Biochemical characterization of contractile proteins of rat cultured mesangial cells

Kidney International, Vol. 39 (1991), pp. /118—1124

Biochemical characterization of contractile proteins of rat cultured mesangial cells TAKAKO ISHINO, Roi KOBAYASHI, HIDEKI WAKUI, YUKITAKA FUKUSHIMA, YAsusH! NAKAMOTO, and AKIR& B. MIuRA Third Department of Internal Medicine, Akita University School of Medicine, Akita, and Department of Pharmacology, Nagoya University School of Medicine, Nagoya, Japan

Biochemical characterization of contractile proteins of rat cultured mesangial cells. We examined characteristics of contractile proteins of rat cultured mesangial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that actomyosin was a major protein component in the extract of mesangial cells. By Western blot, tropomyosin, caldesmon, a-actinin, and vinculin were recognized in mesangial cells. The molecular masses of a-actinin and vinculin were the same as aortic smooth muscle. Mesangial cells contained five tropomyosin isoforms:

In this study, we analyzed contractile proteins of rat cultured

mesangial cells such as actomyosin, tropomyosin including isoforms, caldesmon, a-actinin, and vinculin, by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), Western blot, and immunofluorescent staining. We also compared these proteins with those of rat aortic smooth muscle, cultured smooth muscle cells, and cultured dermal TM-i, -2, -3, -4, and -5. TM-i was present as a major isoform in mesangial cells, and it had an immunological cross reactivity with fibroblasts. Compositions of contractile proteins of mesangial /3-tropomyosin of aortic smooth muscle. On the other hand, the cells were similar to vascular smooth muscle cells, but were molecular mass of caldesmon was similar to dermal fibroblasts, and different from dermal fibroblasts. Our findings suggest that differed from aortic smooth muscle. Immunofluorescent studies showed mesangial cells are contractile cells and are derived from that the staining patterns of tropomyosin and caldesmon between vascular smooth muscle cells. cultured mesangial cells and cultured vascular smooth muscle cells were somewhat different. From these results, we conclude that mesangial cells abound in contractile proteins, and that the compositions of these proteins are similar to those of aortic smooth muscle with minor differences. Thus, this study appears to biochemically support a hypothesis that mesangial cells are derived from vascular smooth muscle cells, but with a minor modification in their evolution.

There are three major classes of cytoskeletal structures in cells: microfilaments, microtubles, and intermediate filaments. Among these structures, microfilaments play central roles in cell motility and cell shape changes. It has been demonstrated that mesangial cells contain bundles of microfilaments similar to

Methods

Isolation and culture of rat mesangial cells, vascular smooth muscle cells, and dermalfibroblasts Glomeruli were isolated from male Wistar rats at the age of four weeks, by the method of Kreisberg, Venkatachalam and Pate! [51. They were placed in culture flasks with a culture medium containing RPMI-1640, L-glutamine (2 mM/mi), penicillin (100 UIml), streptomycin sulfate (100 tg/ml), and 20%

fetal calf serum, and were incubated at 37°C. The routine medium changes were performed twice a week. After four weeks, overgrown cells from the primary culture were detached

those of smooth muscle cells [1], and that actomyosin is from flasks by adding phosphate-buffered saline containing localized in mesangial cells, by using immunofluorescent stud-

0.01% trypsin, 0.008% ethylene diamine tetraacetic acid, and

ies on the human kidney F2, 31. Mesangial cells are thus were substrated into flasks containing the culture medium. Culture of both smooth muscle cells and fibroblasts were also suggested to be contractile cells, and to have a function to established from Wistar rats at the age of four weeks, by the regulate the glomerular perfusion by their contraction. Glomerular mesangial cells from mammalian species have been isolated and propagated, and these cells have recently

method of Yaoita et al [61. Minced pieces of both aortic media

and skin tissue were placed in culture dishes containing the culture medium, and cover strips were placed over the tissues. with smooth muscle cells. For example, cultured mesangial Culture conditions were the same as those for mesangial cells. cells can contract after the addition of angiotensin II or vasoPreparation of proteins from cells and tissues pressin [1, 41. However, biochemical characteristics of contractile proteins of these cells are little known. Among cultured mesangial cells, smooth muscle cells, and fibroblasts, cells only from the second substrate were used. To harvest materials for protein analysis, cell layers were rinsed been shown to have several physiological properties in common

Received for publication May 10, 1990 and in revised form January 3, 1991 Accepted for publication January 11, 1991

three times with physiological saline, and were homogenated by a polytron blender. They were then lysed into the SDS-sample

© 1991 by the International Society of Nephrology

glomeruli and aortic smooth muscle were prepared. Glomeruli

buffer of Laemmli [7]. In addition, rat tissue proteins from 1118

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Fig. 2. Two-dimensional urea-shift gel analysis and Western blot of tropomyosin isoforms of rat cultured meson gial cells and aortic smooth muscle. Proteins were separated in the first dimension on SDS-polyacrylamide (10%) gels containing 8 M urea, and in the second dimension —actrn

Fig. 1. Quantitative densitometric measurements of actomyosin in the

extracts of rat cultured mesangial cells, aortic smooth muscle, and cultured dermal fibroblasts. Quantitative densitometric measurements

were made with use of a Model 1650 scanning densitometer on SDS-polyacrylamide (10%) gels, which were stained with Coomassie blue. A. Rat cultured mesangial cells. B. Rat aortic smooth muscle. C. Rat cultured dermal fibroblasts. Protein peaks of both actin and myosin are indicated in each of the panels.

on SDS-polyacrylamide (10%) gels. A. Two-dimensional urea-shift gel

of cultured mesangial cells. Silver nitrate staining. B. Western blot using rabbit anti-/3-tropomyosin antibody for the first antibody, and peroxidase-conjugated anti-rabbit IgG for the second antibody. C. Two-dimensional urea-shift gel of aortic smooth muscle. Silver nitrate staining. D. Western blot using the antibodies exactly as in B. Numbers on the left in A and C indicate molecular mass standards in kilodalton (kDa). Tropomyosin of cultured mesangial cells consists of 5 isoforms (TM-I, -2, -3, -4, and 5), which are indicated by arrows and numbers in A. TM-i of cultured mesangial cells (B) has an immunological cross reactivity with that of /3-tropomyosin (TM-l) in D. Urea/SOS-PAGE

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isolated by the method of Kreisberg et al [5], minced aortic tissues were homogenated by the blender, and were lysed into the SDS-sample buffer. Proteins from human platelets were also prepared for analysis of tropomyosin isoforms. Human platelets were purified by Sepharose 2B (Pharmacia, Uppsala, Sweden) chromatography. They were homogenated by the blender, and lysed into the SDS-sample buffer.

Production of antibodies against contractile proteins Tropomyosin [8—101, caldesmon [11], a-actinin [12], and vinculin [131 were purified by standard methods or our methods from chicken gizzard, and polyclonal antibodies against these proteins were raised in rabbits immunized with the purified

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proteins. The specificities of each of the antibodies were Fig. 3. Two-dimensional urea-shift gel analysis of tropomyosin isoconfirmed by Western blot according to the following criteria: forms of rat cultured meson gial cells, aortic smooth muscle, cultured

only a single polypeptide band of the antigen among total chicken gizzard proteins reacted with the antibody. We also purified desmin from chicken gizzard by standard methods [14, 151 and raised rabbit polyclonal anti-desmin antibody by the same method.

SDS-PAGE and densitometo,y SDS-PAGE was performed as described by Laemmli [7], and polyacrylamide gels were stained with Coomassie blue or silver nitrate. Two-dimensional urea-shift gel electrophoresis was

dermalfibroblasts, and human platelets. Proteins were separated in the

first dimension on SDS-polyacrylamide (10%) gels containing 8 M urea, and in the second dimension on SDS-polyacrylamide (10%) gel. Gels were stained with silver nitrate. A. Rat cultured mesangial cells. B. Rat

aortic smooth muscle. C. Rat cultured dermal fibroblasts. D. Human

platelets. A through D show a part of enlarged gels, focusing on tropomyosin isoforms. The oblique dot lines indicate the position of other proteins which aligned diagonally. Cultured mesangial cells have

5 isoforms of tropomyosin, with TM-I, -2, -3, and -4 predominating (A). In the extract of aortic smooth muscle, only two isoforms (TM- 1 and -2)

are present (B). Cultured dermal fibroblasts and platelets have 5 isoforms, with TM-4 and -5 predominating (C and D).

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Ishino et a!: Contractile proteins of mesangial cells

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Fig. 4. Indirect immunofluorescent staining of tropomyosin on rat cultured mesangial cells, vascular smooth muscle cells, and dermalfibroblasts. A and B. Rat cultured mesangial cells. C. Rat cultured vascular smooth muscle cells. D. Rat cultured dernial fibroblasts. On B, C, and D, we used rabbit anti-/3-tropomyosin antibody for the first antibody, and fluorescein isothiocyanate-conjugated anti-rabbit IgG for the second antibody. On A we used only the second antibody, as a control. Tropomyosin presents on the stress fibers in mesangial cells, vascular smooth muscle cells, and fibroblasts. Among the three kinds of cells, the staining intensity was weakest in fibroblasts, In vascular smooth muscle cells, an intensive staining on the perinuelear region is also seen.

carried out according to the method of Carmon, Neuman and Yaffe [16], under the condition for the first dimension in which the stacking gel contained 3 M urea and the separating gel contained 8 M urea. Quantative densitometric measurements were made by a Model 1650 scanning densitometer (Bio-Rad, Richmond, California, USA) on SDS-polyacrylamide gels, which were stained with Coomassie blue in the linear range.

Results

Actomyosin Actin, myosin, and other proteins among the extracts from cultured mesangial cells, aortic smooth muscle, and cultured dermal fibroblasts were compared by densitometric scannings of SDS-polyacrylamide gels. The extract of mesangial cells showed three major protein peaks (Fig. 1A). Two of them were from actin and myosin, and another of them was observed Western blot between peaks of actin and myosin. Thus, actomyosin was Western blot was performed by using the method of Towbin, considered to be a major protein component in mesangial cells. Staehelin and Gordon [17]. For the first antibodies, we used Although the amount of actomyosin in fibroblasts was smaller rabbit anti-tropomyosin, anti-caldesmon, anti-a-actinin, anti- than that in mesangial cells, the extract of fibroblasts also vinculin, and anti-desmin antibodies. For the second antibody, showed a protein peak similar to the peak present between we used peroxidase-conjugated anti-rabbit IgG (Dakopatts, peaks of actin and myosin in the mesangial cell extract (Fig. 1 A Glostrup, Denmark). and C). This peak apparently resulted from a protein common to mesangial cells and fibloblasts, which seemed to be unrelated Indirect immunofluorescent study to those known contractile proteins. In consideration of protein The distribution of contractile proteins in rat cultured mesan- peaks other than those of actin and myosin, the relative amount gial cells, cultured smooth muscle cells, and cultured dermal of actomyosin in the extract of fibroblasts was smaller than that fibroblasts were examined by immunofluorescent microscopy. in smooth muscle (Fig. 1 B and C). For the first antibodies, we used rabbit anti-tropomyosin and Tropomyosin anti-caldesmon antibodies. For the second antibody, we used In a two-dimensional urea-shift gel (the first dimensional gel fluorescein isothiocyanate-conjugated anti-rabbit IgG (Cappel, contained SDS plus 8 M urea, and the second dimensional gel West Chester, California, USA).

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Ishino et a!: Contractile proteins of mesangial cells

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vascular smooth muscle cells, the staining on the stress fibers was more intensive than mesangial cells, and an additional staining was observed on the perinuclear region (Fig. 6C). In cultured dermal fibroblasts, there was a faint staining of caldesmon on the stress fibers (Fig. 6D).

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Fig. 5. Immunological cross reactivities of caldesmon from rat cultured mesangial cells, aortic smooth muscle, and cultured dermal fibroblasts. A. SDS-polyacrylamide (9%) gel stained with Coomassie blue. Lane I: Isolated rat glomeruli. Lane 2: Rat cultured mesangial cells. Lane 3: Rat aortic smooth muscle. Lane 4: Rat cultured dermal

reactivity with aortic smooth muscle. Molecular mass of a-actinin in cultured mesangial cells was about 100 kDa, and was the same as both aortic smooth muscle and cultured dermal fibroblasts (Fig. 7). Vinculin

Vinculin in cultured mesangial cells had an immunological cross reactivity with aortic smooth muscle. Its molecular mass was about 110 kDa, which was the same as both aortic smooth conjugated anti-rabbit IgG for the second antibody. Caldesmon of muscle and cultured dermal fibroblasts (Fig. 8). fibroblasts. The bands on the left are molecular-mass standards (same as in Fig. 2). The lowest bands are the dye front. B. Western blot using rabbit anti-caldesmon antibody for the first antibody, and peroxidase-

cultured mesangial cells have an immunological cross reactivity with

aortic smooth muscle cells. Molecular mass of caldesmon in the extracts of both isolated glomeruli and cultured mesangial cells is about 60 kDa, which is the same as cultured dermal fibroblasts, and differs from aortic smooth muscle (about 120 kDa).

Desmin Desmin was detected by Western blot in the cultured mesangial cell extract, and it showed an immunological cross reactivity with aortic smooth muscle. Its molecular mass was the same

as that of smooth muscle cells (about 50 kDa). On the other hand, desmin was not detected in the extract of cultured dermal fibroblasts (Fig. 9).

contained SDS without urea), most peptides become aligned diagonally, whereas tropomyosin, which is retarded in the first Discussion dimentional gel, acquires a position outside the diagonal line In the present study, we biochemically examined several [16]. Two-dimensional urea-shift gel analysis of total proteins extracted from cultured mesangial cells indicated five isoforms contractile proteins of rat cultured mesangial cells, such as of tropomyosin (TM-i, -2, -3, -4, and -5; Fig. 2A). Among them, actomyosin, tropomyosin including isoforms, caldesmon, a-acTM-i and -4 were major isoforms (Fig. 3A). TM-i of cultured tinin, and vinculin, and compared with those of rat aortic mesangial cells had an immunological cross reactivity with smooth muscle and rat cultured dermal fibroblasts. Character/3-tropomyosin (alias TM-i) of aortic smooth muscle (Fig. 2). istics of these proteins demonstrated in our study are summaOn the other hand, in cultured dermal fibroblasts and human rized in Table i. Becker [2] reported that actomyosin was stained on mesanplatelets, TM-4 and -5 were major isoforms (Fig. 3 C and D).

TM-i of fibloblasts and platelets also cross reacted with gial cells, vascular smooth muscle, endothelium of both arteries /3-tropomyosin of aortic smooth muscle, but the reactivity of TM- 1 of fibroblasts was weaker than that of mesangial cells (data not shown). Immunofluorescent studies by using antibody against /3-tropomyosin demonstrated that TM-i was located on the stress fibers in cultured mesangial cells and cultured vascular smooth muscle cells (Fig. 4 B and C). In cultured vascular smooth muscle cells, there was an additional distribution of TM-i in the perinuclear region (Fig. 4C). On the other hand, in

cultured dermal fibroblasts, a weak staining of TM-i was observed on the stress fibers (Fig. 4D). Caldesmon

and veins, and peritubular capillaries in the human kidney. Our results revealed that cultured mesangial cells contain actomyosin as a major protein component, and therefore support his observations. It is known that mRNA of tropomyosin isoforms is produced by the alternative splicing from DNA, accompanying the evolution of cells [i8—20]. In striated and smooth muscle cells, tropomyosin is composed of two subunits, which are called aand f3-tropomyosin (alias TM-2 and -i, respectively). On the

other hand, in rat cultured non-muscle cells, there are five isoforms of tropomyosin (TM-i, -2, -3, -4, and -5), which are named according to their increasing molecular mass. Among

Caldesmon was detected by Western blot in the cultured mesangial cell extract, and it had an immunological cross

these isoforms, TM-i, -2, and -3 show higher affinity for actin

reactivity with aortic smooth muscle. Molecular mass of caldesmon from both isolated glomeruli and cultured mesangial cells was about 60 kilodalton (kDa), which was similar to cultured

detected five isoforms of tropomyosin (TM-i, -2, -3, -4, and -5),

dermal fibroblasts, and differed from aortic smooth muscle

than TM-4 and -5 [21]. In rat cultured mesangial cells, we in which TM-i, -2, -3, and -4 were predominating. We also demonstrated that TM- 1 of cultured mesangial cells had an immunological cross reactivity with /3-tropomyosin of aortic

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Ishino et al: Contractile proteins of mesangial cells

Fig. 6. Indirect immunofluorescent staining of caldesmon on rat cultured mesangial cells, vascular smooth muscle cells, and dermalfibroblasts. A and B. Rat cultured mesangial cells. C Rat cultured vascular smooth muscle cells. D. Rat cultured dermal fibroblasts. On B, C, and D, we used rabbit anti-caldesmon antibody for the first antibody, and fluorescein isothiocyanate-conjugated anti-rabbit IgG for the second antibody. On A we used only the second antibody, as a control. In vascular smooth muscle cells, caldesmon is stained strongly on the stress fibers and the perinuclear region. In mesangial cells, caldesmon is also stained on the stress fibers, but the staining intensity is weaker than that of vascular smooth muscle cells. In fibroblasts, a faint staining of caldesmon is observed on the stress fibers.

smooth muscle. In the extracts of rat cultured dermal fibroblasts and human platelets, we detected five isoforms of tropo-

150 kDa to 70 to 80 kDa in association with dedifferentiation in primary culture. In the present study, we have shown that the

myosin, with TM-4 and -5 predominating. Thus, cultured

molecular mass of caldesmon of both the extracts of rat

mesangial cells contain a larger amount of a muscle cell-related tropomyosin isoform (TM-i) than that of cultured dermal fibroblasts and platelets, although the composition of tropomyosin

cultured mesangial cells and the homogenate of isolated rat glomeruli is equal. This result indicates that the low-molecularmass form of caldesmon from cultured mesangial cells may not

isoforms of mesangial cells resembles both fibroblasts and be the result of culture conditions, and that this form may platelets in that all five isoforms are present. The presence of represent native characteristics of mesangial cells. Moreover, non-muscle forms of tropomyosin in mesangial cells may depend on some function other than contraction. Indirect immunofluorescent studies also demonstrated the tropomyosin distribution in cultured mesangial cells were similar to cultured smooth muscle cells, but were apparently different from cultured dermal fibroblasts. Caldesmon from rat cultured mesangial cells had an immunological cross reactivity with rat aortic smooth muscle cells. However, molecular mass of caldesmon in cultured mesangial cells was about 60 kDa, which was the same as cultured dermal fibroblasts, and differed from aortic smooth muscle. Caldesmon is present in a high-molecular-mass form in the smooth muscle (estimated as 120 kDa by SDS-PAGE), but a non-muscle form has about one half of the molecular mass of the smooth muscle

immunofluorescent studies showed that there are differences in

the caldesmon distribution between cultured mesangial cells and cultured smooth muscle cells. These differences observed between mesangial cells and vascular smooth muscle cells suggest that the functions of caldesmon are somewhat different between the two cells. We revealed that cultured mesangial cells contain a-actinin and vinculin, and that the molecular masses of these proteins are the same as those of aortic smooth muscle and dermal

fibroblasts, by using Western blot. These two proteins of mesangial cells, aortic smooth muscle, and dermal fibroblasts, had an immunological cross reactivity to each other. We examined whether or not the existence of desmin identifies the mesangial cells. By Western blot, we detected desmin in

form. Ueki et al [22] reported that the molecular mass of cultured mesangial cells, and could not detect it in cultured caldesmon of aortic smooth muscle cells was 120 to 150 kDa, and that the molecular mass of these cells reduced from 120 to

dermal fibroblasts. Desmin is classified into intermediate filament protein. Bachmann et a! [23] demonstrated the coexis-

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Fig. 7. Immunological cross reactivities of a-actinin from rat cultured mesangial cells, aortic smooth muscle, and cultured dermalfibroblasts. A. SDS-polyacrylamide (7%) gel stained with Coomassie blue. Lane 1: Rat cultured mesangial cells. Lane 2: Rat aortic smooth muscle. Lane 3: Rat cultured dermal fibroblasts. The bands on the left are molecular mass standards in kDa. The lowest bands are the dye front. B. Western

blot using rabbit anti-a-actinin antibody for the first antibody, and peroxidase-conjugated anti-rabbit IgG for the second antibody. Cultured mesangial cells and cultured dermal fibroblasts have a-actinin, which has an immunological cross reactivity with that of aortic smooth muscle.

A

B

Rat cultured mesangial cells. Lane 2: Rat aortic smooth muscle. Lane 3:

Rat cultured dermal fibroblasts. The bands on the left are molecular mass standards (same as in Fig. 7). The lowest bands are the dye front. B. Western blot using rabbit anti-vinculin antibody for the first antibody, and peroxidase-conjugated anti-rabbit IgG antibody for the second antibody. Cultured mesangial cells and cultured dermal fibroblasts have vinculin, which has an immunological cross reactivity with that of aortic smooth muscle.

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Rat cultured mesangial cells. Lane 2: Rat aortic smooth muscle. Lane 3: Rat cultured dermal fibroblasts. The bands on the left are molecularmass standards (same as in Fig. 2). The lowest bands are the dye front. B. Western blot using rabbit anti-desmin antibody for the first antibody,

and peroxidase-conjugated anti-rabbit IgG for the second antibody. Cultured mesangial cells have desmin, which has an immunological cross reactivity with that of aortic smooth muscle. In cultured dermal fibroblasts, desmin is not detected.

Table 1. Characteristics of contractile proteins of rat cultured mesangial cells

Actomyosin Actin binding proteins Tropomyosin Caldesmon a-Actinin Vinculin

Fig. 8. Immunological cross reactivities of vinculin from rat cultured mesangial cells, aortic smooth muscle, and cultured dermalfibroblasts. A. SDS-polyacrylamide (7%) gel stained with Coomassie blue. Lane 1:

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Fig. 9. Immunological cross-reactivities of desmin from rat cultured mesangial cells, aortic smooth muscle, and cultured dermalfibroblasts. A. SDS-polyacrylamide (9%) gel stained with Coomassie blue. Lane 1:

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Rat cultured mesangial cells

Present as a major component

Smooth muscle form> non-muscle form Non-muscle form Smooth muscle form Smooth muscle form

distinguished in vitro from fibroblasts, by indirect immunofluorescent studies by using antibody against desmin. From their and our results, mesangial cells can be clearly distinguished from fibroblasts by the presence of desmin. In conclusion, mesangial cells abound in contractile proteins,

which have immunological cross reactivities with those of aortic smooth muscle cells, and the composition of these proteins are similar to those of aortic smooth muscle with minor differences. By weighing these similarities and differences between mesangial cells and smooth muscle cells, we suggest that mesangial cells are derived from vascular smooth muscle cells, but with a minor modification in their evolution. Acknowledgment A portion of this study was presented at the XIth International Congress of Nephrology, in Tokyo, Japan, July 1990.

tence of vimentin and desmin only on glomerular and extraglomerular mesangial cells among cells in the bovine glomeruli.

Yaoita et al [61 also stated that mesangial cells could be

Reprint requests to Takako Ishino, M.D., Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita-shi, Akita 010, Japan.

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Ishino et a!: Contractile proteins of mesangial cells

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