Biomedical subjects

J M Tager

Publications and source records attributed to J M Tager.

At least 109 records · Page 6Linked to original sources

PubMed1988

Plasmalogen biosynthesis in peroxisomal disorders: fatty alcohol versus alkylglycerol precursors.

In recent years a growing number of inherited diseases have been recognized to originate from an impairment in one or more peroxisomal functions. Since it is well established that the first two steps in the biosynthesis of plasmalogens proceed in peroxisomes, we studied the biosynthesis of plasmalogens in cultured skin fibroblasts from patients with different peroxisomal and related disorders. When de novo plasmalogen biosynthesis was studied by growing the cells in the presence of [14C]hexadecanol, impaired plasmalogen biosynthesis was found in rhizomelic chondrodysplasia punctata, cerebrohepatorenal (Zellweger) syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. In all these cases, alkyl-acyl phospholipids, the precursors of plasmalogens, did not accumulate and 1-O-[9,10-3H2]octadecylglycerol was converted into plasmalogens with equal efficiency as in controls. This indicated that impaired de novo plasmalogen biosynthesis as measured by [14C]hexadecanol incorporation was due to a deficient formation of the glycero-ether bond. Using this procedure, normal de novo plasmalogen biosynthesis was found in X-linked adrenoleukodystrophy, adrenomyeloneuropathy, X-linked chondrodysplasia punctata, adult Refsum disease, as well as in heterozygotes for Zellweger syndrome and infantile Refsum disease. The data have indicated that the average extent of the deficiency in glycero-ether bond formation is different in Zellweger syndrome, chondrodysplasia punctata, neonatal adrenoleukodystrophy, and infantile Refsum disease.

Cells, Cultured↗

PubMed1987

An improved procedure for the isolation of lamellar bodies from human lung. Lamellar bodies free of lysosomes contain a spectrum of lysosomal-type hydrolases.

We have recently shown that lamellar body fractions purified from human lung contain a distinct acid alpha-glucosidase distinguishable from lysosomal acid alpha-glucosidase in that it does not cross-react with antibodies raised against the lysosomal enzyme and does not bind to concanavalin A (De Vries, A.C.J., Schram, A.W., Tager, J.M., Batenburg, J.J. and Van Golde, L.M.G. (1985) Biochim. Biophys. Acta 837, 230-238). In order to study the relationship between the non-concanavalin A-binding alpha-glucosidase and lamellar bodies more closely a method was developed for the further purification of the organelles. A purified lamellar body preparation isolated from human lung homogenate by discontinuous sucrose density centrifugation was subjected to gel filtration with Sepharose 4B followed by Percoll density gradient centrifugation, which yielded a lamellar body preparation with a phospholipid phosphorus/protein ratio of 12.57 +/- 0.38 (mumol/mg) (n = 3) as compared to a ratio of 3.34 +/- 0.16 (mumol/mg) (n = 3) in the sucrose density gradient preparation. Concomitantly there was a 3.3 +/- 0.1 (n = 3)-fold enrichment in the content of total acid alpha-glucosidase and a 3.2 +/- 0.1 (n = 3) -fold enrichment of non-concanavalin A-binding acid alpha-glucosidase. The new purification method removes adhering proteins without changing the phospholipid composition. During the successive purification steps the concanavalin A-sensitive and -insensitive alpha-glucosidases remained fully lamellar body fraction associated. Differences between a lysosome-enriched fraction and a lamellar body preparation at varying stages of purification with respect to the ratio between soluble acid hydrolases and the membrane-associated lysosomal enzyme glucocerebrosidase indicate that the purified lamellar bodies were not contaminated with lysosomes. The absence of lysosomes in the purified lamellar body fraction was confirmed by experiments with the weak base glycyl-L-phenylalanine-beta-naphthylamide, which is an artificial substrate for the lysosomal enzyme cathepsin C and brings about lysis of lysosomes. Morphological examination by electron microscopy endorses the absence of contaminating vesicles and organelles and showed a structural integrity of the lamellar bodies in the final preparation. The improved isolation procedure strongly suggests that the concanavalin A-insensitive acid alpha-glucosidase is endogenous to lamellar bodies and supports our earlier idea that it can be used as a lamellar body-specific marker enzyme. In addition, the experiments show that lamellar bodies free of lysosomes contain a spectrum of lysosomal-type enzymes.

Cell Fractionation↗

PubMed1987

Acid sphingomyelinase from human urine: purification and characterization.

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).

Humans↗

PubMed1987

Peroxisomal very long-chain fatty acid beta-oxidation in human skin fibroblasts: activity in Zellweger syndrome and other peroxisomal disorders.

Since very long-chain fatty acids with a chain length of 24 carbons or more are known to accumulate in tissues and body fluids from patients with the cerebrohepato-renal (Zellweger) syndrome, infantile Refsum disease, neonatal adrenoleukodystrophy and X-linked adrenoleukodystrophy, we studied very long-chain fatty acid oxidation in cultured skin fibroblasts from these patients. In this paper, we report that in accordance with earlier results the first step in the beta-oxidation of the very long-chain fatty acid lignoceric acid (C24:0) primarily occurs in peroxisomes in control human skin fibroblasts. Furthermore, it was found that peroxisomal lignoceric acid beta-oxidation was strongly deficient in fibroblasts from patients with Zellweger syndrome, infantile Refsum disease, neonatal and X-linked adrenoleukodystrophy, which explains for the accumulation of very long-chain fatty acids in all four disease entities. In Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy the impairment in peroxisomal very long-chain fatty acid beta-oxidation is probably caused by a strong deficiency of all peroxisomal beta-oxidation enzyme proteins due to a deficiency of peroxisomes.

Adrenoleukodystrophy↗

PubMed1987

Prenatal diagnosis of Zellweger syndrome by measurement of very long chain fatty acid (C26:0) beta-oxidation in cultured chorionic villous fibroblasts: implications for early diagnosis of other peroxisomal disorders.

In this paper we show that cultured chorionic villous fibroblasts efficiently catalyse the peroxisomal beta-oxidation of hexacosanoic acid (cerotic acid), a saturated very long chain fatty acid containing 26 carbon atoms. Hexacosanoic beta-oxidation was found to be strongly impaired in cultured chorionic villous fibroblasts from a Zellweger foetus. This finding indicates that measurement of peroxisomal beta-oxidation can be used (in addition to measurement of acyl-CoA:dihydroxyacetone phosphate acyltransferase, de novo plasmalogen biosynthesis, the amount of particle-bound catalase and phytanic acid oxidase) for prenatal diagnosis in the first trimester of Zellweger syndrome, infantile Refsum disease and neonatal adrenoleukodystrophy. The method should be equally applicable to the early prenatal diagnosis of disorders in which there is a deficiency of a single peroxisomal beta-oxidation enzyme. Such diseases include X-linked adrenoleukodystrophy (peroxisomal very long chain fatty acyl CoA ligase deficiency), 'pseudo-Zellweger syndrome' (peroxisomal 3-oxoacyl-CoA thiolase deficiency) and 'pseudo-neonatal adrenoleukodystrophy' (acyl-CoA oxidase deficiency).

Cells, Cultured↗

PubMed1987

Alanine glyoxylate aminotransferase and the urinary excretion of oxalate and glycollate in hyperoxaluria type I and the Zellweger syndrome.

We have studied the urinary excretion of oxalate and glycollate in patients with the Zellweger syndrome and hyperoxaluria type I and have measured the activity of alanine glyoxylate aminotransferase (a peroxisomal enzyme in man) in the patients. In agreement with earlier reports we found that alanine glyoxylate aminotransferase was strongly deficient in liver from a hyperoxaluria type I patient, thus explaining the increased urinary excretion of oxalate and glycollate in these patients. In livers from Zellweger patients, however, in which morphologically distinguishable peroxisomes are absent, the enzyme was not deficient, which is in accordance with our finding that the urinary excretion of oxalate and glycollate was normal in these patients.

Adolescent↗

PubMed1987

X-linked adrenoleukodystrophy: defective peroxisomal oxidation of very long chain fatty acids but not of very long chain fatty acyl-CoA esters.

We investigated the peroxisomal fatty acid beta-oxidation system in liver and cultured skin fibroblasts from patients with X-linked adrenoleukodystrophy known to accumulate very long chain fatty acids. In order to examine whether the deficient peroxisomal oxidation of very long chain fatty acids in these patients results from a deficiency in one of the peroxisomal beta-oxidation enzyme proteins (acyl-CoA oxidase, bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase) we carried out immunoblotting experiments using antibodies directed against the peroxisomal beta-oxidation enzyme proteins from rat liver. Furthermore, we studied the oxidation of palmitoyl-CoA and lignoceroyl-CoA in homogenates of fibroblasts from the patients. The results indicate that the peroxisomal beta-oxidation enzyme proteins are not only present immunologically but also functionally active which suggests that the defect in X-linked adrenoleukodystrophy is, indeed, as recently suggested by Hashmi and coworkers (FEBS Lett 1986;196:247-250) at the level of a deficient peroxisomal activation of very long chain fatty acids.

Acyl Coenzyme A↗

PubMed1987

Studies on the peroxisomal oxidation of palmitate and lignocerate in rat liver.

We have investigated the pathways involved in the peroxisomal oxidation of palmitate and lignocerate, measured as the cyanide-insensitive formation of acetyl units, in rat-liver homogenates. The peroxisomal beta-oxidation of both fatty acids is dependent on the presence of ATP, coenzyme A, NAD+ and Mg2+. However, there is a striking difference in the dependence of the rate of oxidation of the two substrates on the concentration of the individual cofactors, especially ATP. The peroxisomal beta-oxidation of lignocerate was inhibited to a progressively greater extent by increasing concentrations of palmitate and vice versa. Activation of lignoceric acid to lignoceroyl-CoA, however, was not inhibited by increasing concentrations of palmitate, and vice versa. It can be concluded that the peroxisomal palmitate and lignocerate beta-oxidation pathways differ in at least one enzymic reaction (the synthetase), but that the two pathways share at least one common step.

Animals↗

PubMed1987

Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts.

The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase.

Cell Line↗

PubMed1987

Catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome: normal maturation in peroxisome-deficient cells.

We have compared the properties of catalase in cultured skin fibroblasts from patients with the cerebro-hepato-renal (Zellweger) syndrome, in which peroxisomes are deficient, with those of catalase in fibroblasts from control subjects. The enzymes from the two types of fibroblasts are indistinguishable with respect to kinetic properties, subunit size and molecular mass of the native enzyme. The turnover of the enzyme, measured by following the rate of reappearance of catalase activity in fibroblasts after irreversible inactivation of existing molecules by 3-aminotriazole treatment of the cells, was the same in Zellweger fibroblasts as in control cells. These findings indicate that normal maturation of catalase can occur in the soluble cytoplasm and provide an explanation for the occurrence of extra-peroxisomal catalase in tissues and cells.

Brain Diseases↗

PubMed1987

Relationship between the two immunologically distinguishable forms of glucocerebrosidase in tissue extracts.

Extracts of human spleen contain two immunologically distinguishable forms of glucocerebrosidase: form I is precipitable by polyclonal or monoclonal anti-(placental glucocerebrosidase) antibodies, whereas form II is not [Aerts, J. M. F. G., Donker-Koopman, W. E., Van der Vliet, M. F. K., Jonsson, L. M. V., Ginns, E. I., Murray, G. J., Barranger, J. A., Tager, J. M. & Schram, A. W. (1985) Eur. J. Biochem. 150, 565-574]. The proportion of form II glucocerebrosidase was high in extracts of spleen, liver and kidney and low in extracts of brain, placenta and fibroblasts. Furthermore, the proportion of form II enzyme was higher in a detergent-free aqueous extract of spleen than in a Triton X-100 extract of total spleen or splenic membranes. When form II glucocerebrosidase in a splenic extract was separated from form I enzyme by immunoaffinity chromatography and stored at 4 degrees C, a gradual conversion to form I enzyme occurred. The conversion was almost immediate if 30% (v/v) ethylene glycol was present. In the denatured state both forms of glucocerebrosidase reacted with anti-(placental glucocerebrosidase) antibodies. Form I glucocerebrosidase was stimulated by sodium taurocholate or sphingolipid-activator protein 2 (SAP-2), whereas form II enzyme exhibited maximal activity in the absence of the effectors. The pH activity profile of form II glucocerebrosidase was almost identical to that of form I enzyme in the presence of SAP-2. In the native state, form I glucocerebrosidase had a molecular mass of 60 kDa whereas that of form II glucocerebrosidase was about 200 kDa. After gel-permeation high-performance liquid chromatography of splenic extracts, the fractions with form II glucocerebrosidase contained material cross-reacting with both anti-(placental glucocerebrosidase) and anti-(SAP-2) antibodies. Preincubation of form I glucocerebrosidase with SAP-2 at pH 4.5 led to masking of the epitope on glucocerebrosidase reacting with monoclonal anti-(placental glucocerebrosidase) antibody 2C7. Furthermore, preincubation of form I glucocerebrosidase with monoclonal antibody 2C7 prevented activation of the enzyme by SAP-2. We propose that form I glucocerebrosidase is a monomeric form of the enzyme, whereas form II glucocerebrosidase is a high-Mr complex of the enzyme in association with sphingolipid-activator protein 2.

Antibodies, Monoclonal↗

PubMed1987

Determination of the structure of the carbohydrate chains of acid alpha-glucosidase from human placenta.

Acid alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz 1H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man5GlcNAcGlcNAc-ol and Man7GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man2-3GlcNAc[Fuc]0-1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.

Carbohydrate Sequence↗

PubMed1987

Ultrastructural localization of glucocerebrosidase in cultured Gaucher's disease fibroblasts by immunocytochemistry.

The subcellular localization of glucocerebrosidase was studied in cultured skin fibroblasts from eight patients with Gaucher's disease. The enzyme, in situ, was visualized under the electron microscope by incubating ultrathin frozen sections of fibroblasts with antibodies against glucocerebrosidase, followed by a second incubation with goat anti- (rabbit IgG) coupled to colloidal gold. In control cells, most of the gold label was found in lysosomes, associated with the membrane. Labelling of the rough endoplasmic reticulum (RER) and Golgi complex was also observed. In fibroblasts from three Gaucher's disease patients without neurological symptoms (type 1 disease), a near normal amount of cross-reactive material (CRM) was detected in lysosomes, but in a fourth such patient, the lysosomal CRM was reduced. Little lysosomal glucocerebrosidase was detected in cells from patients with the acute neuronopathic form (type 2) or the subacute neuronopathic form (type 3) of Gaucher's disease. CRM in lysosomes correlates with amount of mature, 59 kDA glucocerebrosidase which is undetectable in type 2 and type 3 Gaucher's disease cell lines. These findings demonstrate that different mutations in the gene for glucocerebrosidase result in mutant proteins that have different intracellular localizations. They also suggest that there is a relationship between the amount of cross-reactive material in the lysosomes and the phenotypic expression of the disease.

Cell Line↗

PubMed1987

Inborn errors of cellular organelles: an overview.

Metabolic processes in the cell are catalysed by enzymes and enzyme systems present in discrete intracellular compartments consisting of the cytosol and various intracellular organelles. Three well defined groups of genetic diseases in man can now be recognized in which the functions of an intracellular organelle are impaired: lysosomal storage diseases, mitochondrial disorders and peroxisomal diseases. Extensive studies carried out during the last decade on the biogenesis of intracellular organelles have contributed to an understanding of the molecular basis of the lesions leading to these three groups of genetic disorders. The results of the studies have stressed that such lesions can arise not only through mutations in the structural genes for the proteins in an organelle but also through mutations in the genes coding for components required for the specific transport and incorporation of proteins into organelles.

DNA↗

PubMed1987

Zellweger syndrome: biochemical procedures in diagnosis, prevention and treatment.

In patients with cerebro-hepato-renal (Zellweger) syndrome, the absence of peroxisomes results in an impairment of metabolic processes in which peroxisomes are normally involved. These include the catabolism of very long chain (greater than C22) fatty acids, the biosynthesis of ether-phospholipids and of bile acids, the catabolism of phytanic acid and the catabolism of pipecolic acid. Many diagnostic tests for Zellweger syndrome have become available in recent years. In classic Zellweger syndrome abnormal C27-bile acids, very long chain fatty acids, dicarboxylic acids and pipecolic acid accumulate in the plasma of the patients. Moreover, depending upon the diet, plasma phytanic acid concentrations may be elevated. In platelets the activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase is deficient; in erythrocytes from young (less than 4 months) patients the plasmalogen content of the phospholipids is decreased. In cultured fibroblasts from skin and from chorionic villus and cultured amniotic fluid cells from Zellweger patients the plasmalogen level is lowered; there is a decreased activity of acyl-CoA: dihydroxyacetone phosphate acyltransferase, alkyl dihydroxyacetonephosphate synthase and phytanic acid oxidase; the de novo biosynthesis of plasmalogens and the peroxisomal beta-oxidation of fatty acids are impaired and the intracellular localization of catalase is abnormal. Dietary treatment of patients with Zellweger syndrome has not so far resulted in an objective clinical improvement. As Zellweger syndrome is usually fatal in early life, prenatal diagnosis of the disease is important.

Bile Acids and Salts↗

PubMed1987

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome.

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.

Brain Diseases↗

PubMed1987

Neonatal adrenoleukodystrophy. Impaired plasmalogen biosynthesis and peroxisomal beta-oxidation due to a deficiency of catalase-containing particles (peroxisomes) in cultured skin fibroblasts.

Neonatal adrenoleukodystrophy belongs to the newly recognized group of inherited diseases, the peroxisomal disorders. Based on the reported similarities between neonatal adrenoleukodystrophy and the cerebro-hepato-renal (Zellweger) syndrome, we have studied peroxisomal functions in cultured skin fibroblasts from 5 neonatal adrenoleukodystrophy patients. The results indicate that multiple peroxisomal enzyme activities are deficient in fibroblasts from neonatal adrenoleukodystrophy patients. Digitonin titration experiments revealed that peroxisomes are strongly deficient in these fibroblasts as found earlier in fibroblasts from Zellweger patients. These findings not only explain the generalized loss of peroxisomal functions in neonatal adrenoleukodystrophy, but also provide an explanation for the observed resemblance in clinical and biochemical abnormalities between neonatal adrenoleukodystrophy and Zellweger syndrome. The implications for the pre- and postnatal detection of this disease will be discussed.

Adrenoleukodystrophy↗

PubMed1987

Production of monoclonal antibodies to thyroglobulin by in vitro immunization with a free synthetic peptide.

A synthetic peptide corresponding to amino acids 1-19 of thyroglobulin was used to test the possibility of generating protein-reactive monoclonal antibodies by immunization in vitro with a synthetic peptide as antigen. Splenocytes from non-immunized Balb/c mice were cultured in serum-free medium for 3 days in the presence of thymocyte-conditioned medium and the synthetic peptide prior to fusion with SP2/0 murine myeloma cells. The synthetic peptide was used in its free form, i.e. not coupled to a protein carrier. Hybridomas secreting monoclonal antibodies reactive with the synthetic peptide were obtained after immunization in vitro with as little as 10 ng/ml of the synthetic peptide. Between 50 and 70% of the primary clones obtained in different experiments produced monoclonal antibodies also reactive with the intact protein. Six stable hybridomas were isolated; all produced antibodies of the IgM class. We conclude that immunization in vitro with a free synthetic peptide is an efficient method for the generation of monoclonal antibodies reactive with the intact protein.

Animals↗