Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Cholinergic systems in muscle and brain in vitamin E-deficient rats

Rats fed a vitamin E-deficient diet for 7–8 weeks postweaning showed no change in brain weight or the activity in brain of various enzymes involved in neurotransmitter synthesis and metabolism. Body and muscle weights were markedly reduced. Muscle choline acetyltransferase and acetylcholinesterase activities were significantly elevated on a protein basis, but the total amount of choline acetyltransferase/muscle was essentially normal and total acetylcholinesterase activity was slightly reduced. Total carnitine acetyltransferase and butyrylcholinesterase activities were markedly decreased. The results are quite different from those found in hereditary murine muscular dystrophy and suggest a myogenic etiology for the vitamin E-deficiency-induced condition.     

Genetic,ontogenetic, and tissue-specific variation of dipeptidases in Drosophila melanogaster

Three dipeptidases in Drosophila melanogaster are under independent genetic control and their structural genes have been localized, Dip-A to 2R and Dip-B and Dip-C to 3R (Voelker and Langley, 1978; Ohnishi and Voelker, 1981). These enzymes were characterized with respect to their substrate specificities, genetic variability (electrophoretic mobility and quantitative activity level), ontogeny (activity and isozyme pattern), and tissue localization. The dipeptide substrate specificities of DIP-A and DIP-B overlap each other considerably, but do not overlap with DIP-C. In natural populations, DIP-B and DIP-C are essentially monomorphic electrophoretically whereas DIP-A is polymorphic for three allozymes. Both DIP-A and DIP-B show quantitative genetic variation of activity level within an allozyme class. All three enzymes are expressed at all stages in the life cycle, but DIP-A and DIP-B activities vary considerably according to developmental stage and sex of adult. The tissue localizations of DIP-A and DIP-B activities show similar patterns and a nearly ubiquitous occurrence of both enzymes, but with particularly high values in larval and adult midguts and in the adult female reproductive system. These results suggest a general metabolic role for the enzymes, such as regulation of the concentrated pools of amino acids and oligopeptides found in Drosophila tissues.This work was supported by Public Health Service Grant GM 11546.Paper No. 7066 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh.     

Local thermal adaptation detected during multiple life stages across populations of Drosophila melanogaster

Thermal adaptation is typically detected by examining the tolerance of a few populations to extreme temperatures within a single life stage. However, the extent to which adaptation occurs among many different populations might depend on the tolerance of multiple life stages and the average temperature range that the population experiences. Here, we examined local adaptation to native temperature conditions in eleven populations of the well‐known cosmopolitan fruit fly, Drosophila melanogaster. These populations were sampled from across the global range of D. melanogaster. We measured traits related to fitness during each life stage to determine whether certain stages are more sensitive to changes in temperature than others. D. melanogaster appeared to show local adaptation to native temperatures during the egg, larval and adult life stages, but not the pupal stage. This suggests that across the entire distribution of D. melanogaster, certain life stages might be locally adapted to native temperatures, whereas other stages might use phenotypic plasticity or tolerance to a wide range of temperatures experienced in the native environment of this species.     

Drosophila acetylcholinesterase: Characterization of different mutants resistant to insecticides

Selection of field populations originating from several countries allowed us to isolate 13 strains ofDrosophila melanogaster resistant to parathion.In vitro studies of acetylcholinesterase inhibition by paraoxon have been carried out on purified enzymes: most of the resistant strains harbor an altered acetylcholinesterase. Enzymes with higher resistance levels have been characterized with respect to their cross-resistance toward several insecticides. The patterns obtained have permitted us to group them and to delineate four categories. The existence of four distinct types of protein suggests that several mutations of acetylcholinesterase are responsible for insecticide resistance inDrosophila.     

CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

Ecdysone-induced changes in morphology and protein synthesis in Drosophila cell cultures.

Permanent cell lines derived from embryonic Drosophila melanogaster show pronounced morphological and biochemical changes in response to the addition of β-ecdysone, the moulting hormone, or two of its analogs. Schneider's line 3 cells, and the French line Kc, morphologically differentiate into what appear to be neural-like cells. During this period levels of acetylcholinesterase activity increase 10- to 30-fold, and DNA synthesis ceases. The level of choline acetyltransferase activity remains unchanged in line 3, and is very low. Schneider's line 2 cells show no morphological transformation in response to β-ecdysone, but undergo a marked change in the pattern of protein synthesis. The six major ecdysone proteins are not “heat shock” proteins.     

Identification of a small genetic region that encodes orotate phosphoribosyltransferase and orotidylate decarboxylase in Drosophila melanogaster

Summary A variety of tests indicate that a small genetic region in chromosome 3R encodes at least a part of the bienzyme complex containing the last two enzymes of the pyrimidine biosynthetic pathway in Drosophila melanogaster, Complex U. Electrophoretic variants of the last pathway enzyme map to this region and both Complex U activities are proportionally reduced in flies heterozygous for specific deficeencies of the region. Complex U mutants have been isolated that lack both activities. Characteristics of these mutants are discussed relative to other pyrimidine pathway mutants known in Drosophila melanogaster and in mammals.     

Characteristics of choline acetyltransferase and cholinesterases in two types of cultured cells from embryonic chick brain

Cells that were mechanically dissociated from the brains of 7-day-old chick embryos were grown in culture for 7–8 days. Two major cell populations were observed: (1) cells that aggregated and sent out processes, (2) flat cells that proliferated rapidly and formed a confluent layer by day 4 of culture. Many of the cells of the first type had the morphological, histochemical and biochemical attributes of neurons. They possessed choline acetyltransferase (ChAc) and acetylcholinesterase (AChEs) activities. The flat cells possessed neither of the activities, but did have butyrylcholinesterase (BuChEs) activity and a choline independent acetylase activity (CIA) that may be carnitine acetyltransferase.The activities of ChAc and AChEs in the cultured neurons increased approximately 9-fold and 5-fold, respectively, over an 8-day period. The patterns of change of these enzymes were not unlike those seen in vivo in intact developing chick brain.The addition of thyroxine (10^?6M) to these cultures increased the activities of neuronal AChEs and flat cell BuChEs by 30–70%.     

Early Inhibition of Acetylcholinesterase and Choline Acetyltransferase Activity in Herpes simplex Virus Type 1 Infection of PC12 Cells

Abstract: Early in the course of productive Herpes simplex virus type 1 (HSV-1) infection of PC12 cells, activities of both acetylcholinesterase (AChE) and choline acetyltransferase (CAT) fell. Studies using metabolic inhibitors and a temperature-sensitive mutant of the virus suggested that the decline in activities of both enzymes was associated with events occurring early in the replicative cycle related to expression of the immediate-early (α) group of viral polypeptides. HSV-1 gene products thus may alter specialized cell functions well before the production of viral progeny and initiation of cell lysis. The early clinical manifestations of nervous system viral infection may reflect focal metabolic disturbance rather than, or in addition to, simple cell death.     

Maharishi Amrit Kalash,an ayurvedic medicinal preparation,enhances cholinergic enzymes in aged guinea pig brain

The effect of orally fed Maharishi Amrit Kalash was examined on the activities of cholinergic enzymes in the guinea pig brain. The activity of the cholinergic enzymes viz. choline-acetyltransferase and acetylcholinesterase enzymes was found to be reduced significantly (P<0.05) in the various regions of CNS of the aged guinea pigs. Oral administration of MAK(500 mg/kg body weight daily) for 2 months significantly increased (P<0.05) the activity of choline acetyltransferase and acetylcholinesterase in the older animals. The present study indicates that this food supplement can be helpful in alleviating the cholinergic deficits in the old age.     

Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

Regional distribution of choline acetyltransferase and acetylcholinesterase in rabbit brain

Regional distribution of choline acetyltransferase (CAT) and acetylcholinesterase (AChE) in seventeen regions of the rabbit CNS has been determined by means of the cation exchange radiometric method. A significant correlation has been found between the activities of AChE and CAT which allows computation of their potencies. A comparison between activities of the cholinergic enzymes in the human and rabbit brain has been discussed.     

High activity of choline acetyltransferase induced in neuroblastoma X glia hybrid cells

Hybrids were made between rat glioma X mouse neuroblastoma cell lines and were examined for the specific activities of choline acetyltransferase and acetylcholinesterase. The specific activities of choline acetyltransferase of the hybrids were as high as those in normal brain, whereas neither parent line showed appreciable activities. The electrical excitability of the neuroblastoma cells was retained in the hybrids.     

Dietary consumption of monosodium L‐glutamate induces adaptive response and reduction in the life span of Drosophila melanogaster

Adaptive response is the ability of an organism to better counterattack stress‐induced damage in response to a number of different cytotoxic agents. Monosodium L‐glutamate (MSG), the sodium salt of amino acid glutamate, is commonly used as a food additive. We investigated the effects of MSG on the life span and antioxidant response in Drosophila melanogaster (D. melanogaster). Both genders (1 to 3 days old) of flies were fed with diet containing MSG (0.1, 0.5, and 2.5‐g/kg diet) for 5 days to assess selected antioxidant and oxidative stress markers, while flies for longevity were fed for lifetime. Thereafter, the longevity assay, hydrogen peroxide (H₂O₂), and reactive oxygen and nitrogen species levels were determined. Also, catalase, glutathione S‐transferase and acetylcholinesterase activities, and total thiol content were evaluated in the flies. We found that MSG reduced the life span of the flies by up to 23% after continuous exposure. Also, MSG increased reactive oxygen and nitrogen species and H₂O₂ generations and total thiol content as well as the activities of catalase and glutathione S‐transferase in D. melanogaster (P < .05). In conclusion, consumption of MSG for 5 days by D. melanogaster induced adaptive response, but long‐term exposure reduced life span of flies. This study may therefore have public health significance in humans, and thus, moderate consumption of MSG is advocated by the authors.     

Laminar Distributions of Choline Acetyltransferase and Acetylcholinesterase Activities in the Inner Plexiform Layer of Rat Retina

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.     

Quantitative histochemical studies of the hypothalamus enzymes of acetylcholine metabolism.

The quantitative histochemical distribution of acetylcholinesterase and choline acetyltransferase activity has been measured in individual hypothalamic nuclei and median eminence, as well as in entire hypothalamic sections by a mapping technique. There was an 18-fold range of nuclear choline acetyltransferase activity with highest activities in the lateral preoptic nucleus and median eminence. There was a nine-fold range of nuclear acetylcholinesterase activity with highest activities in the lateral preoptic and magnocellular nuclei and lowest activity in the median eminence. The substantial gradients of choline acetyltransferase activity found in the hypothalamus indicate the importance of using a technique that provides an objective, permanent record of contiguous sample locations thereby allowing detailed analysis of tissue areas using, but not dependent on, anatomical boundaries.     

Life‐history traits and physiological limits of the alpine fly Drosophila nigrosparsa (Diptera: Drosophilidae): A comparative study

Interspecific variation in life‐history traits and physiological limits can be linked to the environmental conditions species experience, including climatic conditions. As alpine environments are particularly vulnerable under climate change, we focus on the montane‐alpine fly Drosophila nigrosparsa. Here, we characterized some of its life‐history traits and physiological limits and compared these with those of other drosophilids, namely Drosophila hydei, Drosophila melanogaster, and Drosophila obscura. We assayed oviposition rate, longevity, productivity, development time, larval competitiveness, starvation resistance, and heat and cold tolerance. Compared with the other species assayed, D. nigrosparsa is less fecund, relatively long‐living, starvation susceptible, cold adapted, and surprisingly well heat adapted. These life‐history characteristics provide insights into invertebrate adaptations to alpine conditions which may evolve under ongoing climate change.     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.