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Biomedical subjects

S S Miller

Publications and source records attributed to S S Miller.

At least 19 recordsLinked to original sources
PubMed1995

The delayed basolateral membrane hyperpolarization of the bovine retinal pigment epithelium: mechanism of generation.

1. Conventional and ion-selective double-barrelled microelectrodes were used in an in vitro preparation of bovine retinal pigment epithelium (RPE)-choroid to measure the changes in membrane voltage, resistance and intracellular Cl- activity (aCli) produced by small, physiological changes in extracellular potassium concentration ([K+]o). These apical [K+]o changes approximate those produced in the extracellular (subretinal) space between the photoreceptors and the RPE following transitions between light and dark. 2. Changing apical [K+]o from 5 to 2 mM in vitro elicited membrane voltage responses with three distinct phases. The first phase was generated by an apical membrane hyperpolarization, followed by a (delayed) basolateral membrane hyperpolarization (DBMH); the third phase was an apical membrane depolarization. The present experiments focus on the membrane and cellular mechanisms that generate phase 2 of the response, the DBMH. 3. The DBMH was abolished in the presence of apical bumetanide (100 microM); this response was completely restored after bumetanide removal. 4. Reducing apical [K+]o, adding apical bumetanide (500 mM), or removing apical Cl- decreased aCli by 25 +/- 6 (n = 8), 28 +/- 1 (n = 2) and 26 +/- 5 mM (n = 3), respectively; adding 100 microM apical bumetanide decreased aCli by 12 +/- 2 mM (n = 3). Adding apical bumetanide or removing apical bath Cl- hyperpolarized the basolateral membrane and decreased the apparent basolateral membrane conductance (GB). 5. DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) blocked the RPE basolateral membrane Cl- conductance and inhibited the DBMH and the basolateral membrane hyperpolarization produced by apical bumetanide addition or by removal of apical Cl-o. The present results show that the DBMH is caused by delta[K]o-induced inhibition of the apical membrane Na(+)-K(+)-2Cl- cotransporter; the subsequent decrease in aCli generated a hyperpolarization at the basolateral membrane Cl- channel.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Alfalfa NADH-dependent glutamate synthase: structure of the gene and importance in symbiotic N2 fixation.

Glutamate synthase (GOGAT), a key enzyme in ammonia (NH+4) assimilation, occurs as two forms in plants: a ferredoxin-dependent form (Fd-GOGAT) and an NADH-dependent form (NADH-GOGAT). These enzymes are encoded by distinct genes as evidenced by their cDNA and deduced amino acid sequences. This paper reports the isolation and characterization of a NADH-GOGAT gene from alfalfa (Medicago sativa L.), the first GOGAT gene to be isolated from a eukaryote. RNase protection and primer extension experiments map the transcription start site of NADH-GOGAT to nearly identical positions. The transcribed region of this gene, 12,214 bp, is comprised of 22 exons separated by 21 introns. The 2.7 kbp region 5' from the translation initiation site confers nodule-specific reporter gene activity when used in a chimeric beta-glucuronidase (GUS) construct and transformed into Lotus corniculatus and Medicago sativa. Both infected and uninfected cells display GUS activity. The abundance of NADH-GOGAT transcripts increases substantially in developing nodules of plants infected with effective rhizobia. However, this increase is not observed when nodules are induced by a variety of ineffective rhizobial strains. Thus, unlike many other plant genes involved in root nodule NH+4 assimilation, high levels of NADH-GOGAT expression are strictly associated with effective nodules indicating that NADH-GOGAT plays a central role in the functioning of effective root nodules. An alfalfa Fd-GOGAT PCR product showing greater than 85% identity to maize Fd-GOGAT was isolated and used to investigate the contribution of this enzyme to NH+4 assimilation in nodules. Fd-GOGAT mRNA was abundant in leaves and cotyledons but was not detected in alfalfa root nodules. Fd-GOGAT in alfalfa does not appear to play a significant role in symbiotic N2 fixation.

Amino Acid Sequence↗
PubMed1995

Intravenous cholangiography and operative cholangiography: implications for laparoscopic cholecystectomy.

There has been a resurgence of interest in the potential role of intravenous cholangiography with the advent of laparoscopic cholecystectomy. A retrospective review of a historical group of 185 patients undergoing cholecystectomy in whom the results of both intravenous cholangiography (IVC) and routine operative cholangiography (OC) were available was carried out. The common bile duct (CBD) was explored in 31 (16.7%) patients and choledocholithiasis confirmed in 25 (81%). IVC had shown calculi in 17 of these 25 patients (sensitivity, 68%) whereas OC demonstrated calculi in 24 (sensitivity 96%). Out of the six negative duct explorations, IVC suggested stones in two patients (specificity 66%) and OC in four patients (specificity 33%). Ultrasound scan had a sensitivity of only 48% for bile duct calculi. The accuracy of IVC was 68% and OC 84%. Intravenous cholangiography has no routine role in the preoperative assessment in patients undergoing elective cholecystectomy. In high-risk patients, alternative imaging techniques should be used.

Case-Control Studies↗
PubMed1994

Proton-lactate cotransport in the apical membrane of frog retinal pigment epithelium.

We studied lactate- and pyruvate-dependent proton transport across the apical membrane of frog RPE. The epithelium was mounted in a modified Ussing-chamber that allowed measurement of transepithelial potential and resistance while intracellular pH was measured with either intracellular microelectrodes or a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). To estimate the rate of lactate influx from the change in intracellular pH, we used the NH4 pulse technique to measure intracellular buffering capacity and its dependence on intracellular pH. We found that the buffering capacity was 16 mM at pH1 = 7.28, and that it increased as intracellular pH decreased. Intracellular pH was monitored with the tissue bathed in nominally HCO3-free (Hepes buffered) Ringer. The perfusate on the apical side of the epithelium was then changed to a Ringer that contained between 5 and 100 mM lactate or pyruvate. When 10-100 mM lactate or pyruvate was added to the apical bath the cells acidified by 0.05-0.50 pH units. For each of these acidifications, the initial acid influx into the RPE cells was calculated from the intracellular buffering capacity and the initial rate of intracellular acidification. These influxes were plotted as functions of the concentrations of lactate or pyruvate and this relationship was analysed using Michaelis-Menten kinetics. The Km values were: 33 +/- 5 mM for lactate and 9 +/- 3 mM for pyruvate. There were no differences in the rates of acid influx caused by L- or D-lactate. The rates of acidification caused by 50 mM apical L-lactate were reversibly reduced by 56% after apical administration of probenecid (2 mM), and irreversibly reduced by 63% after apical administration of the SH-reagent mersalyl acid (2 mM). These results indicate the presence of a proton-lactate cotransport system in the apical membrane of the frog RPE.

Animals↗
PubMed1994

Lactate transport mechanisms at apical and basolateral membranes of bovine retinal pigment epithelium.

The isolated bovine retinal pigment epithelium actively transports lactate from the apical to the basal bath. Net short-circuit [14C]lactate flux in 20 mM lactate was 0.46 +/- 0.09 mu eq.cm-2.h-1 (n = 8). In open circuit, with a physiological lactate gradient, net [14C]lactate flux was 0.66-1.31 mu eq.cm-2.h-1 (n = 3). Lactate in the apical bath caused intracellular acidifications that were saturable, apparently stereospecific, and reduced in magnitude by several H-lactate cotransport inhibitors. In the basal bath, lactate caused intracellular alkalinizations that were dependent on the presence of Na. In short circuit, 20 mM lactate in both baths reversed the direction of net transepithelial 22Na transport from secretion to absorption, suggesting the presence of basolateral Na-lactate cotransport moving lactate out of the cells. Outwardly directed Na-lactate cotransport requires a lactate:Na stoichiometry > 1.4:1, consistent with the coupled movement of Na, lactate, and net negative charge across the basolateral membrane. Intracellular microelectrode recordings showed that basal lactate hyperpolarized and apical lactate depolarized the basolateral membrane. For lactate absorption, this is a novel arrangement of membrane proteins:luminal H-lactate cotransport and serosal electrogenic Na:(n)lactate cotransport. Lactate transport across the retinal pigment epithelium may play an important role in regulating retinal metabolism and subretinal space volume and composition.

Animals↗
PubMed1992

Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules.

Phosphoenolpyruvate carboxylase (PEPC) plays a key role in N2 fixation and ammonia assimilation in legume root nodules. The enzyme can comprise up to 2% of the soluble protein in root nodules. We report here the isolation and characterization of a cDNA encoding the nodule-enhanced form of PEPC. Initially, a 2945 bp partial-length cDNA was selected by screening an effective alfalfa nodule cDNA library with antibodies prepared against root nodule PEPC. The nucleotide sequence encoding the N-terminal region of the protein was obtained by primer-extension cDNA synthesis and PCR amplification. The complete amino acid sequence of alfalfa PEPC was deduced from these cDNA sequences and shown to bear striking similarity to other plant PEPCs. Southern blots of alfalfa genomic DNA indicate that nodule PEPC is a member of a small gene family. During the development of effective root nodules, nodule PEPC activity increases to a level that is 10- to 15-fold greater than that in root and leaf tissue. This increase appears to be the result of increases in amount of enzyme protein and PEPC mRNA. Ineffective nodules have substantially less PEPC mRNA, enzyme protein and activity than do effective nodules. Maximum expression of root nodule PEPC appears to be related to two signals. The first signal is associated with nodule initiation while the second signal is associated with nodule effectiveness. Regulation of root nodule PEPC activity may also involve post-translational processes affecting enzyme activity and/or degradation.

Amino Acid Sequence↗
PubMed1992

Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Ion transport mechanisms in native human retinal pigment epithelium.

Electrophysiologic techniques were used to characterize the electrical properties and the ion transport mechanisms at the apical and basolateral membranes of the human retinal pigment epithelium (RPE). These experiments used fresh native tissue from adult donor and fetal eyes. In the upper range, adult donor RPE had an apical membrane resting potential (VA) of approximately equal to -60 mV and a transepithelial potential (TEP) and resistance (Rt) of 3.5 mV and 148 omega.cm2, respectively. The means were at least 50% of these values. In RPE from fetuses of gestational age 19-23 wk, VA was -56 +/- 4 mV, TEP was 2.2 +/- 1.5 mV, Rt was 206 +/- 151 omega.cm2, and the ratio of apical to basolateral resistance was 0.70 +/- 0.50 (mean +/- standard deviation; n = 15). The apical membrane of the adult donor and fetal RPE contains a large relative K+ conductance (TK > 0.3) that is barium blockable. In fetal RPE, there is evidence for separate K+ and Cl- conductive mechanisms at the basolateral membrane. However, the evidence for the Cl- conductance is indirect. The fetal RPE apical membrane, but not the basolateral membrane, contains a ouabain-sensitive mechanism that exhibits two distinct phases of apical depolarization. The first, rapid phase suggests that the pump is electrogenic. The apical membrane of fetal RPE contains a bumetanide-sensitive mechanism and a receptor activated by nanomolar amounts of epinephrine. In fetal RPE, step changes in apical [K+]o between 5 and 2 mmol/l produced a delayed basolateral membrane hyperpolarization that in situ generates the fast oscillation trough of the electroretinogram.

Adult↗
PubMed1992

Na-dependent pHi regulatory mechanisms in native human retinal pigment epithelium.

This study provides the first information about pHi regulatory mechanisms in human retinal pigment epithelium (RPE). The experiments were carried out on fresh explant tissues from adult donor and fetal eyes, and pHi was measured using fluorescence microscopy techniques and the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In adult donor RPE, the resting pHi is 7.30 +/- 0.14 (mean +/- standard deviation; n = 6) in HCO3 Ringer's solution. In HCO3 Ringer's solution, apical Na removal caused rapid cell acidification with an initial rate of 0.40 +/- 0.10 pH U/min (n = 4). This Na-dependent acidification was partially inhibited by apical amiloride (n = 1) and DIDS (n = 1). In HCO3 Ringer's solution, pHi recovery from an acid load (NH4 prepulse) also was blocked by apical Na removal. In nominally HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells. These results suggest that the apical membrane of adult human RPE contains an Na/H exchanger and possibly a Na-dependent, DIDS-inhibitable pH regulatory mechanism, perhaps a NaHCO3 cotransporter. For the fetal RPE, the resting pHi was 7.16 +/- 0.10 (n = 9) and 7.19 +/- 0.10 (n = 20) in HCO3 and HCO3-free Ringer's solution, respectively. In HCO3 and HCO3-free Ringer's solution, apical amiloride (1 mmol/l) acidified the cells and the removal of apical Na caused cell acidification with an initial rate of 0.30 +/- 0.08 (n = 32) and 0.58 +/- 0.29 (n = 6) pH U/min, respectively. The pHi recovery from an acid load also was blocked by apical amiloride and apical Na removal. These results suggest that the apical membrane Na/H exchanger is the dominant acid extrusion mechanism in human fetal RPE.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium.

1. Intracellular voltage recordings using conventional and double-barrelled chloride-selective microelectrodes have been used to identify several transport mechanisms at the apical and basolateral membranes of the isolated bovine retinal pigment epithelium (RPE)-choroid preparation. Intracellular recordings were obtained from two cell populations, melanotic (pigmented) and amelanotic (non-pigmented). The electrical properties of these two populations are practically identical. For melanotic cells the average apical resting membrane potential (VA) is -61 +/- 2 mV (mean +/- S.E.M., n = 49 cells, thirty-three eyes). For these cells the ratio of apical to basolateral membrane resistance (a) was 0.22 +/- 0.02. The mean transepithelial voltage and resistance were 6 +/- 1 mV and 138 +/- 7 omega cm2, respectively. 2. The apical membrane, which faces the distal retina, contains a Ba(2+)-inhibitable K+ conductance and a ouabain-inhibitable, electrogenic Na(+)-K+ pump. In addition it contains a bumetanide-sensitive mechanism, the putative Na(+)-K(+)-Cl- cotransporter. The basolateral membrane contains a DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid)-inhibitable chloride channel. The relative conductances of the apical and basolateral membranes to K+ and Cl- are TK approximately 0.9 and TCl approximately 0.7, respectively. 3. The ouabain-induced fast phase of apical membrane depolarization (0-30 s) was used to calculate the equivalent resistances of the apical (RA) and basolateral (RB) cell membranes, as well as the paracellular or shunt resistance (RS). They are: 3190 +/- 400, 17920 +/- 2730 and 2550 +/- 200 omega (mean +/- S.E.M., n = 9 tissues), respectively. From these data the equivalent electromotive forces (EMF) at the apical (EA) and basolateral (EB) membranes were also calculated. They are: -69 +/- 5.0 and -24 +/- 5.0 mV, respectively. 4. Intracellular Cl- activity (aiCl) was measured using double-barreled ion-selective microelectrodes. In the steady state aiCl = 61 +/- 4.0 mM and the Nernst potential ECl = -13.5 +/- 1.5 mV (mean +/- S.E.M., n = 4). 5. In the intact eye or in retina, RPE-choroid preparations it has been shown that the transition between light and dark alters the K+ concentration in the extracellular (or subretinal) space between the photoreceptors and the apical membrane of the RPE. These light-induced changes in subretinal [K+]o were qualitatively simulated in vitro by altering apical K+ between 5 and 2 mM. This produced a sequence of voltage changes at the apical and basolateral membranes that had three operationally distinct phases. Phase 1 is generated by the combination of an apical membrane K+ diffusion potential and inhibition of the electrogenic Na(+)-K+ pump.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

pHi regulation in frog retinal pigment epithelium: two apical membrane mechanisms.

This study demonstrates that the apical membrane of frog retinal pigment epithelium (RPE) contains two intracellular pH (pHi) regulatory mechanisms, an electrogenic Na-HCO3 cotransporter blocked by DIDS and an amiloride-inhibitable Na-H antiporter. pHi was studied using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In these cells resting pHi equals 7.26 +/- 0.09 (n = 58). After an acid load (NH4Cl prepulse), pHi recovery required apical extracellular Na concentration ([Na]o) in HCO3 or HCO3-free Ringer. In HCO3 Ringer recovery was completely blocked by 1 mM apical DIDS (n = 5) but was not affected by absence of Cl. In HCO3-free Ringer, recovery was completely blocked by 1 mM apical amiloride (n = 3). At resting pHi, the intrinsic pH-buffering capacity of the cell is approximately 7.1 mM/pH and rises monotonically as pHi decreases. In HCO3 Ringer, the initial rate of acidification caused by apical Na removal, 0.39 +/- 0.03 pH/min (n = 26), was 80-90% inhibited by apical DIDS (n = 5) and 16% inhibited by 1 mM apical amiloride (n = 7), but not affected by absence of Cl. In HCO3 Ringer, initial rates of acidification induced by apical DIDS or amiloride were 0.11 +/- 0.06 (n = 5) and 0.03 +/- 0.02 pH/min (n = 7), respectively. These results indicate that the Na-HCO3 cotransporter accounts for 80-90% of the acid extrusion from frog RPE cells. Increasing apical [K]o from 2 to 5 mM approximates the in vivo apical [K]o changes during a light-dark transition and alkalinizes the cells. [K]o-induced alkalinization had an initial rate of 0.11 +/- 0.02 pH/min (n = 16), which was approximately 75% inhibited by apical DIDS (to 0.04 +/- 0.01 pH/min, n = 7) and completely blocked by HCO3/CO2 removal from both bathing solutions. [K]o-induced pHi changes alter RPE transport mechanisms and may affect RPE-photoreceptor interactions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

Epinephrine stimulates fluid absorption across bovine retinal pigment epithelium.

The authors used a modified capacitive probe technique to simultaneously assess the effect of apical epinephrine on fluid transport rate (Jv), transepithelial potential (TEP), and transepithelial resistance (Rt) across bovine retinal pigment epithelium (RPE). In control Ringer, the RPE absorbed fluid at a rate of 1.42 +/- 0.34 microliters/cm2.hr (mean +/- SEM; 22 tissues). Tissues with the highest TEP (8-9 mV) and Rt (160-220 omega.cm2) had maximum fluid absorption rates (3-4 microliters/cm2.hr). Apical epinephrine (100 nM) stimulated Jv by a factor of 3, from 0.70 +/- 0.18 microliter/cm2.hr to 2.17 +/- 0.24 microliters/cm2.hr and TEP from 4.6 +/- 0.4 mV to 7.0 +/- 0.6 mV (n = 6). The epinephrine-induced transport changes were inhibited by apical bumetanide (0.1 mM). The alpha-1 adrenergic antagonist prazosin (1 microM) completely blocked the epinephrine-induced stimulation of Jv and TEP. In contrast, the beta adrenergic antagonist propranolol (1 microM) had no effect on epinephrine-induced transport changes. These results, coupled with previous studies on bovine RPE, suggest that the mechanisms underlying the epinephrine-induced stimulation of fluid absorption include an apical membrane alpha-1 adrenergic receptor, a bumetanide-inhibitable apical membrane Na-K-2Cl cotransporter, and a basolateral membrane Cl conductance.

Absorption↗
PubMed1991

Adolescent health care: an assessment of referral activities.

The referral activities of 45 agencies (90% of the sample originally polled) in a moderate-sized midwest community were investigated with respect to agency referral patterns and professionals' appraisal of adolescents' health care status. A combined analysis of qualitative and quantitative data provided valuable insight into the problems of unmet adolescent health care needs and underutilization of adolescent health care services. Referral activity characteristics which may hinder the successful networking of services for adolescents are highlighted.

Adolescent↗
PubMed1990

Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2-year study.

Zygote intrafallopian transfer (ZIFT) was used as a treatment for long-standing nontubal infertility for a 2-year period. The overall clinical pregnancy rate for 114 tubal transfers was 40.4% with a delivery/ongoing rate of 34.2%. Concurrent use of in vitro fertilization and embryo transfer (IVF-ET) for tubal factor infertility gave significantly lower clinical pregnancy and delivery/ongoing rates (21.1% and 15.8%, respectively). The use of gamete intrafallopian transfer (GIFT) for nontubal infertility yielded a 32% clinical pregnancy rate and a 26% delivery rate for 53 transfers. Zygote intrafallopian transfer resulted in an implantation rate per zygote of 17% overall compared with 8.1% per embryo for IVF-ET and 11.2% per oocyte for GIFT. The transfer of three zygotes per patient gave the same clinical pregnancy rate as the transfer of four while reducing the incidence of multiple gestation from 19% to 7.8% per transfer. No significant decline in the clinical pregnancy or delivery rate was seen with ZIFT in women aged 25 through 39.

Adult↗
PubMed1990

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport.

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.

Animals↗