Factors influencing the pattern of dopamine secretion in Tetrahymena pyriformis

Dopamine production and secretion by the unicellular eukaryote Tetrahymena pyriformis were examined through the use of high performance liquid chromatography (HPLC) with electrochemical detection and through labeling studies with radioactive precursors. Growing cultures maintained a steady state intracellular level of 1.6 ± 0.3 pmol dopamine/10⁶ cells while secreting dopamine into the medium at a rate of 0.2–0.3 pmol/10⁶ cells per min. Incorporation of [¹⁴C]tyrosine and l-[³H]dihydroxyphenylalanine (DOPA) into dopamine was most successful in a basal medium (1.3 mM Tris-HCl, 1 mM citric acid, and 1 mM Ca(OH)₂, (pH 6.5)). A rapid conversion of added l-[³H]DOPA into dopamine confirmed the dynamic pattern of dopamine synthesis and secretion first indicated by the quantitative chromatographic analyses. The intracellular concentration of dopamine dropped sharply after cells were resuspended in the basal medium at 10⁶ cells/ml, so that by approx. 1 h after resuspension, dopamine dropped below the level detectable by HPLC (0.15 pmol/10⁶ cells). Under these conditions, dopamine secretion continued at a high rate for some time, finally leading to a maximal extracellular concentration of 8.71 ± 1.73 pmol/ml by 1 h. At this concentration, the rate of secretion appears to match that of degradation. Pulse chase experiments confirmed the rapid 3urnover of intracellular dopamine. Approx. 90% of [³H]dopamine and l-[³H]DOPA disappeared from l-[³H]DOPA-prelabeled cells during a 5 min chase, with approx. 50% of this being recovered as [³H]dopamine in the cells' medium. Dopamine secretion could be increased by nearly 100-fold by adding high levels (15 nmol/ml) of l-DOPA to the medium. In contrast, NSD-1015, a potent inhibitor of dopamine synthesis, completely blocked dopamine production. 0.15 mM dibucaine and 0.02 mM reserpine reduced dopamine secretion by approx. 65% over a 25-min incubation, but 5 mM EGTA had no noticeable effect.     

Presence of 3,4-dihydroxyphenylalanine and 3,4,5-trihydroxyphenylalanine in a coelenterate nervous system: Possible tyrosinase-mediated accumulation

3,4-dihydroxyphenylalanine (DOPA), 3,4,5-trihydroxyphenylalanine (5-OH-DOPA), 5-S-cysteinylDOPA (5-SC.D) and 2-S-cysteinylDOPA (2-SC.D) in the tentacles of the sea anemone, Metridium senile, were studied by the combined use of differential centrifugation of tissue homogenates, ultracentrifugation on sucrose density gradients, HPLC and electron microscopy. DOPA, 5-OH-DOPA and o-diphenol:O₂ oxidoreductase (Tyrosinase) were enriched in fractions containing membranes and subcellular particles, and the cysteinylDOPAs in the cytosol fractions. Ultrastructurally studied fractions rich in DOPA and 5-OH-DOPA contained large numbers of highly osmium-reducing vesicles. Identical structures were localized in ectodermal nerves and epidermal sensory cells.The results suggest that previous findings of catecholderivatives in the tentacles of Metridium and other sea anemone species by histochemical methods, are explained by a tyrosinase-based accumulation of DOPA and 5-OH-DOPA in the ectodermal nerve net. These substances are confined in specific compartments (vesicles) in the neurons and sensory cells.     

An Improved HPLC-Electrochemical Detection Method for Measuring Brain Levels of 5-S-Cysteinyldopamine, 5-S-Cysteinyl-3,4-Dihydroxyphenylalanine, and 5-S-Cysteinyl-3,4-Dihydroxyphenylacetic Acid

Brain levels of the 5-S-cysteinyl adducts of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine were determined in several mammalian species. The low levels of the compounds and the risk of artifacts during sample preparation necessitated rather profound modifications of the assaying method. The refined method has made it possible to present more accurate data than those previously reported from this laboratory. The occurrence of low levels of the 5-S-cysteinyl adducts in dopamine-rich brain areas, but not in cerebellum, is indirect evidence of in vivo autoxidation of DOPA, DOPAC, and dopamine. The products generated during catechol autoxidation, including quinones and reduced forms of oxygen, are known to be potentially cytotoxic.     

ALTERATIONS IN RECEPTORS CONTROLLING DOPAMINE SYNTHESIS AFTER CHRONIC ETHANOL INGESTION

The influence of acute and chronic ethanol treatment and withdrawal on regulation of dopamine synthesis in striatal and mesolimbic areas of mouse brain was evaluated. Tyrosine hydroxylase activity was estimated by measuring in vivo DOPA accumulation after inhibition of aromatic amino acid decarboxylase. Eight hours after a single (3 g/kg) dose of ethanol, DOPA synthesis was increased and pimozide, a dopamine receptor antagonist, stimulated DOPA synthesis to the same degree in ethanol-treated and control animals. On the other hand, 8 h after withdrawal of animals from chronic ethanol treatment, endogenous dopamine synthesis was the same in ethanol-withdrawn and control animals, but the stimulation of dopamine synthesis produced by low doses of pimozide or haloperidol was significantly less in the animals that had consumed ethanol. This effect was even more apparent at 24h after withdrawal; by 3 days after withdrawal the decreased response of ethanol-withdrawn animals to the administration of dopamine receptor blockers was no longer statistically significant. At all time points tested, high doses of pimozide or haloperidol stimulated DOPA synthesis equally in control and ethanol-withdrawn animals. Chronic ethanol treatment and withdrawal may alter the coupling between dopamine receptors which regulate dopamine synthesis and tyrosine hydroxylase.     

The effects of 5-hydroxytryptophan and 5-hydroxytryptamine on dopamine synthesis and release in rat brain striatal synaptosomes.

The effects of 5-hydroxytryptophan (5-HTP) and serotonin (5-HT) on dopamine synthesis and release in rat brain striatal synaptosomes have been examined and compared to the effects of tyramine and dopamine. Serotonin inhibited dopamine synthesis from tyrosine, with 25% inhibition occurring at 3 μM-5-HT and 60% inhibition at 200 μM. Dopamine synthesis from DOPA was also inhibited by 5-HT, with 30% inhibition occurring at 200 μ. At 200 μM-5-HTP, dopamine synthesis from both tyrosine and DOPA was inhibited about 70%. When just the tyrosine hydroxylation step was measured in the intact synaptosome, 5-HT, 5-HTP, tyramine and dopamine all caused significant inhibition, but only dopamine inhibited soluble tyrosine hydroxylase [L-tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] prepared from lysed synaptosomes. Particulate tyrosine hydroxylase was not inhibited by 10 μM-5-HT, but was about 20% inhibited by 200 μM-5-HT and 5-HTP. At 200 μM both 5-HT and 5-HTP stimulated endogenous dopamine release. These experiments suggest that exposure of dopaminergic neurons to 5-HT or 5-HTP leads to an inhibition of dopamine synthesis, mediated in part by an intraneuronal displacement of dopamine from vesicle storage sites, leading to an increase in dopamine-induced feedback inhibition of tyrosine hydroxylase, and in part by a direct inhibition of DOPA decarboxylation.     

The influences of ecto- and endoderm in determining the axial polarity ofHydra attenuata Pall. (Cnidaria,Hydrozoa)

Summary Ectoderm and endoderm of the gastric column ofHydra attenuata Pall. were separated from each other and reassembled with either the same (controls) or opposite polar orientation. The controls always regenerated a head and basal disc according to the original polarity. In those specimens in which the polarity of ecta- and endoderm was opposite 33 specimens out of 41 reconstituted a single polyp whose body axis was clearly identifiable. Of these cases 8 followed the polarity of the ectoderm, 3 obeyed that of the endoderm, and 22 formed axes perpendicular to the original longitudinal axis. In 5 cases 2 specimens regenerated from the reassembled specimens. It is thus demonstrated that axial polarity ofHydra is determined by both the ecto- and the endoderm.This work was carried out with the support of the Swiss National Science Foundation (Grant Nr. 3.317-0.78)     

Embryology ofHeliotropium scabrum andH. strigosum (Boraginaceae)

The embryology ofHeliotropium scabrum andH. strigosum has been studied. The development of the anthers follows the dicotyledonous type, the tapetal cells become binucleate. The pollen grains are shed at the two-celled stage. Megaspore tetrads are linear and the development of the megagametophyte corresponds with the Polygonum type. The endosperm is cellular. The embryo development follows the Onagrad Type, i.e. the Capsella variation inH. scabrum and the Nicotiana variation of the Solanad Type inH. strigosum. The pericarp is differentiated into a one-layered epicarp with bulbous-based, unicellular hair, a 5–6-layered chlorenchymatous mesocarp and a 6–7-layered endocarp. The seed coat consists only of the thickened portions of the epidermis.     

Anomalous secondary vascular tissue inHelminthostachys zeylanica (Ophioglossaceae)

The vascular anatomy ofHelminthostachys zeylanica was examined with special reference to anomalous secondary tissue. Primary xylem development gradually takes place centrifugally. In branched rhizomes with destroyed apices, the vascular cylinder apical to the insertion of branch traces is generally composed of primary xylem, accessory xylem, inner parenchyma of radially arranged cells, outer parenchyma of irregularly arranged cells, and partly crushed phloem, listed in order going outwards. The accessory xylem as well as the inner parenchyma ofHelminthostachys zeylanica is probably secondarily produced, partly to contribute to the branch traces, in a position corresponding to that of secondary vascular tissue developed from a normal cambium inBotrychium sensu lato. It is suggested that although a cambium is lacking inHelminthostachys zeylanica, the secondary vascular tissues are comparable between the genera. The phylogenetic implication of this tissue is discussed.     

Quantitative nuclear DNA changes inEleusine (Gramineae)

2C nuclear DNA amounts were determined in 30 collections belonging to 10 species ofEleusine. About a 2.5-fold variation in genome size is evident in the genus. The 2C DNA amount in the diploid species ranged from 2.50 pg inE. verticillata to 3.35 pg inE. intermedia. In contrast, the tetraploid species showed a range from 4.95 pg inE. africana to 6.13 pg inE. floccifolia. At intraspecific level 10 collections ofE. coracana, 6 ofE. indica, 4 ofE. africana, 2 ofE. tristachya, and 2 ofE. kigeziensis did not show any significant variation. However, 2 collections ofE. floccifolia, connected with polyploidy, displayed about 90% variation. Polyploid species showed approximately double the genome size of that of their corresponding diploids. An evolutionary increase in DNA amount is evident inE. coracana during the course of its origin and domestication fromE. africana.     

Effect of dietary proteins and carbohydrates on urinary and sympathoadrenal catecholamines

We previously observed that administration of tyrosine to rats or humans elevated urinary dopamine, norepinephrine and epinephrine levels. The present studies examine the effects on these urinary catecholamines of varying the ratio of protein to carbohydrate in the diets.Rats consumed diets containing 0, 18 or 40% protein (76, 58 and 36% carbohydrate respectively) for 8 days. The stress of consuming the protein-free food was associated with a 16% weight reduction, and with significantly lower serum, heart and brain tyrosine levels than those noted in rats eating the 18 or 40% protein diets. Absence of protein from the diet also decreased urinary levels of dopamine and DOPA but increased urinary norepinephrine and epinephrine, probably by increasing sympathoadrenal discharge; it also increased the excretion of DOPA in animals pretreated with carbidopa, a DOPA decarboxylase inhibitor. Carbidopa administration decreased urinary dopamine, norepinephrine and epinephrine as expected; however, among carbidopa-treated rats urinary norepinephrine and epinephrine concentrations were highest for animals consuming the protein-free diet, again suggesting enhanced release of stored catecholamines from sympathoadrenal cells. The changes in urinary catecholamines observed in animals eating the protein-free diet were similar to those seen in rats fasted for 5 days: dopamine levels fell sharply while norepinephrine and epinephrine increased.These data indicate that the effects of varying dietary protein and carbohydrate contents on dopamine secretion from peripheral structures differ from its effects on structures secreting the other two catecholamines. Protein consumption increases dopamine synthesis and release probably by making more of its precursor, tyrosine, available to peripheral dopamine-producing cells; it decreases urinary norepinephrine and epinephrine compared with that seen in protein-deprived animals, probably by diminishing the firing of sympathetic neurons and adrenal chromaffin cells.     

In vivo release of dopa and dopamine from genetically engineered cells grafted to the denervated rat striatum

Fibroblastic 3T3 and endocrine RIN cells were genetically modified by infection with a recombinant retrovirus encoding the form I of human tyrosine hydroxylase (TH) and selection in tyrosine-free medium. These cells were grafted to rats unilaterally lesioned with 6-hydroxy-dopamine. Both cell types survived implantation into the striatum, expressed TH immunoreactivity, and as assessed by microdialysis 8-9 days after implantation, secreted high amounts of DOPA and/or dopamine into the surrounding host striatum. The modified 3T3 cells secreted large amounts of DOPA that was efficiently decarboxylated to dopamine by the host striatal tissue; the newly synthesized dopamine was stored only to a limited extent in the denervated striatum. The modified RIN cells synthesized dopamine that was stored intracellularly and released in a regulated fashion. The grafted DOPA-secreting cells produced 4-5 times higher extracellular dopamine levels than the dopamine-secreting cells, and they were more efficient in reducing apomorphine-induced rotation. No effect was observed with either cell type on amphetamine-induced turning behavior.     

Characterization of the tyraminergic system in the central nervous system of the locust,Locusta migratoria migratoides

Tyramine occurs in the central nervous system (CNS) of the migratory locust,Locusta migratoria migratoides. The distribution of tyramine within the CNS does not parallel that of octopamine. Tyramine is synthesised from tyrosine in the presence of tyrosine decarboxylase. A second decarboxylase in the CNS is active against 5HTP and DOPA. The locust ganglia incorporate tyramine by high- and low-affinity uptake processes that appear to be independent of dopamine and octopamine. Depolarisation of the locust ganglia by high potassium concentration results in calcium-dependent release of incorporated [³H]tyramine.     

Chromosome analysis ofHaemanthus andScadoxus (Amaryllidaceae)

Chromosome analysis of nine species ofHaemanthus (2n = 16) and four species ofScadoxus (2n = 18), using conventional stains, Quinacrine fluorescence and C-banding, has shown that the two genera do not possess significant amounts of constitutive heterochromatin. The two genera are closely related and differ in respect of a translocation which has resulted in the dysploid reduction in chromosome number from 2n = 18 inScadoxus to 2n = 16 inHaemanthus.     

Ultrastructural localization of [3H]dopamine in neurons of leech (Haemopis sanguisuga) abdominal ganglia

Neurons in the cortex of the segmental ganglia of Haemopis sanguisuga accumulated [3H]DOPA in vitro. Electron microscopic autoradiography revealed deposits of silver grains over clusters of electron dense granules measuring 20-35 nM in diameter. [3H]Dopamine (DA) was localized in the giant glial cells. Dopaminergic terminals were labelled with both DOPA and dopamine and the morphology is similar to other DA synapses. The leech could be a useful model for the study of synaptic events at dopaminergic terminals.     

SHORT-TERM REGULATION OF CATECHOLAMINE BIOSYNTHESIS IN A NERVE GROWTH FACTOR RESPONSIVE CLONAL LINE OF RAT PHEOCHROMOCYTOMA CELLS

Abstract— A clonal cell line (designated PC12) has been previously established from a transplantable rat adrenal medullary pheochromocytoma. Tissue cultures of PC12 cells synthesize, store, release and take up catecholamines. PC12 cells also respond to nerve growth factor (NGF) protein by cessation of mitosis and extension of neurites. The present studies concern the comparison of several aspects of catecholamine metabolism in PC12 cultures with that in normal noradrenergic tissues. One question was why the ratio of dopamine to norepinephrine in PC12 cultures (in contrast to that in normal noradrenergic tissue) is considerably more than one. The presence of exogenous reduced ascorbate (a cofactor for dopamine-β-monooxygenase) enhanced by 5–10-fold the rate at which PC12 cultures converted [³H]tyrosine to [³H]norepinephrine. Under such conditions, the rate of synthesis of [³H]do-pamine was unchanged. It was also found that the ratio of norepinephrine to dopamine increased by 10-fold when the cells were grown in vivo as tumors. Since tissue culture medium is essentially free of reduced ascorbate, it is likely that the absence of this cofactor is responsible for the low norepinephrine to dopamine ratio in PC12 cultures. Experiments were also carried out on short-term regulation of catecholamine synthesis in PC12 cultures. These studies revealed the following: (1) The rate of conversion of [³H]tyrosine to [³H]catechols was increased 2–3-fold (as compared with controls) in the presence of depolarizing levels of K⁺ (51.5 mM), and by 2-fold in the presence of 0.5–2 mM-dibutyryl cyclic adenosine 3′, 5’monophosphoric acid (db-cAMP). (2) Similar increases occurred in cultures which had been treated with (and had responded to) nerve growth factor. (3) The stimulatory effects of 51.5 mM-K⁺ rapidly returned toward control levels when the cultures were returned to control medium and (4) required the presence of Ca²⁺ in the extracellular medium. (5) Stimulation of catechol synthesis by 51.5 mM-K⁺ and db-cAMP also occurred in the presence of an inhibitor of DOPA decar-boxylase. Thus, the ultimate effects of these agents were probably at the level of conversion of tyrosine to dopa by tyrosine 3-monooxygenase. (6) Simultaneous exposure of cultures to 51.5 mM-K⁺ and mM-db-cAMP gave additive levels of stimulation. Such findings demonstrate that catecholamine synthesis in cultures of PC12 cells undergoes short-term regulation which is similar to that previously demonstrated in normal monoaminergic tissues. As a homogeneous tissue culture line, the PC12 bears certain advantages for studying the primary mechanisms of such effects.     

Occurrence of L-DOPA and dopamine in plants and cell cultures ofMucuna pruriens and effects of 2,4-d and NaCl on these compounds

The development of the L-DOPA-content of roots, stems and leaves ofMucuna pruriens during growth of the plants is described. Besides L-DOPA, the leaves, but not the stems and the roots, also contain the related catechol dopamine. The time course of dopamine accumulation is compared to that of L-DOPA. In cell suspension cultures ofM. pruriens dopamine can be detected as well. Its level is strongly increased by addition of the growth regulator 2,4-d to the medium, a condition that suppresses cell growth and L-DOPA-accumulation. Dopamine induction appears to be a specific metabolic effect of 2,4-d. Salt stress, as caused by the addition of NaCl, gives no induction of dopamine formation, whereas L-DOPA is released into the medium.     

Evolution of albinism in cave planthoppers by a convergent defect in the first step of melanin biosynthesis

Albinism, the reduction or loss of melanin pigment, is found in many diverse cave‐dwelling animals. The mechanisms responsible for loss of melanin pigment are poorly understood. In this study we use a melanogenic substrate assay to determine the position where melanin synthesis is blocked in independently evolved cave planthoppers from Hawaii and Croatia. In this assay, substrates of enzymes responsible for melanin biosynthesis are added to fixed specimens in vitro and their ability to rescue black melanin pigmentation is determined. L‐tyrosine, the first substrate in the pathway, did not produce melanin pigment, whereas L‐DOPA, the second substrate, restored black pigment. Substrates in combination with enzyme inhibitors were used to test the possibility of additional downstream defects in the pathway. The results showed that downstream reactions leading from L‐DOPA and dopamine to DOPA‐melanin and dopamine‐melanin, the two types of insect melanin, are functional. It is concluded that albinism is caused by a defect in the first step of the melanin synthesis pathway in cave‐adapted planthoppers from widely separated parts of the world. However, Western blots indicated that tyrosine hydroxylase (TH), the only enzyme shown to operate at the first step in insects, is present in Hawaiian cave planthoppers. Thus, an unknown factor(s) operating at this step may be important in the evolution of planthopper albinism. In the cavefish Astyanax mexicanus, a genetic defect has also been described at the first step of melanin synthesis suggesting convergent evolution of albinism in both cave‐adapted insects and teleosts.     

Increased Neostriatal Tyrosine Hydroxylation During Stress: Role of Extracellular Dopamine and Excitatory Amino Acids

Abstract: We examined the regulation of neostriatal tyrosine hydroxylation during acute stress, testing the hypothesis that excitatory amino acids (EAAs) contribute to the stress-evoked increase in dopamine (DA) synthesis. Dialysis probes implanted into neostriatum permitted delivery of drugs and sampling of extracellular fluid. Rats were exposed to 30 min of intermittent tail shock during infusion of an inhibitor of aromatic amino acid decarboxylase (AAAD), NSD-1015 (100 µM), and DOPA was measured in the dialysate. Tail shock was applied beginning either 15 min after the onset of NSD-1015 treatment (the initial rate of DOPA accumulation) or 75 min after the onset of treatment (when DOPA had approached steady state). Tail shock increased the steady-state levels of extracellular DOPA in neostriatum (+40%). However, there was no change in the initial rate of DOPA accumulation unless animals also received the D₂ receptor antagonist eticlopride (50 nM), in which case an increase was observed (+228%). The impact of tail shock on the steady-state level of DOPA was attenuated by the D₂ agonist quinpirole (100 µM), or by 2-amino-5-phosphonovalerate (APV) (100 µM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (100 µM), EAA antagonists acting at NMDA or d ,l -α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, respectively. These data suggest that acute stress normally has little effect on tyrosine hydroxylation in neostriatum due to the inhibitory influence of DA in the extracellular fluid. However, when that influence is absent (e.g., during extended inhibition of DOPA decarboxylation or blockade of DA receptors), stress increases tyrosine hydroxylation via EAAs acting on NMDA and AMPA receptors. Thus, EAAs released from corticostriatal projections may stimulate DA synthesis and thereby restore dopaminergic activity under conditions in which the availability of DA for release has been compromised.     

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Einfluss möglicher phosphodiesterase-inhibitoren und cAMP auf die betacyan-akkumulation

The influence of cyclic AMP, theophylline, papaverine and NH₄NO₃ on the accumulation of betacyanin in callus of Portulaca grandiflora, var. JR, were studied in relation to amounts of dihydroxyphenylalanine (DOPA), dopamine, phenylalanine, tyrosine, protein, ‘lipid’ and the nucleotides CMP, AMP, GMP and UMP present. Inhibition of betacyanin formation is characterized by reduced amounts of DOPA and dopamine and a constant rise of GMP and CMP (GMP/CMP = 8). Protein accumulation is also inhibited. The increase in pigment accumulation due to theophylline and NH₄NO₃ is characterized by a raised DOPA and protein concentration and a lower GMP/CMP ratio (=3). The increase in betacyanin accumulation is due to de novo synthesis of enzymes. Inhibition is probably due to the regulation of the callus phosphodiesterase by papaverine and theophylline (≦ 10^?5 M/1) which triggers a change in concentration of nucleotides, which eventually regulates tyrosinase biosynthesis.