Amount Of Acetylcholinesterase Research Articles

Anticancer activity of midostaurin in hormone refractory human prostate cancer DU145 cells

<p class="Abstract">We tried a new method of prostate cancer treatment by inducing<em> in vitro</em> differentiation which resulted in reduction of cancer cells growth. A protein kinase inhibitor, midostaurin's ability to trigger the human prostate cancer cell line, DU145 to segregate into nerve cells was studied. Midostaurin (100 nM) suppressed the growth of DU145 cells but without change in the number of dead cells. Midostaurin started to extend neurites on DU145 cells after 24 hours and differentiated into nerve cells by 72 hours. The microtubule was stabilized by tau protein and its mRNA expression showed time-dependent increase in midostaurin-treated DU145 cells. At the same time, the amount of acetylcholinesterase was also increased. The midostaurin-treated DU145 cells showed 40% less activity than control in the colony forming assay. The results suggests that midostaurin can induce differentiation of DU145 cells into nerve cells.</p><p> </p>

Changes in the auditory neuropil after deafferentation in adult grasshoppers ( Schistocerca gregaria)

Nervous systems are capable of structural adjustments. Such plastic changes also occur in the auditory system of the locust Schistocerca gregaria in which a deafferentation leads to compensatory mechanisms, such as collateral sprouting of interneurons. In this study we further investigated lesion related changes in the major auditory neuropil, the median ventral association center (mVAC) of the metathoracic ganglion. The auditory sensory organ of adult locusts was unilaterally extirpated and the mVAC was histologically and immunocytochemically analyzed until 20 days postoperative. Measurements of the neuropil area in transverse sections showed a decrease in size. The putative transmitter of the afferents, acetylcholine, was investigated by acetylcholinesterase histochemistry. Comparisons of staining intensities in the intact and deafferentated mVAC indicated that the amount of acetylcholinesterase in the deafferentated mVAC decreased shortly after the operation. Both, the decreases in size of the mVAC as well as that in acetylcholinesterase histochemistry were only less than 10% compared to the controls. The immunoreactivity against the neurotransmitters γ-amino butyric acid and serotonin was not influenced by the deafferentation.

Cholinesterase activity of human lung tumours varies according to their histological classification

The probable involvement of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in cancer and the relevance of cholinergic responses for lung cancer growth prompted us to study whether cholinesterase activity of human lung is altered by malignancy. Surgical pieces of non-small lung carcinomas (NSLC) and their adjacent non-cancerous tissues (ANCT) were analysed for AChE and BChE activities. AChE activity in adenocarcinoma (AC) was 7.80 +/- 5.59 nmol of substrate hydrolysed per min and per mg of protein (mU/mg), the same as in their ANCT (8.83 +/- 4.72 mU/mg; P = 0.823); in large cell carcinoma (LCC), 7.52 +/- 3.32 mU/mg, approximately 50% less than in their ANCT (15.39 +/- 5.66 mU/mg; P = 0.043); and in squamous cell carcinoma (SCC), 1.39 +/- 0.58 mU/mg, 80% less than in ANCT (6.08 +/- 2.88 mU/mg; P = 0.003). BChE activity was 5.85 +/- 3.20 mU/mg in AC and 9.56 +/- 3.38 mU/mg in ANCT (P = 0.022); 2.94 +/- 2.01 mU/mg in LCC and 6.50 +/- 6.63 mU/mg in ANCT (P = 0.068); and 4.49 +/- 2.30 mU/mg in SCC and ANCT 6.56 +/- 4.09 mU/mg (P = 0.026). Abundant AChE dimers and fewer monomers were identified in lung and, although their distribution was unaffected by cancer, the binding with concanavalin A revealed changes in AChE glycosylation between SCC and their ANCT. The fall in BChE activity affected all molecules, with a strong decrease of the amphiphilic tetramers. Western blotting revealed protein bands with the expected mass of the principal AChE subunits, and the deeper intensity of the protein signal in SCC than in healthy lung, in lanes loaded with the same units of AChE activity, supported an augment in the amount of AChE protein/unit of AChE activity in SCC. The increased availability of acetylcholine in neoplastic lung, resulting from the fall of cholinesterase activity, may enhance cholinergic signalling and contribute to tumour progression.

Physicochemical and Biological Data for the Development of Predictive Organophosphorus Pesticide QSARs and PBPK/PD Models for Human Risk Assessment

ABSTRACTA search of the scientific literature was carried out for physiochemical and biological data [i.e., IC50, LD50, Kp (cm/h) for percutaneous absorption, skin/water and tissue/blood partition coefficients, inhibition ki values, and metabolic parameters such as Vmax and Km] on 31 organophosphorus pesticides (OPs) to support the development of predictive quantitative structure–activity relationship (QSAR) and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for human risk assessment. Except for work on parathion, chlorpyrifos, and isofenphos, very few modeling data were found on the 31 OPs of interest. The available percutaneous absorption, partition coefficients and metabolic parameters were insufficient in number to develop predictive QSAR models. Metabolic kinetic parameters (Vmax, Km) varied according to enzyme source and the manner in which the enzymes were characterized. The metabolic activity of microsomes should be based on the kinetic activity of purified or cDNA-expressed cytochrome P450s (CYPs) and the specific content of each active CYP in tissue microsomes. Similar requirements are needed to assess the activity of tissue A- and B-esterases metabolizing OPs. A limited amount of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) inhibition and recovery data were found in the literature on the 31 OPs. A program is needed to require the development of physicochemical and biological data to support risk assessment methodologies involving QSAR and PBPK/PD models.

Nuclear Autonomy in Multinucleated Cells

Skeletal muscle fibers are large multinucleated cells capable of developing highly specialized regions such as the neuromuscular junction (the site of nerve-muscle synapses). Rossi et al. developed a divided culture system to test whether individual nuclei of myotubes in culture can respond to local signals generated from the plasma membrane. Local membrane depolarization was controlled pharmacologically and the responses of the treated and untreated sides of the myotube compared. The amount of acetylcholinesterase (AChE) that was secreted and that accumulated at the cell surface was monitored. The transcription of AChE from individual nuclei was also determined. The data indicate that each nucleus can respond independently to locally generated signals. Rossi, S.G., Vazquez, A.E., and Rotundo, R.L. (2000) Local control of acetylcholinesterase gene expression in multinucleated skeletal muscle fibers: Individual nuclei respond to signals from the overlying plasma membrane. J. Neurosci. 20: 919-928. [Abstract] [Full Text]

Uptake of Acetylcholinesterase by Neurons in the Substantia Nigra

It is known that acetylcholinesterase is secreted by the dopaminergic neurons of the substantia nigra and has a subsequent action independent of cholinergic transmission. Although non-cholinergic actions of this protein have been demonstrated, the subsequent fate of acetylcholinesterase is unknown. One possibility is that acetylcholinesterase is taken up following secretion into the extracellular space. This hypothesis has been tested in vivo, in both conscious and anaesthetized guinea-pigs. Exogenous acetylcholinesterase (2-20 pM) was infused via a push-pull cannula implanted into either the substantia nigra or the surrounding extranigral regions: the amount subsequently recovered in the perfusate was then compared with control values. Only when the push-pull cannulae were implanted in the substantia nigra was there a marked decrease in the amount of acetylcholinesterase recovered; this selective retention was abolished when the perfusion medium was cooled to 4 degrees C or when the experiment was performed post mortem. Direct visualization of immunocytochemically identified nigral dopaminergic cells revealed co-localized deposits of labelled, exogenous acetylcholinesterase. Moreover, when exogenous acetylcholinesterase was boiled to prevent detection by the assay system and to eliminate any classical enzymatic action, an enhancement in perfusate levels of endogenous acetylcholinesterase was observed from nigral but not from extranigral sites, indicating that endogenous and exogenous acetylcholinesterases were in competition. These results suggest that, within the substantia nigra, secreted acetylcholinesterase may be subject to a temperature- and energy-dependent uptake mechanism.

Drosophila acetylcholinesterase: Mechanisms of resistance to organophosphates

Quantitative and qualitative changes of acetylcholinesterase can affect the sensitivity of insects to insecticides. First, the amount of acetylcholinesterase in the central nervous system is important in Drosophila melanogaster, flies which overexpress the enzyme are more resistant than wild-type flies. On the contrary, flies which express low levels of acetylcholinesterase are more susceptible. An overproduction of acetylcholinesterase outside the central nervous system also protects against organophosphate poisoning, that is, flies producing a soluble acetylcholinesterase, secreted in the haemolymph, are resistant to organophosphates. Second, resistance can also result from a qualitative modification of acetylcholinesterase. Four mutations have been identified in resistant strains: Phe 115 to Ser, Ileu 199 to Val, Gly 303 to Ala and Phe 368 to Tyr. Each of these mutations led to a different pattern of resistance and combinations between these mutations led to highly resistant enzymes.

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.

Effect of acute physostigmine-hyoscine pretreatment on the neurochemical changes produced by soman in the guinea-pig

The protective effects of two dose regimes of the organophosphate pretreatment combination, physostigmine and hyoscine, were assessed on the central neurochemical changes produced following soman intoxication. The lower dose combination (physostigmine 20 ?g/kg, hyoscine 10 ?g/kg, s.c.) inhibited brain regional acetylcholinesterase (AChE) by between 13.5 and 37.6% in all regions except the striatum, where there was no statistically significant inhibition. This low dose pretreatment failed to protect a measureable proportion of brain AChE from soman and did not prevent the neurotransmitter changes produced by soman. Signs of intoxication were indistinguishable from those seen with soman alone, however more animals survived longer than 24 h. The higher dose combination (physostigmine 196 ?g/kg and hyoscine 113 ?g/kg s.c.) inhibited brain regional AChE by between 56.8 and 67.3%, but had significant effects alone on the levels of ACh and NA. The high dose pretreatment effectively protected 20-30% of the enzyme following soman challenge. This protected enzyme became available as the physostigmine was eliminated from the body after 60-120 min. Animals exhibited mild signs of poisoning, such as hyperactivity and chewing, during the first 30-60 min, after which they recovered. Transmitter changes following soman were completely prevented with high dose pretreatment. Both dose levels of pretreatment decreased lethality but only the high dose, which protected a measurable amount of AChE and prevented neurotransmitter changes, decreased incapacitation.

Age-dependent changes in acetylcholinesterase activity in the primary somatosensory cortex of the cat

As cortical reorganization in cat somatosensory cortex has been shown to be age-dependent and acetylcholinesterase and acetylcholine have been implicated in the shaping of sensory responses during the developmental process, we decided to investigate the biochemical changes that occur in acetylcholinesterase during postnatal development of the primary somatosensory cortex in normal cat. Somatosensory cortices were removed from cats at various ages between 4 and 144 postnatal days. Three fractions (total, membrane-bound and soluble) were analyzed for activity (esterase assay and sedimentation analysis) and amount of acetylcholinesterase (electrophoresis). Results indicated that both esterase activity levels and amounts were characterized by 4 distinct phases which included a large step increase in all fractions between postnatal days 10 and 12; a gradual rise between days 12 and 28; a ‘dip’ during the 42- to 82-day interval, and a subsequent recovery. Results may be attributed to concomitant developmental events. Furthermore, we suggest that the observed changes may relate to age-dependent differences in somatosensory cortex reorganization that occur after spinal cord transection.

Dihydropyridine receptor regulation of acetylcholinesterase biosynthesis.

The dihydropyridine calcium channel antagonist nifedipine causes marked reductions in the amounts of acetylcholinesterase (AchE) molecular forms in primary tissue cultures of avian pectoral muscle. These reductions are time-dependent, requiring passage of 3 h prior to any observable response, dose-dependent, with principal actions occurring in the 1-100 nM range, are greater on the 7 S and 19 S forms than on the 11.4 S form, and, based on susceptibility of AchE to irreversible inhibition by a cationic inhibitor, occur almost exclusively with intracellular AchE coincident with a 2-fold reduction in the rate of secretion. The effects are markedly more pronounced in skeletal muscle than in neurons and differ from those observed for verapamil, diltiazem, and the calcium ionophore A23187. These reductions are incompatible with accelerated protein degradation, alterations in posttranslational processing and assembly in the Golgi complex, or enhanced loss of enzyme to the medium, but instead indicate that nifedipine causes a reduction in AchE biosynthesis. Since AchE forms are thought to arise from a single gene, these findings imply a linkage in skeletal muscle between transcription and posttranscriptional processing of mRNA and ligand occupation of the dihydropyridine receptor.

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick, Dermacentor variabilis (Say).

Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.

Architectonics of the thalamus in the echidna (Tachyglossus aculeatus): search for the mediodorsal nucleus.

Architectural characteristics of the thalamus in echidnas and rats were compared in sections stained to reveal cell bodies, myelin, acetylcholinesterase, succinate dehydrogenase and cytochrome oxidase. Numerous species differences were noticed: in general, the thalamus is architecturally more homogeneous in echidnas than in rats, especially anteriorly. In this report we emphasize the presence of a relatively large structure localized in the anteromediodorsal part of the thalamus in echidnas. This structure, previously shown to project to the frontal cortex, contains very small amounts of acetylcholinesterase and the oxidative enzymes; in this respect it resembles the mediodorsal nucleus of rats. The same properties make this formation different from the anterodorsal and anteroventral nuclei in rats, the equivalents of which could not be identified in echidnas. The anteromediodorsal region of the thalamus in echidnas consists chiefly of two cytoarchitecturally different regions: the medial, 'polymorphic' part contains relatively small, densely packed, multiform perikarya, whereas the lateral, 'monomorphic' part is characterised by larger, sparse neurons with little cytoplasm and round, large, empty-looking nuclei in which the nucleolus is clearly seen. We conclude tentatively that this brain structure of echidnas corresponds to the mediodorsal nucleus in placental species. Further studies of connections and chemical properties will be essential to determine the degree of correspondence of the presumed 'frontal lobe system' in echidnas to that in other mammals.

Substance P neurons sprout in the cervical spinal cord of the wobbler mouse: A model for motoneuron disease

The mutant mouse, wobbler, possesses a recessively inherited degeneration of motoneurons and other ventral horn cells in the cervical spinal cord, and therefore it has been proposed as an animal model of human motoneuron disease. Affected mice have been identified by behavioral tests that also determined the extent of the motor deficit. The results from these tests were combined and used to define distinct stages of the disease process that could then be correlated histochemically with the amount of acetylcholinesterase (AChE) staining in the cervical spinal cord. AChE is used as a marker for cholinergic neurons and is known to hydrolyze the neuropeptide modulator substance P (SP). SP, a peptide neuromodulator of primary afferent transmission in the dorsal horn, excites motoneurons in the ventral horn, and may possess secondary functions in neuronal maintenance. Therefore, the levels of immunoreactive (IR) SP and AChE were examined in an attempt to determine the possible interaction between these factors in motoneuron degeneration. By enzyme histochemistry, the cervical spinal cord, taken from wobbler mice at behaviorally identified stages of the motor deficit, exhibited decreased levels of AChE throughout the ventral horn. The decrease detected in the AChe staining intensity was linear and correlated with the decrease in the number of AChE-positive cells in the ventral cervical spinal cord, as the motor deficit progressed. Presumably, the continual decrease in AChE staining represents the degeneration of cholinergic perikarya and neuronal processes in the ventral horn as the motoneuron disease proceeds. At two well-established stages of the motor deficit, the amount of immunoreactive SP increased in the ventral horn compared with the control mice. The elevated levels of immunoreactive SP suggest sprouting may have occurred preceding, or in response to, the motoneuron degeneration. Several additional hypotheses are discussed in respect to phenomena that might contribute to the increase of immunoreactive SP in the degenerating ventral horn of the wobbler mouse.

Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C.

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.

Reduced axonal transport of 10S acetylcholinesterase in dystrophic mice

Extracts of extensor digitorum longus muscle, atria, brain, and sciatic nerve from phenotypically normal and dystrophic ReJ/129 mice were subjected to sucrose density gradient ultracentrifugation, and the amounts of acetylcholinesterase (AChE) activity associated with each major enzyme form were determined. Normal muscle showed approximately equivalent amounts of the 4S, 10S, and 16S forms of AChE, while dystrophic muscle was relatively deficient in 10S AChE and relatively oversupplied with 4S AChE. This abnormality was not present in the other tissues examined. However, as measured by the 24-hour accumulation of enzyme activity proximal to a ligature on the sciatic nerve, the axonal transport of 10S AChE was only about one third as great in dystrophic as in normal nerve. This result is consistent with the view that the reduction in the amount of this enzyme form in dystrophic muscle could be related to disturbances in a transport-dependent trophic interaction between nerve and muscle.

GENETICS OF DACUS OLEAE. VIII. SELECTION FOR THE AMOUNT OF ACETYLCHOLINESTERASE AFTER ORGANOPHOSPHATE TREATMENT.

The main amount of esterase in the adult olive fruit fly Dacus oleae (Gmel) is controlled by two autosomal independently segregating loci, named A and B. Locus A controls the synthesis of an acetylcholinesterase and locus B of a pseudocholinesterase or lipase (Zouros, Tsakas and Krimbas, 1968). Both loci are highly polymorphic. More than 20 active variants plus a silent one (As) have been found for locus A and more than 15 variants plus a silent one (Bs) for locus B. Flies homozygous for A, and B, are viable in nature. Zouros and Krimbas (1970) found a duplication for locus A in a natural population which had not been treated with organophosphates. This duplication had a frequency of 0.8 X 10. From all kinds of duplications we can only detect the electrophoretically different ones, that is the ones carried by flies which upon electrophoresis show three different allozyme bands. If all active variants produce the same amount of acetylcholinesterase and if there is no dosage compensation for this enzyme, then adult flies can be classified as follows according to their amount of acetylcholinesterase:

Ultrastructural pattern of acetylcholinesterase distribution in the cerebellar cortex of the quail.

The ultrastructural localization of acetylcholinesterase (AChE) was studied in the cerebellar cortex of the quail by means of histochemical method. The greater amount of AChE was detected at leve of the molecular layer in the intracellular spaces between parallel fibers and between parallel fibers and dendritic terminals. Many neurons showed intracellular localization of enzyme activity: the AChE positive neurons were all Golgi cells, most stellate the basket cells and different aliquots of Purkinje and granule cells. The enzymatic activity was usually localized in the cisternae of endoplasmic reticulum, in the nuclear envelope (but this last localization was not present in Purkinje cells- and sometimes in the Golgi apparatus; reaction granules were usually scarce in the different dendritic branches ramifying in the molecular layer. On the basis of the ultrastructural pattern of AChE distribution, some considerations are developed on the methodological aspects concerning the reliability of histochemical methods, the differences recorded at light and electron microscope level, the problems related to extracellular localization of enzyme, the difficulty of establishing a precise correlation between AChE localization in a cerebellar neuron and its possible cholinergic and/or cholinoceptive nature.

Distribution of protein-bound N-acetylneuraminic acid in subcellular particulate fractions prepared from rat whole brain

1. 1. A particulate fraction, rich in synaptosomes, was prepared from rat whole brain and centrifuged through a discontinuous sucrose-density gradient consisting of 0.32, 0.9, 1.0, 1.1, and 1.2 M sucrose to obtain five subfractions (P2B-a, P2B-b, P2B-c, P2B-d, and P2B-e). 2. 2. The amount of acetylcholinesterase and gangliosides present, per mg protein, decreased from a high value in the lightest particle fraction (P2B-a) to a low value for the pellet (P2B-e) at the bottom of the centrifuge tube. 3. 3. The amount of cytochrome oxidase, per mg protein, increased from a low value in the lightest fraction (P2B-a) to its highest value in P2B-e. 4. 4. The amount of glycoprotein- N-acetylneuraminic acid (NANA), per mg protein, was tha same in all fractions, with the exception of P2B-a, which had a lower value. 5. 5. Particulate fractions that are enriched in synaptosomes, as well as synaptosome ghosts prepared by osmotic shock, are characterized by a higher ratio of glycoprotein-NANA to gangliosidic NANA as compared to lighter, less dense particulate fractions that contain few synaptosomes. 6. 6. Synaptic vesicles do not contain glycoproteins or gangliosides. 7. 7. Myelin fragments were identified in P2B-a. Synaptosomes, with enclosed mitochondria, were identified in P2B-c, P2B-d, and P2B-e. Free mitochondria were noted in P2B-e. Tubular forms, tentatively identified as axons, were noted in P2B-b and P2B-c, with lesser numbers present in P2B-a and P2B-d.