Biomedical subjects

H A Lardy

Publications and source records attributed to H A Lardy.

At least 37 records · Page 2Linked to original sources

PubMed1989

Adenylate kinase activity in ejaculated bovine sperm flagella.

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activity was detected in the flagella of ejaculated bovine spermatozoa. This activity provided sufficient ATP to produce normal motility in cells permeabilized with digitonin and treated with 0.5 mM MgADP. In the presence of ADP, adenylate kinase activity was inhibited by P1,P5-di(adenosine 5')-pentaphosphate (Ap5A), an adenylate kinase-specific inhibitor, and motility was stopped. ATP-supported motility was not affected by Ap5A. Mitochondrial adenylate kinase activity allowed AMP to stimulate respiration in permeabilized sperm. Adenylate kinase activity in tail fragments was most active in a pH range from 7.6 to 8.4, and a similar pH sensitivity was observed for this enzyme activity in a hypotonic extract of whole sperm. The apparent km of adenylate kinase activity in permeabilized tail fragments was about 1.0 mM ADP in the direction of ATP synthesis. The fluctuation of nucleotide concentrations in normal and metabolically stimulated sperm suggested that adenylate kinase was most active when the cell was highly motile, although adenylate kinase activity did not appear to be coupled strictly with motility.

Adenosine Diphosphate↗

PubMed1989

Sources of intramitochondrial malate.

Liver mitochondria from rats treated with gluconeogenic hormones or subjected to vigorous exercise consume oxygen more rapidly than do mitochondria from control rats. These treatments result in elevated mitochondrial malate concentrations, which facilitate the entry of added substrate into the mitochondria. In this paper we describe experiments conducted to determine the source of the extra malate. Injections of glutamate plus alanine, two amino acids that are increased in blood after exercise and hormone treatment, caused liver mitochondrial malate to be increased. Injections of glucagon, cortisol, or both hormones elevated liver mitochondrial malate concentrations in both adrenalectomized and sham-operated rats.

Adrenalectomy↗

PubMed1989

Glutathione peroxidase and iron-thiol dependent lipid peroxidation.

Role of glutathione peroxidase in iron-thiol-mediated lipid peroxidation was examined. The enzyme was unable to prevent peroxidation of extracted rat liver microsomal lipids. In contrast, when arachidonic acid was the substrate, glutathione peroxidase did decrease the formation of thiobarbituric acid-reactive material. Superoxide dismutase produced a consistent but partial inhibition of peroxidation and catalase was without effect. Our results suggest that iron-thiol-dependent lipid peroxidation cannot be completely blocked by protective enzymes that are effective in other systems.

Animals↗

PubMed1988

Enzymatic down regulation with exercise in rat skeletal muscle.

Maximal activities of rat skeletal muscle mitochondrial citrate synthase (CS), malate dehydrogenase (MDH), and alanine aminotransferase (ALT), as well as several other mitochondrial enzymes involved in various metabolic functions were significantly suppressed after a single bout of acute or exhaustive treadmill running. This enzymatic "down regulation" was maintained 24 and 48 h post exhaustion, especially in the untrained rats. Neither muscle cytosolic nor hepatic enzymes exhibited down regulation after exercise. Proteolysis was increased with exercise as assessed by the clearance of [3H]leucine previously incorporated into the proteins of the rats. Decreased CS, MDH, and ALT activities correlated with a significant loss of mitochondrial total protein sulfhydryl (r = 0.67, 0.68, 0.59, respectively, P less than 0.001) in untrained rats and both CS and MDH could be partially restored by incubation with dithiothreitol. Endurance-tested untrained and trained rats had significantly higher glutathione peroxidase (GPX) activity in both muscle mitochondria and cytosol which correlated significantly with endurance time (r = 0.70 and 0.74, respectively). It is concluded that enzymatic down regulation is not caused by proteolysis alone; i.e., peroxides and oxygen free radicals produced in prolonged exercise may alter the intramitochondrial redox state by oxidizing free thiols that may be required at active sites of these enzymes. Training may enhance the ability of the muscle to resist the toxic oxygen species by increasing GPX activity.

Animals↗

PubMed1988

Antioxidant enzyme systems in rat liver and skeletal muscle. Influences of selenium deficiency, chronic training, and acute exercise.

The influences of selenium deficiency (Se-D), chronic training, and an acute bout of exercise on hepatic and skeletal muscle antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), as well as glutathione S-transferase (GST) and tissue lipid peroxidation, were investigated in post-weaning male Sprague-Dawley rats. Se-D per se depleted GPX in both liver and skeletal muscle but had no effect on SOD or catalase activity. One hour of treadmill running (20 m/min, 0% grade and 27 m/min, 15% grade for untrained and trained rats, respectively) significantly elevated hepatic catalase and cytosolic SOD activity; more prominent activations were found in the Se-D or untrained rats, whereas skeletal muscle antioxidant enzymes were little affected. Ten weeks of training (1 h/day, 5 days/week at 27 m/min, 15% grade) increased hepatic mitochondrial SOD by 23% (P less than 0.05) in Se-D rats. Both hepatic mitochondrial and cytosolic GPX were decreased by training whereas GPX was increased twofold in skeletal muscle mitochondria. Se-independent GPX was elevated by training only in the skeletal muscle mitochondria of Se-D rats. Lipid peroxidation (malondialdehyde formation) was increased by an acute bout of exercise in hepatic mitochondria of the untrained rats and in skeletal muscle mitochondria of the Se-D rats. These data indicate that antioxidant enzymes in liver and skeletal muscle are capable of adapting to selenium deficiency and exercise to minimize oxidative injury caused by free radicals.

Animals↗

PubMed1988

Rat liver gamma-butyrobetaine hydroxylase catalyzed reaction: influence of potassium, substrates, and substrate analogues on hydroxylation and decarboxylation.

Interaction of rat liver gamma-butyrobetaine hydroxylase (EC 1.14.11.1) with various ligands was studied by following the decarboxylation of alpha-ketoglutarate, formation of L-carnitine, or both. Potassium ion stimulates rat liver gamma-butyrobetaine hydroxylase catalyzed L-carnitine synthesis and alpha-ketoglutarate decarboxylation by 630% and 240%, respectively, and optimizes the coupling efficiency of these two activities. Affinities for alpha-ketoglutarate and gamma-butyrobetaine are increased in the presence of potassium. gamma-Butyrobetaine hydroxylase catalyzed decarboxylation of alpha-ketoglutarate was dependent on the presence of gamma-butyrobetaine, L-carnitine, or D-carnitine in the reaction and exhibited Km(app) values of 29, 52, and 470 microM, respectively. gamma-Butyrobetaine saturation of the enzyme indicated a substrate inhibition pattern in both the assays. Omission of potassium decreased the apparent maximum velocity of decarboxylation supported by all three compounds by a similar percent. beta-Bromo-alpha-ketoglutarate supported gamma-butyrobetaine hydroxylation, although less effectively than alpha-ketoglutarate. The rat liver enzyme was rapidly inactivated by 1 mM beta-bromo-alpha-ketoglutarate at pH 7.0. This inactivation reaction did not show a rate saturation with increasing concentrations of beta-bromo-alpha-ketoglutarate. None of the substrates or cofactors, including alpha-ketoglutarate, protected the enzyme against this inactivation. Unlike beta-bromo-alpha-ketoglutarate, beta-mercapto-alpha-ketoglutarate did not replace alpha-ketoglutarate as a cosubstrate. Both beta-mercapto-alpha-ketoglutarate and beta-glutathione-alpha-ketoglutarate were noncompetitive inhibitors with respect to alpha-ketoglutarate.

Animals↗

PubMed1988

Pathway of gluconeogenesis from D- and L-glycerate in rat hepatocytes.

Synthesis of glucose from 10 mM D-glycerate by hepatocytes isolated from rats fasted 24 h was inhibited by 3-aminopicolinate (3AP), 3-mercaptopicolinate (3MP), and aminoxyacetate (AOA). The inhibition by AOA can be exerted over that caused by either of the picolinates. The effects of 3AP and 3MP were not additive, and cannot be ascribed totally to inhibition of reducing equivalent transfer by their inhibitory effect on phosphoenolpyruvate carboxykinase. The time course and dependence on substrate concentration of gluconeogenesis from L-glycerate was similar to those from D-glycerate, even though the cellular content of D-glycerate was markedly less when L-glycerate was the substrate. Gluconeogenesis from L-glycerate was not significantly affected by 3AP or 3MP, but was inhibited by AOA. The degree of inhibition by AOA of glucose synthesis from L-glycerate was greater than that from D-glycerate. Studies employing [14C]paraformaldehyde revealed that hydroxypyruvate may be converted to dihydroxyacetone in the hepatocyte. A hitherto unknown inhibition site of 3AP and 3MP is inferred and a dual pathway of D- and L-glycerate gluconeogenesis consistent with these results is proposed.

Aminooxyacetic Acid↗

PubMed1988

Identification and partial characterization of caltrin-like proteins in the reproductive tract of the guinea pig.

The presence of caltrin-like proteins in reproductive tract fluid (RTF) and seminal vesicle content from male guinea pigs has been determined. Two fractions with electrophoretic mobility corresponding to Mr = 6200 (main band) and 5100 were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Isoelectric focusing in thin-layer agarose gels revealed three bands with acidic pIs of 5.3, 6.0, and 6.2, respectively. RTF prevented the enhancement of calcium permeability induced by incubating guinea pig epididymal spermatozoa in medium for capacitation. Spermatozoa incubated for 2 h in minimal culture medium plus pyruvate and lactate containing RTF accumulated less than 30% of the 45Ca2+ accumulated by cells maintained in absence of this fluid. Calcium uptake by preincubated spermatozoa was also inhibited by RTF. Inhibition of calcium transport activity by RTF and seminal vesicle proteins was not decreased by heating the dialyzed preparations at 60 degrees C for 5 min. After this treatment, the inhibitory activity and the protein pattern were stable for 3 wk when stored at 4 degrees C. Unheated extracts lost calcium transport inhibitory activity after 2 or 3 days at 4 degrees C. In spite of the differences in pIs among the proteins from the guinea pig reproductive tract and bovine caltrin, several features indicate they may play a similar role in both species by controlling Ca2+ movement across the plasma membrane. By this mechanism, these proteins could regulate physiologic events essential for the fertilization process.

Animals↗

PubMed1987

Effects of beta 1- and beta 1 + beta 2-antagonists on training-induced myocardial hypertrophy and enzyme adaptation.

The effects of beta 1- and beta 1 + beta 2-antagonists on the myocardial adaptation to exercise training were investigated in male Sprague-Dawley rats randomly divided into trained (treadmill, 1 hr/day, 5 days/week for 10 weeks at 27 m/min, 15% grade) without drug (TC), sedentary without drug (SC), trained treated with atenolol (TA) (10 mg/kg body wt, i.p.), trained treated with propranolol (TP, 30 mg/kg body wt, i.p.), and sedentary propranolol. Doses of both beta-antagonists were titrated to decrease the exercise heart rate by 25% compared to the controls. The heart weight and heart/body weight ratio were significantly greater in TC (1.28 +/- 0.07 g (P less than 0.01); 296 +/- 12 mg/100 g body wt (P less than 0.05) respectively) than in SC (1.09 +/- 0.04 g and 268 +/- 11 mg/100 g body wt), or in TP and TA. Myocardial mitochondrial protein was unchanged by training or beta-blockade. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities were not altered. Carnitine palmitoyltransferase activity was increased in SP compared to SC. Training increased hexokinase activity only in TC (5.22 +/- 0.12 vs 4.26 +/- 0.23 mumol/min/g wet wt, P less than 0.01). Lactate dehydrogenase activity increased significantly (P less than 0.01) in both TC (383 +/- 14 mumol/min/g wet wt) and TA (372 +/- 14 mumol/min/g wet wt) compared to SC (276 +/- 14 mumol/min/g wet wt), but not in TP versus SP. These data indicate that (1) beta-adrenergic blockade prevents training-induced cardiac hypertrophy; (2) beta-antagonists have little effect on the myocardial oxidative capacity; and (3) while the training induction of myocardial hexokinase is inhibited by both beta 1- and beta 1 + beta 2-antagonists, myocardium may increase its ability to utilize lactate during exercise with training despite beta 1-blockade.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1987

Phosphoenolpyruvate carboxykinase ferroactivator 1. Mechanism of action and identity with glutathione peroxidase.

A cytosolic protein factor (ferroactivator) facilitates the activation of phosphoenolpyruvate carboxykinase by ferrous ions (Bentle, L. A., and Lardy, H. A. (1977) J. Biol. Chem. 252, 1431-1440). We have extended our studies on the interaction of Fe2+ with this enzyme to establish the conditions under which it is an activator or an inhibitor. Preincubation of phosphoenolpyruvate carboxykinase with Fe2+ and dithiothreitol resulted in irreversible loss of enzyme activity within minutes of Fe2+ addition. This was attributed to an active oxygen species produced by aerobic oxidation of the divalent metal ion in the presence of dithiothreitol as suggested by lack of inhibition in preincubation experiments with Fe2+ under mildly acidic pH; ferroactivation by many H2O2 scavenging enzymes; and lack of inhibition on preincubation under anaerobic conditions. We conclude that Fe2+ per se can activate phosphoenolpyruvate carboxykinase and that ferroactivator protein helps to overcome the deleterious effects of aerobic oxidation. Mechanistic details of ferroactivation and a comparison of the known properties of ferroactivator indicated the similarity of this protein with rat liver glutathione peroxidase. The identity of ferroactivator as glutathione peroxidase was confirmed by the demonstration of catalytic activity, selenium content, and immunological cross-reactivity.

Animals↗

PubMed1987

gamma-Butyrobetaine hydroxylase and the protective role of glutathione peroxidase.

The selenoenzyme glutathione peroxidase in the presence of GSH effectively replaced catalase in the in vitro assay for gamma-butyrobetaine hydroxylase. Quantitatively, glutathione peroxidase was an order of magnitude more efficient than catalase, with maximal activity at less than 0.1 microM glutathione peroxidase in a standard reaction. Glutathione peroxidase prevented the loss of gamma-butyrobetaine hydroxylase during preliminary incubation with ferrous ions but without other substrates as well as in the course of the reaction. Regardless of whether glutathione peroxidase or catalase was present in the assay, the ascorbate concentrations needed to achieve half-maximal rates were similar (about 1 mM). Phosphate stimulated the rate of L-carnitine synthesis. Ferrous ion saturation indicated a pronounced effect of phosphate on the maximal velocity of the enzyme-catalyzed reaction, but its mechanism of action remains to be elucidated. Based on the subcellular distribution of gamma-butyrobetaine hydroxylase, catalase, and glutathione peroxidase, the role of glutathione peroxidase assumes importance. However, initial studies indicated that the assayable activity of liver gamma-butyrobetaine hydroxylase and L-carnitine concentrations in liver, blood plasma, and muscle were not significantly altered in selenium-deficient rats.

Animals↗

PubMed1987

Amino acid metabolism during exercise in trained rats: the potential role of carnitine in the metabolic fate of branched-chain amino acids.

The influence of endurance training and an acute bout of exercise on plasma concentrations of free amino acids and the intermediates of branched-chain amino acid (BCAA) metabolism were investigated in the rat. Training did not affect the plasma amino acid levels in the resting state. Plasma concentrations of alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val) were significantly lower, whereas glutamate (Glu), glycine (Gly), ornithine (Orn), tryptophan (Trp), tyrosine (Tyr), creatinine, urea, and ammonia levels were unchanged, after one hour of treadmill running in the trained rats. Plasma concentration of glutamine (Glu), the branched-chain keto acids (BCKA) and short-chain acyl carnitines were elevated with exercise. Ratios of plasma BCAA/BCKA were dramatically lowered by exercise in the trained rats. A decrease in plasma-free carnitine levels was also observed. These data suggest that amino acid metabolism is enhanced by exercise even in the trained state. BCAA may only be partially metabolized within muscle and some of their carbon skeletons are released into the circulation in forms of BCKA and short-chain acyl carnitines.

Alanine Transaminase↗

PubMed1987

Insulin inhibition of hepatic cAMP-dependent protein kinase: decreased affinity of protein kinase for cAMP and possible differential regulation of intrachain sites 1 and 2.

In hepatocytes stimulated with 8-bromo-cAMP, insulin decreases the affinity of the cAMP-dependent protein kinase for cAMP, shifting the Ka without affecting the Vmax activity. This occurs under conditions where cyclic adenine nucleotide concentrations are unchanged. We report here that glycogenolysis stimulated by 8-(4-chlorophenylthio)-cAMP, an analog with 100 times tighter affinity than cAMP for the protein kinase regulatory subunit, was only slightly antagonized by insulin. The tight binding of this analog appears to overcome the protein kinase affinity change induced by insulin. The relative importance of the two intrachain cAMP binding sites of the cAMP-dependent protein kinase regulatory subunit was investigated by using analogs with relative selectivity for each site. Analogs exhibiting preferential binding to site 2 were far less sensitive to insulin antagonism than were analogs binding preferentially at site 1 and less well at site 2. No other property of these analogs, including the rate of hydrolysis by phosphodiesterase, the IC50 for phosphodiesterase, the Ka for protein kinase, or the type I versus type II kinase specificity, could account for the ability of insulin to antagonize glycogenolysis stimulated by these analogs. These data indicate that insulin may act to decrease the affinity of protein kinases for cAMP through a possible regulation of intrachain site 2 binding.

Animals↗

PubMed1987

The effects of beta-adrenergic blockade on body composition in free-fed and diet-restricted rats.

The effects of the non-selective beta-adrenergic blocking agent propranolol (known for its anti-lipolytic activity) on body composition were investigated in growing male rats on normal unrestricted diet (N = 7) and on diet restriction (N = 7, 95% of controls). Three animals in each group were injected i.p. with 30 mg propranolol per kg body weight (bw) dissolved in saline, 5 days/week. This dose attenuates exercising heart rate by 25% and exercise training-induced enzyme activity. The remaining animals received saline. Fat, glycogen, moisture and non-ether extractable residue were determined in the homogenized residue of the whole animal. After 9 weeks on the experimental regimen, bw gain was significantly lower in the diet restricted rats, whereas propranolol had no effect on the bw gain. The percentage of fat, moisture and non-ether extractable residue were unchanged by either propranolol or diet restriction. However, glycogen content was significantly lower in the beta-blocked rats either with or without diet restriction. These data indicated that neither beta-adrenergic blockade nor minimal diet restriction influences the percentage body fat, whereas body glycogen content is decreased under both conditions.

Adipose Tissue↗

PubMed1987

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa.

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.

Animals↗

PubMed1987

Effects of fluoride and caffeine on the metabolism and motility of ejaculated bovine spermatozoa.

Treatment of washed, ejaculated bovine sperm with 30 mM sodium fluoride immobilized the cells in a characteristically rigid form. In cells metabolizing endogenous substrates, fluoride decreased respiration by about 60%, but did not inhibit the cells' ability to produce adenosine-5'-triphosphate (ATP) via oxidative phosphorylation and did not block access to endogenous substrates. Fluoride-immobilized sperm maintained maximal ATP titers for at least 60 min, but oligomycin treatment rapidly depleted ATP, indicating that ATP synthesis and metabolism was occurring in immobilized sperm. The putative phosphodiesterase inhibitor caffeine (2.5 mM) restored motility and increased respiration in fluoride-treated sperm, but 8-bromo-adenosine-3',5'-monophosphate (8-bromo-cAMP) did not, even though 8-bromo-cAMP stimulated respiration in control (untreated) sperm. Carboxyfluorescein analysis of the intracellular pH of untreated sperm indicated a normal pH of 6.3. Fluoride addition decreased the apparent intracellular pH slightly, but this effect was attributable to dilution. Caffeine did not change internal pH in untreated or fluoride-immobilized sperm. Fluoride did not appear to affect cAMP metabolism, but caffeine increased intracellular cAMP titers by about 35% in both untreated and fluoride-inhibited sperm. However, caffeine treatment did not mimic 8-bromo-cAMP, as analyzed by electrophoresis and autoradiography of sperm proteins labeled with 32P from endogenously generated [32P]ATP. Clearly, caffeine is not stimulating motility in fluoride-treated sperm by affecting the cyclic AMP system. Fluoride also inhibited motility in digitonin-permeabilized sperm by a mechanism that may have involved magnesium depletion, but caffeine had no stimulatory effect on either untreated or fluoride-immobilized, permeabilized sperm.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa.

Comparative studies of Ca2+-uptake by guinea pig spermatozoa were performed with fresh epididymal sperm and with cells preincubated in a chemically defined, Ca2+-free medium for capacitation. Calcium uptake was negligible in fresh spermatozoa, but increased dramatically after 20 min of incubation at 37 degrees C in the presence of pyruvate and lactate. Spermatozoa incubated in the absence of these substrates accumulated only 34% as much 45Ca2+ as was taken up by cells in complete medium. The monosaccharides glucose, fructose, and mannose and the nonmetabolizable sugars 2-deoxyglucose and sucrose inhibited the enhancement of Ca2+-permeability. In the presence of 6 mM sucrose 45Ca2+ uptake was not influenced by external sodium chloride concentration between 0 mM and 145 mM. The respiratory activity of the capacitated spermatozoa not only was higher than that of uncapacitated cells, but it was stimulated by Ca2+. No effect of Ca2+ on respiration of fresh spermatozoa was detected. An increase in calcium uptake was associated with increasing pH of the medium. It is possible that a regulatory mechanism through the calcium permeability of the plasma membrane of guinea pig spermatozoa exists and controls the development of physiological events related with the fertilization process. The sugar composition, the availability of the energy substrates lactate and pyruvate, and the pH of the reproductive tract fluids could play an important role in the accessibility of Ca2+ into the cells in vivo, as has been demonstrated in vitro. The enhancement of calcium permeability during the preincubation could be a useful indicator to verify if capacitation has occurred.

Animals↗

PubMed1987

Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma.

Indirect immunofluorescence studies with polyclonal antibodies show that caltrin binds to the plasma membrane over the acrosome and principal tail regions of bovine spermatozoa but not to the postacrosomal area or the midpiece. Calcium influx into bovine epididymal spermatozoa maintained in a simple salt medium containing DL-beta-hydroxybutyrate is prevented by caltrin freshly prepared from bovine seminal plasma through a procedure employing only gel permeation columns. Older preparations, on the other hand, enhance calcium uptake into these cells. Caltrin freshly prepared through a purification scheme that includes a cation exchanger only induces enhancement of calcium uptake into bovine epididymal spermatozoa maintained under identical conditions. It is postulated that early during sperm transit through the female reproductive tract, caltrin bound to the sperm plasma membrane protects the sperm cells from calcium influx. As the cells enter the oviduct where meeting with the egg could take place, factors present in the surrounding milieu may cause caltrin to change from an inhibitor to an enhancer of calcium uptake. The acrosome reaction and possibly hyperactivation, two components of capacitation that require calcium influx as an initial event, then take place.

Animals↗