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R Whalen

Publications and source records attributed to R Whalen.

At least 19 recordsLinked to original sources
PubMed1998

Ischaemia and reperfusion injury of rat liver increases expression of glutathione S-transferase A1/A2 in zone 3 of the hepatic lobule.

Effects of ischaemia-reperfusion injury (I/R) of liver on expression of rat glutathione S-transferase (rGST) isoenzymes that metabolize products of oxidative stress were examined. Rats underwent lobar liver ischaemia for 30 min followed by reperfusion. In ischaemic lobes, rGSTA1/A2 transcript levels increased significantly 12 h after I/R (2.94-fold) and protein levels increased significantly at 24 h (1.45-fold); increased transcript levels were also observed in nonischaemic lobes (1.78-fold). Superoxide dismutase prevented I/R and the increases in transcript and protein levels in ischaemic and non-ischaemic lobes. By in-situ hybridization, increases in transcript levels at 6 h were present in zones 2 and 3 of the ischaemic lobes and peaked at 12 h (2.5-fold zone 2, 4.5-fold zone 3). Significant increases in transcript levels also were observed at 24 h in zones 2 (2.0-fold) and 3 (2.9-fold) of non-ischaemic lobes. Nuclear run-off assays showed a 1.8-fold increase in rGSTA1/A2 transcription rates in ischaemic lobes at 3 h. We conclude that I/R causes increased rGSTA1/A2 expression in the zone of the hepatic lobule most susceptible to oxidative injury and that this expression may be an important defence against injury.

Animals↗
PubMed1998

Human glutathione S-transferases.

Human glutathione S-transferases (GSTs) are a functionally diverse family of soluble enzymes of detoxification that use reduced glutathione (GSH) in conjugation and reduction reactions. Toxic electrophiles, including a variety of carcinogens, are substrates for the GSTs and after conjugation or reduction they are more easily excreted into bile or urine. Many of the GSTs have been cloned, and the three-dimensional structures of GSTs from several species, including humans, have been determined. These data have provided significant insight into how the GSTs function as enzymes. Many GST substrates are inducers of GST gene expression; nonsubstrate inducers include H2O2 and other reactive oxygen species. The regulatory elements of several human GST genes have been partially characterized, and the regulation of the GSTs in humans appears to be very different from that in rodents. Several polymorphisms of GST expression occur commonly in humans and have been associated with an increased susceptibility to certain cancers, particularly when combined with other genetic and environmental factors such as smoking. The role of GSTs in protecting cells from injury by toxic electrophiles continues to be developed.

Animals↗
PubMed1997

Successful nucleic acid based immunization of newborn chimpanzees against hepatitis B virus.

To determine whether DNA based immunization could protect newborn chimpanzees against a challenge infection with hepatitis B virus, two chimpanzees were immunized on the day of birth with a plasmid coding for hepatitis B surface antigen, and boosted at 6 and 24 weeks. Both animals produced transient antibody to the hepatitis B surface antigen. Following challenge with hepatitis B virus at 33 weeks the two immunized animals developed anamnestic antibody responses, however, neither developed detectable hepatitis B surface antigen or antibody to the core protein, the conventional markers of hepatitis B infection. Both of these markers appeared in an unimmunized control animal. We conclude that DNA based immunization can induce protective immunity in newborn chimpanzees.

Animals↗
PubMed1996

Structural studies of a human pi class glutathione S-transferase. Photoaffinity labeling of the active site and target size analysis.

The glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of dimeric proteins that catalyze reactions between glutathione (GSH) and various electrophiles. A partial cDNA for human GST pi was obtained and the open reading frame completed. The completed cDNA was cloned, and GST pi protein was expressed in bacteria. Cloned enzyme was purified and had the same kinetic constants, molecular mass, pI value, and N-terminal sequence as placental GST pi except that some of the polypeptides had N-terminal methionines. A radiolabeled azido derivative of GSH, S-(p-azidophenacyl)-[3H]glutathione, was used to photoaffinity-label the active site of the cloned enzyme. Labeled enzyme did not bind to a GSH-agarose affinity column. Labeling was prevented in the presence of S-hexylglutathione, and noncovalently-bound azido affinity label was a competitive inhibitor towards 1-chloro-2,4-dinitrobenzene and GSH. These results suggest that the azido label was binding at the active site of the enzyme. Photoaffinity-labeled enzyme was trypsinized, and two labeled peptides were purified and sequenced. One peptide corresponded to residues 183-188, whereas the other corresponded to residues 183-186. These residues appear to form part of the hydrophobic (H-site) binding region of human GST pi that has not been shown previously. Cloned enzyme was subjected to radiation inactivation to assess the importance of subunit interactions in the maintenance of catalytic activity. The target size of enzymatic activity (23 kDa) was not significantly different from that of the protein monomer (24 kDa). Therefore, each subunit of human GST pi appears to be capable of independent catalytic activity.

Affinity Labels↗
PubMed1996

Role of interleukin 6 and corticosteroids in the regulation of expression of glutathione S-transferases in primary cultures of rat hepatocytes.

The effect of recombinant interleukin 6 (rIL-6) on the transcript levels of rat glutathione S-transferase (GST) genes rGSTA2, rGSTP1, rGSTM1 and rGSTM2 was examined in primary cultures of rat hepatocytes. rIL-6 had little effect on the increase in expression of rGSTP1 that occurs in cultured hepatocytes. Dexamethasone (DEX), in contrast, prevented the expression of rGSTP1 by hepatocytes, and rIL-6 in combination with DEX had no additional effect. Neither rIL-6 nor DEX alone had a significant effect on the transcript levels of rGSTA2, rGSTM1 and rGSTM2 in cultured hepatocytes. However, when both were present (15 ng/ml rIL-6 and 10(-7) M DEX) the transcript levels of rGSTA2, rGSTM1 and rGSTM2 decreased significantly (P < 0.05) after 48 h in culture. If the rIL-6 was removed from the cultures after 24 h, the levels of transcripts recovered and were the same at 48 h as cells cultured without rIL-6 for the entire period. Dose-response relationships of rIL-6 with 10(-7) M DEX were determined for transcripts of each GST isoenzyme and the IC50 values were between 1.5 and 7.5 ng/ml. Declines in transcript levels of rGSTA2 were observed with rIL-6 plus 10(-8) or 10(-7) M DEX but not with rIL-6 plus 10(-9), 10(-6), or 10(-5) M DEX. To determine if the cytokine and glucocorticoid effects were mediated by sequences in the 5'-flanking sequence of rGSTA2, a plasmid construct containing a 1.6 kb fragment of the 5'-flanking sequence of the rGSTA2 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect rat hepatocytes in primary culture. The addition of rIL-6 and DEX to the culture medium caused a significant (P < 0.05) decrease in CAT activity after 48 h in culture. If rIL-6 was removed after 24 h in culture, CAT activity after an additional 24 h in culture was greater than the CAT activity in cells cultured for 48 h without rIL-6. Therefore cytokines and glucocorticoids may be important physiological regulators of GST expression.

Animals↗
PubMed1994

Cellular sources of glutathione S-transferase P in primary cultured rat hepatocytes: localization by in situ hybridization.

Hepatocytes in vivo express Alpha and Mu but not Pi forms of glutathione S-transferase (GST). GST P (a fetal Pi form) appears in rat hepatocytes after 2 days in primary culture, which suggests that hepatocytes may undergo dedifferentiation [Abramovitz, Ishigaki and Listowsky (1989) Hepatology 9, 235-239]. However, in this and other studies, primary rat hepatocyte cultures were shown by immunohistochemistry to contain significant numbers of lipocytes (Ito cells). Freshly isolated lipocytes contained GST activity when assayed with chlorodinitrobenzene (680 nmol/min per mg), and expression of Alpha, Mu and Pi forms of GST was detected by Western-blot analysis. Expression of GST P persisted during culture of the lipocytes. In situ hybridization of the cultured cells was performed to define whether hepatocytes, lipocytes or both expressed the enzyme. Lipocytes in culture contained abundant GST P transcripts. Hepatocytes contained no GST P transcripts after 12 h in culture, and after 24 h, only a few hepatocytes expressed this enzyme. After 48 h in culture all hepatocytes contained GST P transcripts, and the number of transcripts continued to increase up until 72 h. Therefore, in freshly isolated preparations of hepatocytes and early in hepatocyte culture, measurable levels of GST P protein or message appeared to reflect the presence of lipocytes. After 48 h in culture almost all of the GST P reflected expression by the hepatocytes. Lipocytes constitutively expressed Alpha-, Mu- and Pi-class GSTs and had significant intracellular levels of GSH (5.2 nmol/mg of protein). Lipocytes are capable therefore of detoxifying a number of injurious compounds.

Adipocytes↗
PubMed1993

Nitroglycerin metabolism in vascular tissue: role of glutathione S-transferases and relationship between NO. and NO2- formation.

Nitroglycerin is a commonly employed pharmacological agent which produces vasodilatation by release of nitric oxide (NO.). The mechanism by which nitroglycerin releases NO. remains undefined. Recently, glutathione S-transferases have been implicated as important contributors to this process. They are known to release NO2- from nitroglycerin, but have not been shown to release NO.. The present studies were designed to examine the role of endogenous glutathione S-transferases in this metabolic process. Homogenates of dog carotid artery were incubated anaerobically with nitroglycerin, and NO. and NO2- production was determined by chemiluminescence. The role of glutathione S-transferases was studied by incubating homogenates with nitroglycerin in the presence of 1 mM GSH or 1 mM S-hexyl-glutathione, a potent inhibitor of glutathione S-transferases. Homogenates released 163 pmol of NO./h per mg of protein from nitroglycerin, and 2370 pmol of NO2-/h per mg. Adding GSH decreased NO. production by 82% and increased NO2- production by 98%. S-Hexylglutathione inhibited glutathione S-transferase activity by 96% and decreased NO2- production by 78%, but had no effect on NO. release. A linear relationship between glutathione S-transferase activity and NO2- production was observed, whereas glutathione S-transferase activity and NO. release were unrelated. Western-blot analysis demonstrated that dog carotid vascular smooth muscle contained Pi and Mu forms of glutathione S-transferases, with a predominance of the former. Purified preparations of human Pi and rat Mu isoforms metabolized nitroglycerin only to NO2- and not to NO.. On the basis of these findings, we conclude that (1) glutathione S-transferases do not contribute to the bioconversion of nitroglycerin to NO., but instead act as a degradative pathway for nitroglycerin, and (2) the release of NO. from nitroglycerin is not dependent on the formation of NO2-.

Animals↗
PubMed1992

Bleeding tendency, platelet function, and pharmacokinetics of ibuprofen and zidovudine in HIV(+) hemophilic men.

The use of ibuprofen (IBP) in hemophilic men for chronic hemophilic arthropathy is associated with transient coagulation abnormalities, but usually does not cause bleeding symptoms. However, when hemophilic men are treated with ibuprofen while also receiving zidovudine (ZDV), excess bleeding has occurred in some. In order to evaluate platelet function and pharmacokinetics of combination IBP and ZDV, we measured platelet aggregation, platelet adhesive index, bleeding time, and IBP and ZDV drug levels by high performance liquid chromatography on five patients receiving chronic oral IBP, 400 mg every 6 hr, and on ten patients receiving both IBP and ZDV, 100 or 200 mg every 4 hr five times daily. Samples were obtained at baseline (ZDV alone), acutely (IBP+ZDV acutely), and chronically (2 weeks on IBP and ZDV). Abnormal platelet aggregation with arachidonic acid occurred in four of five (80%) of those receiving IBP alone and in 7 of 10 (70%) at baseline, 9 of 10 (90%) at acute, and 8 of 10 (80%) at chronic IBP and ZDV treatment, most commonly at 2 hr following dosing, when peak IBP levels occurred, and persisting 4 hr in those on chronic dosing. Half or more of those on combination IBP+ZDV showed a lowered platelet adhesive index and/or prolonged bleeding time. Excess bleeding symptoms occurred in three on chronic combination IBP+ZDV, two with increased frequency of spontaneous hemorrhages, and one with prolonged traumatic bleeding. Bleeding tendency was unrelated to the degree of platelet function abnormality, to peak drug levels of IBP or ZDV, or degree of liver function abnormality (SGPT). The clearance of IBP alone and ZDV alone were not different from the clearance of combination IBP and ZDV. Caution is urged regarding potential enhanced bleeding tendency in hemophiliacs receiving both IBP and ZDV in combination.

Acute Disease↗
PubMed1992

Bone mineral density, muscle strength, and recreational exercise in men.

Muscle strength has been shown to predict bone mineral density (BMD) in women. We examined this relationship in 50 healthy men who ranged in age from 28 to 51 years (average 38.3 years). BMD of the lumbar spine, proximal femur, whole body, and tibia were measured by dual-energy x-ray absorptiometry (Hologic QDR 1000W). Dynamic strength using one repetition maximum was assessed for the biceps, quadriceps, and back extensors and for the hip abductors, adductors, and flexors. Isometric grip strength was measured by dynamometry. Daily walking mileage was assessed by 9 week stepmeter records and kinematic analysis of video filming. Subjects were designated as exercisers and nonexercisers. Exercisers participated in recreational exercise at least two times each week. The results demonstrated that BMD at all sites correlated with back and biceps strength (p < 0.01 to p = 0.0001). Body weight correlated with tibia and whole-body BMD (p < 0.001); age negatively correlated with Ward's triangle BMD (p < 0.01). In stepwise multiple regressions, back strength was the only independent predictor of spine and femoral neck density (R2 = 0.27). Further, back strength was the most robust predictor of BMD at the trochanter, Ward's triangle, whole body, and tibia, although biceps strength, age, body weight, and leg strength contributed significantly to BMD at these skeletal sites, accounting for 35-52% of the variance in BMD. Exercisers and nonexercisers were similar for walking (3.97 versus 3.94 miles/day), age (37.8 versus 38.5) years, and weight (80.0 versus 77.7 kg). However, BMD and muscle strength were significantly greater in exercises than in nonexercisers.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗
PubMed1992

Modification of the dystrophic phenotype after transient neonatal denervation: role of MHC isoforms.

While it recently has been demonstrated that it is possible to modify the phenotypic expression of murine dystrophy (dy/dy) (i.e., prevent myofiber loss) by subjecting the extensor digitorum longus (EDL) muscle of 14-day-old dy/dy mice to transient neonatal denervation (Moschella and Ontell, 1987), the mechanism responsible for this phenomenon has not been determined. Since it has been suggested that the effects of dystrophy vary according to fiber type, the fiber type frequency in 100-day-old normal (+/+) and dy/dy EDL muscles subjected to transient neonatal denervation has been determined by immunohistochemical analysis of their myosin heavy chain (MHC) composition. This frequency has been compared with that found in the EDL muscles of 14- and 100-day-old unoperated +/+ and dy/dy mice, in order to determine whether the reinnervation of transiently denervated neonatal muscle results in a preponderance of fibers of the type that might be spared dystrophic deterioration. In unoperated dy/dy muscle there is a progressive decrease in the frequency and in the absolute number of fibers that express MHC2B, with 100-day-old dy/dy muscles having approximately 32% of the number of myofibers fibers containing MHC2B as is found in age-matched +/+ muscles. The number of fibers containing the other fast isoforms (MHC2A and MHC2X) is similar in +/+ and dy/dy muscles at this age, indicating that fibers with MHC2B are most affected by the dystrophic process. Reinnervation following transient neonatal denervation of both the +/+ and the dy/dy EDL muscles results in a similar decrease (approximately 62%) in the number of myofibers containing MHC2B and an increase in myofibers containing the other fast MHC isoforms (MHC2A and MHC2X). The selective effect of dy/dy on fibers containing MHC2B and the sparing of myofibers in transiently denervated dy/dy muscle (which contains a reduced frequency of fibers containing MHC2B) are consistent with, although not direct proof of, the hypothesis that alterations in the fiber type may play a role in the failure of myofibers in transiently denervated dy/dy muscles to undergo dystrophic deterioration. Evidence is presented suggesting that neurons that supply myofibers containing MHC2B may be at a selective disadvantage in their ability to reinnervate neonatally denervated muscles.

Animals↗
PubMed1992

Direct measurement of melphalan conjugation with glutathione: studies with human melanoma cells and mammalian liver.

The rate of formation of the major glutathione conjugate of the antitumor alkylating agent melphalan can be directly measured by high pressure liquid chromatography. Rates of melphalan-glutathione conjugate formation were determined in the presence and in the absence of human melanoma cell homogenates, or cell fractions from various tissue sources, and the relative contributions of enzyme-catalyzed and nonenzymatic conjugate formation to the overall rates of conjugation were determined. Significant rates of conjugation were observed in the absence of any enzyme-containing cell fractions. These rates were not increased by the addition of melanoma cell homogenates, animal liver microsomes or human liver cytosol or microsomes, even though these preparations all enhanced the rate of conjugation of 1-chloro-2,4-dinitrobenzene. Animal liver cytosol contains enzymes that provided a significant contribution to the overall rate of melphalan conjugate formation. We conclude that although liver cytosol contains enzymes that significantly enhance the rate of glutathione conjugation with melphalan, in the case of the tumor cells studied, cellular glutathione S-transferase-catalyzed activity appears to be, at best, a very minor determinant of the overall rate of melphalan-glutathione conjugate formation.

Animals↗
PubMed1990

Inhibition of catalase and epoxide hydrolase by the renal cystogen 2-amino-4,5-diphenylthiazole and its metabolites.

Subchronic feeding of 2-amino-4,5-diphenylthiazole (DPT) to rats results in the development of renal cysts and has been used as a model system to study polycystic kidney disease. Because previous studies revealed changes in renal enzymes following DPT administration, a possible direct effect of DPT and its phenolic metabolites on catalase and a related enzyme, epoxide hydrolase, was examined. Experiments with three in vitro systems (suspensions of rabbit renal tubules, rat kidney homogenates, and commercially obtained bovine liver catalase) revealed direct inhibition of catalase activity by the diphenolic metabolite (diOH- DPT: 2-amino-4,5di(4'-hydroxyphenyl)-thiazole), the known renal cystogen nordihydroquaiaretic acid (NDGA) 2-amino-4(4'-hydroxyphenyl),5-phenyl-thiazole (4OH-DPT), and the known catalase inhibitor 3-amino-1,2,4-triazole; DPT did not inhibit catalase activity. Following oral administration to rats of the DPT congeners, 4OH-DPT caused the greatest decrease in both renal catalase and cytosolic epoxide hydrolase activities and the shortest time to onset of cystic lesions. In vitro, mouse liver cytosolic epoxide hydrolase activity was substantially inhibited by 4OH-DPT and dioH-DPT, and NDGA, but not by 2-amino-4-phenyl,5-(4'-hydroxyphenyl)-thiazole (5OH-DPT) or DPT itself. Microsomal epoxide hydrolase (mEH) activity was inhibited by 4OH-DPT, unaffected by DPT or dioH-DPT, and stimulated 2-fold by 5OH-DPT. Finally, mEH activity was substantially higher in samples of normal human kidney than in samples of kidney derived from a patient with autosomal recessive polycystic kidney disease; no differences were observed in cEH activity in these samples. Although the role of altered catalase and epoxide hydrolase activities in cystogenesis is unknown, DPT-induced cyst formation is associated with loss of these enzyme activities in kidney tissue. To our knowledge, this is the first report of an in vivo diminution of cytosolic epoxide hydrolase activity by xenobiotics.

8,11,14-Eicosatrienoic Acid↗
PubMed1989

Selective inhibition of cytosolic epoxide hydrolase activity in vitro by compounds that inhibit catalase.

The ability of a number of known inhibitors of catalase activity to affect cytosolic and microsomal epoxide hydrolase activities in vitro, measured as enzymatic trans-stilbene oxide hydrolysis and styrene oxide hydrolysis, respectively, was investigated. Catalase and cytosolic epoxide hydrolase activities are inhibited by hydroxylated metabolites of 2-amino-4,5-diphenylthiazole (DPT). The metabolite hydroxylated on the 4-phenyl ring (4OH-DPT) and the metabolite hydroxylated on both phenyl rings (4,5-DIOH-DPT) are potent inhibitors of both enzymes; the metabolite hydroxylated on the 5-phenyl ring (5OH-DPT) is less potent. Unmetabolized DPT has no effect on either enzyme. 4OH-DPT inhibits, but 5OH-DPT enhances, microsomal epoxide hydrolase activity. 4,5-DIOH-DPT and DPT have no effect on this enzyme. Other compounds that inhibit both catalase and cytosolic epoxide hydrolase activities, but do not inhibit microsomal epoxide hydrolase activity, are nordihydroguaiaretic acid and 2-aminothiazole. Microsomal epoxide hydrolase activity is enhanced by 2-aminothiazole and levamisole in vitro. Thus these inhibitors of catalase are selective epoxide hydrolase inhibitors in that they inhibit cytosolic epoxide hydrolase activity in vitro, but have either no effect on, or increase the activity of, microsomal epoxide hydrolase in vitro. Conversely, the selective cytosolic epoxide hydrolase inhibitors 4-phenylchalcone oxide and 4'-phenylchalcone oxide do not inhibit catalase activity, nor does trichloropropene oxide, a selective microsomal epoxide hydrolase inhibitor.

Animals↗
PubMed1989

Similarities between catalase and cytosolic epoxide hydrolase.

Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

Amino Acids↗
PubMed1987

Late vertex positivity in event-related potentials as a guilty knowledge indicator: a new method of life detection.

Subjects were allowed to choose an item to keep from nine items in a box. They then were shown one of nine words randomly selected on a display screen. One of these words described the chosen item, the others described novel items. The subjects were told to try not to react emotionally to any of the words, but to try to defeat this test of deception. It was found that large positive waves with latencies between 400 and 700 ms poststimulus were present in the ERPS to the chosen but not to the novel words.

Adolescent↗
PubMed1985

Immunocytochemistry of luteinizing hormone-releasing hormone during spontaneous and thyroxine-induced metamorphosis of bullfrogs.

Double-bridge peroxidase-antiperoxidase immunocytochemistry was used to compare the developmental appearance of immunoreactive LH-RH (ir-LH-RH) in brains of bullfrog (Rana catesbeiana) tadpoles during either spontaneous or thyroxine-induced metamorphosis. During spontaneous metamorphosis, ir-LH-RH was localized in fibers of the external layer of the median eminence (ME) of stage XIII-XXV animals, while immunoreactive perikarya and other immunostained brain structures were absent. The extent and intensity of ME immunostaining increased concomitantly with measured ME morphological development. Tadpoles induced with thyroxine to metamorphic stages XIX-XXI exhibited ME structural development and neurohypophysial neurosecretory staining similar to spontaneously metamorphosed individuals of equal stages. However, comparable ME ir-LH-RH immunostaining and gonadal size were both less developed in thyroxine-treated animals, although increased relative to non-metamorphic vehicle-injected controls. These results indicate that the hypothalamic LH-RH system changes concurrently with ME structural development during spontaneous metamorphosis. Reduced ME ir-LH-RH staining and gonadal size in thyroxine-treated animals suggest that during prometamorphosis, factors other than thyroxine alone may coordinate the normal maturation of the hypothalamo-pituitary-gonadal axis of the bullfrog.

Animals↗
PubMed1982

[Proliferation and differentiation of a myogenic line in synthetic media].

When plated in plastic culture dishes, muscle cells of the L6 cell line are unable to proliferate or to differentiate in a synthetic medium composed of Dulbecco's Minimal Essential medium (DME), bovine fetuin (1 mg/ml) and bovine insulin (10 micrograms/ml). In contrast, these cells will proliferate and differentiate morphologically and biochemically in this medium if the culture dishes have been previously treated with DME containing fetal Calf serum. Pretreatment of dishes does not increase cell attachment in synthetic medium but seems to act as a factor increasing cell viability.

Animals↗