Biomedical subjects

F Cramer

Publications and source records attributed to F Cramer.

At least 37 records · Page 2Linked to original sources

PubMed1987

Structure of the yeast isoleucyl-tRNA synthetase gene (ILS1). DNA-sequence, amino-acid sequence of proteolytic peptides of the enzyme and comparison of the structure to those of other known aminoacyl-tRNA synthetases.

The ILS1 gene encoding for cytoplasmic isoleucyl-tRNA synthetase from Saccharomyces cerevisiae was subcloned from a 5.4-kb insert of the shuttle vector YEp13 to M13mp8 and M13mp9. Nucleotide sequence analysis of a 4.3-kb BamHI-HpaI fragment revealed a single open reading frame from which we deduced the amino-acid sequence of the enzyme. Independently obtained amino-acid sequence information from ten tryptic peptides of the purified enzyme confirmed the gene-derived structure. The enzyme is comprised of 1073 amino-acids consistent with earlier determinations of its molecular mass. The codon usage of ILS1 is typical of abundant yeast proteins. A significant homology to E. coli isoleucyl- and valyl-tRNA synthetases as well as to yeast valyl-tRNA synthetase was detected. The characteristic amino-acid residues of the aminoacyl-adenylate site and of the potential binding site of the 3'-end of tRNA found in other synthetases are present in the structure.

Amino Acid Sequence↗

PubMed1986

Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa.

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate.

Adenosine Triphosphate↗

PubMed1986

The proofreading of hydroxy analogues of leucine and isoleucine by leucyl-tRNA synthetases from E. coli and yeast.

Three analogues each of leucine and isoleucine carrying hydroxy groups in gamma- or delta- or gamma- and delta-position have been synthesized, and tested in the aminoacylation by leucyl-tRNA synthetases from E. coli and yeast. Hydrolytic proofreading, as proposed in the chemical proofreading model, of these analogues and of homocysteine should result in a lactonisation of these compounds and therefore provide information regarding the proofreading mechanism of the two leucyl-tRNA synthetases. Leucyl-tRNA synthetase from E. coli shows a high initial substrate discrimination. Only two analogues, gamma-hydroxyleucine and homocysteine are activated and transferred to tRNALeu where a post-transfer proofreading occurs. Lactonisation of gamma-hydroxyleucine and homocysteine could be detected. Leucyl-tRNA synthetase from yeast has a relatively poor initial discrimination of these substrates, which is compensated by a very effective pre-transfer proofreading on the aminoacyl-adenylate level. No lactonisation nor mischarged tRNALeu is detectable.

Adenosine Monophosphate↗

PubMed1986

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns.

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.

Adenosine Triphosphate↗

PubMed1986

Biochemical comparison of the Neurospora crassa wild-type and the temperature-sensitive leucine-auxotroph mutant leu-5. Detailed kinetic comparison of the leucyl-tRNA synthetases.

The cytoplasmic leucyl-tRNA synthetases were purified from a wild-type Neurospora crassa and from a temperature-sensitive leucine-auxotroph (leu-5) mutant. A detailed steady-state kinetic study of the aminoacylation of the tRNALeu from N. crassa by the purified synthetases was carried out. These enzymes need preincubation with dithioerythritol and spermine before the assay in order to become fully active. The Kappm value for leucine was lowered by high ATP concentrations and correspondingly the Kappm,ATP was lowered by high leucine concentrations. The Kappm,Leu was lowered by high pH, a pK value of 6.7 (at 30 degrees C) was calculated for the ionizable group affecting the Km. At the concentrations of 2 mM ATP, 20 microM leucine, 0.3 microM tRNALeu, and pH 7 the apparent Km values were Kappm,ATP = 1.3 mM, Kappm,Leu = 49 microM and Kappm,tRNA = 0.15 microM. No essentially altered cytoplasmic leucyl-tRNA synthetase was produced by the temperature-sensitive mutant strain when kept at 37 degrees C. In none of these experiments could we find any difference between the wild-type enzyme and the enzyme from the mutant strain (whether grown at permissive temperature, 28 degrees C, or grown at permissive temperature for 24 h followed by growth at 37 degrees C). We therefore think that the small difference in the Km value for leucine of the wild-type and mutant enzyme, established in some earlier investigations, is not due to a difference in the kinetic properties of the enzyme molecules but to an external influence. The almost total lack of the mitochondrial leucyl-tRNA synthetase in the mutant strain besides the leucine autotrophy remains the only difference between the wild-type and mutant strains.

Adenosine Triphosphate↗

PubMed1986

Phenylalanyl-tRNA synthetase from chloroplasts of a higher plant (Phaseolus vulgaris). Purification and comparison of its structural, functional, and immunological properties with those of the enzymes from the corresponding cytoplasm, the cyanobacterium Anacystis nidulans, and the photosynthetic green sulfur bacterium Chlorobium limicola.

Chloroplastic phenylalanyl-tRNA synthetase from bean leaves is purified under optimal protective conditions over 4,900-fold. Its apparent molecular weight is 78,000, as determined by gel filtration, with a dimeric subunit structure of alpha beta (alpha = 33,000 and beta = 42,000), as determined by sodium dodecyl sulfate gel electrophoresis. This indicates a drastic size reduction of 40% for each subunit compared to the corresponding cytoplasmic enzyme and a unique quaternary structure. Heterologous aminoacylation and substrate properties of ATP analogs indicate substantial differences in the topographies of the substrate binding domains of these two heterotopic intracellular plant enzymes. No common antigenic determinants with the bean cytoplasmic enzyme were detected by polyclonal antibodies against the chloroplastic enzyme. The same negative result applies to the immunological comparison with the partially purified enzymes from the cyanobacterium Anacystis nidulans and the photosynthetic green sulfur bacterium Chlorobium limicola that both have a molecular weight of 260,000.

Amino Acyl-tRNA Synthetases↗

PubMed1986

Carbohydrate-binding proteins of tumor lines with different growth properties. II. Changes in their pattern in clones of transformed rat fibroblasts of differing metastatic potential.

A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weight of 42 kDa. The alpha-glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5-8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5-8#1 and 5-20#20, whereas the metastatic variant 5-20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.

Animals↗

PubMed1986

Localization of endogenous lectins in normal human breast, benign breast lesions and mammary carcinomas.

Specific antisera against three mammalian beta-galactoside-specific lectins of apparent molecular weights 14.5 kDa, 18 kDa and 29 kDa have been used to localize these lectins in normal breast, and in benign and malignant mammary lesions. In normal breast tissue discrete localization of two lectins (Mrs 14.5 kDa and 18 kDa) was demonstrated in fibroblasts, smooth muscle cells, myoepithelial cells and capillary endothelium. Extracellular localization of one lectin (Mr 14.5 kDa) in collagen was apparent. The third lectin (Mr 29 kDa) labelled preferentially luminal cells and their secretory product. Two benign tumours (an analyzed fibroadenoma and a papilloma) revealed strong staining with two lectins (Mrs 18 kDa and 29 kDa). Of the 24 mammary carcinomas examined, the lectin (Mr 14.5 kDa) was expressed by only occasional tumour cells, the lectin (Mr 18 kDa) occurred in many tumour cells and the lectin (Mr 29 kDa) labelled tumour cells in nearly all cases. The expression of these beta-galactoside-specific endogenous lectins therefore appears to be regulated differently in normal breast compared with mammary tumours.

Breast↗

PubMed1986

Phenylalanyl-tRNA synthetases as an example for comparative and evolutionary aspects of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are indispensable components of protein synthesis in all three lines of evolutionary descent, eubacteria, archaebacteria and eukaryotes. Furthermore they are also present in the translational apparatus of the semi-autonomous organelles, mitochondria and chloroplasts, of the eukaryotic cell. Therefore aminoacyl-tRNA synthetases are appropriate objects for comparative molecular biology in order to obtain a comprehensive picture of the evolution of the translational process. The analysis of the phenylalanyl-tRNA synthetase in a large variety of organisms and organelles in this respect is the most advanced. In addition to comparison of quaternary structure, analysis includes functional aspects of accuracy mechanisms (proofreading) and comparison of structural features by means of substrate analogs. Evolutionary relationships are furthermore elucidated using the immunological approach and heterologous aminoacylation.

Amino Acyl-tRNA Synthetases↗

PubMed1986

Pattern of endogenous lectins of a human sarcoma (Ewing's sarcoma) reveals differences to human normal tissues and tumors of epithelial and germ cell origin.

A human sarcoma, Ewing's sarcoma, contains activities of endogenous lectins. Fractionation of salt and detergent extracts by affinity chromatography on columns with immobilized sugars or glycoproteins results in the pattern of endogenous lectins for alpha- and beta-galactosides, alpha-mannosyl- and alpha-fucosyl-moieties. Whereas some lectins are known from normal, non-malignant human tissues or from a human epithelial tumor or a human germ cell tumor, a Ca2+-independent alpha- and beta-galactoside-binding protein at apparent molecular weight of 58 kilodaltons has so far not been characterized from any human source. The patterns for the tumors and human normal tissues reveal various differences in comparison between each other. These differences, documented for the first human sarcoma, human tumors of different histogenetic lineage and normal tissues are a first step to a lectin-based diagnosis and therapy of certain human cancer types.

Apudoma↗

PubMed1986

Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine.

The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated.

Adenosine Monophosphate↗

PubMed1986

Comparison of endogenous lectins in human embryonic carcinoma and yolk sac carcinoma.

Salt and detergent extracts of xenografted human embryonic carcinoma and a human yolk sac carcinoma were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan and fucose. Successive elution by chelating reagent and specific sugar resulted in isolation of endogenous Ca2+-dependent and Ca2+-independent carbohydrate-binding proteins, as analyzed by gel electrophoresis. The salt extracts of the embryonic carcinoma contained a Ca2+-dependent carbohydrate-binding protein at Mr66,000, that was undetectable in yolk sac tumor extracts. Also, a Ca2+-independent fucose-binding protein at Mr62,000 could be purified. In general, the pattern of carbohydrate-binding proteins showed quantitative and qualitative differences as compared to normal tissue. The carbohydrate-binding proteins were assayable as agglutinin and showed no enzymatic activity. They can thus be defined as lectins. Their presence within certain stages of differentiation and developmental regulation may contribute to improvement of the classification, diagnosis and therapy of this tumor class. These new lectins may also be functionally related to the stage-specific embryonic antigens like SSEA-1.

Adult↗

PubMed1986

Differences in the pattern of endogenous lectins from spontaneous rat mammary tumors.

Three spontaneously occurring rat mammary tumors, a fibroadenoma and two tubulopapillary adenocarcinomas, differing in their degree of histologic differentiation and malignancy, were investigated. They revealed significant differences in the pattern of carbohydrate-binding proteins with specificities for alpha- and beta-galactosyl, alpha-mannosyl, and alpha-fucosyl moieties, respectively. Analysis was performed by affinity chromatography on resins with immobilized sugars or glycoproteins and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The patterns were divided into categories according to the dependence of binding activity on the presence of Ca2+ and to dependence on extraction conditions. In comparison with both adenocarcinomas, the fibroadenoma contained alpha- and beta-galactoside-binding proteins at apparent molecular weights of 74,000 and 130,000. These proteins were not found in either adenocarcinoma. Comparison between the two adenocarcinomas revealed qualitative differences for all classes of carbohydrate-binding proteins under investigation. These differences between tumors of the same tumor class (mammary tumors) and tumor type (tubulopapillary adenocarcinoma) allow for the proposal that the pattern of endogenous carbohydrate-binding proteins may be potentially useful as a diagnostic marker.

Adenocarcinoma↗

PubMed1985

Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation.

For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.

Amino Acyl-tRNA Synthetases↗

PubMed1985

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of the cytoplasmic and chloroplastic enzymes of a higher plant (Phaseolus vulgaris).

The phenylalanyl-tRNA synthetases from cytoplasm and chloroplasts of bean (Phaseolus vulgaris) leaves employ different strategies with respect to accuracy. The chloroplastic enzyme that is coded for by the nuclear genome follows the pathway of posttransfer proofreading, also characteristic for enzymes from eubacteria and cytoplasm and mitochondria of lower eukaryotic organisms. In contrast, the cytoplasmic enzyme uses pretransfer proofreading in the case of noncognate natural amino acids, characteristic for higher eukaryotic organisms and archaebacteria. Dependent on the nature of the noncognate amino acid, pretransfer proofreading in this case occurs without tRNA stimulation or with tRNA stimulated with no or little effect of the nonaccepting 3'-OH group of the terminal adenosine. The fundamental mechanistic difference in proofreading between the heterotopic intracellular isoenzymes of the plant cell supports the idea of the origin of the chloroplastic gene by gene transfer from a eubacterial endosymbiont to the nucleus. Origin by duplication of the nuclear gene, as indicated for mitochondrial phenylalanyl-tRNA synthetases [Gabius, H.-J., Engelhardt, R., Schroeder, F.R., & Cramer, F. (1983) Biochemistry 22, 5306-5315], appears unlikely. Further analyses of the ATP/PPi pyrophosphate exchange and aminoacylation of tRNAPhe-C-C-A(3'NH2), using 11 phenylalanine analogues, reveal intraspecies and interspecies variability of the architecture of the amino acid binding part within the active site.

Amino Acyl-tRNA Synthetases↗

PubMed1985

Archaebacterial phenylalanyl-tRNA synthetase. Accuracy of the phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri, Zn(II)-dependent synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate, and immunological relationship of OFFnylalanyl-tRNA synthetases from different urkingdoms.

Phenylalanyl-tRNA synthetase from the archaebacterium Methanosarcina barkeri activates a number of phenylalanine analogues (methionine, p-fluorophenylalanine, beta-phenylserine, beta-thien-2-ylalanine, 2-amino-4-methylhex-4-enoic acid and ochratoxin A) in the absence of tRNA, as demonstrated by Km and kcat of the ATP/PPi exchange reaction. Upon complexation with tRNA, AMP formation from the enzyme X tRNA complex in the presence of ATP, one of the above analogues or tyrosine, leucine, mimosine, N-benzyl-L- or N-benzyl-D-phenylalanine indicates activation of the analogues under conditions of aminoacylation. Natural noncognate amino acids are not transferred to tRNAPhe-C-C-A or tRNAPhe-C-C-A-(3'-NH2). This pretransfer proofreading mechanism, together with the comparatively low ratio of synthetic to successive hydrolytic steps, resembles the mechanism of liver enzymes of vertebrates. In contrast, eubacterial phenylalanyl-tRNA synthetases achieve the necessary fidelity by post-transfer proofreading, a corrective hydrolytic event after transfer to tRNAPhe. Diadenosine 5',5'''-P1,P4-tetraphosphate synthesis is shown to be a common feature for phenylalanyl-tRNA synthetases from all three lineages of descent. The immunological approach demonstrates that aminoacyl-tRNA synthetases do not belong to the group of enzymes in gene expression with high structural conservation.

Adenine Nucleotides↗

PubMed1985

Carbohydrate-binding proteins of tumor lines with different growth properties. I. Differences in their pattern for three clones of rat fibroblasts transformed with a myeloproliferative sarcoma virus.

Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca2+ -dependent and Ca2+ -independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.

Animals↗

PubMed1985

Endogenous tumor lectins: a new class of tumor markers and targets for therapy?

Endogenous lectins of normal tissues can play a functional role in recognition processes and cell adhesion. These functions are areas of particular relevance to tumor growth and metastasis. Our initial results on endogenous lectins of different tumors lead to the working hypothesis that the pattern of endogenous lectins is qualitatively and quantitatively different between different types of tumors and between tumors and normal, nonmalignant tissues. The endogenous lectins may thus prove to be potentially important in establishing a new concept for a rational lectin-based type of diagnosis and therapy of various tumors.

Animals↗