From the
Laboratory of Pharmacology, Brussels University School of Medicine, Bât. GE, 808 route de Lennik, B-1070 Brussels, Belgium and the
Department of Human Physiology and Biophysics, Institute of Biomedical Science, University of São Paulo, São Paulo 05508.900, Brazil
Received for publication, December 4, 2002 , and in revised form, March 27, 2003.
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ABSTRACT |
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INTRODUCTION |
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The PMCA belongs to the P-type family of transport ATPases. Four different genes corresponding to four isoforms PMCA1, PMCA2, PMCA3, and PMCA4 have been evidenced. Diversity among the ATPases is in addition generated by alternative splicing of the primary transcripts that may involve two major sites (for a review, see Refs. 46). In a previous work, we identified the PMCA transcripts expressed in the -cell and characterized them at these two alternative splicing sites (7).
The Na/Ca exchanger (NCX) is a mechanism extruding Ca2+ from the cell in exchange with Na+ without consuming any energy (8). Up to now, three mammalian isoforms of NCX have been cloned, NCX1, NCX2, and NCX3, representing the products of three distinct genes. Although NCX1 is widely distributed in various tissues, NCX2 and NCX3 seem to be restricted to brain and skeletal muscle. Several splice variants of NCX1 and NCX3 have been described, each exhibiting a specific tissue distribution (for a review see Refs. 9 and 10). Rat pancreatic islet cells, purified -cells, and RINm5F and BRINBD11 cells express two NCX1 splice variants: NCX1.3 and NCX1.7 (11).
Na/Ca exchange has a low affinity but a high capacity for Ca2+, whereas the Ca2+-ATPase has a high Ca2+ affinity but low capacity for the divalent cation (12). Therefore, it is considered that Na/Ca exchange takes care of large intracellular Ca2+ loads, whereas the Ca2+-ATPase performs the fine tuning of intracellular Ca2+ level around basal [Ca2+]i, namely 0.10.2 µM (12).
Using antisense oligonucleotides, we recently showed in the rat -cell, that Na/Ca exchange was responsible for up to 70% of Ca2+ removal from the cytoplasm upon membrane repolarization (13). In addition, different lines of evidence suggest that glucose stimulates rat Na/Ca exchange activity (14, 15). Previous work on the PMCA shows, on the contrary, that glucose inhibits PMCA activity (for a review, see Ref. 16). Thus, while two groups out of three found a direct inhibitory effect of glucose on enzyme activity (when added to the assay medium) (1719), a fourth group found no direct effect but showed that the activity of the ATPase was significantly inhibited when measured in islets previously incubated with glucose (20). Although found by three groups, glucose-induced inhibition of PMCA activity, whether by a direct or indirect effect, was found somewhat surprising and/or unexpected. It was suggested that inhibition of PMCA could contribute to the increase in [Ca2+]i that stimulates insulin release (17, 18, 20). However, such inhibition is consistent with the abovementioned view on the respective roles of PMCA and NCX in Ca2+ homeostasis (12), which in the case of the
-cell, could be formulated as follows: when stimulated by glucose, the
-cell is faced with a major increase in Ca2+ inflow and, therefore, switches from a low efficiency Ca2+ extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger.
To ascertain such a view, we measured the effect of glucose on PMCA and NCX1 isoforms transcription, expression, and activity in rat islet cells. The data reveal that such processes are indeed reduced by glucose in the case of the PMCA but increased by the sugar in the case of the NCX.
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EXPERIMENTAL PROCEDURES |
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Design of Polymerase Chain Reaction PrimersPrimer sequences used are described in Table I. To amplify the putative splicing area of NCX1 (GenBankTM accession number X68191
[GenBank]
), the primers were designed based on rat heart cDNA sequence. For PMCA isoform 1 (PMCA 1) (accession number J03753
[GenBank]
), PMCA isoform 2 (PMCA 2) (accession number J03754
[GenBank]
), and PMCA isoform 3 (PMCA3) (accession number J05087
[GenBank]
), the primers were designed based on rat brain cDNA sequence. For PMCA isoform 4 (PMCA 4) (accession number U15408
[GenBank]
), the primers were designed based on rat testis cDNA sequence. For -actin (accession number V01217
[GenBank]
), primers were based on rat cytoplasmic sequence. All primers were synthesized by Amersham Biosciences (Belgium).
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Reverse Transcription and Polymerase Chain ReactionTotal RNA was isolated from rat pancreatic islets using the RNAnowTM method (Biogentex). RNA (2 µg) was heated 10 min at 70 °C, to denaturate the RNA, and reverse-transcribed for 50 min at 42 °C, using 200 units of Superscript II RT (Invitrogen), with 25 µg/ml oligo(dt) primer, 2.5 µg/ml random primer (Promega, Leiden, The Netherlands), and triphosphate nucleosides (0.5 mM each) (Roche Applied Science, Brussels, Belgium) in a 20-µl reaction volume as recommended by the manufacturer. RNA complementary to cDNA was removed using 2 units of Escherichia coli RNase H (Roche Applied Science) for 20 min at 37 °C. The medium was then diluted with 30 µl of 16 mM EDTA, and the reaction was terminated by heating the medium up to 70 °C for 15 min. Three microliters of single-strand cDNA were amplified by PCR in a 50-µl volume using a Pwo DNA polymerase kit (Roche Applied Science), 30 pmol of each primer, and 0.5 units of Pwo DNA polymerase. The amplification was conducted in a thermal cycler (GeneAmp PCR system 2400, PerkinElmer Life Sciences, Zaventem, Belgium) under the following conditions: initial denaturation at 94 °C for 2 min; 10 cycles of 94 °C for 30 s/60 °C for 30 s/72 °C for 45 s and 17 cycles of 94 °C for 30 s/60 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA1; 10 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s and 22 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA2; 10 cycles of 94 °C for 30 s/56 °C for 30 s/72 °C for 45 s and 27 cycles of 94 °C for 30 s/57 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA3; 10 cycles of 94 °C for 30 s/58 °C for 30 s/72 °C for 45 s and 25 cycles of 94 °C for 30 s/58 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for PMCA4; 10 cycles of 94 °C for 30 s/63 °C for 30 s/72 °C for 45 s and 24 cycles of 94 °C for 30 s/63 °C for 30 s/72 °C for 45 s increased by 20 s at each cycle for NCX; and then 72 °C for 7 min (final extension).
Quantitative Comparison of PCR productsTo determine the relative amounts of the NCX1 and PMCAs gene products, the semi-quantitative reverse-transcribed (RT-PCR) method was used (11). At the end of the amplification, a 10-µl aliquot was subjected to electrophoresis in 1% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide in 1x TBE buffer. All experiments were carried out with 10 pmol of -actin primers as internal control. The cDNA bands were quantified by scanning densitometry.
Plasma Membrane Ca2+-ATPase ActivityThe method to measure the PMCA activity was adapted from Gronda et al. (20). After the incubation period, the tubes containing the medium plus the islets were centrifuged for 1 min at 1700 x g (4 °C). The supernatant was discarded, and the precipitated islets were resuspended in 300 mM sucrose and 10 mM Tris-HCl (pH 7.24) at 4 °C. The islet suspension was then transferred to a microhomogenizer, washed twice at 4 °C, and homogenized in 1 ml of the same buffer.
The Ca2+-ATPase activity was assayed in a 0.5-ml reaction volume. The method consists of monitoring the release of 32P from [-32P]ATP by islet homogenates incubated in a solution containing 50 mM Tris-HCl (pH 7.24), 0.1 mM ouabain, 1 mM [
-32P]ATP, 1 mM EGTA, and 20 µmol of thapsigargin. After 45 min of incubation at 37 °C, the tubes were transferred to an ice-cold water bath. After 1 min, 0.75 ml of 0.5% (w/v) ammonium molybdate in 5% (v/v) perchloric acid and then 0.6 ml of isobutanol were added to each tube. The mixture was stirred vigorously and then centrifuged at 1700 x g (4 °C). The radioactivity was measured in an aliquot of the organic phase by liquid scintillation and, from this value, the amount of inorganic phosphate liberated from ATP was calculated. Enzyme activity represents the difference between the activity measured in the medium described above in the presence or absence of 1 µM Ca2+. Protein concentration in the homogenates was determined using the Bio-Rad Dc Protein Assay (Bio-Rad, Nazareth, Belgium) with BSA as standard.
45Ca2+ UptakeThe method used for the measurement of 45Ca2+ uptake in isolated pancreatic islets cells has been described previously (11). In brief, the islets cells were preincubated at 37 °C during 30 min in 1 ml of a non-radioactive solution consisting in a Krebs-Ringer buffer (115 mM NaCl, 1 mM CaCl2,1mM MgCl2,10mM HEPES/NaOH, pH 7.4; gassed with ambient air), containing 2.8 mM glucose (Merck), 10 µM nifedipine (Calbiochem, La Jolla, CA), and then incubated at 37 °C for 5 min in 1 ml of the same medium containing in addition 45Ca2+ (10 µCi/ml). In some experiments, NaCl was iso-osmotically replaced by sucrose (241 mM, Merck). At the end of the incubation, the cells were separated from the incubation medium by using a combined lanthanum and oil technique (14). Na/Ca exchange was evaluated by measuring -dependent 45Cao uptake.
Indirect Immunofluorescence MicroscopyThe islets cells were plated on coverslips and analyzed by indirect immunofluorescence microscopy 48 h after plating. After incubation, cells were washed with TBS (tris-buffered saline: 20 mM Tris, 137 mM NaCl, pH 7.2), fixed for 20 min in 4% formol at pH 7.4 (4 °C), and washed with TBS. The cells were permeabilized in solution containing 0.01% Triton X-100, 197 µM MgCl2, 19.5 µM dithiothreitol, and 10% glycerol (pH 7.4, 4 °C), washed twice with TBS, and incubated in a blocking buffer containing 1% horse serum (Vector Laboratories Inc., Burlingame, CA) in TBS for 20 min. The coverslips were overlaid with the primary antibody (SWant, rabbit anti-Ca2+-ATPase (isoform 1, 2, 3, or 4), and mouse anti-Na/Ca exchanger (isoform NCX1) diluted 1/1000 in TBS-1% bovine serum albumin (BSA) buffer for 1 h. Control cells were incubated in TBS-1% BSA buffer without primary antibody and washed three times with TBS. The cells were then treated with secondary antibody (Alexa FluorTM 594 goat anti-mouse or anti-rabbit IgG (H+L) conjugate, Molecular Probes, Eugene, OR), diluted 1/400 in TBS-1% BSA for 45 min, and washed four times with TBS. The cells were incubated with 300 nM 4',6-diamidino-2-phenylindole solution (Molecular Probes) and washed twice with TBS. The coverslips were mounted with Vectashield® (Vector Laboratories Inc.), and the cells were observed with an Axioplan microscope (Zeiss, Germany) equipped with a HBO 100-watt or XBO 100-watt illuminator and a x100 objective, and photographed with a Dual-mode cooled charge-coupled device camera (C4880, Hamamatsu, Japan). Images were analyzed and quantified using TITN answares software (ALCATEL).
Computer-assisted Microscopy for Quantitative ImmunofluorescenceTo measure PMCA and NCX expression in cells, mean optical density was determined by means of a SAMBA 2005 computer-assisted microscope system (UNILOG, Grenoble, France) with a x40 magnification lens (BX50 microscope, aperture 0.65; Olympus, Tokyo, Japan). Mean optical density (MOD) denotes staining intensity. Twenty fields of between 60,000 and 120,000 µm2 each were scanned for each preparation (see Refs. 2325 for further details and standardization procedures). A negative histological control slide (from which the primary antibody was omitted) was analyzed for each sample. MOD values of the negative control samples were automatically subtracted from their corresponding positive samples.
StatisticsThe results are expressed as means ± S.E. The statistical significance of differences between data was assessed by using analysis of variance followed by Tukey's post test.
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RESULTS |
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Because the amount of RNA obtained from pancreatic islets was very limited, the total RNA was reverse-transcribed in the corresponding cDNA, which was then serially diluted. The data were expressed in proportion to the amount of -actin mRNA present in the respective preparations.
Effect of Glucose on PMCA and NCX1 mRNAPancreatic islets were cultured with 2.8 mM, 11.1 mM, and 22.2 mM glucose for 24 h. Total RNA was isolated, and quantitative RT-PCR was performed to quantify PMCA and NCX1 isoforms mRNAs. Insulinotropic concentrations of glucose (11.1 and 22.2 mM) induced a significant linear decrease in PMCA1 and PMCA2 mRNA but failed to affect PMCA3 and PMCA4 mRNA (Fig. 2). In contrast, glucose induced a linear increase in NCX1.3 mRNA and a trend toward an increase in NCX1.7 mRNA (Fig. 2). At 11.1 mM glucose, PMCA1 and PMCA2 mRNA were reduced by 13 and 28%, respectively, whereas NCX1.3 was increased by 52%. At 22.2 mM glucose, the figures obtained were 31, 51, and 66%, respectively. Representative gels showing the effect of glucose on PMCA2 and NCX1 transcription are shown in Fig. 3.
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To evaluate the time course of the decrease in transcription, the effect of glucose on PMCA2 and NCX1.3 mRNA levels were measured over shorter periods of time (3 and 6 h). Fig. 4 shows that high glucose affected PMCA2 and NCX1.3 mRNA levels after 6 h of incubation. No significant effect of glucose was observed after 3 h (data not shown).
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Effect of Glucose on PMCA and NCX1 Expression at the Protein LevelTo quantify the effect of glucose on PMCA and NCX1 expression at the protein level, quantitative immunofluorescence was used. Again, the cells were exposed to glucose 2.8 or 22.2 mM for 24 h. Exposure to the high concentration of glucose decreased PMCA2 expression by about 28% (p < 0.001) but failed to affect the expression of PMCA1, PMCA3, and PMCA4 (p > at least 0.10, Fig. 5). In contrast, the sugar increased NCX1 expression by about 28% (p < 0.001, Fig. 5). Fig. 6 shows representative images illustrating the effect of glucose on PMCA2 and NCX1 (both NCX1.3 and NCX1.7) immunofluorescence.
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Effect of Glucose on PMCA and NCX1 ActivityCa2+-ATPase activity was measured by monitoring the release of 32P from [-32P]ATP by islet homogenates in the presence of ouabain and thapsigargin to ensure complete inhibition of the Na+/K+-ATPase and sarco(endoplasmic) reticulum Ca2+-ATPase activity, without affecting the PMCA. Na+/Ca2+ exchange activity was measured as intracellular Na+-dependent 45Ca2+ uptake, in the presence of 10 µM nifedipine to block the effect of glucose on voltage-sensitive Ca2+-channels. Fig. 7 shows that Ca2+-ATPase activity was decreased when cultured for 24 h in the presence of 11.1 and 22.2 mM glucose (p < 0.025), whereas Na+/Ca2+ exchange activity was increased under the same experimental conditions (p < 0.025).
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Effect of -Ketoisocaproate on PMCA and NCX1 Expression and ActivityTo confirm the results obtained with glucose, another metabolized insulin secretagogue (
-ketoisocaproate) was used. Fig. 8 shows that
-ketoisocaproate (10 mM) mimicked the effect of glucose to decrease PMCA1 and PMCA2 mRNA level by about 36% (p < 0.02) and 57% (p < 0.001), respectively, while failing to affect those of PMCA3 and PMCA4. Likewise,
-ketoisocaproate increased NCX1.3 mRNA levels by about 60% (p < 0.04) and induced a trend toward an increase in NCX1.7 mRNA.
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PMCA and NCX1 expression at the protein level showed a parallel tendency (Fig. 9). Thus, -ketoisocaproate decreased PMCA1 and PMCA2 expression by 40 and 41% (p < 0.001) while failing to affect PMCA4 expression. Surprisingly, PMCA3 expression was also reduced by
-ketoisocaproate (32%, p < 0.001). Last, like glucose,
-ketoisocaproate increased NCX1 expression by about 20% (p < 0.01).
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Effect of Nifedipine on PMCA and NCX1 ExpressionTo gain further insight into the mechanism by with glucose modulates PMCA and NCX1 expression, the effect of the sugar was examined in the presence of the Ca2+ channel blocker nifedipine. Fig. 10 shows that nifedipine (10 µM) failed to suppress the effect of glucose on PMCA and NCX1 expression. In fact, in the presence of nifedipine, the effect of the sugar was even more marked than in its absence. Thus, in the presence of the antagonist, glucose inhibited PMCA1, 2, and 3, instead of PMCA2 alone in its absence (compare Figs. 5 and 10). Likewise, the effect of the sugar to increase NCX1 expression was of larger magnitude than in its absence, the sugar increasing NCX1 expression by 28 ± 5% and 63 ± 3% in the absence and presence of nifedipine, respectively (p < 0.0001).
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Ca2+ entry into the -cell through voltage-sensitive Ca2+ channels may not be the sole mechanism by which glucose rises [Ca2+]i. Other mechanisms have been identified in
-cell mouse, like activation of store-operated channels and intracellular Ca2+ release through inositol-trisphosphate- or ryanodine-sensitive channels (3437). However, it is unclear to which extent such mechanisms also operate in rat
-cells. Therefore, the effect of nifedipine on glucose-induced increase in [Ca2+]i was examined in rat
-cells. Fig. 11 shows that, in the absence of nifedipine, glucose (11 mM) induced a rapid and oscillating increase in [Ca2+]i. Such increase was completely blocked by 10 µM nifedipine. This indicates that, in the rat
-cell, mechanisms other than voltage-sensitive Ca2+ channels do not contribute significantly to glucose-induced increase in [Ca2+]i, at least in the absence of a significant increase in Ca2+ inflow through such voltage-sensitive Ca2+ channels.
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DISCUSSION |
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The data were rather homogeneous in showing a parallel decrease in mRNA and protein levels of one of the four PMCAs, PMCA2. This is of great interest because PMCA2 is expressed in a limited number of specialized tissues like neurons, at variance with PMCA1 and PMCA4, which are transcribed in most tissues and may thus represent housekeeping enzymes (2931). Furthermore, the -cell is the sole non-neuronal tissue, so far identified, that shows the presence of substantial amounts of PMCA2 at the protein level, a finding that attests the special needs of
-cells in terms of Ca2+ homeostasis (7).
Glucose also decreased PMCA1 mRNA but failed to affect the PMCA1 level of expression, although the metabolized insulin secretagogue, -ketoisocaproate, decreased both PMCA1 and PMCA2 transcription and expression. Likewise, in the presence of the Ca2+ channel blocker nifedipine, glucose reduced the expression of PMCA1, 2, and 3. Therefore, it is conceivable that, together with PMCA2, the expression of PMCA1 and PMCA3 is repressed by the sugar.
In parallel with this decrease in expression, glucose reduced global PMCA activity when measured in islets previously incubated with glucose, in agreement with Gronda et al. (20). In the latter work, these authors observed a significant decrease in Ca2+-ATPase activity during the first 3 min of exposure to glucose with subsequent return toward control values. However, the enzyme activity measured at 60 min was still below the control value, even though this difference was not significant. Our data showing a dose-related inhibition of the ATPase activity after 24 h of incubation confirm that the inhibitory of the sugar is not transient. No attempt was made in the present study to measure any direct effect of glucose on enzyme activity.
In contrast to its effects on PMCA, the sugar induced a dose-related increase in NCX1 transcription, expression, and activity. The isoform that appeared to be up-regulated at the mRNA level was NCX1.3, although there was a trend toward an increase in NCX1.7 transcription, with resulting increase in NCX1 global expression at the protein level (NCX1.3 plus NCX1.7) and activity.
To identify pancreatic -cell genes that are responsive to glucose, Webb et al. (32) recently carried out a microarray analysis of murine
-cell line MIN6 cells exposed to either high (25 mM) or low (5.5 mM) glucose for 24 h. The study demonstrated that many
-cell genes were glucose-responsive, with 78 demonstrating a 2.2-fold or greater change in transcript levels. The largest functional clusters identified were secretory pathway, metabolism, signaling, and transcriptional regulation transcripts. The microarray used in the latter study allowed interrogation of 6500 murine genes and expression sequence tags, including the sequences of NCX1 gene but apparently not those of the PMCA genes (32). With respect to NCX1, the study was negative but the authors only reported genes showing a 2.2-fold or greater change in transcript levels. In the present study, the maximum change did not exceed 66%. Another microarray analysis of intact human pancreatic islets using very stringent criteria also failed to show changes in PMCA and NCX1 transcripts levels (33).
The effects exerted in the present study by the metabolizable insulin secretagogue -ketoisocaproate on PMCA and NCX1 expression and activity were similar to those induced by glucose, and even tended to be more marked than those induced by the sugar. Likewise, the effects of glucose on both PMCA and NCX1 were not suppressed by nifedipine. Although rather preliminary, the latter data suggest that the effects of glucose to modulate PMCA and NCX1 expression and activity do not result from a direct effect of the sugar or from a simple increase in Ca2+ entry or content of the
-cell, but may implicate more complex mechanism such as those evoked in the amplifying pathway of the sugar. Further work is required to identify such mechanisms of regulation.
Taken as a whole, the present data confirm the view that, in response to a stimulation by glucose, the -cell switches from a low efficiency Ca2+-extruding mechanism, the PMCA, to a high capacity system, the Na/Ca exchanger, to better face the increase in Ca2+ inflow. To our knowledge, this is the first demonstration of a reciprocal change in PMCA and NCX1 expression and activity in response to a given stimulus in any cellular preparation or tissue. The up-regulation of NCX1 by glucose may have a further advantage, because NCX1, in addition to being able to extrude Ca2+, may also contribute to Ca2+ entry through reverse Na/Ca exchange and generate an inward current that may prolong the duration of the burst of spikes of electrical activity generated by glucose and hence enhance insulin release (15). In addition, the up-regulation of NCX1 could help to protect the
-cell against the deleterious actions of elevated glucose levels. Indeed, the sugar, when used at a high concentration was observed to trigger apoptosis in
cells, a process that was Ca2+-dependent (38).
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FOOTNOTES |
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¶ Supported by the ALFA program IRELAN of the European Union, coming from the Institute of Biomedical Science of the University of São Paulo, Brazil.
|| A Senior Research Assistant of The Belgian Fund for Scientific Research.
** To whom correspondence should be addressed. Tel.: 32-2-555-6201; Fax: 32-2-555-6370; E-mail: herchu{at}ulb.ac.be.
1 The abbreviations used are: KATP, ATP-dependent K+ channel; KIC, -ketoisocaproate; MOD, mean optical density; NCX, Na+/Ca2+ exchanger; PMCA, plasma membrane Ca2+-ATPase; RT, reverse transcription; BSA, bovine serum albumin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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