Dynamics and Function of the Inositolcycle in Dictyostelium discoideum ANTHONY A. BOMINAAR, JEROEN VAN DER KAAY, AND PETER J.M. VAN HAASTERT Department of Biochemistry, University of Groningen, Groningen, The Netherlands ABSTRACT The inositolcycle in Dictyostelium discoideum was studied under several conditions both in vitro and in vivo. The results are compared with the inositolcycle as it is known from higher eukaryotes: although there is a strong resemblance both cycles are different at some essential points.

target enzymes, such as protein kinases, calcium channels, and cytoskeletal components. The two main cellular functions of extracellular cAMP in Dictyostelium are chemotaxis to bring the amoeboid cells in a multicellular structure, and cell type specific gene expression to induce cell differentiation in this structure. In this paper we describe our recent work on the characterization of the inositol cycle in Dictyostelium. Key words: CAMP, dephosphorylation, phosThe metabolism of Ins( 1,4,5)P3 was investigated in vivo and in vitro, and the stimulation of Ins(1,4,5)P3 phatase production by receptor and G-protein agonists was demonstrated. The function of the inositol cycle was established mainly by using a mutant which appears to INTRODUCTION lack a functional G,-subunit that activates phospholiTransmembrane signal transduction is characterized pase C . largely by the interaction between its components: Metabolism of Ins(1,4,5)P3in vitro ligand, receptor on the surface of cells, G-protein subunits at the inner face of the plasma membrane, and In mammalian cells Ins(1,4,5)P3 is degraded by a 5effector enzymes. The effector enzymes may vary phosphatase yielding Ins( 1,4)P2, which is further dewidely depending on the organism and the ligand, and phosphorylated via Ins4P to inositol. The major part of include adenylate cyclase, quanylate cyclase, phospho- Ins(1,4,5)P,, however, is phosphorylated to Ins( 1,3,4, lipase C, and ion channels. The consequence of these 5)P4 which is then dephosphorylated by the same 5interactions is the production of intracellular second phosphatase to Ins(1,3,4)P3; this InsP, isomer is then messengers, such as cyclic adenosine 3'5' monophos- metabolized by a complex pattern of phosphorylations phate (CAMP), cyclic guanosine 3 ' 5 ' monophosphate and dephosphorylations [See Berridge and Irvine, (cGMP), inositol 1,4,5-trisphosphate [Ins(1,4,5)P31, di- 19891. Pilot experiments in Dictyostelium revealed that acylglycerol, C a 2 + ,and K t . Besides the interaction be- the metabolism of Ins(1,4,5)P3 could be different from tween these proteins t h a t generate second messengers, that in mammalian cells. there also exists a n extensive interaction between the The dephosphorylation of Ins(1,4,5)P3 was elucidated second messenger systems such that one system mod- in homogenates of Dictyostelium, using a mixture of ulates or rules another system. The main problem for [2-3HlIns(1,4,5)P, and [4,5-32P11ns(1,4,5)P,followed by the elucidation of transmembrane signal transduction chromatography of the products on Dowex columns is probably to understand how the flow of information [Van Lookeren Campagne et al., 19881. The rationale of proceeds through this complicated network of interact- this experiment is that the ,H-radioactivity is associing molecules. ated with the inositol structure, whereas the 32P-radioTransmembrane signal transduction has been stud- activity is present predominantly at the 5-position ied extensively in the eukaryotic microorganism Dic- (85%); therefore, detection of 3H-radioactivity detyostelium discoideum, and appears to be very similar scribes the extent of dephosphorylation, whereas the to signal transduction in higher eukaryotes [see Jans- detection of 32P-radioactivity describes the specificity sens and Van Haastert, 19871. cAMP is the extracellu- of the dephosphorylation. It was readily observed that lar signal in Dictyostelium, to be compared with the the major part of Ins( 1,4,5)P, dephosphorylation did hormone in mammalian cells. cAMP is detected by surface receptors that have the classical seven putative transmembrane spanning domains of receptors that in- Received for publication January 2, 1990 teract with G-proteins [Klein et al., 19891. The effector Address reprint requests to Anthony A. Bominaar, Department of enzymes are adenylate cyclase, guanylate cyclase, and Biochemistry, University of Groningen, Nijenborgh 16, 9747 AG phospholipase C; the second messengers interact with Agroningen, The Netherlands.



BOMINAAR ET AL. TABLE 1. Enzymology of Ins(1,4,5)P3Dephosphorylation No. 1 2 3 4 5 6

Enzyme Name 145-5Pase 145-1Pase a

14-1 Pase 45-5 Pase monoPase

Elution 0.1 M Wash 0.3 M 0.1 M 0.1 M 0.1 M

Reaction 145- 14 145 -+ 45 145- 1 14- 4 454 InsxP-Ins

Co-substrate 1345 -


134 -


No substrate 45 1345, 134, 14 1345, 14, 134 1345, 145 1345, 145 x=2

“Ins(1,4,5)P34-5 bisphosphatase. The high speed supernatant of a Dictyostelium lysate was chromatographed over a DEAE cellulose column that was eluted with a gradient of NaCl. The eluting fractions were assayed for the dephosphorylation of Ins(1,3,4,5)P4, Ins(1,4,5)P3, Ins(1,3,4)P3,Ins(4,5)P2, Ins(l,4)P2,InslP, Ins3P, and Ins4P; the product of all reactions was identified (with the exception of the reactions with Ins(1,3,4,5)P4 and Ins(1,3,4)P3.

not occur at the 5-position as in mammalian cells, because essentially all 32P-radioactivity was retained in the InsP, product. We concluded that the product was either Ins( 1,5)P2or Ins(4,5)P2. The InsP, product was purified, further dephosphorylated by a Dictyostelium lysate, and the InsP product was analysed and identified by high performance liquid chromatography (HPLC) as Ins4P. Thus the major route of Ins(1,4,5)P3 dephosphorylation in Dictyostelium was identified a s Ins( 1,4,5)P3 + Ins(4,5)P, Ins4P + Ins. This route of Ins(1,4,5)P3 dephosphorylation is present exclusively in the cytosol and dephosphorylates about 80% of the Ins(1,4,5)P3. The other 20% of Ins( 1,4,5)P3 dephosphorylation is mediated by the mammalian route: Ins(1,4,5)P3+ Ins(l,4)P2- Ins4PIns. Between 30-50% of the Ins(1,4,5)P35-phosphatase is present in the membrane fraction; all the other phosphatases are present in the cytosol fraction [Van Lookeren Campagne et al., 19881. Since Ins(1,4,5)P3 and Ins(l,4)P2 are both dephosphorylated at the 1-position, we investigated the substrate specificity of this enzyme reaction; the same was done for the dephosphorylation of Ins(1,4,5)P3 and Ins(4,5)P2 at the 5-position [Bominaar et al., unpublished research]. The cytosol was partially purified by DEAE-cellulose chromatography, and the dephosphorylation of Ins(1,4,5)P3, Ins(1,4)P2, Ins(4,5)Pz, InslP, and Ins4P was investigated. To get a complete picture of the specificity profile, the dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,3,4)Pe was also determined, but the products of the reaction were not identified. The results are summarized in Table 1. There exist probably at least six enzyme activities that are involved in the dephosphorylation of Ins(1,4,5)P3 to Ins. Enzyme 1 is a n Ins(1,4,5)P3 5-phosphatase that also dephosphorylates Ins(1,3,4,5)P4, but not Ins(4,5)P2; the enzyme may be very similar to the mammalian Ins( 1,4,5)P3 5-phosphatase. Enzyme 2 dephosphorylates exclusively Ins(1,4,5)P3 at the 1-position. Enzyme 3 is a minor activity and not very stabile; preliminary results suggest that it dephosphorylates Ins(1,4,5)P3 first a t the 4-position and then a t the 5position; other inositol phosphates are not degraded


by this column fraction, suggesting that the enzyme is very specific. Enzyme 4 dephosphorylates Ins(l,4)P, a t the 1-position; Ins(1,3,4)P3 is also a substrate, but Ins(1,3,4,5)P4 and Ins(1,4,5)P3 are not. The enzyme may be very similar to the mammalian inositolpolyphosphate 1-phosphatase. Enzyme 5 dephosphorylates Ins(4,5)P, a t the 5-position. This enzyme is probably specific and does not dephosphorylate Ins(1,4,5)P3 or Ins( 1,3,4,5)P4; the separation from the Ins( 1,4,5)P3 5-phosphatase was not complete however. Finally, InslP and Ins4P are dephosphorylated by a n inositolmonophosphate phosphatase; this enzyme is very similar to the mammalian monophosphatase. In summary, Dictyostelium has probably at least six phosphatases that participate in the dephosphorylation of Ins(1,4,5)P3. Three enzymes may be similar to the mammalian counterparts: Ins(1,4,5)P3 5-phosphatase, Ins( 1,4)P, 1-phosphatase, and InsP phosphatase. Three enzymes may be unique for Dictyostelium: the Ins( 1,4,5)P3 1-phosphatase, the Ins(1,4,5)P3 4-5bis-phosphatse, and the Ins(4,5)P2 5-phosphatase. It is likely that this complex dephosphorylation pattern is more than just degradation. InsP2 isomers may have signal transducing functions. Furthermore, the trifurcation in the dephosphorylation of Ins(1,4,5)P3 opens ways for fine-regulation, and the system could be compartmentalized or under developmental control. In contrast to the complex dephosphorylation of Ins(1,4,5)P3, the phosphorylation of Ins(1,4,5)P3 is extremely simple: all experimental evidence indicate that the appropriate kinases are absent. The Ins(1, 4,5)P3 3-kinase could not be detected under conditions where the rat brain enzyme was very active, and Ins(1,3,4,5)P4 and Ins(1,3,4)P3 were not detectable in extracts from [3H]inositol-labelled cells [Van Haastert et al., 19891.

Metabolism of Ins(1,4,5)P, in Permeabilized Cells In collaboration with Cor Schoen, University of Amsterdam, we measured the degradation of [3H]Ins(l,4, 5)P3in electropermeabilized cells. The protocol was op-



column fractions

Fig. 1. Dephosphorylation of [3HlIns(1,4,51P, in electropermeabilized Dictyostelium cells. Permeable cells were incubated with [3HlIns(1,4,5)P, and at the times indicated samples were withdrawn and analysed by HPLC on a Partisil SAX column (Whatman)using gradient elution. The elution of authentic standers is shown

umns in the InsP6 fraction, and elutes only partly from the HPLC SAX column [Van Haastert et al., 19891. It should be mentioned that we have not yet identified the inositol phospholipids in terms of polar headgroup and fatty acid composition. This is probably relevant, since Dictyostelium seems to lack arachidonic acid [MacDonald and Weeks, 19851. The results (Fig. 2) demonstrate that, after pulselabelling with [3H]inositol, the radioactivity was very rapidly incorporated into phosphatidylinositol Metabolism of [3H]Inositol In Vivo (PtdIns); a maximum was obtained after 10 minutes [3H]inositol was introduced into Dictyostelium cells followed by a decline to half peak levels a t about 60 by electroporation; the protocol was optimized to gen- minutes. Radioactivity was subsequently found in the erate very small holes that are just large enough for phospholipids PtdInsP and PtdInsP, with maxima at, inositol to pass, but are impermeable for charged mol- respectively, 45 and 60 minutes after pulse-labelling. ecules such a s Ins(1,4,5)P3or ATP [Van Haastert et al., This kinetics is in perfect agreement with the lipid part 19891. This method of labelling cells with [3H]inositol of the inositol cycle where PtdIns is formed from Ins is very efficient (2.5% within 10 minutes) if compared and cytidine diphosphate-deoxyguanosine(CDP-DG), with metabolic labelling (0.2% in 6 hours). The metab- and PtdInsP and PtdInsP, from the phosphorylation of olism of [3H]inositol was then followed by the analysis PtdIns. The first watersoluble inositolphosphate coof the inositolphospholipids by thin layer chromatog- chromatographed with InslP; the radioactivity was raphy and the analysis of the water soluble compounds maximal at 50 minutes after pulse-labelling, well beby HPLC (Van der Kaay et al., unpublished research). fore Ins(l,4)P2was formed. This suggests that there is Two HPLC systems had to be used: Ins(1,4,5)P3 levels a phospholipase C activity that acts on PtdIns. The were analysed by reversed phase ion-pair chromatog- second watersoluble product co-chromatographed with raphy and the other inositolphosphates by ionexchange Ins(l,4)P2 and reached a maximum a t 75 minutes. chromatography. We have learned that a very signifi- Ins(l,4)P2was formed well before Ins(1,4,5)P3,suggestcant amount of the radioactivity is incorporated in un- ing that it was not formed from the dephosphorylation identified compound(s) that elute from Dowex columns of Ins(1,4,5)P3, but from a phospholipase C reaction in the InsP3/InsP, fraction, from reversed phase col- acting on PtdInsP. [3H]Ins(1,4,5)P3was the next water-

timized for the generation of holes that are sufficiently large for charged small molecule such a s CAMP and adenosine triphosphate (ATP) to pass, but are impermeable to enzymes [Schoen et al., 19891. Intact cells do not dephosphorylate Ins( 1,4,5)P3 a t a detectable rate. Permeable cells dephosphorylate Ins(1,4,5)P3 to Ins(4, 5)P, and Ins4P (Fig. l ) , confirming the results that Ins(1,4,5)P3is dephosphorylated in vitro mainly by the 1-phosphatase.



cpm 4



x 16’ 16





Fig. 3. The inositol cycle of Dictyostelium. A small portion of Ins(1,4,5)P3may also be dephosphorylated to Ins(1)P via Ins(l,5)P2.

Fig. 2. Dynamics of the inositol cycle. Cells were pulse-labelled with [3Hlinositol by electroporation. Samples were withdrawn a t 15 minute interval and intracellular levels were analysed for inositolcontaining compounds. Since there is considerable secretion of [3Hlinositol-containing compounds, the radioactivity in all compounds reaches a maximum. The Figure presents the magnitude and time at which the maximal radioactivity in each compound was reached.










Fig. 4. Ins(1,4,5)P3levels after stimulation with cAMP or GTPyS. A Relative levels of [3H11ns(1,4,5fP3after stimulation of saponinpermeabilized cells with cAMP or GTPyS. B: Absolute Ins(1,4,5)P3 levels of cells after stimulation with CAMP.

soluble radioactive compound that could be detected with a maximum a t 90 minutes, suggesting that i t was formed from PtdInsP,. Finally, the last compound was tope dilution assay [Van Haastert, 19891. The results Ins(4,5)P2, in accordance with its proposed Ins(1,4,5)P3 were similar (Fig. 41, but now absolute Ins(1,4,5)P3levsource. The kinetics of Ins4P formation is presently els were known: the mean intracellular concentration unknown, because i t was not yet possible to obtain a increased from 3.3 pM to a maximum of 5.5 pM. The complete separation of the small amount of Ins4P from increase is relatively small, but the absolute increase is the bulk of InslP. considerable, taking into account the submicromolar The results of the experiments on the metabolism of concentrations of Ins(1,4,5)P3that induce Ca2 release. Ins(1,4,5)P3 in vitro and inositol in vivo provide a Since the pool size of Ins(1,4,5)P3 is about ten times nearly complete picture of the inositol cycle in Dictyosmaller than the pool of PtdInsP,, it is not unexpected stelium; this is summarized in Figure 3. that we have never been able to detect a receptor stimulated decrease of PtdInsP,. Unfortunately this imStimulation of Ins(1,4,5)P3Production by cAMP plies that we have no complete evidence that the inand GTPyS. crease of Ins(1,4,5)P3 is derived from a receptor and Cells were labelled with [3H]inositol as described G-protein stimulated phospholipase C activity. above, incubated for 45 minutes to allow for the incorFunction of Inositol Cycle in Dictyostelium poration of [3H]inositol into PtdJnsP2, permeabilized Several mutants of Dictyostelium were analysed for with saponine [Europe-Finner and Newell, 19861, and then stimulated with the receptor agonist cAMP or the the receptor and G-protein stimulation of Ins(1,4,5)P3 G-protein agonist GTPyS. Both agonists induce a rapid production [Van Haastert e t al., unpublished research]. increase of [3HlIns(1,4,5)P3to about 145%of basal levels Mutants of the fgd A complementation group [Coukell with a maximum at about 6 seconds after stimulation; et al., 19831 are defective in both cAMP and GTPySbasal levels were recovered at 20-30 seconds after stim- stimulation of Ins( 1,4,5)P3 levels (Table 2). Biochemiulation [Van Haastert et al., 19891. The response was cal characterization of this mutant has demonstrated also measured in unlabelled cells from which Ins- defects in the interaction between receptor and G-pro(1,4,5)P3 was extracted and the levels measured by iso- tein [Kesbeke et al., 1988, Snaar-Jagalska et al., 19881, +

INOSITOLCYCLE IN DICTYOSTELIUM DISCOIDEUM TABLE 2. cAMP and GTPyS-Stimulation of Ins(1,4,5)P3Levels in Mutant fgdA*

Ins(1,4,5)P3levels (% of control) Stimulus cAMP GTPyS

Wild-type 146 -f lla 148 2 13"

fgd-4 101 ? 4 NS 102 ? 8 NS

*Wild-type and mutant cells were labelled with [3Hlinositol, permeabilized with saponin and stimulated with cAMP or GTPyS. [3H]Ins(1,4,5)P3levels were determined by HPLC. aSignificant from basal levels a t P < 0.01; n = 4; NS, not significant.

due to the low expression of a gene that codes for a G-protein alpha subunit (Kumagai et al., 1989). These results strongly suggest that the defective G-protein is involved in the receptor mediated activation of phospholipase C. This hypothesis should be confirmed by experiments which measure a GTP-stimulation of phospholipase C activity in vitro. Assuming t h a t the hypothesis is correct, the phenotype of the mutant provides the function of the receptor stimulated inositol cycle in Dictyostelium. The characteristics of mutant fgdA are summarized in Table 3. cAMP receptors are present and functional in terms of binding, covalent modification, and down-regulation. However, none of the second messenger responses is induced in the mutant, and cAMP does not induce chemotaxis or differentiation. Thus there is a complete blockade of all signal transduction in mutant fgdA. It was therefore unexpected to find that GTP-stimulation of adenylate cyclase was still normal in membranes from the mutant [Kesbeke et al., 19881. This suggests that the defective G-protein is not directly involved in the regulation of adenylate cyclase. This observation leads to two conclusions: there must be another G-protein, and the G-protein that stimulates phospholipase C is indirectly involved in the regulation of adenylate cyclase. This last hypothesis was tested by the experiment shown in Figure 5. Mutant fgdA cells were permeabilized with saponin and stimulated with cAMP in the absence or presence of Ins(1,4,5)P3, and the production of cellular cAMP levels was measured. cAMP or Ins(1,4,5)P3 alone did not alter cellular cAMP levels, but a strong increase was observed when both compounds were present. Subsequent experiments suggest that cAMP acts on the surface receptors, whereas Ins(1,4,5)P3 is probably required intracellularly.


TABLE 3. Characterization of mutant fgdA

Surface cAMP receptors are present Normal CAMP-induced receptor phosphorylation Normal CAMP-induced receptor down-regulation No CAMP-induced cAMP formation No CAMP-induced cGMP formation No CAMP-induced chemotaxis No CAMP-induced differentiation Normal GTP y S-stimulation of adenylate cyclase No GTP y S-stimulation of Ins(1,4,5)P3production

fgd A mutant

HC 8 5


dcAMP + lP3







3 minutes



Fig. 5. Rescue of adenylate cyclase stimulation in mutant fgdA by Ins(1,4,5)P3. Saponin-permeabilized mutant cells were stimulated with the receptor agonist 2'dcAMP and Ins(1,4,5)P3and the accumulation of cAMP levels was measured.

Ins(1,4,5)P3 is more complex and involves three additional enzyme activities t h a t have not yet been described in mammalian cells. The inositol cycle is probably the major signal transduction pathway in Dictyostelium as was demonstrated with mutant fgdA that presumably lacks the functional G protein to activate phospholipase C. We have not yet completely identified the inositol CONCLUSIONS cycle of Dictyostelium. During the analysis of the The microorganism Dictyostelium has a n inositol watersoluble compounds we detected large quantities cycle that is reminiscent of the inositol cycle of mam- of radioactivity that could not be identified; these commalian cells. The main differences are the metabolism pounds may contain up to 50% of the watersoluble raof Ins(1,4,5)P3: i) the phosphorylation to 1ns(1,3,4,5)P4 dioactivity. Furthermore, it is still largely unknown seems to be absent (and thus also the complex me- how InsP6 is formed in Dictyostelium. Finally, the identabolism of this compound; ii) the dephosphorylation of tification of some compounds is badly needed, such as of



the fatty acid and polar headgroup of the inositolphospholipids. Whereas the identification of the inositol cycle may be on its way, the identification and characterization of the participating enzymes are still in its infancy. The enzyme phospholipase C has not been detected yet in in vitro assays [Irvine et al., 19801. Finally, the precise function of Ins( 1,4,5)P, in the cell is not completely known; it releases Ca2' from nonmitochondria1 stores [Europe-Finner and Newell, 19861, but other functions cannot be excluded. This is suggested by the complex dephosphorylation of Ins(1, 4,5)P,. These details of the inositol cycle may be relevant since the mutant fgdA suggests that the inositol cycle rules signal transduction, chemotaxis, and differentiation in Dictyostelium.

ACKNOWLEDGMENTS We would like to thank Fanja Kesbeke and Ewa Snaar-Jagalska for stimulating discussions. This study was supported by grants of the C . and C. Huygens Fund and the Organization for Medical and Health Research (Medigon) which are subsidized by The Netherlands Organization for Scientific Research. REFERENCES Berridge MJ, Irvine RF (1989): Inositol phosphates and cell signalling. Nature 341: 197-205. Coukell MB, Lappano S, Cameron AM (1983): Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum. Dev Genet 3: 283-297. Europe-Finner GN, Newell PC (1986):Inositol 1,4,5-trisphosphatein-

duces calcium release from a non-mitochondria1pool in amoebae of Dictyostelium. Biochim Biophys Acta 887: 335-340. Irvine RF, Letcher AJ, Brophy PJ, North MJ (1980): Phosphatidylinositol-degrading enzymes in the cellular slime mould DictyosteZium discoideum. J Gen Microbiol 121: 495-487. Janssens PMW, Van Haastert PJM (1987): Molecular basis of transmembrane signal transduction in Dictyostelium discoideum. Microbiol Rev 51: 396-418. Kesbeke F, Snaar-Jagalska BE, Van Haastert PJM (1988): Signal transduction in Dictyostelium fgdA mutants with a defective interaction between surface cAMP receptor and a GTP binding regulatory protein. J Cell Biol 107: 521-528. Klein P, Sun TJ, Saxe CL, Kimmel AR, Devreotes PN (1989): A chemoattractant receptor controls development in Dictyostelium discoideum. Science 241: 1467-1472. Kumagai A, Pupillo M, Gunderson R, Mike-Lye R, Devreotes PN, Firtel RA (1989): Regulation and function of G, protein subunits in Dictyostelium. Cell 57: 265-275. MacDonald JIS, Weeks G (1985): The biosynthesis and turnover of lipid during the differentiation of Dictyostelium discoideum. Biochim Biophys Acta 834: 301-307. Schoen CD, Arents JC, Bruin T, Van Driel R (1989): Intracellular localization of secretable cAMP in relaying Dictyostelium cells. Exp Cell Res 181: 51-62. Snaar-Jagalska BE, Kesbeke F, Pupillo M, Van Haastert PJM (1988): Immunological detection of G protein 01 subunits in Dictyostelium discoideum. Biochem Biophys Res Commun 156: 757-761. Van Haastert PJM (1989): Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay. Anal Biochem 177: 115-119. Van Haastert PJM, De Vries MJ, Penning LC, Roovers E, Van der Kaay J, Erneux C, Van Lookeren Campagne MM (1989): Chemoattractant and guanosine 5'-[.-thioltriphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3Hlinositol by electroporation. Biochem J 258: 577586. Van Lookeren Campagne MM, Erneux C, Van Eijk R, Van Haastert PJM (1988): Two dephosphorylation pathways of inositol 1,4,5trisphosphate in homogenates of the cellular slime mould Dictyostelium discoideum. Biochem J 254: 343-350.

Dynamics and function of the inositolcycle in Dictyostelium discoideum.

The inositolcycle in Dictyostelium discoideum was studied under several conditions both in vitro and in vivo. The results are compared with the inosit...
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