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Articles |
1
Institute for Clinical Chemistry and Laboratory Medicine and
2
Institute of Pathology, University of Regensburg, 93042 Regensburg, Germany.
3
Department of Clinical Biochemistry, Pomeranian Medical Academy, 70-111 Szczecin, Poland.
aAddress correspondence to this author at: Institute for Clinical Chemistry, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 Regensburg, Germany. Fax 49-941-944-6202; e-mail gerd.schmitz{at}klinik.uni-regensburg.de.
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Abstract |
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Methods: To quantify ABCA1 mRNA, we generated a calibration curve from serial dilutions of in vitro-transcribed RNA corresponding to an amplified ABCA1 cDNA 205-bp fragment (homologous calibrator). Two pairs of fluorescent hybridization probes were used to detect the ABCA1 and porphobilinogen deaminase (PBGD) mRNAs; the latter served as an internal control. PCR was performed as real-time amplification of ABCA1 mRNA in 100 ng of total RNA isolated from various human tissues, and cultured cells were calculated from the calibration curve. In addition, normalized values of target (ABCA1/PBGD ratio) were calculated.
Results: Using this method, we quantified ABCA1 transcripts in various human tissue samples as well as in monocytes, THP-1 cells, fibroblasts, and adipocytes. We demonstrated ABCA1 mRNA up-regulation during human adipocyte and monocyte differentiation. In addition, we examined the effect of cholesterol loading and deloading on ABCA1 expression in monocytes, THP-1 cells, and fibroblasts.
Conclusions: Our RT-PCR assay allows the specific and highly reproducible detection and quantification of minute amounts of human ABCA1 mRNA. This new method is more accurate, more informative, and less laborious than the classic RT-PCR methods and Northern blot; it therefore could simplify all studies on ABCA1 mRNA expression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent results from overexpression studies have demonstrated the importance of ABCA1 as a major determinant of HDL-cholesterol concentrations in plasma and in cellular trafficking of cholesterol and choline phospholipids (5)(6). ABCA1 gene expression is up-regulated by modified LDL (4), cAMP (7)(8), and oxysterols, which act on liver X receptor-
and liver X receptor-ß together with the retinoid X receptor (9)(10). Few data are available concerning inhibitory factors of ABCA1 expression. It was shown that activation of mouse peritoneal macrophages by interferon-
down-regulated ABCA1 along with a substantial reduction of apolipoprotein A-I-mediated cholesterol and phospholipid efflux (11). A zinc finger transcription factor (ZNF202) located within a hypoalphalipoproteinemia locus on chromosome 11q23 acts as a strong repressor on ABCA1 expression and cellular lipid efflux (12).
Quantification of ABCA1 gene expression is critical in studies related to its expression and regulation by metabolic factors, nutritional status, and new antiatherogenic drug candidates. Here we demonstrate that the homogeneous LightCycler-based real-time reverse transcription-PCR (RT-PCR) technique is a reliable and rapid tool that allows the amplification, detection, and quantification of minute amounts of ABCA1 mRNA in a variety of tissues and cultured cells in contrast to classic RT-PCR or Northern blot analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enzymatic modification of ldl
LDL (d = 1.006–1.063 kg/L) and HDL3 (d = 1.125–1.21 kg/L) were prepared from human plasma of healthy donors by standard methods (13).
Enzymatic modification of LDL was performed as described (14)(15)(16). Briefly, LDL was diluted to 2 g/L of protein in HEPES buffer (20 mmol/L HEPES, 150 mmol/L NaCl, 2 mmol/L CaCl2, pH 7.0). Enzyme treatment was with trypsin (6.6 g/L; Sigma) and cholesterol esterase (40 g/L; Roche Biochemica) for 6–8 h at 37 °C. Subsequently, trypsin inhibitor (Sigma) was added, and the pH of the solution was adjusted to 5.5 by the addition of MES buffer, pH 5.0. Neuraminidase (79 U/L; Behring) and magnesium ascorbate solution (30 g/L) were then added for 14 h at 37 °C. Subsequently, the solution was neutralized with 1 mol/L NaOH. The absence of oxidation products in the enzymatically modified LDL (E-LDL) was verified by measurement of the thiobarbituric acid-reactive substances to quantify lipid peroxidation products (17). Modified lipoproteins were stored at 4 °C and used within 1 week. During LDL preparation and subsequent modification, general precautions were taken to avoid lipopolysaccharide contamination. The latter was monitored by the Limulus endotoxin assay (Kinetic-QCL; BioWhittaker).
rna isolation
Total RNA from different human tissues was obtained from Clontech. Total RNA from human preadipocytes, adipocytes, fibroblasts, monocytes, macrophages, and THP-1 cells was isolated from in vitro cultures with Trizol reagent (Sigma). All RNA samples were treated with DNase I (Roche) according to the protocol of Huang et al. (18). The concentration, purity, and integrity of the RNA were assessed on the Agilent 2100 bioanalyzer with the RNA 6000 LabChip® reagent set (Agilent Technologies).
generation of an external abca1 rna calibrator
A 205-bp ABCA1 RT-PCR product was subcloned in the plasmid vector pCR® II-TOPO containing a T7 RNA polymerase promoter (TOPO TA Cloning; Invitrogen) according to the manufacturer’s instructions. Plasmid DNA was isolated and purified with the Qiaprep Spin Miniprep Kit (QIAGEN). The ABCA1 insert sequence was verified by DNA sequencing on an ABI Prism Genetic Analyzer 310 (PE Biosystems). Linearization of purified plasmid DNA was achieved by restriction with BamHI, and RNA was synthesized in vitro with the RiboProbe® In Vitro Transcription System (Promega) and T7 polymerase according to the protocol for large-scale RNA synthesis.
primers and hybridization probes
ABCA1-specific PCR primers and hybridization probes capable of fluorescence resonance energy transfer were used to generate and monitor 205-bp ABCA1, 264-bp porphobilinogen deaminase (PBGD) fragments, and 262-bp glyceraldehyde 3-phosphate dehydrogenase (GAPDH) fragments, respectively (Table 1
). All primers and hybridization probes were synthesized by TIB MOLBIOL. The pairs of hybridization probes were labeled with fluorescein at their 3' ends (donor probes) and with LC-Red 640 at their 5' ends (acceptor probes).
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rt-pcr
First-strand cDNA synthesis was performed in a total volume of 20 µL containing 40 U of AMV Reverse Transcriptase, 2.0 µL of 10x Reaction Buffer, 50 units of RNase Inhibitor, 2 µL of Deoxynucleotide Mix (1 mM), 2 µL of Random Primer p(dN)6 (3.2 µg), 4 µL of MgCl2 (5 mM), and 1 µg of total RNA, as recommended by the manufacturer [1st Strand cDNA Synthesis Kit for RT-PCR (AMV); Roche Molecular Biochemicals]. Serial dilutions of in vitro-synthesized ABCA1 RNA (100, 10, 1, and 0.1 pg) were reverse-transcribed, and 1/10 of each reaction was used for generation of a calibration curve (Fig. 2
). After cDNA synthesis for 60 min at 42 °C and inactivation of the enzyme at 95 °C for 5 min, PCR reactions were performed in the LightCycler instrument with the FastStart DNA Master Hybridization Probes Kit (Roche Molecular Biochemicals). Each reaction mixture consisted of 2 µL of cDNA from the reverse transcription step (unknown samples and each dilution of the calibrator), a pair of flanking PCR primers (0.5 µM each), two pairs of acceptor and detection probes (0.3 µM each), 1.5 µL of "Hot Start" PCR Reaction Mix, 2.4 µL of 25 mM MgCl2, and PCR-grade water up to a final volume of 15 µL. For all samples, a master mixture was prepared, and 13 µL was transferred into each precooled glass capillary. cDNA (2 µL) was then pipetted into all but one of the capillaries; 2 µL of PCR-grade water was added to that capillary as a negative control. Denaturation was performed at 95 °C for 10 min, followed by 45 PCR cycles of 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 5 s in the glass capillaries. For quantification, an external homologous ABCA1 RNA calibrator was generated as described above. The calibration curve (Fig. 2B
) was used to quantify both target mRNAs, ABCA1 and PBGD. Both absolute (pg ABCA1/sample) and normalized (ABCA1/PBGD ratio) values for the target gene were calculated. The data are means of at least three independent measurements. For the efficiency analysis of the target (ABCA1) and control genes (PBGD and GAPDH), cDNA from macrophages was serially diluted in 1:10 steps and amplified in the LightCycler by the same protocol. Crossing point (Cp) cycles vs cDNA dilution input were plotted to calculate the slope. The corresponding real-time PCR efficiencies (E) for each PBGD, GAPDH, and ABCA1 assay were calculated according to the equation: E = 10(-1/slope) (Fig. 3
).
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northern blot analysis
Total RNA (10 µg) isolated from macrophages of three healthy donors and from all human tissues analyzed by real-time PCR was separated on a 1.2% agarose-formaldehyde gel and transferred to a nylon membrane (Schleicher & Schuell) followed by ultraviolet cross-linking (Stratalinker Model 1800; Stratagene). For detection of ABCA1 mRNA, the membranes were hybridized with a 1-kb ABCA1 DNA fragment derived from PCR amplification using the primers described by Langmann et al. (4). The same membranes were reprobed with GAPDH cDNA.
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Results |
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, these primers span across introns 8 and 9, preventing the amplification of genomic DNA templates. The hybridization probes are separated by two nucleotides and are complementary to the sequence within the central region of exon 9. ABCA1 mRNA expression was normalized using specific primers and hybridization probes for a 264-bp PBGD fragment. In addition, for efficiency analysis, specific primers and hybridization probes that detect human GAPDH were used. The primer and hybridization probe sequences and characteristics for ABCA1, PBGD, and GAPDH are shown in Table 1
. Efficiency analysis for the target (ABCA1) and two housekeeping genes (PBGD and GAPDH; Fig. 3
) showed that only PBGD had an amplification efficiency very similar to that of ABCA1 (1.82 and 1.80, respectively); therefore, PBGD was used in our assay instead of the commonly used GAPDH, which had a higher amplification efficiency here (E = 2.00).
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To achieve absolute quantification of ABCA1 mRNA, we used serially diluted homologous in vitro-transcribed ABCA1 RNA as an external control. Four dilutions of ABCA1 cRNA were reverse transcribed in parallel to the sample material to be analyzed, and 2 µL of each reverse transcription reaction was amplified with the probes for ABCA1 and PBGD in the LightCycler. The real-time PCR curves from the amplification of the ABCA1 cRNA dilutions that were used to generate the calibration curve are displayed in Fig. 2A
. The calibration curve (Fig. 2B
) was used for quantification of both the ABCA1 transcript and the housekeeping gene (PBGD) when relative quantities were calculated. In contrast to GAPDH, PBGD was amplified with the same efficiency as the target ABCA1 gene (Fig. 3
) and therefore was chosen as the internal standard. One aliquot of cDNA was used to analyze ABCA1 in each sample, whereas we used another aliquot to determine PBGD expression. Both transcripts were amplified simultaneously during the same run. Depending on the initial concentration of target mRNA, signal intensity began to increase at different cycles. Amplification profiles for the ABCA1 and PBGD targets in various human tissues are shown in Fig. 2, C and D
, respectively. The initial template concentration was calculated by LightCycler software based on the cycle at which the fluorescent signal crossed a threshold in the exponential phase of the PCR reactions (Cp). To characterize the amplified products, melting curves (Fig. 4A
) were generated for all samples after each run and then converted into melting peaks (Fig. 4B
). The melting points (Tm) identified and discriminated between both analyzed products (64.6 and 69.8 °C for ABCA1 and PBGD, respectively).
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ABCA1 mRNA expression in various tissues compared with macrophages is shown in Fig. 5
. The results shown in Fig. 5A
are absolute amounts of ABCA1 mRNA based on a calculation using the calibration curve (pg/sample), whereas the results shown in Fig. 5B
are standardized by the quantification of PBGD. From all samples examined, the highest amounts of ABCA1 mRNA were found in macrophages loaded with E-LDL, followed by liver, lung, placenta, small intestine, and stomach. The lowest amounts of ABCA1 transcripts were detected in pancreas and heart. Fig. 6
shows the Northern blot analysis of ABCA1 mRNA in macrophages from three healthy donors (Fig. 6A
) and from all human tissues analyzed by real-time PCR (Fig. 6B
). Except for macrophages, the amount of ABCA1 mRNA in human tissues was rather low when analyzed with this method; therefore, long-term exposure of the x-ray film was necessary (1 week; Fig. 6B
). For macrophages, overnight exposure was sufficient. Rehybridization with the GAPDH cDNA commonly used for Northern blot analysis revealed variable expression of GAPDH in different tissues; nevertheless, the expression was constant in macrophages in different individuals (Fig. 6A
).
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To examine the effect of monocyte differentiation on ABCA1 expression, we cultured freshly isolated monocytes in vitro for 4 days in serum-free medium supplemented with M-CSF (Fig. 7A
). To examine the effect of E-LDL on ABCA1 mRNA concentrations, we incubated differentiated monocytes (Fig. 7A
) and THP-1 cells (Fig. 7B
) for 2 days in the presence or absence of E-LDL (40 mg/L). In addition to the up-regulation of ABCA1 during differentiation, cholesterol loading (E-LDL) induced ABCA1 expression in both cell types (Fig. 7, A and B
). Expression of ABCA1 mRNA in normal human fibroblasts (Fig. 7C
) was measured under basal conditions and after incubation with HDL3 (100 mg/L) for 1 and 12 h, respectively. We could demonstrate that a 12-h incubation of fibroblasts with HDL3 (cholesterol deloading) caused a substantial decrease in ABCA1 mRNA expression.
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Unlike macrophages, adipocytes store cholesterol mainly in the unesterified form (19). Cholesterol accumulation increases during differentiation, which is paralleled by an increase in triglyceride content (20). We were interested to see whether ABCA1 was expressed in adipocytes and whether there was a differentiation-dependent component in its regulation. Fig. 7D
shows a substantially higher amount of ABCA1 transcripts in differentiated human subcutaneous adipocytes compared with preadipocytes. To our knowledge, this is the first study reporting on the detection and differentiation-dependent up-regulation of ABCA1 mRNA in human subcutaneous preadipocytes and adipocytes.
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Discussion |
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). The melting points (Tm) for the ABCA1 and PBGD products were 64.6 and 69.8 °C, respectively, and no run-to-run variation was observed. Melting curve analysis at the end of the real-time assay enables characterization of the amplified product and obviates gel electrophoresis.
Among all tissues examined in this study, high ABCA1 mRNA expression was identified in the liver, placenta, small intestine, and stomach, but the strongest expression was observed in macrophages loaded with E-LDL (Fig. 5
). The lowest amount of ABCA1 transcripts was detected in the pancreas and heart. Tissue expression of ABCA1 mRNA was previously assessed with conventional technologies, which are less accurate (4). The expression patterns of ABCA1 mRNA displayed in Fig. 5A
(absolute ABCA1 mRNA) and Fig. 5B
(relative amount) show differences. On the basis of the above results, we conclude that standardization with a housekeeping gene (e.g., PBGD) is not completely satisfactory when the expression of a target gene (e.g., ABCA1) in different tissues and different cells needs to be analyzed. Because PBGD is not expressed equally in various tissues, absolute quantification may be more reliable. However, when we normalized the absolute amounts of ABCA1 mRNA with PBGD as a reference gene in the same cell type (e.g., fibroblasts or monocytes), we obtained comparable results. In contrast, the other commonly used internal standard, GAPDH, has been demonstrated to vary quantitatively in response to various factors, rendering interpretation of the results difficult (21)(23). The expression of GAPDH has also been reported to vary across tissues (24), and it can also be seen in our Northern blot data (Fig. 6B
). We rehybridized our Northern blot membrane with PBGD cDNA and obtained only very weak signals (data not shown).
Our initial experiments with GAPDH as an endogenous control prompted us to use PBGD as a reference gene (25). In our hands, unlike GAPDH (E = 2.00), both ABCA1 and PBGD transcripts were amplified with almost the same efficiency (E = 1.80 and 1.82, respectively; Fig. 3
), which is a critical factor for the final calculation of expression values (26). In addition, the amounts of both transcripts expressed were similar, in contrast to GAPDH. Furthermore, unlike for GAPDH, no pseudogene has been reported for human PBGD (27); thus, only cDNA-derived products are amplified by PCR. This may be a critical factor in cases where DNase digestion after RNA extraction is avoided to minimize loss of material. On the basis of our data, we recommend PBGD as a normalization control for mononuclear cells, fibroblasts, adipocytes, HepG2 cells, HeLa cells, and THP-1 cells. When we analyzed RNA samples from liver and lung taken from different individuals, we observed substantial differences in ABCA1 mRNA expression (data not shown). Because ABCA1 shows high expression in macrophages, the accumulation of these cells in some tissues under certain conditions, e.g., inflammation, may be responsible for the observed variations in ABCA1 mRNA expression in these tissues.
As we demonstrated previously by Northern blot analysis (4) and semiquantitative RT-PCR (28), expression of human ABCA1 mRNA in macrophages is regulated in a differentiation-dependent fashion. Here we confirmed these results with our new quantitative assay (Fig. 7A
). E-LDL taken up by human macrophages and smooth muscle cells contributes to the formation of early atherosclerotic lesions (15)(16)(29)(30) and up-regulates ABCA1 mRNA in macrophages and THP-1 cells. A similar up-regulation of ABCA1 mRNA has been reported in human monocyte-derived macrophages loaded with acetylated LDL (4)(28). Conversely, cholesterol efflux mediated by HDL3 down-regulated ABCA1 expression. We have used fibroblasts and monocytes to confirm this effect of HDL3 as an exogenous lipid acceptor in down-regulating ABCA1 mRNA (Fig. 7C
). The expression and function of ABCA1 in adipocytes have been not investigated to date. Adipose tissue serves not only as the main triglyceride storage site in humans, but also represents the largest deposit of unesterified cholesterol (19)(31). The amounts of both lipids increase during adipocyte differentiation, and Prattes et al. (32) recently demonstrated that most of the intracellular pool of free cholesterol in adipocytes is tightly associated with triglyceride droplets where it forms an outer lipid layer. Cholesterol efflux experiments revealed that a triglyceride-associated cholesterol pool is mobilized from these cells when stimulated with extracellular cholesterol acceptors (32). On the basis of those data and our results showing up-regulation of ABCA1 mRNA during adipocyte differentiation, we suggest that ABCA1 is involved in cholesterol trafficking in adipocytes.
Our quantitative real-time RT-PCR assay with hybridization probes allowed us to quantify the absolute amounts of ABCA1 transcripts and to determine the relative expression (ABCA1/PBGD ratios). This novel assay for the quantification and monitoring of ABCA1 transcripts is rapid, reliable, highly reproducible, and therefore applicable for (a) monitoring drugs effects, (b) epidemiologic studies, (c) studies assessing correlation between ABCA1 expression and susceptibility to lipid disorders, and (d) exploring the effects of polymorphisms in the promoter region of the ABCA1 gene.
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Acknowledgments |
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Footnotes |
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References |
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induces downregulation of Tangier disease gene (ATP-binding-cassette transporter 1) in macrophage-derived foam cells. Arterioscler Thromb Vasc Biol 2000;20:1565-1571.The following articles in journals at HighWire Press have cited this article:
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  A. V. Lebedev, N. Paul, J. Yee, V. A. Timoshchuk, J. Shum, K. Miyagi, J. Kellum, R. I. Hogrefe, and G. Zon Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance Nucleic Acids Res., November 1, 2008; 36(20): e131 - e131. [Abstract] [Full Text] [PDF]
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 V. Paul, H. H. D. Meyer, K. Leidl, S. Soumian, and C. Albrecht A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells J. Lipid Res., October 1, 2008; 49(10): 2259 - 2267. [Abstract] [Full Text] [PDF]
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 S. R. Bates, J.-Q. Tao, K. J. Yu, Z. Borok, E. D. Crandall, H. L. Collins, and G. H. Rothblat Expression and Biological Activity of ABCA1 in Alveolar Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2008; 38(3): 283 - 292. [Abstract] [Full Text] [PDF]
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 J. Wong, C. M. Quinn, I. C. Gelissen, and A. J. Brown Endogenous 24(S),25-Epoxycholesterol Fine-tunes Acute Control of Cellular Cholesterol Homeostasis J. Biol. Chem., January 11, 2008; 283(2): 700 - 707. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 J. F. Oram and A. M. Vaughan ATP-Binding Cassette Cholesterol Transporters and Cardiovascular Disease Circ. Res., November 10, 2006; 99(10): 1031 - 1043. [Abstract] [Full Text] [PDF]
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|
 |
|
 C. Farke, E. Viturro, H. H. D. Meyer, and C. Albrecht Identification of the bovine cholesterol efflux regulatory protein ABCA1 and its expression in various tissues J Anim Sci, November 1, 2006; 84(11): 2887 - 2894. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takata, V. Chu, A. R. Collins, C. J. Lyon, W. Wang, F. Blaschke, D. Bruemmer, E. Caglayan, W. Daley, J. Higaki, et al. Transcriptional Repression of ATP-Binding Cassette Transporter A1 Gene in Macrophages: A Novel Atherosclerotic Effect of Angiotensin II Circ. Res., October 28, 2005; 97(9): e88 - e96. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. Oram and J. W. Heinecke ATP-Binding Cassette Transporter A1: A Cell Cholesterol Exporter That Protects Against Cardiovascular Disease Physiol Rev, October 1, 2005; 85(4): 1343 - 1372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yoshinaga, H. Inoue, T. Utsunomiya, H. Sonoda, T. Masuda, K. Mimori, Y. Tanaka, and M. Mori N-Cadherin Is Regulated by Activin A and Associated with Tumor Aggressiveness in Esophageal Carcinoma Clin. Cancer Res., September 1, 2004; 10(17): 5702 - 5707. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. K. Callahan, M. S. Halleck, S. Krahling, A. J. Henderson, P. Williamson, and R. A. Schlegel Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells J. Leukoc. Biol., November 1, 2003; 74(5): 846 - 856. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Takeuchi, A. Bilchik, S. Saha, R. Turner, D. Wiese, M. Tanaka, C. Kuo, H.-J. Wang, and D. S. B. Hoon c-MET Expression Level in Primary Colon Cancer: A Predictor of Tumor Invasion and Lymph Node Metastases Clin. Cancer Res., April 1, 2003; 9(4): 1480 - 1488. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Takeuchi, C. Kuo, D. L. Morton, H.-J. Wang, and D. S. B. Hoon Expression of Differentiation Melanoma-associated Antigen Genes Is Associated with Favorable Disease Outcome in Advanced-Stage Melanomas Cancer Res., January 15, 2003; 63(2): 441 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. R. Su, M. F. Linton, and S. Fazio Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction J. Lipid Res., December 1, 2002; 43(12): 2180 - 2187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Rainen, U. Oelmueller, S. Jurgensen, R. Wyrich, C. Ballas, J. Schram, C. Herdman, D. Bankaitis-Davis, N. Nicholls, D. Trollinger, et al. Stabilization of mRNA Expression in Whole Blood Samples Clin. Chem., November 1, 2002; 48(11): 1883 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Langmann, M. Porsch-Ozcurumez, S. Heimerl, M. Probst, C. Moehle, M. Taher, H. Borsukova, D. Kielar, W. E. Kaminski, E. Dittrich-Wengenroth, et al. Identification of Sterol-independent Regulatory Elements in the Human ATP-binding Cassette Transporter A1 Promoter. ROLE OF Sp1/3, E-BOX BINDING FACTORS, AND AN ONCOSTATIN M-RESPONSIVE ELEMENT J. Biol. Chem., April 19, 2002; 277(17): 14443 - 14450. [Abstract] [Full Text] [PDF]
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