Báo cáo Y học: Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues

Eur. J. Biochem. 269, 3313–3320 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03018.x Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues Araceli del Arco1,3, Julian Morcillo2, Juan Ramon Martınez-Morales2, Carmen Galian1, Vera Martos1, Paola Bovolenta2 and Jorgina Satrustegui1 1Departamento de Biologıa Molecular, Centro de Biologıa Molecular Severo Ochoa, Universidad Autonoma de Madrid Spain; 2Departamento de Neurobiologıa del Desarrollo, Instituto Cajal, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain; 3Facultad de Ciencias del Medio Ambiente, Universidad de Castilla La Mancha, Toledo, Spain Aralar1andcitrinaremembersofthesubfamilyofcalcium-binding mitochondrial carriers and correspond to two iso-forms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. I n situhybridization analysis indicates that both isoforms are expressed strongly in the branchialarches,dermomyotome,limbandtailbudsatearly embryonicstages.However,citrinwasmoreabundantinthe ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal Metabolitesaretransportedthroughtheinnermitochondrial membranebyproteinsbelongingtothemitochondrialcarrier (MC) superfamily [1]. The structure of these carriers (molecularmass30 kDa)consistsofathreefoldrepetition ofasequenceofabout100aminoacids[2,3]withtwoputative transmembrane domains. In the last few years, a number of newMCshavebeenidentified[3–5],includingasubfamilyof Ca2+-binding mitochondrial carriers (CaMCs) with new structural characteristics [6–8]. The CaMC subfamily mem-bers have a bipartite structure. Their C-terminal domains havethefeaturesoftheMCsuperfamilyandtheirN-terminal extensions harbor EF-hand Ca2+-binding motifs [6]. Aralar1andcitrin,twomembersoftheCaMCsubfamily, are nuclear-encoded proteins, with genes in human Correspondence to J. Satrustegui, Departamento de Biologıa Molecular, Centro de Biologıa Molecular Severo Ochoa, Universidad Autonoma de Madrid, 28049-Madrid, Spain. Fax: 00 34 91 3974799, Tel.: 00 34 91 3974872, E-mail: jsatrustegui@cbm.uam.es Abbreviations: AGC, aspartate/glutamate carrier; MC, mitochondrial carrier; CaMC, calcium-binding mitochondrial carrier; CTLN2, adult-onset type II citrullinemia; ASS, argininosuccinate synthetase. (Received 14 February 2002, revised 19 April 2002, accepted 23 May 2002) musclewasdetectedatE18andthatintheheartbeganearly in development (E11) and was preferentially localized to auricularmyocardiuminlateembryonicstages.Aralar1was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hemato-poieticsystem.Botharalar1andcitrinwereexpressedinfetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1isenrichedinlungandinsulin-secretingbcells.These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues. Keywords: aspartate/glutamate carrier; calcium; citrulline-mia; development; mitochondria. chromosome 2 (SLC25A12 [9,10]) and 7 (SLC25A13 [8,11]), respectively. As recently demonstrated, aralar1 and citrinareisoformsofthemitochondrialaspartate/glutamate carrier (AGC) [12] which catalyzes a 1 : 1 exchange of aspartate for glutamate and plays an important role in the malate/aspartateshuttle,ureasynthesisandgluconeogenesis fromlactate[13–15].ThesetwoAGCisoformsareactivated by Ca2+ on the external face of the inner mitochondrial membrane [12]. Mutations in the human gene coding for citrin are responsible for adult-onset type II citrullinemia (CTLN2: 603471) [8,16], an autosomal recessive disease caused by a liver-specific deficiency in argininosuccinate synthetase (ASS). In the liver, the AGC plays an important role in the urea cycle by providing aspartate for incorporation into argininosuccinate [17]. The mutations in the citrin gene in patients affected by CTLN2 cause either truncation of the protein or deletion of a loop between the transmembrane spans [8,16], impairing the function of citrin as an AGC in mitochondria. This impairmentwouldpresumably lead to a failure in the supply of aspartate from mitochondria for argininosuccinate synthesis, with consequent alterations in the stability/activity of liver ASS, one of the symptoms of CTLN2. Citrin is strongly expressed in both liver and kidney [6–8,18]. However, CTLN2 is a liver-specific metabolic 3314 A. del Arco et al. (Eur. J. Biochem. 269) deficiency, and ASS levels are normal in other tissues such as kidney [8,19]. This difference can be explained by the observation that aralar1, the second human AGC isoform, isalsoexpressedinkidney[6,18]andhumankidneycelllines [12,18], therefore it may compensate for the loss of citrin in the kidney of patients with CTLN2. This raises a general question of whether the two isoforms are expressed in the same tissues and cell types and whether these isoforms play the same role. To address the first of these questions, we have now studied the expression of aralar1 and citrin throughout mouse development and in tissues of the adult rat with the use of isoform-specific probes and antibodies. Our results indicate that the two isoforms are widely expressed throughout embryogenesis with a dynamic expression pattern. The characteristic expression of aralar1 in skeletal muscle and citrin in liver is only manifested at E18 or after birth, respectively. Aralar1, but not citrin, is expressed early (E11) in heart and it is preferentially localized to auricular myocardium in late embryonic stages. Whereas citrin is preferentially expressed in liver and kidney, the classical gluconeogenic organs, a number of adult tissues and cell types were found to express aralar1 preferentially over citrin, including the adult lung and hematopoietic cells. MATERIALS AND METHODS Animals and tissues mRNAexpression wasexamined inBalb/cmiceandWistar rats.Animalswerekeptinclimate-controlledquartersunder a 12-h light cycle with free access to water and standard chow diet. The animal facilities fulfilled the requirements of theEuropeanlaws,andthehigheststandardsofanimalcare were met in all experimental protocols. Mouse embryos were collected from timed pregnant Balb/c mice. The day of vaginal plug appearance was considered embryonic day 0.5 (E0.5). The following tissues and organs were dissected from 3-month-old rats: liver, forebrain, cerebellum, heart, small intestine, stomach, lung, kidney, testis, ovary, white adipose tissue, pancreas, bone marrow, spleen and muscle. Bone marrow was obtained from the tibia bone. Rat pancreatic bislets were isolated by collagenase digestion and standard procedures [20]. Brown adipose tissue was collected from 1-day-old pups. Rat fetuses staged at embryonic day 18 (E18) delivered by cesarian section and newborn pups (1–6 h after sponta-neousdelivery)wereusedtostudypostnataldevelopmentof the liver. Cell lines HEK-293Tcells were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum (Gibco-BRL) at 37 °C in a 7% CO2 atmosphere. RAW 264 and Jurkat cells were grown in RPMI 1640 medium with 5% fetal bovine serum under identical conditions. Probes The mouse aralar1 probe used was a 381-bp PstI fragment obtained from the mouse ESTclone W82002 (ATCC). A probe specific for mouse citrin was generated by RT-PCR Ó FEBS 2002 using 2 lg total RNA obtained from adult mouse liver as template. The oligonucleotides used, ara2-rat5 (5¢-AT CTGTCCTGTGTGCTCCGG-3¢) and ara2-mouse3 (5¢-TCCATGGGTGTAACCTGACC-3¢), were designed from mouse citrin cDNA sequence [11]. The amplified fragment was subcloned into the blunted pSTBlue-1 (Novagen) and verified by sequencing. In situ hybridization The 381-bp and 557-bp fragments of aralar1 and citrin cDNA were transcribed to generate digoxigenin-labeled antisense and sense cRNA probes. Whole-mount in situ hybridizations were performed as described [21]. Briefly, hybridizationswerecarriedoutat65 °Cin50%formamide. Post-hybridization washes were performed at the same temperature and in the same buffer. Embryos staged at embryonic day 11 (E11) were hybridized in toto. After hybridization, embryos were photographed, dehydrated, embedded in Paraplast, and sectioned with a microtome at 18 lm. For older embryos, E18.5, hybridizations were carried out on tissue sections. Embryos were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, at 4 °C overnight and then cryoprotected by immersion in 30%sucrosesolutioninphosphatebuffer.Cryostatsections 16–20 lm thick were mounted on 2% 3-aminopropyltri-ethoxysilane-coatedslides,air-dried,andpermeabilizedwith proteinase K (10 lgÆmL)1 in NaCl/Pi containing 0.1% Tween) for 5–10 min at room temperature. Sections were then postfixed in 4% paraformaldehyde in phosphate buffer, prehybridized for 1 h at 65 °C in 50% formamide, and incubated with probes for 16 h at 65 °C. All the staining patterns described below were obtained only with antisense riboprobes and not with control sense riboprobes. RNA analysis Total RNA was extracted from rat tissues using the guanidine isothiocyanate method. Northern blot analysis was carried out using 20 lg total RNA from different rat tissues as previously described [7]. As human and rat nucleotide sequences are highly homologous ( 90% identity), we used fragments of human citrin and aralar1 1cDNAs as probes. The blot was stripped on 0.1% SDS at 100 °Cfor30 min,andreprobedunderidenticalconditions. Antibodies An antibody to the N-terminal half of aralar1 (amino acids 12–343) was described previously [6]. A citrin-specific antibody was generated to amino acids 9–278 of the N-terminalhalfofcitrinexpressedinbacteria.Theconstruct for bacterial expression has been previously described [7]. In addition,selectedregionsofhumancitrin(aminoacids305– 319) and human aralar1 (amino acids 507–520), with Jameson and Wolf antigenic indexes of 1.7, as predicted by the peptidestructure program from the CGC (Genetic Computer Group, Madison, WI, USA) package, were used to generate epitope-specific antibodies. These regions of human aralar1 and citrin are conserved in the mouse proteins. The citrin 305–319 peptide was conjugated with mcKLH (mariculture keyhole limpet hemocyanin) using an Imject Immunogen EDC conjugation kit (Pierce). The Ó FEBS 2002 Development of aspartate/glutamate mitochondrial carriers (Eur. J. Biochem. 269) 3315 aralar1 507–520 peptide was conjugated with maleimide-activated mcKLH (Pierce) through a cysteine added at the N-terminus ofthepeptide, as recommended by thesupplier. The purified citrin protein (amino acids 9–278) and mcKLH-conjugated peptides were injected into rabbits using standard immunization procedures. Westerns blots Rat tissues were homogenized in 250 mM sucrose/10 mM Tris/HCl(pH 7.4)/proteaseinhibitors(1 mM iodoacetamide and 1 mM phenylmethanesulfonyl fluoride) and centrifuged at 750 g (10 min). The supernatant was then centrifuged at 10 000 g (15 min), and the pellets were collected to obtain the crude mitochondrial fractions. Cells were scraped into 250 mM sucrose/20 mM Hepes/10 mM KCl/1.5 mM MgCl2/ 1 mM EDTA/1 mM EGTA/1 mM dithiothreitol/protease inhibitors, pH 7.4, homogenized and subjected to differen-tial centrifugations as described above. Mitochondrial fractions were analysed by Western blot-ting using an Enhanced Chemiluminiscence (ECL) kit (Amersham). Antibody to the N-terminus of aralar1 was used at a dilution of 1 : 5000, and antibodies to the N-terminus of citrin, citrin 305–319 and aralar1 507–520 wereusedatadilutionof1 : 2000.Tocontrolfortheamount of mitochondrial protein loaded, blots were stripped and incubated with an antibody to the mitochondrial protein b-F1ATPase (a gift from J. M. Cuezva, Centro de Biologia Molecular devero Ochoa, JAM, Spain) at a dilution of 1 : 5000. The densities of the bands were evaluated with a Bio-Rad GS-710 calibrated imaging densitometer. Immunocytochemistry The animals were anesthetized with sodium pentobarbital, and perfused through the cardiac ventricle, first with 50 mL 0.9%NaClfollowedby250 mLfixativesolutioncontaining 4%paraformaldehydein0.1 M phosphatebuffer,pH 7.4,at room temperature. The tissues were removed, postfixed at 4 °C for 24 h, and cryoprotected by immersion in 30% sucrose. Free-floating cryostat 40-lm-thick sections were firstquenchedwith3%H2O2 in10%methanolfor20 minin potassium phosphate-buffered saline (NaCl/Pi). After this treatment,thesectionswerepreincubatedfor2–3 hinNaCl/ Pi containing 5% horse serum and 0.25% Triton X-100 and incubated overnight with antiaralar1 antibody at a dilution of 1 : 100 in 1% horse serum and 0.25% Triton X-100 in NaCl/Pi. Secondary biotinylated antibody (goatanti-rabbit; Vector; 1 : 150 dilution) was then incubated for 1–2 h, followed by a 1-h reaction with avidin–biotin–peroxidase complexes (regular ABC kit Vectastain; Vector). Sections were developed using 0.05% 3,3-diaminobenzidine (Sigma) in the presence of 0.03% H2O2 in NaCl/Pi for 1–2 min. Sections were mounted on to polylysine-coated slides, dehydrated, delipidated, and mounted in DPX (BDH). RESULTS Expression of aralar1 and citrin during embryonic mouse development The expression of aralar1 and citrin was studied by using in situ hybridizations in toto or on cryostat tissue sections, depending on the stage of the embryos. The data obtained partially confirmed and further extended those reported by Sinasac et al. [11] on embryonic expression of citrin in mouse. AtE11,theearlieststageanalyzed,botharalar1andcitrin were expressed throughout the developing embryo, with stronger expression in the branchial arches, the developing dermomyotome,thelimbandthetailbuds(Fig. 1A–E,a–e). In spite of the apparent similarities in distribution, citrin expression was predominantly, although not uniquely, associated with the ectodermal, whereas aralar1 expression was more abundant in the mesenchymal components of thesestructures(Fig. 1B–E,b–e).Inparticular,citrinbutnot aralar1 was strongly expressed in the apical ectodermal ridge of the limb and on the tip of the tail bud (Fig. 1A,B). As an additional difference between the two genes, aralar1 butnotcitrintranscriptswerefoundintheheart(Fig. 1C,c). Atlaterstagesofdevelopment(E13–E15),themRNAsof thetwogeneswerealsodetectedinneuraltissue.Afewdays later (E18), the expression of aralar1 and, to a lesser extent, citrin became clearly localized to selected brain regions such as the cortex and hippocampus, the ventromedial thalamus, the mitral cell layer of the olfactory bulb (Fig. 1L–M,l–m), and the developing striatum (not shown). In the peripheral nervoussystem,aralar1andcitrinmRNAsweredetected,at similar levels, in the trigeminal ganglia (Fig. 1L,l). At E18, when organogenesis becomes a predominant event in embryonic development, the mRNAs of the two genes became differentially localized to particular organs and tissues. Skeletal muscle showed high levels of aralar1 expression, whereas the detection of citrin was negligible in this tissue (compare Fig. 1K with 1k). Similarly, aralar1 but not citrin transcripts were present in the heart, mainly confined to the auricular myocardium (compare Fig. 1I with 1i). The gut endothelium expressed both citrin and aralar1 (Fig. 1f–g,F–G), aralar1 transcripts being localized to the basolateral region of the enterocytes (Fig. 1g). Gut endothelium is a site where arginine biosynthesis occurs in thesucklingrat[22]andwheretheAGCsprobably function to provide aspartate for argininosuccinate synthesis. Both genes were also expressed in the kidney but with a differential distribution (Fig. 1H,h). In particular, only high levelsofcitrintranscriptswerefoundintheepitheliumofthe tubules, whereas the expression of aralar1 was associated with mesenchymal components (Fig. 1H,h). In summary, the two AGC isoforms have a partially overlapping expression pattern at early stages of embryo-genesis. At later stages, the expression domain of the two genes diverges, and aralar1 distribution becomes predom-inant in brain, heart and skeletal muscle, whereas citrin expression only predominates in kidney. Distribution of AGC isoforms in tissues from the adult rat In adult rat tissues, the distribution of aralar1 and citrin transcripts was analysed by Northern blot. Two aralar1 transcripts of 2.7 and 3.8 kb were detected in all positive tissues (Fig. 2A) as in humans [6]. The hybridization signal was higher for the 2.7-kb than the 3.8-kb mRNA. Expres-sion was stronger in heart and skeletal muscle, followed by brain,andlowerinkidney.Noaralar1mRNAwasdetected in liver. On the other hand, the rat citrin gene presented a 3316 A. del Arco et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 1. Comparison of expression pattern of citrin and aralar1 during murine embryonic development. Whole embryos from embryonic day (E) 11 (A–E,a–e),intotoE18isolatedorgans(H–I,h–i),ortransverseE18cryostattissuesections(F,G,L,M;f,g,l,m)werehybridizedwithdigoxigenin-labeledprobesspecific for the citrinoraralar1genes. Images in (A–E)and (a–e)showE11 embryos,in toto(A, a)and in transverse paraffin sections (B–E, b–e) taken from the embryos in (A and a) at the axial levels indicated by the dotted lines. Note the strong expression in the limb (B) and tail buds (arrowhead in A), in the branchial arches (D) and dermomyotome (E), more strongly localized in the ectodermal component for citrin (A–E), while to the mesenchyme for aralar1 (a–e). At E11, aralar1 (c) but not citrin (C) was expressed in the heart. Images in (F–M; f–m) illustrate the comparativeexpressionofcitrinand aralar1inthegut(F–G;f–g),kidney(H,h),heart(I,i),skeletalmuscle(K,k),cortex(L,l),olfactorybulbs(M, m) from E18 embryos. Note the stronger expression of aralar1 in skeletal muscle, heart and neural tissue. Note also the basolateral localization of the in situ hybridization signal in the cells ofthe gut. Abbreviations: aer, apical ectodermalridge; am, auricular myocardium; ba, branchial arch; cx, cortex; dm, dermomyotome; h, heart, hc, hippocampus; lb, limb bud; mcl, mitral cell layer; smf, skeletal muscle fiber; tg, trigeminal ganglia; vmt; ventromedial thalamus. Scale bars ¼ 500 lm (B, b; D, d; F, f; H, h; I, i; L, i); 250 lm (C, c; E, e; M, m); 100 lm (K, k); 50 lm (G, g). single transcript of about 3 kb, consistent with the size of the mouse citrin cDNA and with the data reported for human [7,11,18]. Citrin mRNA was abundant in liver, kidney and heart but was notably absent from brain or skeletal muscle. Therefore, the expression pattern of both citrin and aralar1 in rat is consistent with that described for human and mouse tissues [6,8,18]. The data on the mRNA distribution of the AGC isoforms were complemented by the analysis of the content of the respective proteins in mitochondria-enriched extracts using Western blots with isoform-specific antibodies. As shown in Fig. 2B, aralar1 levels were highest in heart, forebrain, cerebellum and skeletal muscle, in agreement with its mRNA distribution. Mitochondrial extracts from two types of skeletal muscles, the fast-twitch glycolytic extensor digitorum longus and the slow-twitch oxidative soleus, showed similar levels of aralar1 protein. In heart, aralar1 levels were higher in auricular than ventricular Ó FEBS 2002 Development of aspartate/glutamate mitochondrial carriers (Eur. J. Biochem. 269) 3317 Fig. 2. Pattern of expression of aralar1 and citrin in adult rat tissues. (A) Northern blot analysis of tissue-specific expression patterns of AGC isoforms. Northern blots with 20 lg total RNA from adult rat heart, kidney, brain, liver and skeletal muscle were hybridized with a 32P-labeled DNAprobeofhumanaralar1underhigh-stringencyconditions.Theblotwassubsequentlystrippedandreprobedunderidenticalconditionswitha human citrin probe The size of the specific transcripts is indicated. Staining with ethidium bromide was carried out to verify the amount of RNA loaded (lower panel). (B) Distribution of AGC isoforms in rat tissues; 20–30 lg mitochondrial protein was used for all tissues except for bislets where 20 lg total protein extract obtained from 500 pancreatic islets was used. Blots for citrin and aralar1 were performed in parallel and reincubated with anti-(b-F1ATPase). Aralar1 was detected with an antibody directed against its N-terminus, or against Aralar1 amino acids 507– 520 (results not shown). Citrin antibodies were against its N-terminus (upper panels) or against citrin amino acids 305–319 (lower panels), both at 1 : 2000 dilution. Bands correspond to 70 kDa (aralar1 and citrin) and 52 kDa for b-F1ATPase. (C) Western blot analysis of aralar1 and citrin in auricular and ventricular myocardium. 20 lg of atria (A) and ventricle (V) mitochondrial extracts from two different animals were analysed. The blot was incubated with anti-(aralar N-terminus) (1 : 5000) and anti-(b-F1ATPase) (1 : 5000), stripped and probed again with anti-(citrin N-terminus) (1 : 2000). The amount of aralar1 (standardized to that of b-F1ATPase) was 1.5 and 0.9 in atria and ventricles, respectively. No significant changes between atria and ventricle are observed for citrin levels (0.86 and 0.64 standardized values, in atria and ventricles, respectively). (D) Immunoblotting of increasing amounts of recombinant citrin and aralar1. Known amounts (as indicated) of bacterially expressed aralar1 and citrin N-terminal regions were loaded on to gels and blotted and processed in parallel with an identical dilution (1 : 2000) oftheir respective specific antiserum antibodies. Note the different amounts of recombinant aralar1 and citrin used. myocardium (Fig. 2C). Lower levels of aralar1 were also present in a wide range of tissues, including lung, kidney, ovary,spleen,pancreas(particularlyinbislets)andstomach and to an even lower extent in mitochondrial extracts of testis, intestine and liver. Citrin was absent from the central nervous system, skeletal muscle and lung but abundant in liver, kidney and heart, in agreement with its mRNA distribution. Weaker expression was also observed in ovary, testis, spleen, stomach and pancreas, but not in bislets (Fig. 2B). Citrin and aralar1 were hardly observed at all in the adult intestine (Fig. 2B), where their expression was instead high at embryonic day 18–19 (Fig. 1F–G,f–g and [18]). Neither aralar1 nor citrin were detectable in either brown or white adipose tissue (Fig. 2B). It is interesting to note that aralar1 protein is present in lung, where aralar1 mRNA was not detectable [6,8,18]. Similarly, citrinand aralar1 proteins, but not their corresponding mRNAs [18], were detected in spleenandtestis.Altogether,theseresultsshowthatmRNA levels are poor indicators of the levels of the AGC proteins. To assess the relative expression of aralar1 and citrin, mitochondrial fractions from a few representative tissues were processed in parallel with either known amounts of recombinant citrin [7] and aralar1 [6] or mitochondrial fractions from HEK-293Tcells overexpressing either aralar1 or citrin, or control HEK-293Tcells, which have citrin/aralar1 ratios of about 0.5, 12, and 2.4, respectively [12].Serialdilutionoftherecombinantproteins(Fig. 2D)or mitochondrial extracts from HEK-293Tcells overexpress-ing aralar1 or citrin (not shown) revealed a linear relation between the amounts of the protein and the densities of the immunoreactive bands. The analysis indicated that spleen, heart (particularly the ventricle), ovary, and stomach have similar levels of citrin and aralar1. Liver, kidney, whole pancreas and testis clearly have higher levels of citrin than aralar1. In contrast, central nervous system tissue, skeletal muscle, lung and possibly auricular myocardium (Fig. 2C) predominantly have aralar1. Expression of aralar1 in cells from the hematopoietic system Citrin isexpressed athigh levelsin the liver.This moleculeis the liver-specific AGC isoform, as citrin deficiency causes ... - tailieumienphi.vn