Biomedical subjects

R Tokunaga

Publications and source records attributed to R Tokunaga.

At least 55 records · Page 3Linked to original sources

PubMed1994

Molecular cloning, sequencing and expression of cDNA encoding human coproporphyrinogen oxidase.

A complete cDNA clone encoding human coproporphyrinogen (coprogen) oxidase, the sixth enzyme in the heme biosynthetic pathway, has been isolated from a human placenta cDNA library. The cDNA had an open reading frame of 1062 base pairs encoding a protein of 354 amino acid residues (M(r) 40,291). Amino acid sequencing showed that the mature enzyme consists of 323 amino acid residues (M(r) 36,842) with a putative leader peptide of 31 amino acid residues. The human enzyme showed an 86% identity to the mouse enzyme. In addition, the recombinant enzyme which did not contain leader peptide was actively expressed in Escherichia coli. The isolation and expression of cDNA for human coprogen oxidase should facilitate studies of the structure of the gene as well as characterization of molecular lesions causing hereditary coproporphyria.

Amino Acid Sequence↗

PubMed1994

Increased cell surface expression of a newly identified heterodimer on activated blastic T cells.

A hybridoma (F6C7) was established by fusing NS1 cells with spleen cells of MRL/Mp-+/+ (MRL/+) mice suffering from lpr-GVHD. This F6C7 mAb (IgG2b, kappa) stains a broad spectrum of blood cells at varying intensities in mice and rats. In normal (BALB/c) mice, granulocytes and B cells are highly positive for F6C7-reactive Ag (F6C7-Ag). Thymocytes and peripheral (CD4+ and CD8+) T cells show negative to low intensities. These staining profiles are similar in C57BL/6, AKR/J, C3H/HeJ, and MRL/+ mice. When spleen cells were activated in vitro, a blastic cell population of autoactivated CD4+ and CD8+ T cells showed increased F6C7-Ag expression. Alloactivated CD4+ blastic T cells also showed increased expression of F6C7-Ag, whereas alloactivated CD8+ blastic T cells as well as Con A-activated CD4+ and CD8+ blastic T cells remained at the level of small (nonblastic) cells. These findings suggest that the surface expression of F6C7-Ag is up-regulated in some activation processes of T cells, particularly in autoactivation. Young (2-month-old) MRL/Mp-lpr/lpr (MRL/lpr) mice show staining profiles of F6C7-Ag similar to those of normal mice, except that many more blastic (CD4+ and CD8+) T cells show high F6C7-Ag expression than those of normal mice. A small but significant number of CD4+F6C7-Ag(high) and a much higher number of CD8+F6C7-Ag(high) blastic T cells were observed in the spleen cells of MRL/+ mice suffering from lpr-GVHD. These blastic T cells may exert autoreactivity and participate in the initiation of autoimmune diseases, lymphadenopathy, and lpr-GVHD. Immunoprecipitation and SDS-PAGE revealed that F6C7-Ag is a heterodimer comprised of approximately 78- and 70-kDa molecules without disulfide bonds.

Animals↗

PubMed1994

Functional analysis of thymic B cells.

The expression of low- and high-affinity interleukin-5 receptors (IL-5Rs) on thymic B cells and reactivity of thymic B cells to IL-5 was investigated. Thymic B cells consist of two populations (CD5+ and CD5- B cells), as previously described. Three-color FACS analyses using anti-CD5, anti-IgK, and anti-IL-5R mAbs reveal that approximately 60% of both populations (CD5+ and CD5-) in the thymus possess IL-5R, detected by mAb H-7. In Scatchard plot analyses, IL-5Rs on thymic B cells are observed as low affinity receptors; the high-affinity IL-5R, which is known to be expressed on some IL-5-activated splenic B blasts or some IL-5-dependent cell line cells, is not clearly detected on thymic B cells. The reactivity of thymic B cells to IL-5 is found to be significantly lower than that of splenic B cells both in proliferative responses and LPS-induced IgM and IgA antibody responses. These findings are compatible with the expression of the low-affinity IL-5R on thymic B cells. The responsiveness of thymic B cells to either IL-6 or the combination of IL-4, IL-5 and IL-6 is also lower than that of splenic B cells. Furthermore, the thymic B cells are found to induce neonatal tolerance. Therefore, thymic B cells act as antigen-presenting cells in the negative selection of thymocytes, rather than as antibody-producing cells under the influence of foreign antigens and/or regulatory cytokines.

Animals↗

PubMed1994

A murine nephritogenic monoclonal antibody binds to both single-stranded deoxyribonucleic acid and glomerulus.

BACKGROUND: Autoantibodies such as anti-DNA and antimyeloperoxidase (MPO) antibodies have been shown to cause glomerulonephritis in experimental animal models. To analyze pathogenic autoantibodies, we developed hybridomas from spleen cells of nontreated FGS mice, in which focal segmental glomerular sclerosis develops spontaneously. EXPERIMENTAL DESIGN: Reactivity and specificity of a monoclonal antibody (FG1H5) were examined using enzyme-linked immunosorbent assay and cryosections of mouse organs as substrates. Immunoprecipitation was performed to analyze reactive antigens. Hybridoma cells were injected ip into severe combined immunodeficiency (SCID) mice to examine their nephritogenicity in vivo. RESULTS: The binding of FG1H5 to single-stranded DNA (ssDNA) was inhibited by ssDNA and also MPO. The binding of FG1H5 to MPO was weak, not inhibited by MPO, and markedly enhanced by the presence of ssDNA. This marked enhancement of the binding to MPO was abolished by DNase I-treatment of the mixture of FG1H5 and ssDNA. When MPO was introduced into ssDNA-coated wells, the binding of FG1H5 to ssDNA was inhibited. On the other hand, when ssDNA was introduced into MPO-coated wells, the binding of FG1H5 to MPO was markedly enhanced. Inhibition tests using double-stranded DNA revealed that FG1H5 is specific for ssDNA. Histologic examination of FG1H5-reactive antigen using SCID mouse kidney showed positive stainings in the nucleus and glomerulus (mainly the mesangium). These positive stainings were abolished after the incubation of FG1H5 with ssDNA. The DNase I treatment of kidney sections markedly reduced the nuclear staining, but the staining of the glomerulus was preserved. Immunoprecipitation of a soluble fraction of SCID mouse kidney with FG1H5 revealed that FG1H5-reactive antigen in the glomerulus is an approximately 28-kilodalton molecule. When FG1H5 hybridoma cells were injected ip into SCID mice, the mice showed glomerulonephritis with the increases in mesangial cells and matrix as well as immunoglobulin M deposition mainly in the mesangium. CONCLUSIONS: Data demonstrate that FG1H5 binds strongly and specifically to ssDNA (but weakly and nonspecifically to MPO), and that ssDNA and MPO bind to each other. One monoclonal antibody reacts with both the nucleus and glomerulus (mainly the mesangium), and glomerular staining is not caused by nonspecific DNA binding. FG1H5, which binds to ssDNA, can induce glomerulonephritis, probably because of a direct crossreactivity to glomerular components.

Animals↗

PubMed1993

Coproporphyrinogen oxidase. Purification, molecular cloning, and induction of mRNA during erythroid differentiation.

Coproporphyrinogen oxidase (EC 1.3.3.3), the enzyme involved in the sixth step of heme biosynthesis, was purified to apparent homogeneity from bovine liver; it has a molecular mass of 37,000 daltons. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequences of trypsin-digested peptides were used in a polymerase chain reaction to amplify a 198-base pair fragment of coproporphyrinogen oxidase DNA, using bovine kidney cells cDNA as a starting template. This fragment was used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library. Sequence analysis revealed that coproporphyrinogen oxidase comprises 354 amino acid residues (M(r) 40,647), with a putative leader sequence of 31 amino acid residues, the result being a mature protein of 323 amino acid residues (M(r) 37,255). RNA blot analysis revealed a 3.0-kilobase coproporphyrinogen oxidase mRNA in mouse liver and in MEL cells. Treatment of MEL cells with dimethyl sulfoxide led to an increase in coproporphyrinogen oxidase mRNA within 10 h, the induction reached a maximum at 24 h, and was in parallel with the induction of ferrochelatase mRNA. The cDNA allows for the expression of active coproporphyrinogen oxidase, the activity of which is mainly present in mitochondria of transfected cultured cells, thereby indicating that mammalian coproporphyrinogen oxidase is mitochondrial enzyme.

Amino Acid Sequence↗

PubMed1993

A monoclonal antibody reactive with a glycophosphatidylinositol-anchored molecule on T cells defines CD4+ T cell subsets.

A hybridoma, 25T3 (IgM, kappa), was established from MRL/+ mice immunized with an autoreactive T cell line (l/+T1). The antigenicity of the antigen recognized by hybridoma 25T3 (25T3-Ag) expressed on thymic and splenic cells was abolished by treatment with phosphatidylinositol-specific phospholipase C, showing that 25T3-Ag is a glycophosphatidylinositol-anchored Ag. 25T3-Ag was expressed on approximately 90% of thymocytes. Double-negative, double-positive and CD8 single-positive cells were highly positive for the expression of 25T3-Ag, whereas CD4 single-positive cells were weakly positive (approximately 40%) or negative (approximately 60%). In the spleen, only CD3+ cells (and not B220+ nor Mac-1+ cells) reacted with 25T3 monoclonal antibody (mAb), indicating that 25T3 mAb is specific for T cells. The majority of splenic CD8+ T cells were positive for the expression of 25T3-Ag, although the intensity was weaker than that of thymocytes. In contrast, splenic CD4+ T cells were divided into negative (60-70%) and positive (30-40%) populations. Similar staining profiles were observed in BALB/c, C57BL/6, C3H/HeN and AKR/J mice. When BALB/c CD4+ T cell subsets were sorted and cultured with irradiated (25 Gy) antigen-presenting cells, stimulation with immobilized anti-CD3 mAb for 2 days resulted in CD4+25T3+ cells secreting more interleukin-2 and less interleukin-4 than did CD4+25T3- subsets, although the proliferative responses of the cells on day 2 of culture were similar. This suggests that CD4+ T cells can be divided into two populations and relatively defined as T helper 1 and T helper 2 cells using this 25T3 mAb. Immunoprecipitation and SDS-PAGE revealed that 25T3-Ag was approximately 70 kDa. These findings are discussed in relation to CD4+ T cell subsets.

Animals↗

PubMed1993

The effect of lead on iron uptake from transferrin in human erythroleukemia (K562) cells.

The effect of lead on cellular iron metabolism has been investigated using human erythroleukemia (K562) cells. When the cells were cultured with 100 microM Pb2+ for 48 h, the rate of cellular iron uptake from transferrin decreased to 46% of that in untreated cells. Scatchard analysis of the binding data revealed that this reduction was the result of a decrease in the number of transferrin receptors rather than an alteration in ligand-receptor affinity. The results of immunoprecipitation of transferrin receptors on the cell surface also confirmed the decreased expression of transferrin receptors by lead-treated cells. The down-regulation of transferrin receptors by treatment with lead did not result from a decrease in the total amount of the receptor, as determined by immunoblotting. Moreover, the biosynthesis of the receptor was unaffected by lead treatment. Thus, the down-regulation of surface transferrin receptors in lead-treated cells might be due to a redistribution of receptors rather than an actual loss of receptors from the cell. Using kinetic analysis, it was shown that redistribution of the receptor did not result from the alteration in the rates of transferrin receptor recycling. A comparison of the amounts of transferrin receptor on the cell surface and in the cycling pool revealed that the sequestration of the receptor from normal flow through the cycle might cause down-regulation of the surface receptor.

Biological Transport↗

PubMed1992

Expression of haptoglobin receptors in human hepatoma cells.

The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.

Biological Transport↗

PubMed1992

Structure of the human ferrochelatase gene. Exon/intron gene organization and location of the gene to chromosome 18.

We have determined the structure of the human ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. This gene was assigned to human chromosome 18 at region q21.3, by fluorescent in situ hybridization. The gene contains a total of 11 exons and has a minimum size of about 45 kb. The exon/intron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to encode functional protein domains. A major site of the transcription initiation, determined by S1 nuclease mapping, was assigned to an adenine base 89 bases upstream from the adenine base of the translation initiation ATG. The promoter region contains a potential binding site for Sp1, NF-E2 and erythroid-specific transcriptional factor GATA-1, but not a typical TATAA or CCAAT sequence. Analysis of primer extension showed that the transcription starts at the same position between hepatoma HepG2 and erythroleukemia K562 cell mRNA, thereby suggesting that there can be a single transcript in erythroid and non-erythroid cells.

Base Sequence↗

PubMed1992

Ribosomal protein P2, a novel iron-binding protein.

We examined the properties of a new iron-binding protein purified previously from rat liver (T. Furukawa, S. Taketani, H. Kohno, and R. Tokunaga, 1991, Biochem. Biophys. Res. Commun. 181, 409-415). The protein was digested with trypsin and the peptides were analyzed by reverse-phase high-performance liquid chromatography. The partial amino acid sequences of the tryptic peptides coincided with that of rat ribosomal protein P2. Immunoblot analysis and iron-binding assay confirmed that the iron-binding protein and ribosomal protein P2 are identical. Then the iron binding ability of ribosomal protein P2 was examined in rat hepatoma H4IIEC3 cells incubated with radioactive iron. When immunoprecipitation with anti-iron-binding protein serum was performed using cells incubated with 59Fe-citrate, about 4% of the 59Fe radioactivity in cells was associated with the iron-binding protein through 30 to 90 min of incubation. About 1.5% of radioactive iron in cells incubated with 59Fe-transferrin was found in immunoprecipitates with anti-iron-binding protein serum during 1 to 5 h of incubation, and 4 to 7% of the radioactivity was found in immunoprecipitates with a monoclonal antibody against ribosomal P proteins in the same incubation. These results demonstrate that ribosomal proteins P2 binds iron taken up by the cells.

Amino Acid Sequence↗

PubMed1992

Iron deprivation decreases ribonucleotide reductase activity and DNA synthesis.

The effects of the iron-chelator, desferrioxamine, and monoclonal antibodies against transferrin receptors on DNA synthesis and ribonucleotide reductase activity were examined in human leukemia K562 cells. Treatment of the cells with desferrioxamine resulted in decreases of ribonucleotide reductase activity, DNA synthesis, and cell growth. Exposure of the cells to anti-transferrin receptor antibody, 42/6, which blocks iron supplement into cells caused decreases of ribonucleotide reductase activity and DNA synthesis, in a parallel fashion. Decreases of ribonucleotide reductase activity and DNA synthesis by 42/6 were restored by the addition of ferric nitriloacetate. These results indicate that ribonucleotide reductase activity is dependent on the iron-supply and also regulates cell proliferation.

Antibodies, Monoclonal↗

PubMed1991

A newly identified iron-binding protein in rat liver: purification and characterization.

A novel iron-binding protein from rat liver homogenates was purified 1,800-fold with a 5.7% yield, to apparent homogeneity. The molecular weight of the protein was estimated to be 16,000, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein exhibited 0.43 mol of iron binding per mol of protein with a dissociation constant (Kd) of 3.5 x 10(-6) M. Al3+ inhibited the iron-binding and the binding was also slightly inhibited by Ni2+. Other divalent metal ions such as Cu2+, Zn2+ and Mn2+ were without effect. Immunoblot analysis of the iron-binding protein revealed that the protein is located mainly in microsomes. This newly identified iron-binding protein may be involved in intracellular transport of iron.

Animals↗

PubMed1991

Endothelin-3 inhibits ganglionic transmission at preganglionic sites through activation of endogenous thromboxane A2 production in dog cardiac sympathetic ganglia.

The effects of endothelin-3 (ET-3) on ganglionic transmission of dog cardiac sympathetic ganglia and possible mechanisms involved were investigated in vivo and in vitro. Positive chronotropic responses to preganglionic stellate stimulation and those to dimethylphenylpiperazinium as well as McN-A-343 administered to the ganglia were inhibited by ET-3. The amount of acetylcholine released by preganglionic stimulation was reduced dose dependently after exposure to ET-3. The reduction elicited by ET-3 was antagonized by pretreatment with phospholipase A2 inhibitors (dexamethasone and methylprednisolone) and cyclooxygenase inhibitors (aspirin and indomethacin). In addition, the reduction of acetylcholine release was similarly induced by exposure to exogenously applied STA2, a stable thromboxane A2 analogue; U-46619, a TXA2/PGH2 receptor agonist; and prostaglandin E2. Furthermore, the reduction produced by ET-3 was antagonized by pretreatment with a thromboxane A2 synthetase inhibitor (OKY-046) and a specific thromboxane A2 receptor antagonist (S-145), but not by a specific prostaglandin E2 receptor antagonist (SC-19220). These results indicate that ET-3 inhibits the sympathetic ganglionic transmission via reducing acetylcholine release from the presynaptic nerve terminals of ganglia and that this inhibition involves the activation of endogenous thromboxane A2 production.

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗

PubMed1991

Selenium antagonizes the induction of human heme oxygenase by arsenite and cadmium ions.

Effects of selenium compounds on the induction of heme oxygenase in human cells exposed to sodium arsenite or cadmium chloride have been investigated by an immunoblotting technique. Exposure of HeLa cells to arsenite or cadmium ions caused a marked increase in the synthesis of heme oxygenase, and the presence of sodium selenite suppressed the induction. DL-Selenocystine was an effective suppressor, and sodium selenate was less effective. DL-Selenomethionine had no effect. Northern blot analysis showed that selenite abolished the induction of heme oxygenase mRNA in the cells exposed to arsenite or cadmium ions. These results indicated that selenium antagonizes the induction of heme oxygenase by heavy metals ions.

Arsenic↗

PubMed1990

Molecular cloning and sequence analysis of cDNA encoding human ferrochelatase.

The cDNA encoding human ferrochelatase [EC 4.99.1.1] was isolated from a human placenta cDNA library in bacteriophage lambda gt11 by screening with a radiolabeled fragment of mouse ferrochelatase cDNA. The cDNA had an open reading frame of 1269 base pairs (bp) encoding a protein of 423 amino acid residues (Mr. 47,833) with alternative putative polyadenylation signals in the 3' non-coding regions and poly (A) tails. Amino acid sequencing showed that the mature protein consists of 369 amino acid residues (Mr. 42,158) with a putative leader sequence of 54 amino acid residues. The human enzyme showed an 88% identity to mouse enzyme and 46% to yeast enzyme. Northern blot analysis showed two mRNAs of about 2500 and 1600 bp for ferrochelatase in K562 and HepG2 cells. As full-length cDNA for human ferrochelatase is now available, molecular lesions related to erythropoietic protoporphyria can be characterized.

Amino Acid Sequence↗

PubMed1990

Molecular cloning, sequencing, and expression of mouse ferrochelatase.

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.

Amino Acid Sequence↗

PubMed1990

Hemopexin-dependent down-regulation of expression of the human transferrin receptor.

To investigate the regulation mechanism of the uptake of iron and heme iron by the cells and intracellular utilization of iron, we examined the interaction between iron uptake from transferrin and hemopexin-mediated uptake of heme by human leukemic U937 cells or HeLa cells. U937 cells exhibited about 40,000 hemopexin receptors/cell with a dissociation constant (Kd) of 1 nM. Heme bound in hemopexin was taken up by U937 cells or HeLa cells in a receptor-mediated manner. Treatment of both species of cells with hemopexin led to a rapid decrease in iron uptake from transferrin in a hemopexin dose-dependent manner, and the decrease seen in case of treatment with hemin was less than that seen with hemopexin. The decrease of iron uptake by hemopexin contributed to a decrease in cell surface transferrin receptors on hemopexin-treated cells. Immunoblot analysis of the transferrin receptors revealed that the cellular level of receptors in U937 cells did not vary during an 8-h incubation with hemopexin although the number of surface receptors as well as iron uptake decreased within the 2-h incubation. After 4 h of incubation of the cells with hemopexin, a decrease of the synthesis of the receptors occurred. Thus, the down-regulation of transferrin receptors by hemopexin can be attributed to at least two mechanisms. One is a rapid redistribution of the surface receptor into the interior of the cells, and the other is a decrease in the biosynthesis of the receptor. 59Fe from the internalized heme rapidly appeared in non-heme iron (ferritin) coincidently with the induction of heme oxygenase. The results suggest that iron released from heme down-regulates the expression of the transferrin receptors and iron uptake.

Biological Transport↗

PubMed1990

Characterization of ferrochelatase in kidney and erythroleukemia cells.

Ferrochelatase from bovine kidney mitochondria has been purified 1600-fold with a 6.5% yield, exhibiting a specific activity of 490 nmol mesoheme formed/mg of protein per min. The Km values for mesoporphyrin IX and protoporphyrin IX with iron were 12.5 and 12.7 microM, respectively. The Km values for iron and zinc with mesoporphyrin IX were 3.51 and 3.17 microM, respectively. The purified enzyme showed a single band with an apparent molecular mass of 42,000 daltons (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The rabbit antibody against the purified enzyme markedly inhibited activities of the enzyme from both the kidney and liver. Immunoblot analysis showed that the antibody reacted with the renal as well as the hepatic enzymes showing the same molecular weight. Peptide mapping with trypsin or alpha-chymotrypsin showed that digested peptides of renal enzyme were similar to those of hepatic enzyme. Ferrochelatase activity in mouse erythroleukemia (MEL) cells increased in parallel with an increase of heme synthesis by treatment with dimethylsulfoxide. Using immunoblotting techniques, the amount of the enzyme in the MEL cells has been shown to increase by the induction, showing a molecular mass of 41 kDa which was the same as that of the mouse hepatic enzyme. Comparative structural analysis of the enzyme of MEL cells and that of mouse liver by peptide mapping showed that the partial digestive peptides of both enzymes exhibited a similar pattern. These results strongly suggest that ferrochelatase in kidney, liver and erythroid cells can be of one type.

Animals↗