Biomedical subjects

M F White

Publications and source records attributed to M F White.

At least 199 records · Page 11Linked to original sources

PubMed1989

Tyrosine phosphorylation of the insulin receptor during insulin-stimulated internalization in rat hepatoma cells.

We have studied the phosphorylation state of the insulin receptor during receptor-mediated endocytosis in the well-differentiated rat hepatoma cell line Fao. Insulin induced the rapid internalization of surface-iodinated insulin receptors into a trypsin-resistant compartment, with a 3-fold increase in the internalization rate over that seen in the absence of insulin. Within 20 min of insulin stimulation, 30-35% of surface receptors were located inside the cell. This redistribution was half-maximal by 10.5 min. Similar results were obtained when the loss of surface receptors was measured by 125I-insulin binding. Tyrosyl phosphorylation of internalized insulin receptors was measured by immunoprecipitation with antiphosphotyrosine antibody. Immediately after insulin stimulation, 70-80% of internalized receptors were tyrosine phosphorylated. Internalized receptors persisted in a phosphorylated state after the dissociation of insulin but were dephosphorylated prior to their return to the plasma membrane. After 45-60 min of insulin stimulation, the tyrosine phosphorylation of the internal receptor pool decreased by 45%, whereas the phosphorylation of surface receptors was unchanged. These data suggest that insulin induces the internalization of phosphorylated insulin receptors into the cell and that the phosphorylation state of the internal receptor pool may be regulated by insulin.

Algorithms↗

PubMed1989

Cascade of autophosphorylation in the beta-subunit of the insulin receptor.

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.

Animals↗

PubMed1989

Interleukin 1 stimulates prostaglandin synthesis and cyclic AMP accumulation in Swiss 3T3 fibroblasts: interactions between two second messenger systems.

Biochemical events elicited by interleukin 1 (IL-1) were studied in Swiss 3T3 fibroblasts. One hour after its addition, IL-1 stimulated synthesis of prostaglandin E2 (PGE2), which continued to accumulate for 4 days. IL-1 also stimulated cAMP accumulation. Indomethacin blocked cAMP accumulation in response to IL-1, suggesting that PGE2 was responsible for the increase. Addition of exogenous PGE2 to indomethacin-treated cells restored cAMP accumulation. IL-1 enhanced thymidine incorporation, and indomethacin attenuated responses to lower concentrations. Thus, PGE2 appeared to play a role in the ability of low concentrations of IL-1 to stimulate thymidine incorporation. PGE2 augmented thymidine incorporation by increasing cAMP accumulation because in the presence of indomethacin addition of exogenous cAMP enhanced thymidine incorporation in response to low concentrations of IL-1. Elevated cAMP further stimulated PGE2 synthesis. Thus, PGE2 and cAMP interacted to potentiate their mutual accumulation. In summary, IL-1 stimulates PGE2 synthesis. PGE2, in turn, stimulates cAMP accumulation which potentiates IL-1-stimulated PGE2 synthesis and thymidine incorporation.

Animals↗

PubMed1989

Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells.

The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or [32P]phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of 125I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.

2,4-Dinitrophenol↗

PubMed1989

Human insulin receptors expressed in insulin-insensitive mouse fibroblasts couple with extant cellular effector systems to confer insulin sensitivity and responsiveness.

When cDNA for human kidney insulin receptors was used to transfect NIH3T3 mouse fibroblast cells with few or no endogenous insulin receptors, a resultant cell line, 3T3/HIR, expressed more than 6 million receptors/cell. Results of the present study demonstrated that these human receptors in murine cells mediated a diverse group of responses, including insulin binding and internalization as well as insulin-stimulated tyrosine phosphorylation of the receptor and a putative cellular substrate pp185. In addition, the cells were stimulated by insulin in various acute and long term metabolic processes, including glucose transport, glycogen formation, amino acid uptake, and thymidine uptake and incorporation into DNA. There were weak or no responses to insulin in control fibroblasts transfected only with the pSV2Neo plasmid containing a bacterial gene for neomycin resistance (3T3/NEO cells). These findings indicated that transfection of insulin receptor cDNA conferred insulin sensitivity to the target cells in a broad range of cellular responses and further demonstrated that effector molecules for mediating such responses were present in cells that normally lacked sensitivity to this hormone. Expressed receptors readily coupled with the effector systems to become fully functional.

Amino Acids↗

PubMed1989

Differentiation-dependent phosphorylation of a 175,000 molecular weight protein in response to insulin and insulin-like growth factor-I in L6 skeletal muscle cells.

Insulin and insulin-like growth factor-I (IGF-I) effects on protein phosphorylation were investigated in intact skeletal muscle cells at different stages of differentiation. In undifferentiated L6 myoblasts, stimulation by either insulin or IGF-I, but not IGF-II, led to a 3- to 5-fold increase in phosphorylation of insulin and IGF receptor beta-subunits and the appearance of a 175,000 mol wt (Mr) phosphoprotein (pp175). These effects reached a maximum within 3 min, were maintained for 12 min, and then declined. Dose-response curves for pp175 phosphorylation in response to insulin (ED50 = 2 nM) and IGF-I (ED50 = 0.2 nM) were consistent with occupancy and stimulation of each receptor kinase by its specific hormone. The 175,000 Mr phosphoprotein was not precipitated by antireceptor antibodies, and the phosphoamino acid composition differed markedly from that of insulin and IGF-I receptors, with a 10-fold lower phosphotyrosine/phosphoserine ratio after insulin stimulation. In contrast to insulin and IGF-I receptors, pp175 was not extracted by the nonionic detergent Triton X-100, but required sodium dodecyl sulfate for solubilization. When experiments were carried out with L6 cells after differentiation into skeletal muscle myotubes, hormone-induced phosphorylation of pp175 was almost undetectable. We conclude that pp175 is a phosphoprotein distinct from insulin and IGF-I receptors that is involved in the early phosphorylation events that follow the activation of the insulin and IGF-I receptor kinases. Its disappearance after terminal differentiation of the L6 cells is consistent with a role in hormonal stimulation of cell proliferation.

Amino Acids↗

PubMed1989

Tyrosine-kinase defect of the insulin receptor in cultured fibroblasts from patients with lipoatropic diabetes.

Postbinding defects in insulin action were described previously in cultured fibroblasts from six patients with lipoatropic diabetes. To define the contribution of the insulin receptor tyrosine kinase in these defects, we studied autophosphorylation and kinase activity of lectin purified receptors from these six patients and six normal cell lines. The patients' insulin receptors, prepared by precipitation with polyethylene glycol, had normal insulin binding characteristics and autophosphorylation properties, but a 56% decrease in the tyrosine kinase activity toward an exogenous substrate. To identify more subtle qualitative defects in autophosphorylation, insulin receptors were sequentially immunoprecipitated and analyzed for their phosphoaminoacid content. The phosphorylated receptors precipitated with an antiphosphotyrosine antibody contained labeled phosphotyrosine, whereas those in the supernatant, when further precipitated with an antireceptor antibody, contained only phosphoserine. Under these conditions, the insulin-stimulated autophosphorylation of tyrosine was significantly decreased by 54% in the patient receptors compared to normal subjects' receptors. In addition, insulin-like growth factor-I stimulation of autophosphorylation of its receptor was reduced by 59% in the patients' cells compared to those from normal subjects. We conclude that fibroblasts from patients with lipoatropic diabetes have defects in the tyrosine kinase activity of their insulin and their insulin-like growth factor-I receptors that might give rise to the in vitro hormone resistance and be related to the in vivo hormone resistance that occurs in these patients.

Adolescent↗

PubMed1989

Recombinant human müllerian inhibiting substance inhibits epidermal growth factor receptor tyrosine kinase.

Autophosphorylation of the epidermal growth factor (EGF) receptor in A-431 cells and plasma membrane fractions was inhibited by partially purified recombinant human Müllerian Inhibiting Substance (MIS). Immunoprecipitation of the EFG receptor using anti-EGF receptor or anti-phosphotyrosine antibodies, and phosphoamino acid analysis of this receptor, demonstrated that MIS specifically inhibited EGF-induced tyrosine phosphorylation. Inhibition of EGF receptor autophosphorylation by MIS in membrane preparations was not affected by increasing concentrations of EGF, manganese or [gamma-(32)P] ATP. Thus, it is unlikely that MIS competes for EGF binding sites or sequesters substrate. Immunoabsorption of MIS with anti-human MIS antibody blocked the MIS inhibition of EGF receptor autophosphorylation, indicating that the inhibition was due to MIS. Our data suggest that MIS regulates the activity of the EGF receptor tyrosine kinase in A-431 cells.

Amino Acids↗

PubMed1989

Eye injury: prevalence and prognosis by setting.

Although an estimated 1 million Americans suffer ocular injuries each year, the setting of injury and its prognostic implications have not been closely examined. Using data compiled by the Eye Injury Registry of Alabama (EIRA) from 514 cases of serious eye injury, we examined the demographics and prognosis of ocular injury by setting of injury. Work-related injuries accounted for only 28% of total injuries, and injuries occurring at home accounted for 27%, followed by situations related to recreation (25%), assault (11%), travel (5%), and "other" (school, unknown, etc) (4%). The poorest initial vision, poorest final vision, and highest rate of enucleation occurred in patients injured by assault, whereas the lowest rate of enucleation and loss of light perception was found in patients who had work-related injuries. Patients in the "other" category had the highest rate of return to 20/100 or better vision.

Accidents, Home↗

PubMed1988

Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity.

Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.

Amino Acid Sequence↗

PubMed1988

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells.

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.

Animals↗

PubMed1988

Tryptic activation of the insulin receptor. Proteolytic truncation of the alpha-subunit releases the beta-subunit from inhibitory control.

Trypsin exerts insulin-like effects in intact cells and on partially purified preparations of insulin receptors. To elucidate the mechanism of these insulinomimetic effects, we compared the structures of insulin- and trypsin-activated receptor species with their functions, including insulin binding, autophosphorylation, and tyrosine kinase activity. In vitro treatment of wheat germ agglutinin-purified receptor preparations with trypsin resulted in proteolysis of both alpha- and beta-subunits. The activated form of the receptor had an apparent molecular mass of 110 kDa under nonreducing conditions, compared to the 400-kDa intact receptor, and was separated following reduction into an 85-kDa beta-subunit related fragment and a 25-kDa alpha-subunit related fragment. Treatment of whole cells with trypsin prior to isolation of the insulin receptor resulted in proteolytic modification of the alpha-subunit only. In this case, the total molecular mass of the activated species was 116 kDa, comprised of an intact 92-kDa beta-subunit and again a 25-kDa alpha-subunit related fragment. Values of Km for peptide substrate phosphorylation and Ki for inhibition of receptor autophosphorylation, and sites of autophosphorylation within the beta-subunits were similar for receptors activated either by insulin or trypsin. Insulin had no additional effect on the rate of autophosphorylation of the truncated receptor, and no binding of insulin by the truncated receptor was detected either by direct assay or cross-linking with bifunctional reagents. Based on the deduced amino acid sequence of the insulin receptor and the structural studies presented here we concluded that this activated form of the receptor resulted from tryptic cleavage at the dibasic site Arg576-Arg577. This was accompanied by loss of the insulin binding site and separation of alpha-beta heterodimers. As truncation of the alpha-subunit results in beta-subunit activation, it appears that the beta-subunit is a constitutively activated kinase and that the function of the alpha-subunit in the intact receptor is to inhibit the beta-subunit.

Cell Line↗

PubMed1988

Sequence of the gene encoding phosphoglycerate mutase from Saccharomyces cerevisiae.

The gene encoding yeast phosphoglycerate mutase was isolated, and its sequence was determined. The gene specifies a protein of 246 amino acids, and contains no introns. The sequence shows a strong codon bias. The upstream untranslated portion of the gene contains a CT-rich block such as is found in many highly expressed yeast genes, but does not have the associated CAAG sequence.

Amino Acid Sequence↗

PubMed1988

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function of the insulin receptor.

Adenosine Triphosphate↗

PubMed1988

A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor.

We identified the major autophosphorylation sites in the insulin receptor and correlated their phosphorylation with the phosphotransferase activity of the receptor on synthetic peptides. The receptor, purified from Fao hepatoma cells on immobilized wheat germ agglutinin, undergoes autophosphorylation at several tyrosine residues in its beta-subunit; however, anti-phosphotyrosine antibody (alpha-PY) inhibited most of the phosphorylation by trapping the initial sites in an inactive complex. Exhaustive trypsin digestion of the inhibited beta-subunit yielded two peptides derived from the Tyr-1150 domain (Ullrich, A, Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) called pY4 and pY5. Both peptides contained 2 phosphotyrosyl residues (2Tyr(P], one corresponding to Tyr-1146 and the other to Tyr-1150 or Tyr-1151. In the absence of the alpha-PY additional sites were phosphorylated. The C-terminal domain of the beta-subunit contained phosphotyrosine at Tyr-1316 and Tyr-1322. Removal of the C-terminal domain by mild trypsinolysis did not affect the phosphotransferase activity of the beta-subunit suggesting that these sites did not play a regulatory role. Full activation of the insulin receptor during in vitro assay correlated with the appearance of two phosphopeptides in the tryptic digest of the beta-subunit, pY1 and pY1a, that were inhibited by the alpha-PY. Structural analysis suggested that pY1 and pY1a were derived from the Tyr-1150 domain and contained 3 phosphotyrosyl residues (3Tyr(P] corresponding to Tyr-1146, Tyr-1150, and Tyr-1151. The phosphotransferase of the receptor that was phosphorylated in the presence of alpha-PY at 2 tyrosyl residues in the Tyr-1150 domain was not fully activated during kinase assays carried out with saturating substrate concentrations which inhibited further autophosphorylation. During insulin stimulation of the intact cell, the 3Tyr(P) form of the Tyr-1150 domain was barely detected, whereas the 2Tyr(P) form predominated. We conclude that 1) autophosphorylation of the insulin receptor begins by phosphorylation of Tyr-1146 and either Tyr-1150 or Tyr-1151; 2) progression of the cascade to phosphorylation of the third tyrosyl residue fully activates the phosphotransferase during in vitro assay; 3) in vivo, the 2Tyr(P) form predominates, suggesting that progression of the autophosphorylation cascade to the 3Tyr(P) form is regulated during insulin stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗

PubMed1988

Field evaluation of polycarbonate versus conventional safety glasses.

Of all the causes of eye injury, gunshot has recently been cited as resulting in the highest rate of blindness and the lowest rate of visual recovery. Many of these cases are hunting accidents in which safety glasses could have prevented or lessened ocular damage. To evaluate the effectiveness of various types of safety glasses, we fired a Remington automatic shotgun at mannequin heads fitted with one of four types of safety lenses. From a distance of 10 yards, only polycarbonate lenses provided even partial protection. At 30 yards, polycarbonate and heat-treated lenses provided the best protection, whereas chemically treated and CR39 lenses provided significantly less protection. We recommend that hunters using shotguns in areas of poor visibility wear widely available polycarbonate safety glasses as well as "hunter's orange" to minimize their risk of devastating ocular gunshot injury.

Accidents↗

PubMed1988

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.

Animals↗

PubMed1988

Decreased autophosphorylation of EGF receptor in insulin-deficient diabetic rats.

We have previously reported that despite an increase in receptor concentration, there is a decrease in autophosphorylation and tyrosine kinase activity of the insulin receptor in insulin-deficient diabetic rats. To determine if other tyrosine kinases might be altered, we have studied the epidermal growth factor (EGF) receptor kinase in wheat germ agglutinin-purified, Triton X-100-solubilized liver membranes from streptozotocin (STZ)-induced diabetic rats and the insulin-deficient BB rat. We find that autophosphorylation of EGF receptor is decreased in proportion to the severity of the diabetic state in STZ rats with a maximal decrease of 67% (P less than 0.01). A similar decrease in autophosphorylation was observed in diabetic BB rats that was partially normalized by insulin treatment. Separation of tryptic phosphopeptides by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. A parallel decrease in EGF receptor phosphorylation was also found by immunoblotting with an anti-phosphotyrosine antibody. EGF receptor concentration, determined by Scatchard analysis of 125I-labeled EGF binding, was decreased by 39% in the STZ rat (P less than 0.05) and 27% in the diabetic BB rat (not significant). Thus autophosphorylation of EGF receptor, like that of the insulin receptor, is decreased in insulin-deficient rat liver. In the case of EGF receptor, this is due in part to a decrease in receptor number and in part to a decrease in the specific activity of the kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗