The ultrastructural localization of a cholinesterase in the body muscle of plaice (Pleuronectes platessa)

Summary By means of a histochemical method adapted for electron microscopy a cholinesterase in body muscle cells of plaice (Pleuronectes platessa) has been localized to the sarcolemma. The cholinesterase activity disappeared from the sarcolemma after the muscle tissue had been incubated with a bacterial enzyme, which had earlier been shown, by biochemical methods, to be able to liberate this cholinesterase activity from plaice muscle.The provision of live plaices from Kristineberg Zoological Station, Fiskebäckskil, Sweden, is gratefully acknowledged. We are greatly indebted to Dr. Åke Bovallius, FOA, who provided the starting material for the bacterial enzyme. We would like to express our sincere thanks to Prof. Lennart Nicander for valuable discussions and for placing the resources of the Department of Anatomy and Histology, Royal Veterinary College, to our disposal.     

Histochemischer Cholinesterase-Nachweis während der Abfaltungsbewegungen des Hühnerembryos

Zusammenfassung An Hühnerembryonen der Stadien 5–21 wurde das Auftreten von Cholinesterase-Aktivität in Zellverbänden verfolgt, die bei der Abfaltung des Embryos von Bedeutung sind.Im Neuroektoderm treten während der Neuralrohrbildung Cholinesterase-positive Zellen auf. Diese Aktivität verschwindet, wenn das Neuralrohr ausgebildet ist. Anschließend lassen sich Cholinesterase-positive Neuroblasten darstellen.Im Oberflächenektoderm tritt Cholinesterase-Aktivität während der Ausbildung der Kopffalte und der Grenzrinnen auf. Die Aktivität verschwindet, wenn sich die Kopfanlage vom extraembryonalen Teil der Keimblätter getrennt hat.Bei der Ausbildung der Entodermwülste und während des Einwanderns derselben zum Darmrohrschluß bilden sich palisadenartige Epithelverdickungen aus, die Cholinesterase aufweisen. Wenn das Darmrohr geschlossen ist, verschwindet die Aktivität wieder. Während der Darmrohrbildung wird das nach ventral und medial einwachsende Entoderm von palisadenartig strukturiertem Mesoderm begleitet, das Cholinesterase-Aktivität aufweist. Nach Schluß des Darmrohres löst sich die palisadenartige Anordnung auf und die Cholinesterase-Aktivität verschwindet.

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Histochemical demonstration of cholinesterase during the folding off of the chick embryo

Summary The localization of cholinesterase activity in chick embryo of stage 5 up to stage 21 has been studied in cell layers that are involved in the formation of the primitive body tube.During the formation of the neural tube, cholinesterase activity can be demonstrated in cells of the neural plate. This pattern of enzyme activity disappears after formation of the neural tube. Thereafter, the enzyme is localized in neuroblasts.In the ectodermal layer, cholinesterase activity is found during the formation of the head fold and the lateral body folds. Again, the enzyme disappears after the development of the cephalic outgrowth.Formation of the entodermal folds and their fusion in the midline — thus establishing the gut tube — is associated with cholinesterase activity in palisade cell layers. There is no enzyme activity in the completed gut.During the formation of the gut tube, the mediad invading entodermal folds are covered by mesodermal cell palisades, which are positive for cholinesterase. After the formation of the gut tube, the palisades disappear as does the enzyme activity.

     

Characterization of plasma cholinesterase activity in the Eurasian Griffon Vulture Gyps fulvus and its in vitro inhibition by carbamate pesticides

The legal and illegal use of organophosphorus and carbamate pesticides represents one of many threats to birds. The activity of the cholinesterase enzyme in plasma is used as a non‐destructive biomarker to diagnose the exposure of birds to these pesticides. Scavengers are one of the most important bird groups threatened by the use of baits poisoned with anticholinesterase pesticides. Knowledge of the characteristics of this enzyme in each bird species is crucial, as several studies indicate that more than one cholinesterase form may be present in the plasma of birds. In this study, cholinesterase activity was characterized in the plasma of the Eurasian Griffon Vulture Gyps fulvus by using several substrates and inhibitors of the enzyme, and its normal activity value was also determined. The in vitro sensitivity of Gyps fulvus plasma cholinesterase to carbamate insecticides (aldicarb, carbaryl and methomyl) was also investigated. The results indicated that propionylthiocholine iodide was the preferred substrate to determine plasma cholinesterase activity, followed by acetylcholine iodide and S‐butyrylcholine iodide, and acetylcholinesterase was the predominant enzymatic activity in Gyps fulvus plasma. Aldicarb was the most potent in vitro inhibitor of plasma cholinesterase activity in this species. However, cholinesterase enzymatic activity was significantly inhibited by all tested carbamates, providing further evidence that this biomarker is a suitable tool to monitor the exposure to these poisons in the field, highlighting its utility in conservation programmes.     

Epithelio-mesenchymal interface and fibronectin in the differentiation of the rat mesonephric and paramesonephric ducts

Abstract. The distribution of fibronectin and the morphological differentiation of the genital ducts was studied in rat fetuses at ages from 15 to 21 days. Fibronectin was localized with the peroxidase-antiperoxidase and avidin-biotin method at the electron- and light-microscope level. In 15-day-old male and female fetuses, fibronectin was localized as a continuous lamella around the mesonephric duct and as a discontinuous lamella around the paramesonephric duct. During the differentiation of the female paramesonephric duct, the fibronectin layer became continuous and remained so after the age of 16 days. The fibronectin layer of the male mesonephric duct remained continuous at all ages. The accumulation of mesenchymal cells on the outer surface of the female mesonephric duct and the concomitant detachment of the fibronectin layer around the duct suggests that mesenchymal regulation plays a role in the regression of the mesonephric duct. In the regressing male paramesonephric duct fibronectin was simultaneously lost in the condensed periductal mesenchyme, the places of epithelio-mesenchymal contact, and the epithelial cytoplasmic protrusions towards the mesenchyme. Ultrastructurally, fibronectin was localized in the basal laminae, on the cell membrane in contact with the extracellular material, and on the surface of the fibrillar and flocculent extracellular material. In addition to auto- and heterophagy, epithelio-mesenchymal interactions seem to play an important role in the regression of the genital ducts, although in different ways in males and females. The present results give additional support to the theory of the possible migration of epithelial cells into the surrounding mesenchyme during the regression of the paramesonephric duct.     

Cholinesterase in the parasitic nematode, Stephanurus dentatus. Characterization and sex dependence of a secretory cholinesterase

An antigenic secretory protein with cholinesterase activity was isolated from the excretory gland cells of Stephanurus dentatus and was purified by gel filtration and ion exchange chromatography. The antigenicity of the cholinesterase was demonstrated by an esterase-active immunoprecipitate formed with S. dentatus antiserum and by the ability of the antiserum to protect the enzyme from heat inactivation. The enzyme was found to be secreted by the adult nematodes during in vitro cultivation. The level of cholinesterase activity and its release from the excretory gland cells of the parasite were 27-fold greater in the male than in the female. Ninety per cent of the enzyme activity was localized in the soluble fraction of the gland cells. The molecular weight of the enzyme, estimated by sucrose density gradient centrifugation, was 100,000. Two molecular forms were separated by isoelectrofocusing, with isoelectric points of 7.0 and 6.9. At optimum substrate concentrations, the rate of hydrolysis of acetylthiocholine was 8 times greater than that of butyrylthiocholine; the Michaelis constants were 560 microM and 81 microM for acetylthiocholine and butyrylthiocholine, respectively. The enzyme exhibited substrate inhibition at substrate concentrations greater than 10 mM and was inhibited by eserine sulfate, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, Tris, and acetone. The enzyme was highly unstable in dilute protein solutions.     

Immunofluorescent localization of tenascin during development of the mouse urogenital sinus: Possible involvement in genital duct morphogenesis

Tenascin is a compound of the mesenchymal extracellular matrix and has been proposed as a possible mediator in epithelial-mesenchymal interactions, because of its characteristic distribution in tissues during fetal development. In the present study, we have investigated by immunofluorescence the changes in the distribution of tenascin during development of the mouse urogenital sinus, a process in which tissue interactions were found to be essential. Tenascin first appears in dorsal mesenchyme on days 13-15 of gestation, coinciding with morphological changes of the epithelium. During male development, tenascin accumulates in the dorsal mesenchyme around the junction of Wolffian ducts, but not in the ventral mesenchyme, into which prostatic buds (prostate gland anlagen) project from the sinus epithelium. During female development, the mesenchyme that participates in the downgrowth of the vagina (derived from Müllerian ducts) stains intensively for tenascin. In both of these tenascin-positive areas, the epithelium undergoes conspicuous morphogenetic changes. The results suggest that mesenchymal tenascin could be involved in the epithelial morphogenesis of the sinus, especially in the morphogenesis of the genital ducts.     

Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

Histochemical Investigations of Tardigrada. IV. Alkaline Phosphatase (ALP), Aminopeptidase (AMP) and Gonad Development in Eutardigrades

In tardigrades of the genus Macrobiotus histochemically localized alkaline phosphatase (ALP) and aminopeptidase (AMP) are taken as markers of absorptive/secretory cells. The enzyme reaction intensity is also related to different metabolic activities of the body tissues and compared with other histochemical and histological features of the cells. ALP and AMP distribution pattern is investigated in the Malpighian tubules, the gut, ovary, testicle and the storage and epidermal cells during female and male gonad development. The results strongly support the hypothesis that the Malpighian tubules are osmoregulatory-excretory organs and may also perform secretory functions. They show morpho-functional variations which are related to sexual maturity and differ in the female and the male: possible physiological roles are discussed. In the female all the tissues here examined undergo changes in size, enzyme activities and other features that show a relationship with phases of both oogenesis and molt: a physiological explanation is suggested that takes into account previously obtained evidence of neuroendocrine regulation. In the male the stages of testicle maturity and molt seem to be independent of each other and the changes of the body tissues found in the female are less or not evident.     

Gender-related differences in circadian rhythm of rat plasma acetyl- and butyrylcholinesterase: Effects of sex hormone withdrawal

The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2–30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.     

Involvement of a matrix metalloproteinase in MIS-induced cell death during urogenital development

Programmed cell death of the Müllerian duct eliminates the primitive female reproductive tract during normal male sexual differentiation. Müllerian inhibiting substance (MIS or AMH) triggers regression by propagating a BMP-like signaling pathway in the Müllerian mesenchyme that culminates in apoptosis of the Müllerian duct epithelium. Presently, the paracrine signal(s) used in this developmental event are undefined. We have identified a member of the matrix metalloproteinase gene family, Mmp2, as one of the first candidate target genes downstream of the MIS cascade to function as a paracrine death factor in Müllerian duct regression. Consistent with a role in regression, Mmp2 expression was significantly elevated in male but not female Müllerian duct mesenchyme. Furthermore, this sexually dimorphic expression of Mmp2 was extinguished in mice lacking the MIS ligand, suggesting strongly that Mmp2 expression is regulated by MIS signaling. Using rat organ genital ridge organ cultures, we found that inhibition of MMP2 activity prevented MIS-induced regression, whereas activation of MMP2 promoted ligand-independent Müllerian duct regression. Finally, MMP2 antisense experiments resulted in partial blockage of Müllerian duct regression. Based on our findings, we propose that similar to other developmental programs where selective elimination or remodeling of tissues occurs, localized induction of extracellular proteinases is critical for normal male urogenital development.     

Modification of flower sex and acid phosphatase activity by phthalimides in female plants of Morus nigra L.

Phthalimide treatments at 125, 250 and 500 mg 1^–1 to female plants of dioecious Morus nigra L. induced intersex and male flowers. Qualitative and quantitative analyses of acid phosphatase in male and female flower buds showed that the male flower had significantly higher levels of the enzyme activity than the female flower buds. The level of acid phosphatase activity significantly increased in intersex and male flowers induced on female plants after phthalimide treatments.     

Nano-Intercalated Organophosphorus-Hydrolyzing Enzymes in Organophosphorus Antagonism

A dendritic poly(2-alkyloxazoline)-based polymer was studied as a new carrier system for the organophosphorus-hydrolyzing recombinant enzymes, organophosphorus acid anhydrolase and organophosphorus hydrolase. Paraoxon (PO) and diisopropylfluorophosphate (DFP) were used as model organophosphorus compounds. Changes in plasma cholinesterase activity were monitored. The cholinesterase activity was proportional to the concentrations of DFP or PO. Plasma cholinesterase activity was higher in animals receiving enzyme and oxime before the organophosphates than in the oxime-only pretreated groups. These studies suggest that cholinesterase activity can serve as an indicator for the in vivo protection by the nano-intercalated organophosphorus acid anhydrolase or organophosphorus hydrolase against organophosphorus intoxications. These studies represent a practical application of polymeric nano-delivery systems as enzyme carriers in drug antidotal therapy.     

20-OH-Ecdysone-induced morphogenetic movements in a Chironomus cell line are accompanied by expression of an embryonic muscarinic system

The permanent epithelial insect cell line used was derived from Chironomus tentans embryos. Cells are maintained in suspension culture, where they grow as single-layered vesicles. On treatment with the moulting hormone 20-OH-ecdysone cell division ceases. Patches of cuboidal epithelium appear in the vesicles which finally become multilayered and form bud-like protrusions at the outside. In the present study, we localized cholinesterase activity in the cell protrusions by histochemistry and demonstrated coexpression of cholinergic muscarinic receptors by immunofluorescence. Muscarinic receptors were visualized with the monoclonal antibody M35. Six hours after treatment with 20-OH-ecdysone, muscarinic receptors appeared in a few individual cells of the epithelial vesicles before morphological changes became visible. After 24 h, immunofluorescence was concentrated in multilayered patches which now also showed cholinesterase activity. After 3 days, muscarinic receptors and cholinesterase activity were localized in the epithelium protrusions. The results are discussed in the context of an embryonic cholinergic muscarinic system the expression of which has been described in vertebrate and non-vertebrate embryos and is correlated with phases of morphogenesis.     

Cholinesterase inhibitory effects of Rhizophora lamarckii,Avicennia officinalis,Sesuvium portulacastrum and Suaeda monica: Mangroves inhabiting an Indian coastal area (Vellar Estuary)

Alzheimer's disease is a progressive neurodegenerative illness accounting for approximately 50% of all types of dementia in elderly people. The only symptomatic treatment proven effective to date is the use of cholinesterase inhibitors to augment surviving cholinergic activity. The purpose of this study is to investigate cholinesterase inhibitory activity of mangroves as an alternative medicine for the treatment of Alzheimer's disease. About nine mangrove plants, which were used as folk medicine in tropical countries, were collected from Parangipettai, Vellar estuary, Tamilnadu, India. Nile Tilapia muscle homogenate was used as source of enzyme. Inhibitory effect of methanolic leaf extract was assessed under in vitro condition by incubating various concentration of the extract with total cholinesterase and butyryl cholinesterase and assessing their residual activities by Ellman's colorimetric method. The results showed that of the nine plants screened Rhizophora lamarckii, Suaeda monica, Avicennia officinalis and Sesuvium portulacastrum showed 50% inhibitory activity to both TChE and BChE at concentrations less than 2 mg/mL when compared to other plant extracts, which was comparable to the standard drug Donepezil. Phytochemical analysis showed the presence of alkaloids in high concentration which might be correlated to its cholinesterase inhibitory activity.     

The role of nucleotide pyrophosphatase in Mullerian duct regression

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn²⁺ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from to rat fetuses (n = 77) were incubated with a-naphthyl thymidine-5′-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male (n = 16) Mullerian duct cells at days of gestation, but no hydrolysis was detected around female (n = 17) Mullerian duct cells at any stage. Controls (n = 24) incubated without substrate did not stain. Addition of exogenous ATP (n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro (n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.     

CHOLINESTERASE ACTIVITY OF THE MOTOR ENDPLATE IN ISOLATED MUSCLE MEMBRANE

Abstract— The cholinesterase activity of motor endplates in tibialis anterior muscle of rats accounted for about 20 per cent of the total cholinesterase activity of the muscle. In the isolated muscle membrane preparation of rat intercostal muscle, the cholinesterase activity was localized solely in the motor endplate, as shown by cholinesterase staining. The cholinesterase activity of the membrane per unit of nitrogen was 26·9 times that of the muscle homogenate. The membrane (endplate) cholinesterase had an optimal pH of 8, K_m value of 3·1 m m , and was stable at 4° for at least 13 days. Cholinesterase of a motor endplate hydrolysed 2·69 × 10⁸ acetylcholine molecules in 1 msec. Since it is estimated that 10⁸ cholinesterase active sites are present in a motor endplate, the turnover time (time necessary for one enzyme site to hydrolyse one acetylcholine molecule) is calculated to be 372 μ sec, and the turnover number (molecules of acetylcholine hydrolysed by one enzyme site/min) to be 1·61 × 10⁵. From studies with cholinesterase inhibitors, cholinesterase activity was estimated to be due mostly to acetylcholinesterase, and only a minor part to pseudocholinesterase. The muscle membrane preparation seems to be useful for the study of other properties of the motor endplate.     

Immunological comparison of the usual and atypical human serum cholinesterase phenotypes

Antiserum prepared against highly purified usual human serum cholinesterase (the most common phenotype) cross-reacted identically with the atypical serum cholinesterase. The level of circulating atypical enzyme protein, determined immunologically, was about 30% lower when the enzyme came from an atypical rather than a usual phenotype, and the level of enzyme activity measured enzymatically atV _max with eithero-nitrophenylbutyrate or benzoylcholine as substrate showed approximately the same degree of reduction. The average specific activity (activity atV _max per microgram of enzyme protein) in sera from 28 usual and 20 atypical individuals did not differ significantly. These findings suggest that the atypical enzyme not only has altered catalytic properties (K)_mbut also might be synthesized more slowly, or clearedin vivo more rapidly, than the usual enzyme. This work was supported by U.S. Public Health Service Grants NS 15871 and GM 27028 and by a grant from the Hoffmann-La Roche Foundation.