©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ceramide Reverses Brefeldin A (BFA) Resistance in BFA-resistant Cell Lines (*)

(Received for publication, May 31, 1994; and in revised form, December 21, 1994)

Tatsuya Oda (§) Chao-Hua Chen Henry C. Wu (¶)

From the Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have found that C(6) ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C(6) ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [^3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C(6) ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C(6) ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C(6) ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD of C(6) ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C(6) ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C(6) ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C(6) ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.


INTRODUCTION

Brefeldin A (BFA)^1, a fungal antibiotic, has been shown to affect the structure and function of the Golgi apparatus, resulting in an inhibition of protein secretion and the redistribution of the Golgi proteins into the endoplasmic reticulum (1, 2) . An early event in the action of BFA is the dissociation of the beta-COP (110-kDa protein) from the Golgi membranes(3, 4, 5, 6, 7) . Yoshida et al.(8, 9) have found that BFA inhibits the cytotoxicity of ricin, Pseudomonas toxin, and modeccin in Vero and other cell lines, and these results suggest an involvement of the Golgi apparatus in the intoxication process of these toxins. Increasing evidence suggests that BFA may modulate the functions of GTP-binding proteins that are involved in intracellular vesicular transport system (10, 11, 12, 13, 14, 15) .

Recently, it has been reported that a water-soluble, cell-permeable ceramide analog, C(6) ceramide (C(6)Cer), inhibits the viral glycoprotein transport through the Golgi complex and reduces the number of infectious virions released from infected cells in a concentration-dependent manner(16) . These results suggest that ceramide may regulate intracellular membrane traffic. In the present study, we found that incubation of BFA-resistant cell lines, BER-40 and MDCK cells, with C(6)Cer substantially restored the sensitivities of these cell lines to BFA as measured by the inhibition of DNA synthesis and the inhibition of colony formation, the protection against ricin cytotoxicity, the inhibition of bulk protein secretion, and the redistribution of beta-COP by BFA in these BFA-resistant cell lines. The enhancement of BFA sensitivity in BER-40 and MDCK cells appear to be specific for ceramide as related sphingolipids have no effect.


EXPERIMENTAL PROCEDURES

Materials

Ricin, arachidonic acid, sn-1,2-dioctanoylglycerol, sphingomyelinase (Streptomyces species) and fluorescein-labeled goat anti-mouse antibodies were obtained from Sigma. N-Hexanoyl-D-erythro-sphingosine (C(6) ceramide), C(6) dihydro-ceramide, N-acetyl-D-erythro-sphingosine (C(2) ceramide), N-octanoyl-erythro-sphingosine (C(8) ceramide), natural N-stearoyl-sphingosine (C(18) ceramide), sphingomyelin (stearoyl), D(+)-erythro-2-amino-4-trans-octadecene-1,3-diol (sphingosine), and lactosylceramide were obtained from Matreya, Inc. (Chalfont, PA). Complex of ceramide with delipidated bovine serum albumin (BSA) was prepared as described(17) . BFA was obtained from Epicentre Technologies (Madison, WI). Mouse monoclonal antibody M3A5 against the beta-COP was a generous gift from Dr. T. Kreis (Geneva, Switzerland). [^3H]Leucine (60 Ci/mmol) and [^3H]thymidine (2 Ci/mmol) were obtained from DuPont NEN. [^14C]Serine (120 mCi/mmol) was obtained from Moravek Biochemicals and [^14C]choline (55 mCi/mmol) was obtained from ICN Radiochemicals.

Cell Culture

Vero, MDCK, and PtK(1) cells were from the American Type Culture Collection. The BFA-resistant mutant cell line, BER-40, was isolated from Vero cells following ethyl methanesulfonate mutagenesis(18) . All these cell lines were cultured as described(19) .

Measurement of Protein Synthesis Inhibition

Cells were inoculated at a density of 1 times 10^5 cells/well for Vero and BER-40 or 2 times 10^5 cells/well for MDCK and PtK(1) cells in 0.5 ml of medium using 24-well plates. One day later, cells were pretreated with or without various ceramide analogs in alpha-MEM containing 35 µM delipidated BSA for 30 min at 37 °C. After further incubation with or without 0.01-10 µg/ml of BFA for 1 h, varying concentrations of ricin were added, and the incubation was continued for 3 h at 37 °C with or without BFA and/or ceramide analog. After removal of the medium, cells were incubated with 0.5 µCi/ml of [^3H]leucine for 45 min at 37 °C in leucine-free medium. The incorporation of [^3H]leucine into perchloric acid/phosphotungstic acid-insoluble materials was determined as described previously(8) .

Measurement of DNA Synthesis Inhibition

Cells grown in 24-well plate were pretreated with or without indicated concentrations of C(6)Cer in alpha-MEM containing 35 µM delipidated BSA for 2 h at 37 °C. Varying concentrations of BFA were added, and incubation was continued for 17 h at 37 °C with or without BFA and/or C(6)Cer. After removal of the medium, cells were incubated with 1 µCi/ml of [^3H]thymidine for 2 h at 37 °C in thymidine-free medium. The incorporation of [^3H]thymidine into perchloric acid/phosphotungstic acid-insoluble materials was determined(8) .

Measurement of Bulk Protein Secretion

Cells grown in 24-well plates were preincubated at 37 °C for 30 min with or without C(6)Cer at the indicated concentrations in alpha-MEM containing 35 µM delipidated BSA. After further incubation with or without appropriate concentrations of BFA for 1 h, cells were pulse-labeled for 20 min with 25-50 µCi/ml of [^3H]leucine at 37 °C in leucine-free medium and chased for 1 h in alpha-MEM containing 35 µM delipidated BSA. At the end of the chase period, the medium was collected from each well, and trichloroacetic acid-precipitable radioactivity was measured as described(19) . C(6)Cer and/or BFA, when indicated, was present in the medium throughout the pulse and chase.

Measurement of the Cytotoxicity of BFA and Ceramide

The cytotoxicity of BFA or ceramide was measured by the inhibition of colony formation. 250-300 cells/well in a six-well plate were cultured with varying concentrations of BFA or ceramide in the growth medium for 7-8 days. The number of colonies formed was counted after staining with 1% methylene blue in 50% methanol. Clusters of 40 or more cells were considered as colonies.

Immunofluorescence Microscopy

Cells grown on glass coverslips were incubated in 2 ml of alpha-MEM containing 35 µM delipidated BSA in 6-well plates with indicated concentrations of C(6)Cer and/or BFA for 30 min at 37 °C. The cells were fixed with 2% formaldehyde in PBS for 10 min at room temperature and washed with PBS containing 10% fetal bovine serum (PBS/serum). For permeabilization, cells were treated with methanol at 0 °C for 1 min, washed with PBS/serum, and incubated with the primary antibody diluted with PBS/serum and 0.15% saponin for 1 h at room temperature, washed three times with PBS/serum, and incubated with fluorescein-labeled secondary anti-mouse IgG in PBS/serum. The coverslips with stained cells were mounted on a glass slide with Fluoromount G, and viewed with an S plan 40 PL lens on an Olympus BH-O microscope.

Lipids Analysis

Monolayers of confluent cells (1.5-2.0 times 10^6 cells/well) in 6-well plate were labeled with 1 µCi/ml of [^14C]serine or [^14C]choline for 24-48 h in serine- or choline-free MEM containing 10% fetal bovine serum. After removal of radioactive medium, cells were washed three times with PBS, and normal growth medium was added and incubated for 1 h at 37 °C before BFA treatment. After treatment with indicated concentration of BFA or sphingomyelinase for 1 h at 37 °C, cells were washed twice with ice-cold PBS and were scraped off and pelleted by centrifugation. The resulting pellet was suspended in 0.8 ml of chloroform:methanol (1:2, v/v) and lipids were extracted. Insoluble materials were removed by centrifugation at 14,000 rpm for 10 min, the supernatant containing lipids was dried under N(2) gas. The resulting lipid film was resuspended in 50 µl of chloroform; 20 µl was spotted onto a thin layer chromatography plate (Whatman). The TLC plate, which had been prerun in chloroform:methanol:water (65:35:8, v/v), was developed in chloroform:methanol: water (65:25:4, v/v) for the analysis of [^14C]choline-labeled phosphatidylcholine (PC) and sphingomyelin (SM)(20) . For the analysis of [^14C]serine-labeled ceramide (Cer), the TLC plate was developed in chloroform:methanol (95:5, v/v)(21) . Individual lipids were identified and quantitated with FUJIX Bio-Imaging Analyzer Bas 2000 (Fuji Photo Film Co., LTD).


RESULTS AND DISCUSSION

To examine the effect of C(6)Cer on BFA cytotoxicity, Vero, BER-40, and MDCK cells pretreated with or without C(6)Cer (10 µM) for 2 h at 37 °C were incubated with varying concentrations of BFA in the presence or absence of C(6)Cer for 17 h. After removal of the medium, the incorporation of [^3H]thymidine into perchloric acid/phosphotungstic acid-insoluble materials was determined. From the dose-response curves of BFA in the presence or absence of C(6)Cer, ID value of BFA in each cell line was estimated. As shown in Table 1, both BER-40 and MDCK cells were rendered BFA-sensitive by the pretreatment with C(6)Cer, as evidenced by a significant reduction in ID of BFA in C(6)Cer-treated BER-40 and MDCK cells as compared with the respective controls; incubation with 10 µM C(6)Cer alone for 19 h did not significantly inhibit [^3H]thymidine incorporation (see legend to Table 1). Moreover, the LD of BFA in BER-40 and MDCK cells, based on its inhibition of colony formation, was reduced 5-8-fold in the presence of sublethal concentration of C(6)Cer (1 µM); the viabilities of Vero, BER-40, and MDCK cells at 1.25 µM C(6)Cer were 87, 78, and 95%, respectively. In contrast, C(6)Cer did not alter the ID or LD of BFA in Vero cells.



BFA has been shown to inhibit ricin cytotoxicity in Vero cells in a dose-dependent manner, and 1 µg/ml of BFA completely abolished ricin cytotoxicity in Vero cells. Even at 0.1 µg/ml of BFA, the ID of ricin increased from 4.4 to >1000 ng/ml (Table 2). However, 1 µg/ml of BFA had no significant effect on ricin cytotoxicity in BFA-resistant BER-40 and MDCK cells(9, 18, 22, 23, 24, 25) (Table 2). To further characterize the effect of C(6)Cer on BFA action, we examined the effect of C(6)Cer on the protection of ricin cytotoxicity by BFA in Vero, BER-40, and MDCK cells at different concentrations of BFA which by itself did not protect each cell line from ricin cytotoxicity. C(6)Cer (35 µM) restored strong protective effects of BFA on ricin cytotoxicity in BFA-resistant BER-40 and MDCK cells, whereas C(6)Cer by itself had a very small protective effect on ricin cytotoxicity in Vero and BER-40 cells; C(6)Cer alone increased ID of ricin 2-3-fold in Vero and BER-40 cells but not in MDCK cells (Table 2). In the presence of 35 µM C(6)Cer, BFA caused an 26.3- and 87-fold increase in the ID of ricin in BER-40 and MDCK cells, respectively. Such a synergistic effect between BFA and C(6)Cer was not observed in Vero cells (Table 2). Especially in MDCK cells, marked protective effect of BFA against ricin cytotoxicity was evident in C(6)Cer-treated cells, even though BFA or C(6)Cer alone had no effect. Furthermore, lower concentration of C(6)Cer (15 µM), which did not have any protective effect on ricin cytotoxicity, restored the protective effect of BFA against ricin cytotoxicity in BER-40 and MDCK cells but not in Vero cells (Table 2).



To gain insight into the relationship between the structure of ceramide and its effect on BFA-induced protection of ricin cytotoxicity in BFA-resistant cell lines, we examined various ceramide analogs with different acyl chain lengths. C(2)Cer and C(8)Cer also restored the protective effect of BFA on ricin cytotoxicity in BER-40 and MDCK cells, albeit less effectively than C(6)Cer (Table 2). However, dihydro-C(6)Cer and natural ceramide (C(18)Cer) were ineffective. These results suggest that the restoration of BFA sensitivity by ceramide is dependent on the presence of a double bond in the sphingoid backbone as well as an optimal acyl chain length. Sphingomyelinase, which has been shown to increase the intracellular ceramide level(26, 27) , also had no effect. This may be related to the lack of an effect of C(18)Cer on BFA sensitivity, since C(18)Cer would be the predominant species produced by sphingomyelinase treatment. Moreover, other related sphingolipids, including sphingosine, sphingomyelin, lactosylceramide, and unrelated lipid second messengers such as 1,2-diacylglycerol and arachidonic acid, did not show any significant effect on ricin cytotoxicity, nor on the protective effect of BFA against ricin cytotoxicity (Table 2). These results suggest that short chain ceramide is a relatively specific lipid effector on BFA action and ceramide itself, rather than its metabolites or other related lipid second messengers, is responsible for restoring BFA sensitivity in BFA-resistant cell lines. It is interesting to note that the activation of heterotrimeric protein phosphatase 2A by ceramide is also dependent on the presence of a double bond in the sphingoid backbone and is influenced by acyl chain composition(28) . These data suggest that a double bond in the sphingoid base and short hydrophobic acyl chain are important structural features for ceramide-mediated bioactivity.

One of the characteristic biological effects of BFA is its inhibition of bulk protein secretion(1, 2, 29) , and such an inhibition is not elicited by BFA in BFA-resistant cell lines(18, 21) . As shown in Table 3, pretreatment of BER-40 and MDCK cells with C(6)Cer rendered these cell lines susceptible to BFA in inhibiting bulk protein secretion. Such an enhancement of BFA-induced inhibition of protein secretion by C(6)Cer was not observed in Vero cells.



To ascertain whether or not the failure of C(6)Cer to alter BFA sensitivity in Vero cells is due to a difference in sensitivity toward C(6)Cer between Vero and BER-40/MDCK cells, we compared the LD of C(6)Cer in these cell lines. The LD values of C(6)Cer of Vero, BER-40, and MDCK cells were found to be 1.97, 1.56, and 1.76 µM, respectively. It is therefore unlikely that the differential enhancement of BFA effect in BER-40 and MDCK cells by C(6)Cer and its lack thereof in Vero cells are due to an intrinsic resistance of Vero cells to C(6)Cer. In fact, bulk protein secretion was as strongly inhibited by C(6)Cer in a dose-dependent manner in Vero cells as in BER-40 and MDCK cells (data not shown).

In contrast to BER-40 and MDCK cells, no significant enhancement of BFA effect by C(6)Cer was observed in PtK(1) cells, another naturally BFA-resistant cell line(23) . An intrinsic resistance of PtK(1) cells to C(6)Cer action may be one reason for the inability of C(6)Cer to enhance the BFA action; the LD of C(6)Cer for PtK(1) cells was 7-8-fold higher than those for BER-40 and MDCK cells, and higher concentrations of C(6)Cer were required to inhibit bulk protein secretion in PtK(1) cells (data not shown).

An early event in the action of BFA is the dissociation of a 110-kDa Golgi-associated protein (beta-COP) from the Golgi membrane to cytosol, resulting in the dispersed distribution of beta-COP in the cytosol in a dose-dependent manner. The BFA resistance in BER-40 and MDCK cells is also manifested in the inability of BFA to cause the redistribution of beta-COP. 0.005 µg/ml of BFA induced a dispersed distribution of beta-COP in BFA-sensitive Vero cells (data not shown), whereas 0.1 µg/ml BFA did not affect the beta-COP distribution in BER-40 and MDCK cells (Fig. 1). To examine the effect of C(6)Cer on BFA-induced redistribution of beta-COP, we used suboptimal concentration of BFA in each cell line, i.e. 0.001 µg/ml, 0.1 µg/ml, and 10 µg/ml in Vero, BER-40, and MDCK cells, respectively. Fluorescence microscopic studies revealed that low concentration of C(6)Cer (1 µM) rendered both BER-40 and MDCK cells susceptible to BFA in causing a dispersed distribution of beta-COP, whereas C(6)Cer (1 µM) alone had no effect on the distribution of beta-COP in these cell lines (Fig. 1). However, C(6)Cer (1 µM) did not render Vero cells susceptible to 0.001 µg/ml BFA in causing a redistribution of beta-COP (Fig. 1). Higher concentrations of C(6)Cer itself induced redistribution of beta-COP in Vero, BER-40, and MDCK cells; treatment of these cell lines with 35 µM C(6)Cer resulted in a dispersed distribution of beta-COP in all three cell lines (data not shown).


Figure 1: Dissociation of beta-COP from the Golgi apparatus by BFA in C(6)Cer-treated BFA-resistant cell lines. Cells were treated with BFA, C(6)Cer (1 µM), or BFA plus C(6)Cer (1 µM) for 30 min in alpha-MEM. The localization of beta-COP was examined using immunofluorescence microscopy as described under ``Experimental Procedures.'' Concentrations of BFA were 0.001 or 0.1 µg/ml in Vero cells, 0.1 µg/ml in BER-40, and 10 µg/ml in MDCK cells.



In the present study we have shown that C(6)Cer partially restores BFA sensitivity in BER-40 and MDCK cells based on four distinct effects of BFA; i.e. the cytotoxicity of BFA, the protection of ricin cytotoxicity by BFA, the inhibition of bulk protein secretion by BFA, and the BFA-induced morphological alterations of the Golgi apparatus. It is worth noting that like BFA, C(6)Cer inhibits the transport of vesicular stomatitis virus G protein through the Golgi complex, and electron microscopic studies have revealed the disruption of the Golgi apparatus in C(6)Cer-treated cells (16) . On the other hand, unlike BFA(9) , C(6)Cer by itself has a marginal protective effect on the cytotoxicity of ricin in Vero cells, and BFA-resistant cell lines are not cross-resistant to C(6)Cer. Although C(6)Cer by itself affects the structure and function of the Golgi apparatus, its mode of action appears to be distinct from those of BFA and epidermal cell differentiation inhibitor. The mode of action of epidermal cell differentiation inhibitor is also distinct from BFA and C(6)Cer, since it does not inhibit bulk protein secretion (15) .

It has been postulated that BFA may mimic an endogenous lipid ligand in regulating a heterotrimeric G protein-coupled receptor that controls intracellular membrane traffic and lipid metabolism(30) . It has been demonstrated that an inhibitor of sphingolipid synthesis affects protein and lipid trafficking through the secretory pathway, suggesting that vesicular transport along the secretory pathway is coupled to sphingolipid metabolism(31) . A linkage between lipid composition and protein transport is also suggested by the defective protein secretion in yeast mutants deficient in phospholipid transfer protein(32, 33) . Related to these findings is the recent study showing that BFA reduces cellular SM level in HL 60 cells without affecting PC level(34) . We have examined the effect of BFA on SM and ceramide level in Vero, BER-40, and MDCK cells. As shown in Fig. 2, SM level was slightly increased by BFA treatment in Vero cells without affecting PC level, whereas SM levels in BER-40 and MDCK cells were not affected by BFA. In addition, BFA decreased ceramide levels in Vero and BER-40 cell, whereas MDCK cells were resistant to these effects of BFA. Interestingly, SM levels in BER-40 and MDCK cells were significantly higher than that in Vero cells, whereas the PC and ceramide levels were similar in these three cell lines (data not shown), suggesting that an alteration in SM metabolism may be associated with increased BFA resistance in BER-40 and MDCK cells. On the other hand, sphingomyelinase at 1 unit/ml decreased SM levels and concomitantly increased ceramide levels in Vero and BER-40 cells as reported in other cell lines(35) . MDCK cells were resistant to sphingomyelinase as well as BFA. Since recent studies suggest that SM is synthesized in the Golgi apparatus from endoplasmic reticulum-derived ceramide(36, 37, 38) , the effects of BFA on sphingolipids metabolism in Vero cells may result from a perturbation of vesicle trafficking of sphingolipids through the Golgi complex by BFA, and such processes may be BFA-resistant in MDCK cells. Ceramide is a precursor for sphingolipid synthesis(39) , and the Golgi apparatus is the major site of sphingomyelin metabolism(37) . Exogenously added ceramide, known to accumulate in the Golgi complex (38, 40, 41, 42) , may alter the physical properties of the Golgi membranes in BFA-resistant cell lines to render them susceptible to BFA action. These results suggest that the Golgi membranes are structurally and/or functionally altered in these BFA-resistant cell lines and consequently are refractory to BFA. BFA has shown to form cation channels in planar lipid bilayers(43) , and a change in lipid composition or lipid/protein ratio in the Golgi membranes may affect the susceptibility of the endomembrane systems to structural and functional perturbation by BFA. Ceramide reverses the BFA resistance in BER-40 and MDCK cells by restoring the Golgi membranes to the ``normal'' structural or functional state. On the other hand, it has been shown that ceramide activates okadaic acid-sensitive cytosolic protein phosphatases (44) as well as a membrane-bound protein kinase activity(45, 46) . C(6)Cer may exert its effect through the modulation of phosphorylation/dephosphorylation-related signal transduction that regulates vesicle trafficking through the Golgi region. Further studies of the relationship between ceramide and BFA action may provide new insights to our understanding of the mode of action of BFA and ceramide as well as the mechanism of BFA resistance in BFA-resistant cell lines.


Figure 2: Effect of BFA and sphingomyelinase (SMase) on SM, PC, and Cer levels in Vero, BER-40, and MDCK cells. Cells, labeled with [^14C]choline or [^14C]serine as described under ``Experimental Procedures,'' were treated with indicated concentrations of BFA or sphingomyelinase. After 1 h, lipids were extracted with chloroform:methanol (1:2, v/v) and analyzed for lipids content by TLC as described. Results are expressed as percent of control. These data (mean ± S.E.) represent triplicate determinations from three experiments. Upper panel, sphingomyelin; middle panel, phosphatidylcholine; lower panel, ceramide. Open columns, Vero cells; hashed columns, BER-40 cells; shaded columns, MDCK cells.




FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM28810 and Uniformed Services University of the Health Sciences Grant R07317. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan.

To whom correspondence should be addressed. Tel.: 301-295-3312; Fax: 301-295-1545.

(^1)
The abbreviations used are: BFA, brefeldin A; MDCK, Madin-Darby canine kidney; beta-COP, beta-coat protein; C(2)Cer, N-acetyl-D-erythro-sphingosine; C(6)Cer, N-hexanoyl-D-erythro-sphingosine; C(8)Cer, N-octanoyl-erythro-sphingosine; C(18)Cer, N-stearoyl-sphingosine; SM, sphingomyelin; PC, phosphatidylcholine; MEM, minimal essential medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin.


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