scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

scholarly journals Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin

1993 ◽  
Vol 291 (3) ◽  
pp. 861-867 ◽  
Author(s):  
H Katagiri ◽  
T Asano ◽  
H Ishihara ◽  
J L Lin ◽  
K Inukai ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Competitive Inhibitor ◽  
Tryptophan Residue ◽  
Wild Type ◽  
Turnover Number ◽  
Glut1 Glucose Transporter

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

scholarly journals Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity

1995 ◽  
Vol 311 (2) ◽  
pp. 699-704 ◽  
Author(s):  
A Muraoka ◽  
M Hashiramoto ◽  
A E Clark ◽  
L C Edwards ◽  
H Sakura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Structural Features ◽  
Transport Activity ◽  
Wild Type ◽  
C Terminus ◽  
Wild Type Clone

C-terminally truncated and mutated forms of GLUT1 have been constructed to determine the minimum structure at the C-terminus required for glucose transport activity and ligand binding at the outer and inner binding sites. Four truncated mutants have been constructed (CTD24 to CTD27) in which 24 to 27 amino acids are deleted. In addition, point substitutions of R468-->L, F467-->L and G466-->E have been produced. Chinese hamster ovary clones which were transfected with these mutant GLUT1s were shown, by Western blotting and cell-surface carbohydrate labelling, to have expression levels which were comparable with the wild-type clone. Wild-type levels of 2-deoxy-D-glucose transport activity were retained only in the clone transfected with the construct in which 24 amino acids were deleted (CTD24). The CTD25, CTD26 and CTD27 clones showed markedly reduced transport activity. From a kinetic comparison of the CTD24 and CTD26 clones it was found that the reduced transport was mainly associated with a reduced Vmax. value for 2-deoxy-D-glucose uptake but with a slight lowering of the Km. These data establish that the 24 amino acids at the C-terminus of GLUT1 are not required for the transport catalysis. However, the point mutations of F467L and G466E (26 and 27 residues from the C-terminus) did not significantly perturb the kinetics of 2-deoxy-D-glucose transport. The substitution of R468L produced a slight, but significant, lowering of the Km. The ability of the truncated GLUt1s to bind the exofacial ligand, 2-N-4-(1-zai-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yl-oxy) -2-propylamine (ATB-BMPA), and the endofacial ligand, cytochalasin B, were assessed by photolabelling procedures. The ability to bind ATB-BMPA was retained only in the CTD24 truncated mutant and was reduced to levels comparable with those of the non-transfected clone in the other mutant clones. Cytochalasin B labelling was unimpaired in all four mutated GLUT1s. These data establish that a minimum structure at the C-terminus of GLUT1, which is required for the conformational change to expose the exofacial site, includes amino acids at positions Phe-467 and Arg-468; however, these amino acids are not individually essential.

scholarly journals Increased sensitivity to oxidative injury in chinese hamster ovary cells stably transfected with rat liver S-adenosylmethionine synthetase cDNA

1996 ◽  
Vol 319 (3) ◽  
pp. 767-773 ◽  
Author(s):  
Estrella SÁNCHEZ-GÓNGORA ◽  
John G. PASTORINO ◽  
Luis ALVAREZ ◽  
María A. PAJARES ◽  
Concepción GARCÍA ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Rat Liver ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Transfected Cells ◽  
In The Wild ◽  
Adenosylmethionine Synthetase

Chinese hamster ovary cells were stably transfected with rat liver S-adenosylmethionine synthetase cDNA. As a result, S-adenosylmethionine synthetase activity increased 2.3-fold, an effect that was accompanied by increased S-adenosylmethionine, a depletion of ATP and NAD levels, elevation of the S-adenosylmethionine/S-adenosylhomocysteine ratio (the methylation ratio), increased DNA methylation and polyamine levels (spermidine and spermine), and normal GSH levels. By contrast, the transfected cells showed normal growth curves and morphology. Exposure to an oxidative stress by the addition of H2O2 resulted in a greater consumption of ATP and NAD in the transfected cells than in the wild-type cells. In turn, cell killing by H2O2 was greater in the transfected cells than in the wild-type cells. This killing of Chinese hamster ovary cells by H2O2 involved the activation of poly(ADP-ribose) polymerase with the resultant loss of NAD and ATP. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, but not the antioxidant N,N´-diphenylphenylenediamine, prevented the killing of Chinese hamster ovary cells by H2O2 and maintained the contents of NAD and ATP. The results of this study indicate that a moderate activation of the synthesis of S-adenosylmethionine leads to ATP and NAD depletion and to a greater sensitivity to cell killing by oxidative stress.

scholarly journals Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-0-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose

1993 ◽  
Vol 290 (2) ◽  
pp. 497-501 ◽  
Author(s):  
A Schürmann ◽  
K Keller ◽  
I Monden ◽  
F M Brown ◽  
S Wandel ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
Binding Domains ◽  
Glucose Transporter Glut1 ◽  
Transporter Glut1

The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.

scholarly journals Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

2004 ◽  
Vol 279 (44) ◽  
pp. 46035-46045 ◽  
Author(s):  
Fengyu Qiao ◽  
Jun Mi ◽  
James B. Wilson ◽  
Gang Zhi ◽  
Natalie R. Bucheimer ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Site Directed Mutagenesis ◽  
Mass Spectrometry Analysis ◽  
Wild Type ◽  
Mutant Cells

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others (Qiao, F., Moss, A., and Kupfer, G. M. (2001)J. Biol. Chem. 276, 23391–23396 and Futaki, M., Watanabe, S., Kajigaya, S., and Liu, J. M. (2001)Biochem. Biophys. Res. Commun. 281, 347–351) have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in anin vitrokinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-typeFANCGcDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.

scholarly journals Characterization of GLUT3 protein expressed in Chinese hamster ovary cells

1992 ◽  
Vol 288 (1) ◽  
pp. 189-193 ◽  
Author(s):  
T Asano ◽  
H Katagiri ◽  
K Takata ◽  
K Tsukuda ◽  
J L Lin ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Glucose Transporter ◽  
Chinese Hamster Ovary Cells ◽  
Broad Band ◽  
Chinese Hamster ◽  
Transport Activity ◽  
Ovary Cells ◽  
Glut1 Protein ◽  
Km Value

We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the Km value of GLUT3 protein for 3-O-methylglucose uptake was approx. 35% of that of GLUT1 protein, whereas the Km value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The Vmax. value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos -4-yloxy)-2- propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower Km and Vmax. values for 3-O-methylglucose uptake.

Endofacial competitive inhibition of the glucose transporter 1 activity by gossypol

2009 ◽  
Vol 297 (1) ◽  
pp. C86-C93 ◽  
Author(s):  
Alejandra Pérez ◽  
Paola Ojeda ◽  
Ximena Valenzuela ◽  
Marcela Ortega ◽  
Claudio Sánchez ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Competitive Inhibition ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Competitive Inhibitor ◽  
Human Erythrocytes ◽  
Cellular Transport ◽  
Protein Fluorescence ◽  
Glucose Transporter 1

Gossypol is a natural disesquiterpene that blocks the activity of the mammalian facilitative hexose transporter GLUT1. In human HL-60 cells, which express GLUT1, Chinese hamster ovary cells overexpressing GLUT1, and human erythrocytes, gossypol inhibited hexose transport in a concentration-dependent fashion, indicating that blocking of GLUT1 activity is independent of cellular context. With the exception of red blood cells, the inhibition of cellular transport was instantaneous. Gossypol effect was specific for the GLUT1 transporter since it did not alter the uptake of nicotinamide by human erythrocytes. Gossypol affects the glucose-displaceable binding of cytochalasin B to GLUT1 in human erythrocyte ghost in a mixed noncompetitive way, with a Kivalue of 20 μM. Likewise, GLUT1 fluorescence was quenched ∼80% by gossypol, while Stern-Volmer plots for quenching by iodide displayed increased slopes by gossypol addition. These effects on protein fluorescence were saturable and unaffected by the presence of d-glucose. Gossypol did not alter the affinity of d-glucose for the external substrate site on GLUT1. Kinetic analysis of transport revealed that gossypol behaves as a noncompetitive inhibitor of zero- trans (substrate outside but not inside) transport, but it acts as a competitive inhibitor of equilibrium-exchange (substrate inside and outside) transport, which is consistent with interaction at the endofacial surface, but not at the exofacial surface of the transporter. Thus, gossypol behaves as a quasi-competitive inhibitor of GLUT1 transport activity by binding to a site accessible through the internal face of the transporter, but it does not, in fact, compete with cytochalasin B binding. Our observations suggest that some effects of gossypol on cellular physiology may be related to its ability to disrupt the normal hexose flux through GLUT1, a transporter expressed in almost every kind of mammalian cell and responsible for the basal uptake of glucose.

Regulation of intestinal Cl−/HCO3− exchanger SLC26A3 by intracellular pH

2009 ◽  
Vol 296 (6) ◽  
pp. C1279-C1290 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Kazuhito Suruga ◽  
Yukari Yamashita
Keyword(s):  
Intracellular Ph ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Functional Coupling ◽  
Intestinal Epithelial ◽  
Transport Activity ◽  
Ovary Cells ◽  
Nacl Absorption ◽  
Reverse Mode

SLC26A3, a Cl−/HCO3− exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na+/H+ exchanger and Cl−/HCO3− exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pHi). We therefore investigated whether SLC26A3 is regulated by pHi. We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy- N-ethylquinolinium iodide (diH-MEQ). pHi was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl−/HCO3− and Cl−/NO3− exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl−/HCO3− exchange was steeply inhibited with pHi decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl−/NO3− exchange was essentially insensitive to pHi within physiological ranges. We also characterized the pHi dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pHi sensitivity. These results suggest that Cl−/HCO3− exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na+/H+ exchanger 3 (NHE3) through the alteration of pHi under physiological conditions.

scholarly journals Subcellular trafficking kinetics of GLU4 mutated at the N- and C-terminal

1996 ◽  
Vol 315 (1) ◽  
pp. 153-159 ◽  
Author(s):  
Satoshi ARAKI ◽  
Jing YANG ◽  
Mitsuru HASHIRAMOTO ◽  
Yoshikazu TAMORI ◽  
Masato KASUGA ◽  
...  
Keyword(s):  
Cell Surface ◽  
Distribution Ratio ◽  
Glucose Transporter ◽  
Chinese Hamster ◽  
Internal Surface ◽  
Transport Activity ◽  
Wild Type ◽  
Subcellular Trafficking ◽  
Surface Distribution ◽  
Intracellular Pool

The glucose transporter isoform, GLUT4, has been expressed in Chinese hamster ovary clones and its subcellular trafficking has been determined following labelling at the cell surface with the impermeant bis-mannose photolabel, 2-N-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA). ATB-BMPA-tagged GLUT4 leaves the cell surface rapidly and equilibrates to give an internal/surface distribution ratio of approx. 3.5 after 60 min. GLUT4 in which the N-terminal phenylalanine-5 and glutamine-6 are mutated to alanine N-(FQ-AA) and in which the C-terminal leucine-489 and -490 are mutated to alanine C-(LL-AA) have low internal/surface ratios of 0.64 and 1.24 respectively. If all cell-surface transporters are able to recycle, as would be the case for a two-pool recycling model with a single intracellular pool, then analysis suggests that the wild-type GLUT4 distribution ratio is dependent on endocytosis and exocytosis rate constants of 0.074 and 0.023 min-1. These values are similar, but not identical, to those found for GLUT4 trafficking in adipocytes. The distribution of the N-(FQ-AA) transporter appears to be due to a decrease in endocytosis with reduced intracellular retention, while the distribution of the C-(LL-AA) transporter appears to be mainly due to poor intracellular retention. These results are also considered in terms of a consecutive intracellular pool model in which GLUT4 targeting domains alter the distribution between recycling endosomes and a slowly recycling compartment. In this case the more rapid apparent exocytosis of the mutated GLUT4s is due to their failure to reach a slowly recycling compartment with a consequent return to the plasma membrane by default. It is suggested that overexpression of transporters increases the proportion that are recycled in this way. Wortmannin is shown to decrease glucose transport activity and cell-surface photolabelled transporters in a manner consistent with an inhibition of transporter recycling. Studies on the rate of loss of transport activity and ATB-BMPA-tagged transporter in wortmannin-treated cells confirm that the N-(FQ-AA) mutant is endocytosed more slowly than the wild-type GLUT4. Taken together, these results suggest that mutation at either the N- or the C-terminal domain can reduce movement to a slowly recycling intracellular compartment but that neither domain alone is entirely sufficient to produce wild-type GLUT4 trafficking behaviour.

scholarly journals Identification of amino acid residues in the C-terminal tail of big endothelin-1 involved in processing to endothelin-1

1998 ◽  
Vol 21 (3) ◽  
pp. 307-315 ◽  
Author(s):  
C Brooks ◽  
A Ergul
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Ovary Cells ◽  
Endothelin 1 ◽  
Recombinant Peptides ◽  
The Media ◽  
Potent Vasoconstrictor

Big endothelin-1 (big ET-1) is converted to 21-amino acid residue endothelin-1 (ET-1) via a specific cleavage at Trp21-Val22 by endothelin converting enzyme (ECE). This conversion is an essential step to produce bioactive ET-1 and represents a regulatory site in the biosynthesis of this potent vasoconstrictor. ECE-1a, a unique membrane-bound enzyme, processes big ET-1 more efficiently than other big ET isoforms, which mainly differ in the C-terminal tail (residues 22-38). In this study, each of the highly conserved residues, Val22, Pro25, Pro30, Gly32, Leu33, and Gly34 were replaced with Ala in the preproendothelin-1 (PPET-1) cDNA using site-directed mutagenesis. The mutant and wild-type cDNAs were transiently transfected into Chinese hamster ovary cells along with ECE-1a cDNAs, and concentrations of the resulting recombinant peptides, ET-1 and big ET-1, in the transfection media were then measured. The concentration of immunoreactive ET-1 in the media from Val22, Pro25, Pro30, Gly32, and Leu33 mutant PPET-1-transfected cells was 4- to 6-fold lower than that of wild type and (Gly34-->Ala)PPET-1. Moreover, with the exception of Gly34 there was a corresponding increase in the concentrations of immunoreactive big ET-1 in the media from mutants. Similar results were obtained when His27, Val28, and Ser35 of big ET-1( )were substituted with the corresponding residues in big ET-2 and big ET-3. These findings suggest that the C-terminal tail has an important role in the intracellular processing of big ET-1 by ECE-1a. Herein we also report that a recombinant big ET-1 analog we previously generated and characterized, (Ala21)big ET-1, inhibits ECE-1a activity in a dose-dependent (K i=1 microM) and competitive manner.

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