Some properties of frog vestibular choline acetyltransferase and acetylcholinesterase

The amount and some properties of choline acetyltransferase (ChAT) and of acetylcholinesterase (AchE) were investigated in the frog vestibule. Enzyme activities were found to be of the same order of magnitude as in frog nervous tissue and various properties of vestibular ChAT (dependence on pH, chloride and Triton X-100 activation, phosphate sensitivity) and AchE (inhibition by eserine but not by Tetraisopropylpyrophosphoramide) were also similar as those of the homologous central nervous system enzymes. Although the precise localization of ChAT and AchE is not yet certain the efferent neurotransmitter in the vertebrate vestibular sensory periphery is believed to be acetylcholine and thus the enzymes responsible for its synthesis and degradation may participate in regulating inner ear function.     

The effect of exposure of acetylcholinesterase to 2,450-MHz microwave radiation

The effect of 2,450-MHz pulsed microwave radiation on the enzyme activity of membrane-free acetylcholinesterase was studied while the enzyme was in the microwave field. We found no significant effect of microwave radiation on enzyme activity using a wide variety of power densities, pulse widths, repetition rates, and duty cycles. This suggests that simple, direct modification by microwave energy of acetylcholinesterase structure and enzymic activity is not related to microwave alteration of acetylcholinesterase central nervous system levels.     

Acetylcholinesterase activity in the rat brain after pneumococcal meningitis

Pneumococcal meningitis is a life-threatening disease characterized by acute purulent infection of the meninges causing neuronal injury, cortical necrosis and hippocampal apoptosis. Cholinergic neurons and their projections are extensively distributed throughout the central nervous system. The aim of this study was to assess acetylcholinesterase activity in the rat brain after pneumococcal meningitis. In the hippocampus, frontal cortex and cerebrospinal fluid, acetylcholinesterase activity was found to be increased at 6, 12, 24, 48 and 96 hr without antibiotic treatment, and at 48 and 96 hr with antibiotic treatment. Our data suggest that acetylcholinesterase activity could be related to neuronal damage induced by pneumococcal meningitis.     

Cholinergic development in chick brain reaggregated cell cultures

Reaggregated cell cultures from dissociated 7-day-old chick embryo whole brains were prepared, and the developmental profiles of acetylcholinesterase and choline acetyltransferase, in the aggregates, determined over a 30-day period. Enzyme activities in vitro, at different times of culture, typically lie between 30 and 60% of the values obtained for embryos or chicks of the same developmental age, up to day-10 posthatching. The increase in acetylcholinesterase activity over a 24-day period of culture/incubation is fourfold in the aggregates vs. sixfold for embryos, while the choline acetyltransferase values increase, during the same period of time, 32-fold in the aggregates vs. 17-fold in vivo. Choline acetyltransferase activity seems to be more dependent on good cell-to-cell contact than acetylcholinesterase activity. On the other hand, morphological studies on the aggregates with light and electron microscopy reveal a number of structural features characteristic of well-developed nervous tissue. It is suggested that aggregate cultures of chick brain cells are an adequate model system that is especially useful in analyzing developmental phenomena requiring free tridimensional interaction.Abbreviations AChE acetylcholinesterase - ChAT choline acetyltransferase - BW284 C51 dibromide 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide - ACh acetylcholine     

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins cause an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19–31, 1998. © 1998 Wiley-Liss, Inc.     

Some components of the cholinergic system in the prawn Palaemonetes varians (Leach)

1. An enzyme similar to mammalian acetylcholinesterase is found in high activity in the nervous tissue of Palaemonetes varians, i.e. eyes plus stalks, brain, suboesophageal ganglion and ventral cord. Acetylcholinesterase is also found associated with the abdominal muscles. Multiple enzyme forms are found in extracts of nervous tissues and muscles by electrophoresis and isoelectric focusing. 2. Cholinesterase is present in high activity in the stomatogastric system of P. varians. Three electrophoretically separable forms are found, having isoelectric points at pH4.2, 4.5 and 5.4. 3. Approx. 50% of the total acetylcholinesterase activity, approx. 80% of the choline acetyltransferase activity and 100% of the acetylcholine equivalents are found associated with the nervous tissue. Among the tissues examined, eyes plus stalks were the richest source of both choline acetyltransferase and acetylcholine equivalents. Suboesophageal ganglion and brain also contained large amounts of these components. 4. The distribution of these components could support the function of acetylcholine as a central and/or sensory transmitter in P. varians.     

Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.     

Acetylcholinesterase activity in Clytia hemisphaerica (Cnidaria)

Cholinesterase activity is known in representatives of all living organisms phyla but the origin of the cholinergic system as known in bilaterian animals is still undeciphered. In particular the implication of cholinesterases in the nervous system of non-bilaterian Metazoa is not well known. We thus chose to investigate this activity in the Clytia hemisphaerica (Cnidaria) medusa. In toto histochemical staining revealed an acetylcholinesterase activity in the tentacle bulbs but not in the nervous system. Sequences homologous to acetylcholinesterase were searched within Clytia ESTs and compared to other sequences found in public databases.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Demonstration of acetylcholinesterase by semipermeable membrane technique: Estimation of soluble and fixation-labile portions in different regions of the central nervous system

Summary A histochemical method for the demonstration of acetylcholinesterase using semipermeable membranes is described. This technique prevents any loss of enzyme activity caused by dissolution and/or fixation. The soluble and fixation-labile portions of acetylcholinesterase were estimated in several regions of the central nervous system of the rat and differences were found. The method improves the accuracy of the histochemical demonstration of the acetylcholinesterase on the light microscopical level.The research reported in this paper was supported by the Ministerium für Wissenschaft und Technik der DDR     

Regional distribution of pyroglutamyl peptidase II in rabbit brain, spinal cord, and organs.

Pyroglutamyl peptidase II (PPII) is a narrow specificity ectoenzyme that degrades thyrotropin-releasing hormone (TRH). We detected the enzyme in the brain of various mammals, with highest specific activity in rabbit brain. In this species, activity was heterogeneously distributed in the central nervous system. There was a 28-fold difference between regions of highest and lowest PPII activity. Enzyme activity was highest in the olfactory bulb and posterior cortex. In the spinal cord, activity was low but unevenly distributed, with highest values detected in the thoracic (T) region. Segments T1 and T2 activities were particularly high. Other organs contained low or undetectable levels of activity. The levels of TRH-like immunoreactivity (TRH-LI) in spinal cord segments were greatest in T3-T4 and lumbar L2-L6. Low concentrations were found in T1 and T9-T12. There was a partial correlation between the distribution of PPII activity and TRH receptors but not with TRH-LI levels. These results demonstrate that PPII is predominantly a central nervous system enzyme, and they support the hypothesis that PPII is responsible for degrading TRH released into the synaptic cleft.     

Solubilization of 20S acetylcholinesterase from chick retina

A 20S form of acetylcholinesterase has been solubilized from young chick retinas by means of a buffered salt-detergent solution containing EDTA. The release of this fast-sedimenting form of the enzyme is selectively blocked by the presence of even small amounts of Ca⁺⁺ in the homogenization medium. The collagen-tailed nature of this molecular species of acetylcholinesterase has been ascertained by collagenase digestion. This finding suggests that the avian central nervous system contains asymmetric, collagen-tailed quaternary structural forms of acetylcholinesterase as is the case in skeletal muscle and cholinergic ganglia.     

Acetylcholinesterase. Two types of modifications confer resistance to insecticide.

Quantitative and qualitative changes in acetylcholinesterase confer resistance to insecticides. We have constructed several Drosophila melanogaster strains producing various amounts of enzyme by P-mediated transformation. Toxicological analysis of these strains demonstrates that resistance to organophosphorus insecticides is correlated with the amount of acetylcholinesterase in the central nervous system. Resistance may also be qualitatively determined. Comparison of the Drosophila acetylcholinesterase gene between a resistant strain caught in the wild and a wild type susceptible strain only revealed one nucleotide transition resulting in the replacement of a phenylalanine by a tyrosine. Flies mutant for acetylcholinesterase and rescued with a minigene mutagenized for this same transition produced an altered enzyme which renders flies resistant to pesticides.     

Acetylcholinesterase activity in Clytia hemisphaerica (Cnidaria)

Cholinesterase activity is known in representatives of all living organisms phyla but the origin of the cholinergic system as known in bilaterian animals is still undeciphered. In particular the implication of cholinesterases in the nervous system of non-bilaterian Metazoa is not well known. We thus chose to investigate this activity in the Clytia hemisphaerica (Cnidaria) medusa. In toto histochemical staining revealed an acetylcholinesterase activity in the tentacle bulbs but not in the nervous system. Sequences homologous to acetylcholinesterase were searched within Clytia ESTs and compared to other sequences found in public databases.     

Electron microscopical demonstration of acid phosphatase activity in the central nervous system

Summary A modified technique is described for the demonstration of acid phosphatase activity in the central nervous system by means of electron microscopy. Enzyme activity can be demonstrated in lysosomes, pigment bodies, and the Golgi zone of cortical neurons. Glial and endothelial cells also contain acid phosphatase active lysosomes. They are located in the pericarya, and in the processes of the glial cells, respectively.The authors express their sincere appreciation to FräuleinS. Luh and FräuleinW. Komp for their assistance and help.     

Phosphatases in the central nervous system of spiders (Arachnida, Araneae).

The central nervous systems of web-building spiders (Araneidae, Agelenidae) and hunting spiders (Lycosidae, Salticidae) were tested for non-specific and specific phosphatases. Acid phosphatase exhibited weakly to moderately positive reactions in the neuronal cell bodies and in the neuropile fibre mass of all species investigated. Alkaline phosphatase could only be demonstrated in the external and internal neural lamellae of the brain and ventral cord of several specimens of the araneid species investigated. Tests for thiamine pyrophosphatase were negative with both the lead and calcium-cobalt methods. Distinctive positive reactions for adenosine triphosphatase were visible in the nervous system of all the species used, being especially strong in the optic ganglia of the hunting spiders. The demonstration of adenosine triphosphatase was only possible when applying the calcium-cobalt method after Padykula and Herman, while the lead method after Wachstein and Meisel did not produce any staining reaction at all. Controls of the histochemical reaction showed that the enzyme was activated by Ca2+ and inhibited by sulphydryl destroying reagents (e.g. PCMB), but was insensitive to ouabain. It could be probably classified as a mitochondrial proton-translocating adenosine triphosphatase.     

ACETYL- AND BUTYRYLCHOLINESTERASE ACTIVITY OF SELECTED BRAIN AREAS IN DEVELOPING RATS AFTER NEONATAL X-IRRADIATION*

Acetylcholinesterase and butyrylcholinesterase activities in sensori-motor cortex, hypothalamus, cerebellum, and brain stem were compared in normally developing Long-Evans rats and after neonatal whole-body exposure to 450 r X-radiation. Enzyme activities were measured on three postnatal days: day 10, when brain is still immature; day 24, when it has reached functional and morphological maturity; and day 64, after sexual maturation. In controls, acetylcholinesterase and butyrycholinesterase activities increased with age in all areas, especially between 10 and 24 days; e.g., in sensori-motor cortex acetylcholinesterase activity increased 60 per cent from 10 to 24 days and 12 per cent from 24 to 64 days. At all ages acetylcholinesterase activity was highest in the brain stern, followed in decreasing order by the hypothalamus, cerebellum, and sensori-motor cortex. Butyrylcholinesterase activity was higher in subcortical than in cortical areas. In neonatally irradiated rats, acetylcholinesterase activity was significantly decreased in the ontogenetically newer structures at 10, but not at 64, days; in the hypothalamus, it remained normal at 10 days but was significantly decreased at 24 and 64 days. Butyrylcholinesterase activity was significantly decreased in some areas 1 week after radiation but returned to normal at 24 days. Total esterase activity in whole blood was signtficantly decreased at 10 days in irradiated rats but returned to control levels by the end of the experiment. The greatest post-radiation decline in acetylcholinesterase activity (60 per cent below controls) did not result in spontaneous gross behaviour alterations, but may be related to disturbances in functional brain maturation evidenced by specific tests. If the role of acetycholine as a central neurotransmitter is accepted, these data suggest that radiation alters acetycholine/acetylcholinesterase ratios and thereby cholinergie synaptic transmission.     

On the role of normal acetylcholine metabolism in the formation and maintenance of the Drosophila nervous system

We have examined the requirement for normal acetylcholine metabolism in the formation and maintenance of the larval and adult central nervous system in Drosophila melanogaster. By using mutations at the Ace and Cha loci, which respectively encode the degradative and synthetic enzymes for acetylcholine (ACh), acetylcholinesterase (AChE), and choline acetyltransferase (ChAT), we have been able to disrupt acetylcholine metabolism in situ. An ultrastructural analysis of embryonic nervous tissue lacking either enzymatic function has indicated that while neither function is required for the formation of the larval central nervous system, each is required for the subsequent maintenance of its structural integrity and function. Using temperature sensitive mutations at the Cha locus, the normal developmental profile of ChAT activity during the late larval and pupal stages was disrupted. Subsequent examination of the morphology and behavior of the treated animals has indicated that normal acetylcholine metabolism is not required for the initial formation of the adult nervous system, but is required for the subsequent maintenance of its structural integrity and function. The results obtained in these studies are discussed with respect to data presented on the adult distribution of the cholinergic markers' AChE activity and ChAT immunoreactivity. The projections of adult peripheral neurons innervating Ace+ tissue from Ace cuticular clones has been examined to address the nature of the structure of Ace neuropil. Normal projections are apparently achieved and maintained, suggesting that the defects seen in adult Ace mosaics arise as an aberrant intracellular organization of morphologically normal cells.     

Phosphatases in the central nervous system of spiders

Summary The central nervous systems of web-building spiders (Araneidae, Agelenidae) and hunting spiders (Lycosidase, Salticidae) were tested for non-specific and specific phosphatases. Acid phosphatase exhibited weakly to moderately positive reactions in the neuronal cell bodies and in the neuropile fibre mass of all species investigated. Alkaline phosphatase could only be demonstrated in the external and internal neural lamellae of the brain and ventral cord of several specimens of the araneid species investigated. Tests for thiamine pyrophosphatase were negative with both the lead and calciumcobalt methods. Distinctive positive reactions for adenosine triphosphatase were visible in the nervous system of all the species used, being especially strong in the optic ganglia of the hunting spiders. The demonstration of adenosine triphosphatase was only possible when applying the calcium-cobalt method after Padykula and Herman, while the lead method after Wachstein and Meisel did not produce any staining reaction at all. Controls of the histochemical reaction showed that the enzyme was activated by Ca²⁺ and inhibited by sulphydryl destroying reagents (e.g. PCMB), but was insensitive to ouabain. It could be probably classified as a mitochondrial proton-translocating adenosine triphosphatase.     

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).