FACSCalibur, with acquisition gated on CD41+ particles.
Cell isolation and culture Mouse fetal livers were aseptically removed from embryos at day 15 and mechanically disrupted. The cells were suspended in RPMI, 10% FBS complete medium and passed through a 40-µm cell strainer (Becton Dickinson, Franklin Lakes, NJ) to remove aggregated cells. Spleens and thymuses were isolated from 6- to 8-week-old mice, and cells were prepared as described for fetal liver. For bone marrow, femurs and tibia were removed from 6- to 8-week-old mice. Bone marrow cells were flushed out and suspended in RPMI complete medium and passed through a cell strainer.Bone marrow cells (105/mL) were first cultured for 6 days
in T75 flasks in MethoCult 3234 (1% methylcellulose, 15% FBS, 1% BSA, 10 µg/mL bovine pancreatic insulin, 200 µg/mL human
transferrin, 100 µM Induction of PLSCR1 expression by cytokines One million cultured bone marrow progenitor cells were mixed with 2 mL MethoCult 3234, and either mouse SCF (20 ng/mL) or G-CSF (60 ng/mL) was added. The cells were incubated at 37°C, 5% CO2 for 3 days, harvested at different times during this period, washed twice with phosphate-buffered saline (PBS), and lysed at 4°C for 1 hour with cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, and protease inhibitor cocktail). Cell lysates were centrifuged at 14 000 rpm for 10 minutes at 4°C, and protein was measured using the bicinchonic acid method (Pierce).Colony-forming assays For the committed progenitor colony-forming assay, fetal liver cells (105), adult mouse spleen cells (2 × 105), or bone marrow cells (5 × 104) were added to 1.5 mL MethoCult 3434 (1% methylcellulose, 50 ng/mL mouse SCF, 10 ng/mL IL-3, 20 ng/mL IL-6, and 3 U/mL Epo, 15% FBS, 1% BSA, 10 µg/mL bovine pancreatic insulin, 200 µg/mL human transferrin, 100 µM -mercaptoethanol in IMDM). For single cytokine-mediated colony assays, the cells were mixed with 1.5 mL
MethoCult 3234 supplemented with either 20 ng/mL mouse SCF, 60 ng/mL
G-CSF, 20 ng/mL GM-CSF, 10 ng/mL mouse IL-3, or 20 ng/mL IL-7. To
promote committed progenitor colonies, G-CSF and SCF were used for
progenitors of granulocyte, Epo, and SCF for erythrocytes, and Tpo,
IL-3, and IL-6 for megakaryocytes. Assays were performed in 35-mm Petri
dishes. The cells were cultured at 37°C in a humidified atmosphere of
5% CO2 in air. Colonies were scored as clusters containing
more than 50 cells. Cells were then stained in the dish overnight at
37°C, 5% CO2 with 2 mL of 0.05% nitroblue tetrazolium in PBS, followed by fixing with 4% formaldehyde. The colony size was
recorded by digital camera.
Cell proliferation assay Thymus T-cell proliferation was performed using BrdU Cell Proliferation Assay Kit (Roche Molecular Biochemicals) as directed by the manufacturer. Briefly, thymus T cells were suspended in RPMI 1640, 10% FBS with 5 µg/mL concanavalin A, or both IL-2 (2 ng/mL) and concanavalin A (5 µg/mL), or RPMI complete medium without cytokine and mitogen, respectively. A total of 100 µL of the cell suspension (106 cells per milliliter) was grown in a 96-well tissue culture plate in triplicate at 37°C, 5% CO2. Two days later, 10 µL 5-bromo-2-deoxyuridine (BrdU) (100 µM) was added to each well and incubated for 4 hours. The plates were centrifuged at 1200 rpm for 10 minutes and heated at 60°C for 60 minutes. Cells were fixed with FixDenat for 30 minutes. Incorporated BrdU was detected using HRP-conjugated anti-BrdU specific antibody and HRP substrate, and OD 370 nm was measured in an ELISA reader.Apoptosis assay Apoptosis of neutrophils was detected by the TUNEL method using the ApoAlert DNA fragmentation assay kit (Clontech, Palo Alto, CA). Neutrophils were isolated from mouse blood (collected by heart puncture) by treatment with dextran and centrifugation through Ficoll-Hypaque.19 The cells were incubated in RPMI complete medium supplemented with 60 ng/mL mouse G-CSF and harvested after 20 and 44 hours. Cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and transferred to glass slides by cytospin. Neutrophils were stained with biotin-labeled anti-Gr-1 and rhodamine-labeled streptavidin and then reacted with terminal deoxynucleotidyl transferase enzyme and nucleotide mix. Apoptotic neutrophils were counted under the fluorescent microscope (total of 1000 Gr-1+ cells per slide).Cell lineage analysis by flow cytometry Blood, bone marrow, spleen, and fetal liver cells were collected as described above. RBCs in samples were lysed with 0.15 M ammonium chloride solution. Cells were washed and resuspended in PBS, 2% BSA. Nonspecific binding to cell surface Fc receptors was blocked by incubating cells with CD16/CD32 Fc Block (PharMingen) for 10 minutes on ice. Cells were stained using phycoerythrin- or FITC-labeled rat antibodies directed against the cell lineage-specific cell surface markers Mac-1, Gr-1 (myeloid); CD45R/B220, CD3, CD4, CD8 (lymphoid); Ter-119 (erythroid); CD41 (platelet); or IgG isotype as control. Fluorescence-activated cell sorter (FACS) analysis was performed on a FACSCalibur flow cytometer.Induced granulocytosis by G-CSF treatment Male 9- to 10-week-old mice were used in the reported study. Human recombinant G-CSF was injected subcutaneously into both PLSCR1 / (n = 5) and WT mice (n = 5) at doses of 120 µg/kg/d in approximately 230 µL saline. G-CSF was administered
daily for 5 consecutive days. Blood samples were collected by eye
puncture before and 18, 66, and 114 hours after the initial G-CSF
injection. WBCs were counted, blood smears prepared, and differential
counts performed (see above).
Statistics Statistical comparisons were made using the unpaired bilateral Student t test. Results are expressed as mean ± SD.
Mice deficient in PLSCR1 Targeted disruption of the PLSCR1 gene locus and elimination of PLSCR1 protein expression in bone marrow cells of the targeted PLSCR1/ mice was confirmed by Southern, Northern, and
Western blotting (Figure 1).
Similar results were obtained in Northern and Western blots of
embryonic fibroblasts, peripheral blood cells, and various tissues
derived from these animals using the murine PLSCR1-reactive mab 219.1 and rabbit antibody specific for either N-terminal or C-terminal
peptides of murine PLSCR1 (data not shown). The PLSCR1
Normal PL scramblase activity in PLSCR1 / platelets
and erythrocytes to mobilize cell surface PS and to promote hemostasis.
We detected no evidence of defective platelet function or impaired
mobilization of cell surface PS in platelets or erythrocytes obtained
from PLSCR1/ animals (Figures
2 and
3).
These mice also showed no apparent hemostatic abnormality, with tail
bleeding times of 98 ± 30 seconds in PLSCR Evidence for reduced granulocyte production in newborn
PLSCR1 / and
PLSCR1+/ mice were distinctly reduced relative to matched
WT animals. This decrease in blood leukocytes at birth was accompanied
by a comparable decrease in the relative number of Gr-1+
cells detected in livers of 2-week-old fetuses (Figure
4).
Defective response to SCF and G-CSF in cultured
PLSCR1 / animals, we next evaluated cytokine-supported
colony formation using hematopoietic precursor cells derived from fetal
liver and from the spleens or bone marrows of age-matched adult animals (Figure 5).
As these data indicate, the hematopoietic precursor cells derived from
PLSCR1 When cultured in the presence of G-CSF and SCF, hematopoietic precursor
cells derived from PLSCR1
These data imply that gene deletion of PLSCR1 affects both hematopoietic precursor cell proliferation (Figure 5) and maturation (Figure 6; Table 3) as selectively regulated through these 2 cytokines. PLSCR1 / hematopoietic
precursor cells when cultured in vitro was also reflected in the
myeloproliferative response of the PLSCR1/ mice to in
vivo challenge with this cytokine. Age- and sex-matched WT and
PLSCR1/ animals were injected daily with recombinant
human G-CSF, and blood neutrophil counts were determined (Figure
7).
WT mice responded to G-CSF with a rapid increase in blood neutrophils,
reaching 10-fold basal levels at 114 hours. By contrast, in the
PLSCR1 PLSCR1 expression is induced by select hematopoietic growth factors Expression of PLSCR1 is known to be highly induced by both type I and II interferons, a response dependent upon Stat-1 and a single interferon-stimulated response element located in the first untranslated exon of the PLSCR1 gene.10,14 The depressed response to certain hematopoietic growth factors observed for cells derived from the PLSCR1/ mice raised the possibility
that PLSCR1 itself normally participates in the cascade of events
underlying growth factor-stimulated cellular proliferation and
differentiation and led us to consider whether this response might also
include a cytokine-induced increase in PLSCR1 expression. As shown in
Figure 8, G-CSF and SCF each induced a
marked increase in the cellular content of PLSCR1. These results are
consistent with a role for newly synthesized PLSCR1 in an effector
pathway mediating the cellular response to these cytokines. By contrast
to the increased expression of PLSCR1 in WT bone marrow cells observed
in response to G-CSF and SCF, there was no apparent change in PLSCR1
levels in response to culture in either TPO or EPO (data not shown).
The results of these experiments provide the first direct evidence
that the expression of PLSCR1, and potentially other members of the
PLSCR gene family, can influence the proliferative and differentiation
response of cells to cytokine stimulation, which in the case of murine
hematopoietic precursors selectively affects granulocyte production as
stimulated by SCF and G-CSF. These data add to the growing body of
evidence that PLSCR1 functions in signaling or effector pathways
potentially involved with cell proliferation, differentiation, or
apoptosis, presumably through its interaction with the various
intracellular kinases that have been reported to phosphorylate this
protein.4-6 Interestingly, despite the attenuated myeloid
colony growth in response to both SCF and G-CSF observed for
PLSCR1 Our data also indicate that PLSCR1, despite its putative identification
as plasma membrane PL scramblase, is not required for normal movement
of PS from the inner to outer leaflet of the plasma membrane under
conditions of either receptor-initiated platelet activation or direct
elevation of intracellular Ca++. As previously reported, we
also found that both the mRNA sequence and level of protein expression
of PLSCR1 in blood cells obtained from a patient with Scott syndrome
were identical to that of normal controls, implying that this bleeding
disorder that was shown to reflect diminished function of the PL
scramblase pathway in platelets and other blood cells does not reflect
either deficiency or defect in PLSCR1.15 Together with the
results of the current experiments, these data imply that PLSCR1 may
not function in the plasma membrane PL scramblase pathway as was
presumed, although we cannot exclude the possibility that PLSCR1
provides redundant function with another member of the PLSCR gene
family in mediating this activity. Of the 4 PLSCR genes that have been
identified in human beings and mice, only PLSCR1 and PLSCR3 are readily
detected in blood and bone marrow. PLSCR3 shares 47% identity with
PLSCR1.3 Of interest in this context, examination of
blood, bone marrow, spleen, and thymus of PLSCR1 The mechanism by which PLSCR1 affects cell proliferative and
differentiation responses to select growth factors remains to be
determined. Of particular interest are previous observations that
PLSCR1 is the substrate of kinases implicated in participating in
regulation of growth, differentiation, and apoptosis, including protein
kinase C The defective proliferation and differentiation of
PLSCR1
The authors acknowledge the superb technical assistance of Ms Hongfan Peng and Ms Lilin Li. We are most grateful to Dr Robert McMillan for generously providing recombinant G-CSF. We also wish to acknowledge the advice, comments, suggestions, and generous assistance of Drs Dong-Er Zhang, Bruce Torbett, Yuzhong Yuan, and Ming Yan. This is manuscript no. 14654-MEM from the Scripps Research Institute.
Submitted December 19, 2001; accepted February 5, 2002.
Prepublished online as Blood First Edition Paper, April 17, 2002; DOI 10.1182/blood- 2001-12-0271.
Supported in part by National Institutes of Health grants HL36946, HL63819, and HL61200.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Peter J. Sims, Dept of Molecular and Experimental Medicine, MEM-275, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037; e-mail: psims{at}scripps.edu.
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K.-W. Zhao, D. Li, Q. Zhao, Y. Huang, R. H. Silverman, P. J. Sims, and G.-Q. Chen Interferon-{alpha}-induced Expression of Phospholipid Scramblase 1 through STAT1 Requires the Sequential Activation of Protein Kinase C{delta} and JNK J. Biol. Chem., December 30, 2005; 280(52): 42707 - 42714. [Abstract] [Full Text] [PDF]
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Q. Zhou, I. Ben-Efraim, J.-L. Bigcas, D. Junqueira, T. Wiedmer, and P. J. Sims Phospholipid Scramblase 1 Binds to the Promoter Region of the Inositol 1,4,5-Triphosphate Receptor Type 1 Gene to Enhance Its Expression J. Biol. Chem., October 14, 2005; 280(41): 35062 - 35068. [Abstract] [Full Text] [PDF]
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C. Albrecht, J. H. McVey, J. I. Elliott, A. Sardini, I. Kasza, A. D. Mumford, R. P. Naoumova, E. G. D. Tuddenham, K. Szabo, and C. F. Higgins A novel missense mutation in ABCA1 results in altered protein trafficking and reduced phosphatidylserine translocation in a patient with Scott syndrome Blood, July 15, 2005; 106(2): 542 - 549. [Abstract] [Full Text] [PDF]
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M.-H. Chen, I. Ben-Efraim, G. Mitrousis, N. Walker-Kopp, P. J. Sims, and G. Cingolani Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin {alpha} J. Biol. Chem., March 18, 2005; 280(11): 10599 - 10606. [Abstract] [Full Text] [PDF]
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S. C. Frasch, P. M. Henson, K. Nagaosa, M. B. Fessler, N. Borregaard, and D. L. Bratton Phospholipid Flip-Flop and Phospholipid Scramblase 1 (PLSCR1) Co-localize to Uropod Rafts in Formylated Met-Leu-Phe-stimulated Neutrophils J. Biol. Chem., April 23, 2004; 279(17): 17625 - 17633. [Abstract] [Full Text] [PDF]
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R. V. Warke, K. Xhaja, K. J. Martin, M. F. Fournier, S. K. Shaw, N. Brizuela, N. de Bosch, D. Lapointe, F. A. Ennis, A. L. Rothman, et al. Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells J. Virol., November 1, 2003; 77(21): 11822 - 11832. [Abstract] [Full Text] [PDF]
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M. Nanjundan, J. Sun, J. Zhao, Q. Zhou, P. J. Sims, and T. Wiedmer Plasma Membrane Phospholipid Scramblase 1 Promotes EGF-dependent Activation of c-Src through the Epidermal Growth Factor Receptor J. Biol. Chem., September 26, 2003; 278(39): 37413 - 37418. [Abstract] [Full Text] [PDF]
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S. Kametaka, M. Shibata, K. Moroe, S. Kanamori, Y. Ohsawa, S. Waguri, P. J. Sims, K. Emoto, M. Umeda, and Y. Uchiyama Identification of Phospholipid Scramblase 1 as a Novel Interacting Molecule with beta -Secretase (beta -Site Amyloid Precursor Protein (APP) Cleaving Enzyme (BACE)) J. Biol. Chem., April 18, 2003; 278(17): 15239 - 15245. [Abstract] [Full Text] [PDF]
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D. L. Daleke Regulation of transbilayer plasma membrane phospholipid asymmetry J. Lipid Res., February 1, 2003; 44(2): 233 - 242. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, c-Abl, and both
immunoglobulin E (IgE) and EGF receptor-coupled
kinase(s).
) and PLSCR1
) mice were incubated with the agonists indicated on the abscissa
and processed for flow cytometry as described in "Materials and
methods." ADP/Epi indicates ADP plus epinephrine; Thr/collagen,
)
mice were incubated in the presence of 1 µM A23178, and PS exposure
at the times indicated was detected by the specific binding of factor
Va as detailed in "Materials and methods." Data shown are
representative of 3 experiments so performed.
using oligonucleotide arrays.
Proc Natl Acad Sci U S A.
1998;95:15623-15628