Eliseo O. Campos

Blood markers in Alzheimer disease: Subnormal acetylcholinesterase and butyrylcholinesterase in lymphocytes and erythrocytes

In patients with the clinical diagnosis of Alzheimer disease (AD), we searched for systemic changes in components of the blood as a diagnostic tool. The acetylcholine-related enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were measured in plasma, erythrocytes, platelets and lymphocytes. Results did not show a general effect; notwithstanding, specific cell types presented alterations either in AChE or BuChE but not in both enzymatic activities. In AD patients, AChE of lymphocytes was reduced by 60% compared with the age-matched controls. However, when patients were divided, the sporadic but not the familial subgroup exhibited a significant reduction. In erythrocytes the BuChE activity was reduced by 45% in sporadic AD. The molecular forms of the lymphocyte AChE were characterized by velocity sedimentation. Both globular forms were subnormal, more so the tetrameric G4 AChE form than the G2 form.

Brain acetylcholinesterase promotes amyloid-β-peptide aggregation but does not hydrolyze amyloid precursor protein peptides

It has been suggested that acetylcholinesterase (AChE) has both a putative proteolytic activity against the amyloid precursor protein (APP), and a capacity to accelerate the assembly of amyloid-β-peptide (Aβ) into Alzheimers fibrils. Here, we have studied the ability of bovine brain AChE to share both activities. Results indicate that AChE purified through acridinium was able to process the APP peptides, however after further purification by an edrophonium column, the protease activity was lost. Under both conditions the capacity of the enzyme to promote amyloid formation was maintained. Kinetic studies of the Aβ aggregation process using edrophonium-AChE, indicated that the lag phase of the aggregation process was smaller than the one observed with the esterase purified by acridinium alone. Considering that the total amount of amyloid formed, measured by thioflavine-T fluorescence, was similar for both AChE preparations, our results suggest that the edrophonium-AChE possesses an higher intrinsic capacity to stimulate the aggregation of Aβ1–40 peptide.

Behavioral Responses of Concholepas concholepas (Bruguière, 1789) Larvae to Natural and Artificial Settlement Cues and Microbial Films

The behavioral responses of veliger larvae of the gastropod Concholepas concholepas were studied in the presence of different natural and artificial settlement cues and microbial films. Early pre-competent larvae stopped swimming, sank (due to ciliary arrests, retraction of the velum into the shell, or both), and remained inactive on the substratum when exposed to conspecific mucus and hemolymph. In both cases the effect was time-dependent and the number of larvae showing these behaviors decreased over time. Larvae exposed to NH4Cl (ammonium ion) showed a similar time- and dose-dependent response. A positive and time-dependent response was also observed when larvae were exposed to different extracellular matrix (ECM) components (i.e., collagen, gelatin, and fibronectin) and sulfated polysaccharides (i.e., carrageenan, heparin, and chondroitin sulfate). In this case the larvae remained attached to the substratum. However, the effect of sulfated polysaccharides on C. concholepas larval behavior was faster than that observed with other ECM molecules. We also studied the responses of premetamorphic C. concholepas larvae exposed to different microbial films. In chemotaxis experiments with different films, with glass as the substratum, larvae showed a significant preference for multispecific and diatoms films. When shells of C. concholepas were used as the substratum, the preference for multispecific films was clear and significant. Likewise, larvae showed velar contractions in the presence of all the films tested. Larvae exposed to multispecific films and to the microalga Prasinocladus marinus showed an increased ciliar movement. The finding that mucus and hemolymph of conspecific adults and ECM molecules (mainly sulfated polysaccharides) induce the cessation of swimming of C. concholepas larvae suggests a possible role for cell-surface receptors in mediating the larval response of marine organisms. Likewise, the positive chemotaxis responses of C. concholepas larvae to different microbial films suggest that microorganisms may have a role in bringing larvae close to settlement inducers on the marine benthos.

Monoamines and the release of gametes by the scallop Argopecten purpuratus

Levels of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were measured fluorometrically in the ganglia, gonads and adductor muscle of the functionally hermaphroditic scallop Argopecten purpuratus at different times during the induced release of gametes. The levels of these amines were analyzed in samples before stimulation, when animals looked stimulated, during the release of sperm, during the release of oocytes, and 24 hours after the end of spawning. Levels in muscles of the three monoamines changed during spawning and the greatest change was detected in the level of NA. In nervous tissue, the level of 5-HT decreased, whereas those of DA and NA increased significantly with the release of sperm. In both the female and male portions of gonad, levels of DA and NA increased during spawning while those of 5-HT decreased. However, levels of 5-T in the female portion of gonads did not show any changes in a group of scallops that did not release sperm before releasing oocytes. Separate analysis of levels of monoamines in the visceral ganglion from those in the cerebropedal ganglion, showed that, in spawning animals, levels of DA and ANA did not change in cerebropedal ganglia but decreased in the visceral ganglia, whereas levels of 5-HT decreased only n the cerebropedal complex. The results are consistent with the existence of two different mechanisms for the control of the release of sperm ad oocytes by hermaphroditic pectinids.

Developmental regulation of mouse brain monomeric acetylcholinesterase

Acetylcholinesterase (AChE) molecular forms were studied during mouse brain development. Mouse embryos expressed a monomeric (G1) and a tetrameric (G4) AChE form. Our results indicate that G4 AChE expressed at embryonic day (ED) 9 and ED15 could be purified by acridinium‐Sepharose chromatography and shared similar biochemical and kinetic properties with the adult form. However, the G1 form expressed at either embryonic stage did not bind to acridinium, was not inhibited by excess substrate, and possessed higher Km and lower Vmax values than the adult G1 form. Two peripheral anionic binding site inhibitors, fasciculin and propidium, had a significantly lower affinity for the monomeric form at ED9. Results are discussed in terms of the biological significance of the embryonic G1 form, and its resemblance to the AChE activity found, associated with the senile plaques present in the brains of Alzheimers patients.

Acetylcholinesterase changes in hearts with sinus rhythm and atrial fibrillation

1. Clinical and experimental evidence suggest that changes in the autonomic tone play a role in the pathogenesis of atrial fibrillation. 2. We have studied the distribution of molecular forms of acetylcholinesterase (AChE) in atrial biopsies obtained from individuals without arrhythmias and in patients with atrial fibrillation. 3. Analysis of the distribution of globular and asymmetric AChE forms, showed a decrease in the amount of the globular forms of biopsies taken from atria during fibrillation. 4. This study is the first attempt to characterize the molecular forms of AChE in the human heart of patients with sinus rhythm and chronic atrial fibrillation.

Localization, specific activity, and molecular forms of acetylcholinesterase in developmental stages of the cestode Mesocestoides corti

The nervous system of flatworms is quite simple although there is increasing evidence indicating that it is chemically complex. Studies of the nervous system in these animals have only been performed in the larval stage or in the adult worms, which are easy to obtain in nature, while the description of the nervous system in developing stages of these organisms is missing. Mesocestoides corti is a parasitic platyhelminth whose larvae can be induced in vitro to develop to adult, sexually mature worms, opening the possibility of studying the nervous system of a flatworm in different stages of development. Here, we describe the presence, activity, location, and molecular forms of acetylcholinesterase (AChE) in different stages of development of M. corti, from the larvae to adult forms of this endoparasite, obtained in in vitro cultures after induction of the larval stage with trypsin. Our results point to AChE as a molecular marker of the nervous system in platyhelminthes. The change in molecular forms of this enzyme and the increase in its activity during development from larvae to adult worm may reflect the presence of a more complex nervous system, necessary to adjust and coordinate the movement of a much bigger structure. A relationship between the development of the reproductive apparatus in segmented and adult worms with a more complex nervous system in these stages is also apparent. Finally, our study opens the possibility of applying anti‐AChE as more effective therapeutic strategies against cestode parasites. J. Cell. Physiol. 206: 503–509, 2006.

Metamorphosis of laboratory-reared larvae of Concholepas concholepas (Mollusca; Gastropoda)

Abstract Metamorphosis of laboratory-reared larvae of the gastropod Concholepas concholepas was observed under the influence of K+ ions. After 64 h of incubation at 10°C, 58% of larvae metamorphosed in response to 20 mM K+ final concentration in seawater, 11 mM above ambient. No effect was observed at 15 mM K+. Likewise, larvae were unable to survive when exposed to 25 mM K+. Metamorphosed individuals of C. concholepas migrated to the water-air interface within 28 h of their metamorphosis. This study reports the first successful experiment on the induction of metamorphosis in laboratory-reared larvae of C. concholepas. The advantages of mass production of larvae with metamorphic capacity of this economically important resource, related to its eventual culture on a pilot scale are discussed.

Molecular changes induced by metamorphosis in larvae of the prosobranch Concholepas concholepas Bruguiere (Mollusca; Gastropoda; Muricidae)

Abstract Metamorphosis may be the most critical point in the life history of marine molluscs. Excess potassium was used to trigger the metamorphic process in competent larvae of the prosobranch Concholepas concholepas Bruguiere. The specific, irreversible stimulation of complex morphogenetic events by this exogenous factor was used to study some molecular changes that occur during metamorphosis. Results show that metamorphosis entails several molecular changes, including: (1) a modification in the pattern of protein synthesis measured by incorporation of [35S]methionine to newly synthetized polypeptides, (2) an increase of [35S]methionine incorporation in heparin-binding proteins or the induction of heparin-binding proteins (i.e. growth factors?), (3) a decrease (20 times) in the larval levels of the second messenger cyclic AMP, and (4) the appearance of a new form of the neurotransmitter-related enzyme, acetylcholinesterase (AChE). To our knowledge this is the first attempt to characterize some of the molecular changes that take place during molluscan metamorphosis.

Characterization of acetylcholinesterase from human heart auricles: Evidence for the presence of a G-form sensitive to phosphatidylinositol-specific phospholipase c

1. Acetylcholinesterase (AChE) is an important enzyme of the cholinergic system in mammals. 2. We report here the subcellular association of the AChE molecular forms in the normal human heart auricle. 3. Both globular (G) and asymmetric (A) forms were identified using velocity sedimentation and sequential extraction procedures. 4. G forms corresponds to 84% and A forms account for 16% of the total AChE activity. 5. Of G forms 64% of AChE activity correspond to the G1 monomer and of the A forms the class I-A account for 80% of AChE activity. 6. In addition, treatment of the cardiac membranes with the enzyme phosphatidylinositol-specific phospholipase c (PIPLC) results in the solubilization of AChE activity. 7. This means that a G2 AChE dimer with a glycolipid anchoring domain is present in the human heart.