Histochemically demonstrable esterase activity in the hypothalamus of the developing rat

Summary The distribution of the acetylcholinesterase, non-specific cholinesterase and non-specific esterase activity has been investigated histochemically in the hypothalamic neurons during the ontogenic development of the rat.Acetylcholinesterase activity is located in the supra-optic and para-ventricular nuclei mostly, but some activity is present in the other nuclei and in the median eminence of the adult rat, as well. The supra-chiasmatic neurons are always negative. The activity of non-specific cholinesterase was encountered in the endothelial cells of the capillaries, in the glia and in the ependymal cells especially around the supra-optic and para-ventricular neurons. The localization of the non-specific esterase was similar to that of the non-specific cholinesterase, but in addition activity is seen in the supra-optic and para-ventricular perikarya, in the parvo-cellular neurons of the tuberal area and in the median eminence. No sexual differences were seen in the distribution of the estrase activity.The appearance of acetylcholinesterase took place already before birth. At about the 16th post-coital day the area from which the arcuate and ventro-medial nuclei will differentiate was positive for acetylcholinesterase. A strong activity in these nuclei was observed during the critical period of the sexual differentiation of the rat hypothalamus (0–10 postnatal days). In the development of the non-specific cholinesterase and esterase no similar variation was seen. Acetylcholinesterase and non-specific esterase were seen in the neurosecretory nuclei before birth, non specific cholinesterase after birth, and non-specific esterase in the parvo-cellular neurons during the first post-natal week.Supported by a grant from The Finnish Medical Society Duodecim.     

Histochemical evidence of three types of esterases in the adrenal medulla of the rat

Summary Esterases of the adrenal medulla have been studied histochemically using alpha-naphthyl acetate and butyrate as substrates, Blue RR Salt as a coupler and eserine and E, 600 as inhibitors. Three types of esterase activity were thus demonstrated: (1) cholinesterase activity in the nerve fibres, ganglion cells and secretory medullary cells; (2) eserine resistant but E. 600 sensitive esterase activity in the ganglion cells and secretory cells; (3) E. 600 resistant activity in strongly positive, unidentified cells scattered in the medulla. The histochemical picture was essentially similar in sections of formalin-fixed tissue and in fresh sections subjected to the voltage gradient employed for electrophoretic separation of esterases. It is concluded that esterases histochemically demonstrable in sections are desmo-enzymes and at least to a major part different from the lyo-enzymes which can be separated by starch gel electrophoresis.With 6 Figures in the Text     

Ultrastructural distribution of cholinesterase activity in the ventral nerve cord of the earthworm Lumbricus terrestris

Summary The fine structure and distribution of cholinesterase (ChE) activity in the ventral nerve cord of the earthworm (Lumbricus terrestris) was studied, using acetyl- and butyrylthiocholine iodides as substrates and iso-OMPA, 284C51 and eserine as inhibitors to discriminate between acetylcholinesterase (AChE) and other cholinesterase (ns.ChE) activities.The earthworm ventral nerve cord exhibits intense ChE activity. Both AChE and ns.ChE were present and they had identical distribution, being located mainly in the supportive glial cells. Most neurones of the ventral nerve cord contained no histochemically demonstrable activity. The ventral giant nerve cells were observed with the electron microscope to exhibit AChE activity. The enzyme was situated in the membranes of the rough-surfaced endoplasmic reticulum and in peculiar lamellated bodies but not in the membranes of the Golgi complex.     

THE HISTOCHEMICAL LOCALIZATION OF PHOSPHATASES AND ESTERASES IN AUST RALORBIS GLABRATUS

The presence and distribution of two groups of enzymes has been determined histochemically on sections of the schistosome-bearing snail Australorbis glabratus. By the use of specific inhibitors, attempts have been made to characterize further the enzymes occurring in the various organs and tissues.
As a result of this study it has been found that alkaline and acid phosphatase are widely distributed but have identical localizations only in the kidney and albumen gland. Both enzymes react typically to the action of the usual inhibitors.
Among the non-specific esterases. an enzyme corresponding to the mammalian A-type esterase (aromesterase) is present in the brain; while a B-type esterase (aliesterase) is located in the digestive gland, intestine, and on the glandular region of the foot surface.
A "true" lipase (an esterase acting on an undissolved substrate) is found principally in the albumen gland, with an indication of its presence in the digestive gland and in portions of the digestive tract.
An enzyme with the properties of an acetylcholinesterase occurs in the radula sac, oesophagus, preputium, junction of carrefour and oviduct, and amoebocytes. A positive reaction for cholinesterase is also obtained with frozen sections of brain but, although this enzyme has been shown biochemically to hydrolyse acetylcholine and is inhibited by low concentrations of eserine. it is remarkably resistant to the action of organophosphorous compounds, and its true nature cannot he stated with certainty.     

Specific demonstration of acetylcholinesterase and non-specific cholinesterase in the adrenal gland of the rat

Summary Distribution of cholinesterase in the adrenal medulla of the rat was studied using acetylthiocholine, butyrylthiocholine and -naphtyl acetate as substrates and eserine, di-isopropylfluorophosphate (DFP), 1:5-bid-(4-trimethylammoniumphenyl)-pentan-3-one di-iodide (62. C. 47) and tetra-isopropylpyrophosphoramide (iso-OMPA) as inhibitors.Acetylcholinesterase was observed in the nerve trunks, the ganglion cells, the coarse and the fine nerve fibers. The fine medullary network showed along the fibers small strongly positive ovoid bodies.Non-specific cholinesterase was detected in the capsule, the nerve trunks, the coarse nerve fibers and the fibers surrounding the noradrenaline-containing, fluorescent medullary cell islets. A weak reaction was also seen in the cytoplasm of the medullary cells. The fine medullary fibers with the ovoid bodies were essentially negative.A method was developed to demonstrate first non-specific cholinesterase and then acetylcholinesterase in the same section. The different distributions of the two cholinesterases were confirmed with this method.With 8 Figures in the Text, of which 2 in ColourThis work has been supported by a research grant from the National Institute of Arthritis and Metabolic Disease, the U.S. Public Health Service (A-1725) and by a grant from Finland's Cultural Fund.     

A comparative study of the effect of vinyl phosphoric acid esters on cholinesterase and carboxylesterase activities in mammals and arthropods

Studies have been made of the effect of organophosphorus inhibitors on cholinesterase and carboxylesterase from various mammals (human erythrocytes, mouse brain, blood serum of mouse and rat, blood serum of horse) and arthropods (Calliphora vicina, Schizaphis graminum, Myzus persicae, Sitophilus oryzae, Pseudococcus maritimus, Tetranychus urticae). Organophosphorus inhibitors were presented by esters of vynylphosphoric acid containing normal and branched alkyls in the phosphoryl part of the molecule. The increase of the radical up to a propyl one increased the effect of organophosphorus inhibitors with respect to cholinesterase from the majority of the arthropods investigated. Organophosphorus compound with an isopropyl radical was found to be weaker for all the enzymes studied. Extremely high sensitivity of carboxylesterase from all arthropods to all organophosphorus inhibitors was noted; in some of the cases, anticarboxylesterase activity of all drugs was 2-3 orders higher than anticholinesterase one (P. maritimus, T. urticae). Regularities established for cholinesterase practically completely were confirmed on carboxylesterase. This finding evidently reveals similar structure of catalytic surface at the vicinity of esterase center in both enzymes.     

The activity of non-specific esterase in the thyroid epithelial cells of the guinea pig as influenced by various inhibitors and activators

Summary The action of various inhibitors and activators upon esterase activity in the thyroid epithelial cells is demonstrated. The agents used were triorthocresylphosphate (TOCP), parachloromercuribenzoate (PCMB), Arsanillic acid, p-nitrophenyl dimethyl carbamate and bis p-nitrophenyl phosphate.TOCP was found to inhibit selectively the activity in the follicle cells proper when naphthyl acetate was used as a substrate.Arsanillic acid (0,001 M) activated the follicle cells proper selectively, but if the concentration was raised to 0,01 M the effect was that of inhibition while the activity in the para-, inter- and intrafollicular cells was unchanged.The results obtained are related to previous biochemical and histochemical observations and the nature of esterases in the thyroid is discussed.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Adrenal oxidative enzymes in ox and sheep. The histochemical demonstration of some pathways involved in chromaffin granule synthesis

Summary Catechol amine secretion is achieved by exocytosis. In this, ATP and protein are also lost from the chromaffin cells. Histochemically various specific coenzyme linked dehydrogenases associated with ATP production have been demonstrated in the adrenals of ox and sheep. These included cytochrome oxidase, DPN and TPN diaphorases, isocitric dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, succinic dehydrogenase, lactic dehydrogenase, alcohol dehydrogenase, -glycerophosphate dehydrogenase and -hydroxybutyrate dehydrogenase. Enzymes of the pentose shunt were found histochemically and biochemically. The RNA content of the adrenal medulla and cortex was also investigated.     

Tissue Carboxylesterases and Chlorpyrifos Toxicity in the Developing Rat

Young animals are more sensitive than adults to the neurotoxic effects of some organophosphorus insecticides. Many investigators attribute this difference in sensitivity to the immaturity of the detoxification capacity of preweanling rats. Chlorpyrifos [O,O-diethylO-(3,5,6-trichloro-2-pyridyl)phosphorothionate] is an organophosphorus insecticide that demonstrates considerable age-related sensitivity. The carboxylesterases are a group of related enzymes that detoxify organophosphorus insecticides by stoichiometrically binding these molecules before they can inhibit acetylcholinesterase. This study presents in vitro and in vivo evidence demonstrating that the carboxylesterases are critical for explaining the age-related sensitivity of chlorpyrifos. The data show that the fetal rat and the postnatal day 17 (PND17) rat pup have fewer molecules of carboxylesterase (less activity), less sensitive molecules of carboxylesterase, and a larger proportion of chlorpyrifos-insensitive molecules of carboxylesterase. An in vitro mixing experiment, using adult striatum as a source of acetylcholinesterase and liver homogenates as a source of carboxylesterase, demonstrates that the adult liver carboxylesterases are superior to the PND17 liver carboxylesterases for detoxifying chlorpyrifos. In the in vivo experiments the time course profiles of carboxylesterase and cholinesterase activity following a maximum tolerated dose of chlorpyrifos also suggest that the carboxylesterases of the PND17 rat were less capable of detoxifying chlorpyrifos. Carboxylesterase activity in the preweanling rat was not as severely inhibited as in the adult, but decrements in cholinesterase activity as a result of chlorpyrifos treatment were comparable. These in vitro and in vivo findings support the previously proffered postulate that the carboxylesterases are critical for determining the age-related sensitivity of chlorpyrifos. In addition, these detailed experiments allow us to propose that the detoxification potential of these enzymes is multifaceted, and depends on the (1) amount of activity (i.e., number of molecules), (2) affinity for the insecticide or metabolite, and (3) amount of carboxylesterase activity that is refractory to inhibition by the insecticide or metabolite.     

The Genetic Basis of Malathion Resistance in Housefly (Musca domestica L.) Strains From Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdE7 gene is probably playing a role in detoxification of xenobiotic esters. In our research, we have isolated, cloned and sequenced the MdE7 gene from five different Turkish housefly strains. High doses of malathion (600 g/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana, and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp²⁵¹ Ser substitution was found in the product of MdE7 gene in all malathion-resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all five strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion-resistant strains.     

Histochemical studies of some enzymes in the tissues of the schistosome vector snailBulinus truncatus (Audouin) with special reference to the effects of a molluscicide. II. Hydrolases

Summary Accomparative study of six hydrolases, acid and alkaline phosphatases, aryl sulphatase, -gluchronidase cholinesterase, and non-specific esterase, was carried out on the tissues of normal healthy and Frescon-treatedBulinus. The presence and activity of these enzymes in the tissues of normal animals were taken to indicate the probale functions of the tissues concerned. Frescon administration caused inhibition of acid phosphatase and also induced the release of cholinesterase and non-specific esterase in some tissue. It is concluded that the most important effects of Frescon on snail physiology are the disorganization of neuronal function and disturbance of olfactory activity.     

Acid phosphatase and esterase activity in orchid mycorrhiza

Summary A cytochemical study was made to examine the possibility that acid phosphatase may be specifically involved in the digestion of endophytic hyphae in orchid mycorrhiza. Esterase activity was studied for comparison. Frozen sections of unfixed or glutaraldehyde-fixed protocorms of Dactylorhiza purpurella infected by Thanatephorus cucumeris (Rhizoctonia solani) were reacted for acid naphthol AS BI phosphatase, acid -glycerophosphatase or naphthol AS D esterase.A marked increase in particulate acid naphthol AS BI phosphatase activity was observed during infection of host, central, parenchyma cells shortly before hyphae lysed; a diffuse reaction of high activity was localised on lysed fungus. Acid -glycerophosphatase was present at particulate sites only in fungal cytoplasm and as a diffuse reaction on lysed fungus.Naphthol AS D esterase showed highest activity at hyphal apices. Esterase seems to be associated with growth and differentiation of hyphae in orchid cells, rather than lysis of the fungus.     

Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone

Undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone was strongly inhibited by organophosphates and other esterase inhibitors. Also, p-chloromercuribenzoate at 1 x 10(-4) M showed a 70% inhibition of esterase activity. The enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M. Under these conditions the highest reaction rate was toward undecyl acetate. No lipase or proteolytic activity was demonstrated. Undecyl acetate esterase was classified as a carboxylesterase (B-esterase). Cell-free activity studies on the production of undecyl acetate esterase grown on different carbon sources plus zymogram studies demonstrated that the enzyme was inducible when 2-tridecanone, 2-tridecanol, undecyl acetate and, to a lesser extent, 1-undecanol were growth substrates. Induction of undecyl acetate esterase during oxidation of 2-tridecanone supports the view that undecyl acetate is an intermediate in the degradation of the methyl ketone.     

Cholinesterases in the hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow,Zonotrichia leucophrys gambelii

Summary The distribution of cholinesterases in hypothalamo-hypophysial neurosecretory system of the White-crowned Sparrow has been examined histochemically. The perikarya of the neurosecretory cells of the paraventricular and supraoptic nuclei have a high acetylcholinesterase activity. Acetylcholinesterase activity also occurs in the cells of the infundibular nucleus. The proximal parts of the axons of the cells of the neurosecretory and infundibular nuclei have strong acetylcholinesterase activity and weak non-specific cholinesterase activity. In the median eminence, the activity of acetylcholinesterase is strongest in the palisade layer. In the pars nervosa, there is definite, although weak, acetylcholinesterase activity.This investigation was supported by grants from the National Institutes of Health to Professor Farner (B-1353) and to Dr. Kobayashi (A-3678).     

Genetics of a tissue esterase polymorphism (Est-6) in the rabbit (Oryctolagus cuniculus)

Genetic analysis of a polymorphic tissue esterase revealed a new locus (Est-6) with two alleles (Est-6 ^a andEst-6 ^b) on linkage group VI of the rabbit.Est-6 is closely linked to theEst-1,2,4 cluster. Esterase ofEst-6 is found in many organs, particularly in liver and small intestine, but not in erythrocytes and serum.Est-6 esterase hydrolyzes -naphthyl acetate and butyrate, naphthol AS-D acetate, indoxyl acetate, and butyrate as well as 5-bromoindoxyl acetate,N-acetyl-l-alanine--naphthyl ester but not 4-methylumbelliferyl acetate and fluorescein diacetate. The enzyme is inhibited by bis-p-nitrophenyl phosphate and eserine but not byp-chloromercuribenzoate. It was classified as a carboxylesterase (EC 3.1.1.1). Based on chromosomal localization, tissue distribution, substrate specificity, inhibitor sensitivity, and range ofpI's, rabbitEst-6 is assumed to be homologous with mouseEs-7.The contribution of Dr. O. von Deimling (No. 59) was supported by the Deutsche Forschungsgemeinschaft (De 315/2-2).     

Biochemical characterization of insecticide resistance in the German cockroach, Blattella germanica, from Malaysia

The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.     

Isoenzymes and histochemistry of non-specific esterases in normal and cancerous skin

Synopsis Non-specific esterases in normal and carcinomatous skin of the mouse have been investigated electrophoretically and histochemically. Three esterase bands were obtained on electrophoresis from homogenates of normal skin; homogenates of carcinomas showed an accumulation of esterase-Ia and esterase-Ib.^* However, using several ester substrates, substrate-specific patterns were demonstrated in the electrophoresis separations and histochemically in tissue sections. On the electrophoresis separations, -naphthyl acetate, -naphthyl acetate, 6-bromo-2-naphthyl acetate, naphthol AS acetate, naphthol AS-D acetate and naphthol AS-LC acetate gave rise to similar patterns, but with -naphthyl propionate as subsmate, more esterase-Ib was indicated and with 5-bromo-indoxyl acetate a distinctive preponderance. Peripheral or uniformly distributed staining was found histochemically in tumour epithelium using -naphthyl acetate, -naphthyl propionate and -naphthyl acetate, whereas with the substrates of naphthol AS acetate, naphthol AS-D acetate and indoxyl acetate an intermediate pattern of staining related to keratinization was obtained.     

Cholinesterases from plant tissue: v. Cholinesterase is not pectin esterase

Several properties of the cholinesterase from Phaseolus aureus Roxb. and of pectin (methyl) esterases from both Phaseolus aureus and Lycopersicon esculentum (L.) Mill. are contrasted. Cholinesterase activity is inhibited by all of the concentrations of NaCl tested, from 0.05 m to 0.9 m, a property which differs sharply from published data pertaining to pectin esterase. Although crude preparations of cholinesterase contain pectin esterase activity, further purification by gel filtration of the cholinesterase results in a nearly complete elimination of the pectin esterase activity. The activity of neither the pectin esterase from Lycopersicon esculentum nor that from Phaseolus aureus is affected by 25 μm neostigmine, a potent inhibitor of the cholinesterase activity extracted from Phaseolus aureus.     

A novel geminal diol as a highly specific and stable in vivo inhibitor of insect juvenile hormone esterase

Thio-containing and acetylenic trifluoromethyl ketones were potent inhibitors of insect juvenile hormone (JH) esterase with greater inhibitory activity than aliphatic and α,β-unsaturated homologs. Octylthio-1,1,1-trifluoropropan-2-one was the most potent inhibitor with the greatest equilibrium hydration constant in pure water. However, a keto/hydrate equilibrium was not necessary for JH esterase inhibition. The carbonyl tautomer of 1-octyl [1-(3,3,3-trifluoropropan-2,2- dihydroxy)] sulfone (OTPdOH-sulfone) was not detectable, and yet OTPdOH-sulfone was a potent in vitro inhibitor of JH esterase with an I₅₀ of 1.2 nM. The mechanism of JH esterase inhibition by these compounds is discussed. OTPdOH-sulfone inhibited JH esterase with minimal activity toward insect 1-naphthyl acetate esterase and electric eel acetylcholinesterase. The inhibitor was also active in vivo, selective for JH esterase, and persistent for over 32 h. OTPdOH-sulfone when topically applied to larval and adult cabbage loopers, Trichoplusia ni, elicited juvenoid activity apparently because of the specific in vivo inhibition of JH metabolism. Arch. Insect Biochem. Physiol. 36:165–179, 1997. © 1997 Wiley-Liss, Inc.