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S Subramaniam

Publications and source records attributed to S Subramaniam.

At least 19 recordsLinked to original sources
PubMed1996

Modulation of arrestin release in the light-driven regeneration of Rh1 Drosophila rhodopsin.

We report studies of the in vitro regeneration of Rh1 Drosophila rhodopsin using immunochemical and spectroscopic probes for the release of arrestin (49 kDa). Upon illumination of metarhodopsin-containing membrane suspensions isolated from homogenized Drosophila heads, arrestin was released into the aqueous medium. In contrast, no release of arrestin was observed upon illumination of metarhodopsin in lipid/detergent micellar extracts. The spectroscopic changes associated with the transition from metarhodopsin to rhodopsin were, however, similar in membrane suspensions and in micellar extracts. The light-driven release of arrestin was restored in reconstituted liposomes formed by dialysis of detergent from the micellar extracts. We conclude that micellar solubilization of membranes decouples the light-driven release of arrestin from rhodopsin structural changes which are responsible for altering the lambda max of the chromophore. The finding that arrestin release from rhodopsin can be modulated by changes in the local membrane environment provides an opportunity to further characterize the nature of rhodopsin conformational changes during regeneration.

Animals↗
PubMed1996

Modeling the structure of the combining site of an antisweet taste ligand monoclonal antibody NC10.14.

We report the predicted combining site structure of the monoclonal antibody fragment, NC10.14, which is specific for the superpotent sweetener, N-(p-cyanophenyl-N'-(diphenylmethyl) guanidine acetic acid, using computer-aided molecular modeling and experimental methods, such as fluorescence spectroscopy and circular dichroism. This is the first computer-aided modeling study on a lambda-chain antibody fragment. We have also identified the amino acids that are involved in ligand binding. Aromatic residues, L:91(W), L:96(W), and H:100G(Y) are predicted to make van der Waals contacts with the p-cyanophenyl moiety of the ligand. Residue H:56(K) is predicted to provide a counterion for the acetic acid moiety, and H:50(E) provides the negatively charged potential for interaction with the positive guanidinium group. We also make a comparison of the binding site architecture of NC10.14 with that of a related monoclonal antibody fragment NC6.8.

Acetates↗
PubMed1996

Modeling of the D1/D2 proteins and cofactors of the photosystem II reaction center: implications for herbicide and bicarbonate binding.

A three-dimensional model of the photosystem II (PSII) reaction center from the cyanobacterium Synechocystis sp. PCC 6803 was generated based on homology with the anoxygenic purple bacterial photosynthetic reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis, for which the X-ray crystallographic structures are available. The model was constructed with an alignment of D1 and D2 sequences with the L and M subunits of the bacterial reaction center, respectively, and by using as a scaffold the structurally conserved regions (SCRs) from bacterial templates. The structurally variant regions were built using a novel sequence-specific approach of searching for the best-matched protein segments in the Protein Data Bank with the "basic local alignment search tool" (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990, J Mol Biol 215:403-410), and imposing the matching conformational preference on the corresponding D1 and D2 regions. The structure thus obtained was refined by energy minimization. The modeled D1 and D2 proteins contain five transmembrane alpha-helices each, with cofactors (4 chlorophylls, 2 pheophytins, 2 plastoquinones, and a non-heme iron) essential for PSII primary photochemistry embedded in them. A beta-carotene, considered important for PSII photoprotection, was also included in the model. Four different possible conformations of the primary electron donor P680 chlorophylls were proposed, one based on the homology with the bacterial template and the other three on existing experimental suggestions in literature. The P680 conformation based on homology was preferred because it has the lowest energy. Redox active tyrosine residues important for P680+ reduction as well as residues important for PSII cofactor binding were analyzed. Residues involved in interprotein interactions in the model were also identified. Herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was also modeled in the plastoquinone QB binding niche using the structural information available from a DCMU-binding bacterial reaction center. A bicarbonate anion, known to play a role in PSII, but not in anoxygenic photosynthetic bacteria, was modeled in the non-heme iron site, providing a bidentate ligand to the iron. By modifying the previous hypothesis of Blubaugh and Govindjee (1988, Photosyn Res 19:85-128), we modeled a second bicarbonate and a water molecule in the QB site and we proposed a hypothesis to explain the mechanism of QB protonation mediated by bicarbonate and water. The bicarbonate, stabilized by D1-R257, donates a proton to QB2- through the intermediate of D1-H252; and a water molecule donates another proton to QB2-. Based on the discovery of a "water transport channel" in the bacterial reaction center, an analogous channel for transporting water and bicarbonate is proposed in our PSII model. The putative channel appears to be primarily positively charged near QB and the non-heme iron, in contrast to the polarity distribution in the bacterial water transport channel. The constructed model has been found to be consistent with most existing data.

Amino Acid Sequence↗
PubMed1996

Alteration of pulmonary surfactant proteins in rats chronically exposed to cigarette smoke.

Surfactant proteins (SP) play an important role in enhancing the surface properties of pulmonary surfactant and participate in host-defense mechanism(s) of the lung. Although it is known that cigarette smoking alters both pulmonary surfactant lipid composition and function, its effect on SPs is unknown. The present study was carried out to determine if chronic exposure to cigarette smoke alters pulmonary SPs, namely, SP-A and SP-B, in a rat model. Sprague-Dawley rats were exposed to cigarette smoke in a nose-only exposure system twice a day, every day for 70 weeks. At termination, bronchoalveolar lavage (BAL) fluid and the lung tissues were collected from room control, sham-treated (SH), and smoke-exposed (SM) animals for analyses. The total protein levels in the BAL fluid of SM rats tended to be higher but were not statistically different from those of the SH group. However, the albumin content of BAL fluid in SM rats, measured by quantitative immunoblotting, was significantly higher than in control groups. Compared to control groups, SP-A and SP-B levels in the BAL fluid of SM rats were significantly reduced by 25 and 50%, respectively, when expressed as units per microgram of BAL fluid protein. However, when calculated as total BAL fluid SP recovered per rat, only the SP-B levels of SM rats were significantly different from the control groups. Further analysis by ELISA confirmed the reduced levels of SP-B in SM rats. In contrast to BAL fluid, the lung tissue levels of SP and their respective mRNAs were not significantly different between the control and smoke-exposed groups. These results show a selective reduction in SP-B content on the bronchoalveolar surface following chronic exposure to cigarette smoke and suggest an inhibitory effect of cigarette smoke on surfactant secretory processes and/or a localized destruction of SPs on the bronchoalveolar surface.

Animals↗
PubMed1996

Modification of kainate-induced behavioral and electrographic seizures following inhibition of nitric oxide synthase in mice.

We assessed the effects of N(omega)-nitro-L-arginine-methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), on behavioral and electrographic seizures elicited in mice by convulsant doses of kainate. In Expt. 1, L-NAME dose-dependently potentiated the convulsant effects of kainate (44 mg/kg s.c.), transforming long-latency clonic convulsions into short-latency fits of wild-running, and increased the incidence of kainate-induced mortality. The proconvulsant effects of L-NAME (5 mg/kg i.p.) did not reflect shortened latency to kainate-induced epileptiform afterdischarge recorded via electrodes chronically implanted into the hippocampus, amygdala, frontal cortex or mesencephalic reticular formation (Expt. 2). We also observed a dramatic uncoupling of behavioral and electrographic seizures in mice treated with L-NAME 30 min prior to kainate: 4/6 mice treated with L-NAME failed to express afterdischarge from any of the sites assessed during fits of wild-running. The proconvulsant effects of L-NAME were dependent on the route of administration of kainate, as the inhibitor of NOS failed to alter behavioral (clonic) or electrographic seizures elicited by intrahippocampal kainate (1 nmol, Expt. 3) yet shortened latency to fits of wild-running following i.c.v. kainate (1 nmol, Expt. 4) and reduced the dose of systemic kainate required for either clonic convulsions or wild-running (Expt. 5). The observations that L-NAME potentiates kainate-induced wild-running but not necessarily clonus suggest the involvement of tectopontine mechanisms.

Animals↗
PubMed1996

Factors determining proconvulsant and anticonvulsant effects of inhibitors of nitric oxide synthase in rodents.

Although a majority of studies suggest that inhibitors of nitric oxide synthase (NOS) are proconvulsant, a substantial minority indicate the opposite (i.e. that inhibitors of NOS are anticonvulsant). As a consequence, the role of endogenous nitric oxide (NO) in the expression of seizures is unclear. In the present series of experiments, we therefore assessed factors governing pro- and anticonvulsant effects of inhibitors of NOS. In mice receiving systemic injections of kainate or picrotoxin, we confirmed the hypothesis that the effects of inhibitors of NOS vary with the model of seizure: Whereas 7-nitroindazole (7-NI) reduced the latency and increased the severity of kainate-induced convulsions (Expt. 1), both 7-NI and N(omega)-nitro-L-arginine methyl ester (L-NAME) slightly delayed clonus following the systemic administration of picrotoxin at doses > or = 3.5 mg/kg but not at doses < or = 3.0 mg/kg (Expts. 2-5). Paradoxically, L-NAME but not 7-NI significantly reduced the CD50 of picrotoxin, which was approximately 2 mg/kg in control mice (Expt. 4), revealing inhibitor-specific interactions with the dose of the convulsant. Finally, we determined in rats that the effects of L-NAME on kainate-induced seizures vary as a function of genetic factors: L-NAME significantly potentiated kainate-induced convulsions in Sprague-Dawley rats but not in Wistar rats (Expt. 6).

Animals↗
PubMed1996

Explicit solvent models in protein pKa calculations.

Continuum methods for calculation of protein electrostatics treat buried and ordered water molecules by one of two approximations; either the dielectric constant of regions containing ordered water molecules is equal to the bulk solvent dielectric constant, or it is equal to the protein dielectric constant though no fixed atoms are used to represent water molecules. A method for calculating the titration behavior of individual residues in proteins has been tested on models of hen egg white lysozyme containing various numbers of explicit water molecules. Water molecules were included based on hydrogen bonding, solvent accessibility, and/or proximity to titrating groups in the protein. Inclusion of water molecules significantly alters the calculated titration behavior of individual titrating sites, shifting calculated pKa values by up to 0.5 pH unit. Our results suggest that approximately one water molecule within hydrogen-bonding distance of each charged group should be included in protein electrostatics calculations.

Animals↗
PubMed1996

Block of the N-methyl-D-aspartate receptor by remacemide and its des-glycine metabolite.

The anticonvulsant and neuroprotective properties of remacemide [(+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)acetamide] and its active des-glycine metabolite [(+/-)-1-methyl-1,2-diphenylethylamine] may result in part from blockade of N-methyl-D-aspartate (NMDA) receptors. The blocking actions of the remacemide enantiomers and their des-glycinates were investigated in whole cell voltage-clamp recordings from cultured rat hippocampal neurons and in binding studies with [3H]dizocilpine in rat forebrain membranes. (+/-)-Remacemide caused a rapid and reversible inhibition of NMDA-evoked current; the R(+)- and S(-)-enantiomers were roughly equipotent (IC50 values at -60 mV, 67 and 75 microM, respectively). In contrast, the block by the S(+)- and R(-)-des-glycine analogs was slower, more potent and occurred in a stereoselective fashion (IC50 values, 0.7 and 4 microM). The block by S(+)-des-glycine remacemide was strongly use- and voltage-dependent, and, in addition, could be occluded by Mg++, indicating that it occurs by an open channel mechanism. In contrast, the block by R(+)-remacemide was only partially voltage-dependent, suggesting that it occurs by both channel blocking and nonchannel blocking (allosteric) mechanisms. Support for an allosteric mechanism was obtained in nonequilibrium [3H]dizocilpine binding studies where it was observed that 100 microM R(+)-remacemide slowed the dissociation of the radioligand [whereas 10 microM S(+)-des-glycine remacemide did not]. Neither R(+)-remacemide nor S(+)-des-glycine remacemide inhibited currents evoked by kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate or gamma-aminobutyric acid. We conclude that des-glycine remacemide is a potent and selective channel blocking NMDA receptor antagonist, whereas remacemide is weaker and inhibits NMDA receptors by both channel blocking and nonchannel blocking actions.

Acetamides↗
PubMed1996

A knowledge-based method for protein structure refinement and prediction.

The native conformation of a protein, in a given environment, is determined entirely by the various interatomic interactions dictated by the amino acid sequence (1-3). We describe here a knowledge-based approach for protein structure assessment and prediction. Using a well-defined set of high-resolution protein structures, we have derived statistical potentials, in the form of atom-pairwise distance probability density functions. These provide a description of pairwise interatomic interactions of native proteins. When applied to highly randomized and noisy structures of proteins distinct from the basis set, native-like structures were obtained to very high precision (< or = 2A). The examples tested include proteins of all sizes (from 38 up to 461 amino acids long) and diverse topological structures (alpha, beta and alpha-beta classes). The potentials appear to be sensitive enough to recognize subtle distortions from a native packing structure and in optimization of structures drive them consistently to a higher probability. Therefore they provide a powerful tool for refinement of X-ray and NMR derived structures at arbitrary degrees of initial precision.

Crystallography, X-Ray↗
PubMed1995

Molecular mechanism of protein-retinal coupling in bacteriorhodopsin.

Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93-->Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93-->Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93-->Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.

Bacteriorhodopsins↗
PubMed1995

Effect of eugenol on drug-metabolizing enzymes of carbon tetrachloride-intoxicated rat liver.

The chemoprotection extended by eugenol against carbon tetrachloride (CCl4) intoxication was established by studies on drug-metabolizing phase I and phase II enzymes. An overall decrease in drug-metabolizing enzymes, namely NADPH-cytochrome c reductase, NADH-cytochrome reductase, coumarin hydroxylase, 7-ethoxy coumarin-O-deethylase, UDP-glucuronyltransferase and glutathione-S-transferase, was observed with CCl4 intoxication, with a subsequent decrease in cytochrome P450 and cytochrome b5 content. CCl4 caused a significant decrease in microsomal phospholipids and the marker enzymes glucose-6-phosphatase and 5'-nucleotidase, and an increase in thiobarbituric acid reactive substances (TBARS). Simultaneous administration of eugenol with CCl4 inhibited the accumulation of TBARS and the decrease in the microsomal phospholipids and marker enzymes. Further, the chemical onslaught imposed by CCl4 on the drug-metabolizing system was removed successfully by eugenol. Eugenol appears to act as an in vivo antioxidant and as a better inducer of phase II enzymes than phase I enzymes. It is therefore suggested that eugenol could be an interesting basic structure for drug design.

5'-Nucleotidase↗
PubMed1995

Biochemical and biophysical characterization of pulmonary surfactant in rats exposed chronically to cigarette smoke.

The pulmonary surfactant plays an important role in the gas exchange functions of the lungs. Although previous studies suggest that cigarette smoking alters the pulmonary surfactant system in human smokers, the nature of such changes is poorly understood. The aim of the present study was to determine if biochemical and biophysical properties of pulmonary surfactant are affected in rats following chronic exposure to cigarette smoke. Female Sprague-Dawley rats were exposed daily to smoke from the University of Kentucky high tar/high nicotine reference cigarettes, twice a day, for 60 weeks in a nose-only exposure system. Blood carboxyhemoglobin, plasma cotinine, and pulmonary aryl hydrocarbon hydroxylase activity measurements showed that animals effectively inhaled smoke during exposures. At termination, the bronchoalveolar lavage fluids (BALF) and the lung tissues were collected for biochemical and biophysical analyses of surfactant. The total phospholipid content of the BALF and the lung tissues from room control (RC), sham-treated (SH), and smoke-exposed (SM) animals were the same among the different groups. However, disaturated phosphatidylcholine (DSPC) levels in the BALF were significantly decreased in SM rats compared to RC or SH groups. In contrast, the lung tissue DSPC content in SM rats was not significantly different from that of control groups. Phospholipid profile analysis of the BALF also did not reveal any significant differences among other major constituents of surfactant from control and SM animals. The organic extracts of BALF obtained from different animal groups were assessed for surface activity using a Wilhelmy balance. The results showed an increase in surface compressibility and a reduction in respreadability index in SM group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.

Acetates↗
PubMed1995

Incorporation of surface tension into molecular dynamics simulation of an interface: a fluid phase lipid bilayer membrane.

In this paper we report on the molecular dynamics simulation of a fluid phase hydrated dimyristoylphosphatidylcholine bilayer. The initial configuration of the lipid was the x-ray crystal structure. A distinctive feature of this simulation is that, upon heating the system, the fluid phase emerged from parameters, initial conditions, and boundary conditions determined independently of the collective properties of the fluid phase. The initial conditions did not include chain disorder characteristic of the fluid phase. The partial charges on the lipids were determined by ab initio self-consistent field calculations and required no adjustment to produce a fluid phase. The boundary conditions were constant pressure and temperature. Thus the membrane was not explicitly required to assume an area/phospholipid molecule thought to be characteristic of the fluid phase, as is the case in constant volume simulations. Normal to the membrane plane, the pressure was 1 atmosphere, corresponding to the normal laboratory situation. Parallel to the membrane plane a negative pressure of -100 atmospheres was applied, derived from the measured surface tension of a monolayer at an air-water interface. The measured features of the computed membrane are generally in close agreement with experiment. Our results confirm the concept that, for appropriately matched temperature and surface pressure, a monolayer is a close approximation to one-half of a bilayer. Our results suggest that the surface area per phospholipid molecule for fluid phosphatidylcholine bilayer membranes is smaller than has generally been assumed in computational studies at constant volume. Our results confirm that the basis of the measured dipole potential is primarily water orientations and also suggest the presence of potential barriers for the movement of positive charges across the water-headgroup interfacial region of the phospholipid.

Crystallography, X-Ray↗
PubMed1995

Computer modeling of electrostatic steering and orientational effects in antibody-antigen association.

Brownian dynamics simulations are performed to investigate the role of long-range electrostatic forces in the association of the monoclonal antibody HyHEL-5 with hen egg lysozyme. The electrostatic field of the antibody is obtained from a solution of the nonlinear Poisson-Boltzmann using the x-ray crystal coordinates of this protein. The lysozyme is represented as an asymmetric dumbell consisting of two spheres of unequal size, an arrangement that allows for the modeling of the orientational requirements for docking. Calculations are done with the wild-type antibody and several point mutants at different ionic strengths. Changes in the charge distribution of the lysozyme are also considered. Results are compared with experiment and a simpler model in which the lysozyme is approximately by a single charged sphere.

Animals↗
PubMed1995

Anticonvulsant efficacy of ADCI (5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) after acute and chronic dosing in mice.

ADCI (5-aminocarbonyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine), a low-affinity uncompetitive N-methyl-D-aspartate (NMDA) antagonist, is a broad-spectrum anticonvulsant with a favorable side-effect profile. In the present study, we sought to determine if tolerance develops to the anticonvulsant activity of ADCI, using the maximal electroshock (MES) test to assess seizure protection. Mice were treated with three daily injections of a 2 x ED50 dose for MES protection (18 mg/kg, intraperitoneally, i.p.) or vehicle for 7 or 14 days. On the day after the chronic treatment protocol, all animals received a challenge dose of ADCI (18 mg/kg) and 15 min later were evaluated in the MES test. In control animals, 83-94% of animals were protected and the ADCI plasma levels immediately after the MES test were 5.5-9.7 micrograms/ml. In treated animals, 29 and 0% of animals were protected at 7 and 14 days, respectively, and the ADCI plasma levels were 77 and 52% of the control values. [3H]Dizocilpine binding to brain NMDA receptors was unaltered by the chronic drug treatment. In subsequent experiments, we determined that 14-day chronically treated animals could be completely protected by increased doses of ADCI (ED50 28.9 mg/kg). In both naive and chronically treated animals receiving a challenge dose of ADCI, plasma drug levels decreased in two phases, the first with a time constant of approximately 55 min and the second with a much slower rate. The estimated plasma concentrations of ADCI reflecting threshold (3-5 micrograms/ml) and 50% protection (5-7.5 micrograms/mg) were similar in naive and chronic animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Felbamate block of the N-methyl-D-aspartate receptor.

The anticonvulsant felbamate may act as an N-methyl-D-aspartate (NMDA) receptor antagonist, but the mechanism of block has not been fully characterized. We sought to identify the sites at which felbamate exerts its NMDA receptor blocking action using radioligand binding to rat forebrain membranes and whole-cell voltage clamp and single-channel recordings from cultured rat hippocampal neurons. Equilibrium binding isotherms for [3H]dizocilpine, a channel blocking ligand, were obtained in the presence of saturating glutamate and glycine. At a concentration of 1 mM, felbamate competitively inhibited specific [3H]dizocilpine binding, indicating that felbamate interacts with the channel blocking site. At a higher concentration (3 mM), felbamate also reduced the maximal saturation binding, demonstrating an additional allosteric action. The dissociation constant (Kb), estimated from fits to the binding isotherms, was 0.7-1.1 mM. It has been proposed that felbamate block of NMDA receptors occurs via competitive glycine site antagonism. However, the slowing of [3H]dizocilpine dissociation by felbamate, unlike the slowing produced by 7-chlorokynurenic acid, was not reversed by increasing the glycine concentration. In addition, felbamate did not reduce specific binding of [3H]5,7-dichlorokynurenic acid, a glycine site ligand. In whole-cell voltage clamp recordings of NMDA receptor currents, its blocking time constant (69 +/- 0.4 msec) was substantially faster than the dissociation time constant of glycine (390 +/- 23 msec), whereas the time constant for 5,7-dichlorokynurenic acid (390 +/- 20 msec) was similar. These observations indicate that felbamate block of NMDA receptors does not occur by an action at the glycine site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗