Iron requirement for isolated rat liver mitochondrial protein synthesis

136

Biochimica et Biophysica Acta, 6 0 7 ( 1 9 8 0 ) 1 3 6 - - 1 4 4 © Elsevier/North-Holland

Biomedical Press

BBA 99609

IRON REQUIREMENT FOR ISOLATED RAT LIVER MITOCHONDRIAL PROTEIN SYNTHESIS

D A V I D L. M A R C U S , N A D E R G. I B R A H I M M I C H A E L L. F R E E D M A N * *

*, N A O M I G R U E N S P E C H T

and

Department of Medicine, New York University Medical Center, 550 First Avenue, New York, N Y 10016 (U.S.A.) (Received July 16th, 1979)

Key words: Protein synthesis; Iron requirement; Heroin; (Rat liver mitochondria)

Summary Isolated rat liver mitochondrial protein synthesis was severly inhibited by ~,a-dipyridyl (a ferrous iron-chelating agent), chloramphenicol and hemin (10-7 M or greater). In contrast, 7,7-dipyridyl (a non-iron-chelating analogue of a,~-dipyridyl), cycloheximide and lower concentrations of hemin were noninhibitory. The inhibitory action of a,a-dipyridyl was reversed by addition of Fe(NH4)2(SO4)2 while ZnC12, CuC12 and CoC12 were ineffective. Hemin, however, did not protect against the a,a-dipyridyl inhibition of mitochondrial protein synthesis. These results indicate that ferrous iron is required for mitochondrial protein synthesis and suggests that it is through a mechanism independent of hemin concentration.

Introduction

Previous work from this laboratory has shown that iron is required f o r mammalian cell cytoplasmic protein synthesis by way of its requirement for heme synthesis [1]. Heme has been shown to be necessary for maximal cytoplasmic protein synthesis in a wide variety of mammalian cells, including reticulocytes (both globin and the non-globin proteins) [2--6], Krebs II ascites t u m o r cells [6], platelets [7] and brain and liver cells [8]. It is not yet clear where in the heme synthetic pathway iron exerts its * Present address: D e p a r t m e n t of Medicine, New York Medical College, Valhalla, NY 10595, U.S.A. ** To w h o m reprint requests should be addressed. Abbreviation: SDS, s o d i u m dodeeyl sulfate.

137 greatest effect. Ferrous iron has been reported in various cell types to be necessary for maximal activity of several heme synthetic enzymes including 8-aminolevulinic acid synthetase [9,10], 8-aminolevulinic acid dehydmtase [11], coproporphyrinogen oxidase [12,13] and ferrochelatase [14]. It is of note that 5-aminolevulinic acid synthetase [15,16], coproporphyrinogen oxidase [14] and ferrochelatase [17] are mitochondrial enzymes whose activities are intimately dependent upon the integrity of the inner mitochondrial membrane. Mitochondria contain their own unique protein-synthesizing system which produces at least some of the structural proteins of the inner mitochondrial membrane [18--22]. Thus, any inhibition of mitochondrial protein synthesis ultimately may lead to an interference of the function of the mitochondrial inner membrane and the heme synthetic enzymes. In the present study we investigated the role of ferrous iron in rat liver mitochondrial protein synthesis and have found that iron-deficient mitochondria have diminished protein synthetic capability.

Experimental procedures Preparation of rat liver mitochondria. Isolated rat liver mitochondria were prepared under sterile conditions in 0.25 M sucrose containing 10 mM Tris-HC1, pH 7.4, and 1.0 mM EDTA as previously described [23]. In brief, this procedure involved decapitation of the rats and removal of the liver. The liver was sliced and gently homogenized in a Teflon-glass homogenizer. The homogenate was then centrifuged twice at 650 × g for 10 min. The supernatant was decanted and the pellet centrifuged at 9500 X g for 10 min. The resultant pellet was then washed twice and resuspended in the sucrose media for study of amino acid incorporation. Amino acid incorporation studies. The amino acid incorporation media was similar to that previously described [24] and contained 90 mM KC1, 5 mM potassium phosphate buffer, pH 7.4; 10 mM MgC12; 50 mM bicine buffer, pH 7.4; 22.5 /~g of a complete amino acid mixture minus leucine, 15 /~M (0.5 ~uCi/ml L-[U-14C]leucine; 2 mM ATP; 2 mM phosphoenolpyruvate; 10 pg pyruvate kinase; and 2--3 mg mitochondrial protein in a final volume of 2 ml. The incubations were for 30 min at 37°C in a shaking waterbath with air as the gas phase. Incubations were terminated by addition of 10 mM non-radioactive L-leucine followed by precipitation of protein with 5% trichloroacetic acid. The labeled proteins were prepared for counting by previously described methods [25]. The counting was performed in a Beckman scintillation counter in 'Aquasol' [25]. Protein concentration was determined by the method of Lowry et al. [26]. The preparation and addition of different agents to the incubation media are described in the tables. Identification of mitochondrial membrane proteins. In these experiments the incubation conditions were identical to those described above except that L-[3,4,5-3H]leucine was substituted for L-[U-14C]leucine. The concentration of L-[3,4,5-3H]leucine was 15 /~M and 10 #Ci]ml were added. After 30 min incubation the mitochondria were suspended in a sucrose/Tris/EDTA buffer (0.25 M sucrose 0.01 M Tris-HC1, 10-3M EDTA, pH 7.8) and disrupted by

138 sonication for 15 s in a Branson Sonifier (model No. W185) at a power o u t p u t of 40 W. The suspension was then centrifuged at 150 000 × g at 4 ° for 30 min in a Beckman L-2 65 ultracentrifuge. The membrane pellet was washed successively twice with 5% trichloroacetic acid, four times with ether and dissolved in a minimum volume of 10% sodium dodecyl sulfate (SDS). SDS-polyacrylamide gel electrophoresis was performed b y the m e t h o d of Weber and Osborn [27]. 100 /zg of protein were electrophoresed in each tube (9 cm) at room temperature with a constant current of 3.0 mA/gel for 16 h. Protein bands were visualized by staining with Coomassie brilliant blue and the gels scanned at 570 nm in a Gilford model 240 recording spectrophotometer equipped with a linear transporter. Identical gels were sliced into 1.6mm sections, dissolved by heating with 0.5 ml of 30% H202 at 50°C for 2 h, and counted in 'Aquasol' in a Beckman scintillation counter. Recovery of counts from the gels was 95%. Materials. Chloramphenicol, cycloheximide, adenosine triphosphate, phosphoenolpyruvate, pyruvate kinase, hemin and the metals were obtained from the Sigma Chemical Co.; a,a-dipyridyl and 7,7-dipyridyl from Fisher Scientific Co.; and L-[U-~4C]leucine 350 Ci/mol was obtained from Amersham, and L-[3,4,5,-3H]leucine 120 Ci/mmol from New England Nuclear Corporation. All other reagents were of the highest grade commercially available. Results

Effect o f a,a-dipyridyl on rat liver mitochondrial protein synthesis The effect of a,a-dipyridyl on rat liver mitochondrial protein synthesis is shown in Table I. The incubations were performed under sterile conditions and periodical samples were cultured to exclude bacterial contamination. In addition, in each experiments tubes minus mitochondria were incubated and processed identically to the experimental tubes to show that incorporation did n o t

TABLE I EFFECT OF A FERROUS IRON-CHELATING CHONDRIAL PROTEIN SYNTHESIS

A G E N T (~,o~-DIPYRIDYL) O N R A T L I V E R M I T O -

I n c u b a t i o n s in d u p l i c a t e w e r e for 30 rain at 3 7 ° C in t h e m e d i a d e s c r i b e d u n d e r E x p e r i m e n t a l p r o c e d u r e s . D u p l i c a t e s w e r e w i t h i n 1 0 % o f e a c h o t h e r . E a c h v a l u e w a s c o r r e c t e d b y s u b t r a c t i n g a s m a l l zero t i m e . T h e m e a n specific activity (cpm/mg protein) of the controls without additives was 3697 cpm/mg protein. Each o f t h e five e x p e r i m e n t s w a s p e r f o r m e d w i t h s e p a r a t e rat livers o n d i f f e r e n t days. Addition

Final c o n c e n t r a t i o n

% control

S.E.

~,~-Dipyridyl

1 0 -6 M 1 0 -s M 10 -4 M 2 • 10-4 M 10 -6 M 10 -5 M 10 -4 M 2 • 10-4 M 100/~g/ml 100/~g/ml

87.6 80.4 59.8 56.4 98.8 109.0 107.1 97.3 12.6 99.6

6.76 4.25 7.66 6.37 2.56 3.72 4.64 2.64 0.87 0.24

7,7-Dipyridyl

Chloramphenicol Cyclohe ximide

139

occur in the absence of the mitochondria. Furthermore, 100 /~g/ml erthromycin did not inhibit protein synthesis in our system. It has previously been shown [24] that this concentration of antibiotic inhibits yeast and bacterial protein synthesis b u t does n o t inhibit rat liver mitochondria p r o t e i n s y n t h e s i s when the mitochondrial membrane is intact. When isolated rat liver mitochondria were incubated at 37°C for 30 min, chloramphenicol, a specific inhibitor of mitochondrial protein synthesis, inhibited this system b y 87.4% while the cytoplasmic protein synthesis inhibitor, cycloheximide [25], had no effect. When a,a-dipyridyl, a ferrous iron chelator [28] was added to the medium significant inhibition was apparent at 10 -s M. At a concentration of 2 • 10 -4 M there was 44% inhibition in these experiments. In contrast %v-dipyridyl which is n o t an i r o n chelator [28], had no inhibitory effects on this system. It should be noted that there was some variability between rats in their susceptibility to ~,a-dipyridyl inhibition. Some preparations required concentrations of 2 • 10 -4 M to show significant inhibition. This p h e n o m e n o n appears to be related to the age of the animal with old rats showing greater inhibition with the iron chelator [29]. In all experiments, therefore, the results are expressed as percent of its own control.

Ferrous iron protection of a,~-dipyridyl inhibitions In order to demonstrate that a,a-dipyridyl does inhibit mitochondrial protein synthesis as a result of its chelation of iron, it was necessary to show protection of its effect b y ferrous iron. The protective results of Fe(NH4)2(SO4)2 against a,~-dipyridyl are shown in Table II. In these experiments 5" 10 -4 M Fe(NH4)2(SO4)2 significantly protected against 2 . 1 0 -4 M ~,~-dipyridyl inhibition of mitochondrial protein synthesis ( P < 0.001 by Student's t-test analysis). (NH4)2SO4 was neither inhibitory nor protective. Thus, it appears that ~,a-dipyridyl inhibition of mitochondrial protein synthesis is due to chelation of iron.

Ferrous iron reversal of a,a-dipyridyl inhibition The experiments in Table III show that ferrous iron reversed the inhibition o f a,a-dipyridyl. In these experiments 2 • 10 -4 M a,a-dipyridyl inhibited mitochondrial protein synthesis b y 29% at 15 and 30 min. When 5 . 1 0 -4 M

T A B L E II F E R R O U S (NH4)2(SO4) 2 P R O T E C T I O N OF ~ , ~ - D I P Y R I D Y L I N H I B I T I O N OF R A T L I V E R MITOCHONDRIAL PROTEIN SYNTHESIS

I n c u b a t i o n s in duplicate w e r e for 3 0 m i n at 3 7 ° C as d e s c r i b e d in E x p e r i m e n t a l p r o c e d u r e s and T a b l e I. The m e a n specific activity o f t h e c o n t r o l s w a s 4 7 6 3 c p m / m g protein. ~,0~-Dipyridyl c o n c e n t r a t i o n w a s 2 • 1 0 - 4 M. R e s u l t s a r e f r o m t e n e x p e r i m e n t s . Addition

Final c o n c e n t r a t i o n

% control

S.E.

~,~-Dipyridyl F e ( N H 4 )2 ( S O 4 ) 2 ~,~-Dipyridyi + Fe(NH4)2 (SO4)2

2 5 2 5

51.6 94.2 91.1

5.24 3.36 4.25

• 1 0 -4 • 10-4 • 10-4 + • 10-4

140 TABLE III REVERSAL THESIS

OF ~,tv-DIPYRIDYL

INHIBITION

OF RAT

LIVER

Incubations i n d u p l i c a t e w e r e f o r 3 0 m i n a t 3 7 ° C a s d e s c r i b e d ~ , a - D i p y r i d y l was P r e s e n t f r o m t i m e zero. The m e t a l s were all ity of the controls at 15 min was 2234 cpm/mg protein and at the mean value of the ~,~-dipyridyl alone was 71% of the 15 ments. Addition

Final concentration

MITOCHONDRIAL

PROTEIN

SYN-

i n E x p e r i m e n t a l p r o c e d u r e s a n d T a b l e I. added at 15 rain. The mean specific activ30 min 3938 cpm/mg protein. At 15 min m i n c o n t r o l . R e s u l t s are f r o m six experi-

% control

S.E.

71.3 89.7 68.2 53.0 65.6 109 107 105 103

1.93 2.44 3.12 2.63 2.87 2.58 2.96 2.39 3.20

(M) ~,a-Dipyridyl a,o~-Dipyridyl + cz,a-Dipyridyl + a,a-Dipyridyl + ~,a-Dipyridyl + Fe(NH4)2(SO4) ZnCl2 CuCl 2 COC12

Fe(NH4)2(SO4) 2 ZnC12 CoC12 CoCI 2 2

2 2 2 2 2 5 5 5 5

• • • • • • • • •

10-4 10-4 10-4 10-4 10-4 10-4 10-4 10-4 10-4

+ + + +

5 5 5 5

• • • •

1 0 -4 10-4 10-4 10-4

Fe(NH4)2(SO4)2 was added at 15 min there was reversal of the inhibition at 30 min (P<~ 0.01 b y Student's t-test analysis). However, zinc, copper and cobalt were ineffective in reversing the inhibition. The specific reversal by iron of a,a-dipyridyl inhibition is strong evidence for an iron requirement in mitochondrial protein synthesis.

a,a-Dipyridyl inhibition of mitochondrial membrane protein synthesis In order to further demonstrate that the effect of a,a-dipyridyl was on mitochondrial protein synthesis, the mitochondrial membranes were isolated as described in Experimental procedures. In Fig. 1 (upper panel) a representative labeling pattern from a 30 min incubation in the presence of 2 - 10 -4 M a,a-dipyridyl plus 5" 10 -4 M Fe(NH4)2(SO4)2 is shown. Control labeling patterns either with or without iron were essentially identical. In the middle panel the labeling pattern with 2 • 10 -4 M a,a-dipyridyl (no iron) is shown. As can be seen, there is a marked reduction in labeling of all peaks with virtual absence of labeling of peaks below 25 000 daltons. The protein-staining pattern with Coomassie blue (lower panel) was identical for control mitochondria incubated with or without iron, non-incubated mitochondria or those incubated with ~,~-dipyridyl with or w i t h o u t iron. In these experiments 100 /~g/ml cycloheximide did n o t alter the labeling pattern while 100 #g/ml chloramphenicol almost totally inhibited incorporation confirming that we were indeed measuring the mitochondrial portion of synthesis of mitochondrial membrane proteins.

Inability of hemin to protect against ~,a-dipyridyl inhibitions In intact reticulocytes and other cells the iron chelator inhibition of cytoplasmic protein synthesis may be prevented by addition of hemin (5 • 10 - s -

141

60k 40W 36k 25W 18W

600500400-

~3oo200100I

I

2O 40 60 GEL SLICE NUMBER 300-

o

200-

100-

¢n
0

2

4 "

6

8

10

DISTANCE FROM TOP OF GEL (cm)

Fig. 1. E f f e c t o f c~,~-dipyrldyl o n L - [ 3 , 4 , 5 - 3 H ] l e u c i n e i n c o r p o r a t i o n i n t o m i t o c h o n & d a i m e m b r a n e p r o t e i n . T h e i n c u b a t i o n s , p r e p a r a t i o n o f m e m b r a n e P r o t e i n s , a n d p d l y a c r y l a m i d e gel e l e e t r o p h o r e s i s w e r e c a r r i e d o u t as d e s c r i b e d i n t h e t e x t . U p p e r t r a c i n g : 2 • 1 0 - 4 M a , ~ * d l p y r i d y l p l u s 5 • 1 0 - 4 M f e r r o u s ( N H 4 ) 2 ( S O 4 ) 2. M i d d l e t r a c i n g : 2 • 1 0 - 4 M 01,a-dipyridyl. B o t t o m t r a c i n g : t h e p r o t e i n - s t a i n i n g P a t t e r n o f t h e u p p e r gel. T h e P a t t e r n o b t a i n e d f r o m t h e m i d d l e gel w a s i d e n t i c a l t o t h a t o f t h e u p p e r gel. T h e m o l e c u l a r w e i g h t o f t h e v a r i o u s p e a k s w a s e s t i m a t e d f r o m a p l o t o f t h e M r vs. m i g r a t i o n d i s t a n c e e m p l o y i n g t h e following markers of known molecular weight: bovine serum albumin (68 000); aidolase (40 000); chymotrypsinogen (25 700); ribonuelease (14 000).

142 NORMAL

~ I

IRON DEFICIENCY

Y

MITROCHONDRIAL PROTEIN SYNTHESIS I

¢ie

e

1

I H~-MESYNTH"-S,S1 ele t(HEME) I HCRI -

•\ L.-

-

I HCRA

/e

I

CYTOPLASMIC

I PROTEINSYNTHESIS I

~--J

Fig. 2. P r o p o s e d i n t e r r e l a t i o n s h i p b e t w e e n m i t o c h o n d r i a l a n d c y t o p l a s m i c p r o t e i n s y n t h e s i s . U n d e r n o r m a l c o n d i t i o n s (+) t h e r e is m a x i m a l m i t o c h o n d r i a l p r o t e i n s y n t h e s i s (i'), w h i c h a l l o w s m a x i m a l h e i n e s y n t h e s i s (1") a n d s u f f i c i e n t c y t o p l a s m i c h e i n e t o m a i n t a i n t h e h e i n e - c o n t r o l l e d r e p r e s s o r ( H C R ) i n t h e n o n - i n h i b i t o r y f r o m ( H C R I ) . T h u s u n d e r n o r m a l c o n d i t i o n s (+) c y t o p l a s m i c p r o t e i n s y n t h e s i s is m a x i m a l ( ? ) . I n i r o n d e f i c i e n c y (--) m i t o c h o n d r i a l p r o t e i n s y n t h e s i s falls (4), h e i n e s y n t h e s i s a n d c o n c e n t r a t i o n d r o p s ( 4 ) a n d H C R ( 1 - - 2 ) is a c t i v a t e d ( H C R A ) t o i n h i b i t c y t o p l a s m i c p r o t e i n s y n t h e s i s . I f h e i n e levels are s u p r a n o r m a l (i.e. w h e r e t h e r e is p r i m a r y i n h i b i t i o n o f c y t o p l a s m i c p r o t e i n s y n t h e s i s ) m i t o c h o n d r i a l p r o t e i n s y n t h e s i s w o u l d be i n h i b i t e d b y h e i n e (--). As s h o w n ( . . . . . . ) t h e r e a p p e a r s t o be a m e c h a n i s m f o r c y t o p l a s m i c p r o t e i n s y n t h e s i s t o s i g n a l m i t o c h o n d r i a l p r o t e i n s y n t h e s i s . T h i s c o u l d c o n c e i v a b l y be v i a t h e c y t o p l a s m i c p r o t e i n s t h e m s e l v e s , H C R or s o m e o t h e r m e c h a n i s m .

1 " 1 0 -4) [ 6 , 7 , 3 0 - - 3 5 ] . In the present study, we therefore attempted to overc o m e ~,~-dipyridyl inhibition with hemin. Hemin itself was inhibitory to mitochondrial protein synthesis in concentrations of 1 . 1 0 - 7 M or greater (Table IV). Non-inhibitory concentrations of 10 -8 and 10 -9 M hemin resulted in no significant protection against ~,a-dipyridyl. Hemin at 1 . 1 0 - 9 M was slightly stimulatory to mitochondrial protein synthesis (P < 0.01 by Student's t-test analysis), but was ineffective in protection against ~,~-dipyridyl. The possible mechanism of this slight stimulation is presently unknown.

TABLE IV INABILITY OF HEMIN TO PROTECT DRIAL PROTEIN SYNTHESIS

c~¢-DIPYRIDYL INHIBITION OF RAT LIVER MITOCHON-

I n c u b a t i o n s i n d u p l i c a t e w e r e f o r 3 0 r a i n at 3 7 ° C as d e s c r i b e d in E x p e r i m e n t a l p r o c e d u r e s a n d T a b l e I. T h e m e a n s p e c i f i c a c t i v i t y o f t h e c o n t r o l s w a s 3 7 2 5 c p m / m g p r o t e i n . H e r o i n w a s d i s s o l v e d in 0 . 2 m l 1 N K O H , a n d d i l u t e d a p p r o x i m a t e l y 1 0 - f o l d i n d e i o n i z e d w a t e r . T h e p H w a s a d j u s t e d t o 7 . 4 w i t h 1 N HCI. T h e h e r o i n c o n c e n t r a t i o n ( t e n t i m e s t h e f i n a l c o n c e n t r a t i o n ) w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y as previously described [33]. Addition

Final c o n c e n t r a t i o n (M)

% control

S.E.

~,~-Dipyridyl Hemin

1 • 1 0 -`4 1 • 10 -9 1 • 10 -8 1 • 10 -7 1 • 1 0 --4 + 1 • 10 -9 1 • 10 .-4 + 1 • 10 -8 1 • 1 0 --4 + 1 • 10 -7

56.5 111.4 98.7 68.5 61.0 59.8 49.9

4.36 3.40 2.00 5.18 6.22 5.43 2.54

~-Dipyridyl ~-Dipyridyl ~-Dipyridyl

+ hemin + hemin + hemin

143

Discussion Much of the work regarding mitochondrial protein synthesis has been directed toward elucidating the biogenesis of the functional mitochondrial inner membrane. Two distinct genetic systems are involved, one mitochondrial and one nuclear. Hence, there are two intracellular sites involved, one mitochondrial and one cytoplasmic [36,37]. The vast majority of mitochondrial proteins are synthesized in the cytoplasm and transported into the mitochondria [16]. The integration of these proteins into the mitochondrial membrane requires the presence of 8--12 hydrophobic proteins which are synthesized within the mitochondria on mitochondrial ribosomes [18,19]. In the present study, the results of our SDS-polyacrylamide gel electrophoresis of mitochondrial membranes show the presence of 8--12 protein staining bands, as has been demonstrated b y o~hers [36,37]. Previous work in this and other laboratories has shown that the inhibition of cytoplasmic protein synthesis in a variety of iron-deficient mammalian cells results from a decrease in cellular heme [1--8,28,30--35]. This inhibition may be overcome b y hemin. In contrast, in the present study with isolated rat liver mitochondria the inhibitory effects of a,a-dipyridyl were n o t overcome b y hemin. Indeed, hemin at concentrations of 10 -7 M or greater was severly inhibitory to mitochondria and has been shown to inhibit b o t h mitochondrial ~-aminolevulinic acid synthetase [38] and ferrochelatase [17] activities by end product inhibition. Since we are dealing with isolated mitochondria, this inhibition cannot be secondary to a lack of cytoplasmic proteins. Rather it appears that iron deficiency by itself produces a profound inhibition of the synthesis of the mitochondrial proteins in a manner different from that seen with cytoplasmic protein synthesis. On the basis of these and other experiments [1--2] in our laboratory, we tentatively propose the following scheme for the integration of synthesis of mitochondrial and cytoplasmic proteins (Fig. 2). In iron deficiency one possible lesion is the decrease in mitochondrial protein synthesis and a lowered concentration cellular heme. The heme controlled repressor [1--2] then would be activated and cytoplasmic protein synthesis would be diminished. Similarly, a primary decrease in cytoplasmic protein synthesis also signals the mitochondria to decrease protein synthesis [22]. One possibility is that this is due to a rise in heme concentration which then inhibits mitochondrial protein synthesis. It is n o t y e t known whether the signal from the cytoplasm to the mitochondria is via heme controlled repressor, or other proteins, or heme or iron concentrations.

Acknowledgements This research was supported by a grant from the National Institutes of Health, AM 13532. Portions of this work were presented at the National Meeting of the American Society of Hematology (New Orleans, LA) Blood 52 Suppl. 1, 101. D.L.M. is a recipient of a National Research Service Award from

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the National Institutes of Health, HL 05462. N.G.I. is a recipient of a National Service Award from the National Institutes of Health, AM 05558. M.L.F. is a recipient of a Research Career Development Award from the National Institutes of Health, AM 49358. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

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