Cholinesterase as an inflammatory marker of subclinical infection of dairy cows infected by Neospora caninum and risk factors for disease.

The aim of this study was to evaluate the seroprevalence of Neospora caninum in dairy cows, as well as to verify the risk factors for N. caninum infection and inflammatory response in dairy cows. Using the indirect immunofluorescence reaction, the seroprevalence of neosporosis was determined to be 32.82%. Based on regression analysis, the presence of dogs at a farm increased the probability of a cow testing positive for N. caninum (OR = 20.01 [5.21-123.12]). These data suggest that N. caninum has a relevant prevalence in dairy cows of the Microregion of Rio do Sul (Brazil), with elevated frequencies of anti-N. caninum IgG. The data also suggest that the parasite is widely distributed in dairy herds of the micro-region, because 94.4% of properties screened had at least one seropositive animal. The principal risk factors for disease maintenance in herds may be considered the presence of dogs and absence of a diagnostic test when introducing new animals. Blood from these cows was used to measure variables related to the inflammatory response. Serum cholinesterase activity, as well as serum levels of globulins and C-reactive protein were higher in seropositive to N. caninum than in seronegative cows. Furthermore, the infection by parasite causes an intense inflammatory process, contributing to disease pathophysiology.

Changes on the activity of cholinesterase's in an immunomodulatory response of cattle infected by Listeria monocytogenes.

The aim of this study was to evaluate whether Listeria monocytogenes infection alters the activity of cholinesterases in cattle to module their inflammatory response and neurotransmission. Thus, ten male bovines (Holstein) were divided into two groups of five animals each: uninfected (control) and L. monocytogenes infected. Blood samples were collected on days 0, 7 and 14 post-infection (PI) to evaluate the activity of acetylcholinesterase (AChE) in the blood, and seric butyrylcholinesterase (BChE) activity, as well as total protein, albumin, globulin and C-reactive protein (CPR) levels in serum. The AChE activity and acetylcholine (ACh) levels were measured in the central nervous system on day 14 PI, and histopathological analyses were also performed. The infected animals did not show apparent clinical signs of listeriosis, however histopathological alterations were seen in the intestines and spleens. On days 7 and 14 PI, AChE activity in the blood was lower in infected animals, as well the seric BChE activity on day 7 PI. In the cerebral cortex and cerebellum, AChE activity was lower in infected animals compared to the control group, while the ACh levels were higher in the cerebral cortex compared to uninfected animals. Moreover, seric levels of total protein, globulin and CRP were higher in infected animals on days 7 and 14 PI compared to the control group. Therefore, we concluded that acute infection by L. monocytogenes alters the cholinergic system through the reduction of cholinesterase enzymes in the blood, serum and cerebral tissues as an adaptive response to an anti-inflammatory effect in order to increase the ACh levels, an anti-inflammatory molecule with an important role in the host immunomodulation.

Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes.

The present study aimed to investigate the association of cholinesterase activity with trypanosomosis in buffaloes. Thirty-three clinical cases of trypanosomosis in water buffaloes, found positive for trypomastigotes of T. evansi on blood smear examination, were divided into two groups based on clinical manifestations. Twenty diseased buffaloes revealing only common clinical signs were allocated to Group I, while the remaining 13 buffaloes showing common clinical manifestations along with neurological disturbances were allocated to Group II. Twelve clinically healthy buffaloes, free from any haemoprotozoa infection, were kept as healthy control (Group III). Blood samples were collected from buffaloes of all three groups to determine serum cholinesterase activity. Compared to buffaloes of healthy control group, cholinesterase activity in T. evansi-infected buffaloes of Group I and II was significantly (P<0.001) lower. However, no significant difference was observed in cholinesterase activity between the T. evansi-infected buffaloes exhibiting neurological disorders and no neurological disorders. Summing up, reduced cholinesterase activity seems to be associated with the pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes possibly by evading immune response. Further studies are warranted on association of cholinesterase activity in T. evansi-infected buffaloes with neurological disorders.

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 µg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

Tick Salivary Cholinesterase: A Probable Immunomodulator of Host-parasite Interactions.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is the most economically important cattle ectoparasite in the world. Rhipicephalus microplus and Rhipicephalus annulatus (Say) continue to threaten U.S. cattle producers despite eradication and an importation barrier based on inspection, dipping of imported cattle in organophosphate (OP) acaricide, and quarantine of infested premises. OP acaricides inhibit acetylcholinesterase (AChE), essential to tick central nervous system function. Unlike vertebrates, ticks possess at least three genes encoding AChEs, differing in amino acid sequence and biochemical properties. Genomic analyses of R. microplus and the related tick, Ixodes scapularis, suggest that ticks contain many genes encoding different AChEs. This work is the first report of a salivary cholinesterase (ChE) activity in R. microplus, and discusses complexity of the cholinergic system in ticks and significance of tick salivary ChE at the tick-host interface. It further provides three hypotheses that the salivary ChE plausibly functions 1) to reduce presence of potentially toxic acetylcholine present in the large bloodmeal imbibed during rapid engorgement, 2) to modulate the immune response (innate and/or acquired) of the host to tick antigens, and 3) to influence transmission and establishment of pathogens within the host animal. Ticks are vectors for a greater number and variety of pathogens than any other parasite, and are second only to mosquitoes (owing to malaria) as vectors of serious human disease. Saliva-assisted transmission (SAT) of pathogens is well-known; however, the salivary components participating in the SAT process remain to be elucidated.

Myasthenia gravis Lambert-Eaton overlap syndrome.

INTRODUCTION: To assess whether a myasthenia gravis (MG) Lambert-Eaton overlap syndrome (MLOS) exists. METHODS: Case reports that met the universally accepted diagnostic criteria for MG and Lambert-Eaton myasthenic syndrome (LEMS) were sought through a PubMed search. Fifty-five possible cases of MLOS were identified. RESULTS: Thirty-nine cases met the diagnostic criteria for MG and LEMS. Analysis of clinical features showed that these patients have common MG and LEMS symptoms: oculo-bulbar paresis and good response to anti-cholinesterase for MG and limb weakness and decreased or absent reflexes for LEMS. All had the classical LEMS pattern in the repetitive nerve stimulation test: low compound muscle action potential amplitude and incremental response > 60% with brief exercise or at high rate of stimulation. Eight patients had combined positive acetylcholine receptor antibody (AChR-ab) or muscle-specific kinase-ab and voltage-gated calcium channel- ab tests. CONCLUSIONS: A myasthenia gravis Lambert-Eaton overlap syndrome (MLOS) does exist.

Specific oligobodies against human brain acetylcholinesterase.

In order to develop the specific oligobodies against human brain acetylcholinesterase (AChE) and distinguishes between human erythrocyte and brain AChEs, we applied the strategy of 'target switching' to obtain the specific polyclonal and monoclonal oligobodies. The specificity between human brain AChE and other ChEs was identified by Western blotting, dot blotting and enzyme protein binding assay (EPBA). The results showed that the oligobodies against the human brain AChE specifically immunoreacted with the human brain AChE and Torpedo AChE, not showing significant binding to AChE from human erythrocyte and butyrylcholinesterase (BChE) from human serum.

Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators.

Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.

Localization of a novel adhesion-promoting site on acetylcholinesterase using catalytic antiacetylcholinesterase antibodies displaying cholinesterase-like activity.

A monoclonal antibody (MAb) raised against human acetylcholinesterase was found to have catalytic activity. A similar phenomenon was observed in a polyclonal antibody raised against the same antigen. The antibodies were demonstrated to be pure, and no contamination with either acetylcholinesterase or butyrylcholinesterase was found. Both antibodies hydrolyzed acetylthiocholine, an acetylcholinesterase substrate, and the MAb followed Michaelis-Menten kinetics. Six other MAbs and one other polyclonal antibody showed no evidence of catalytic activity. Acetylcholinesterase is a key component in the transmission of the nerve impulse, and is also expressed nonsynaptically during embryonic development, and abnormalities in expression are seen in neural tumors and degenerative disorders. This unusual expression is believed to be associated with a novel function of the enzyme related to differentiation and cell adhesion. Autoantibodies to acetylcholinesterase have been observed in a variety of neurologic, muscular, and autoimmune disorders. In an investigation of the possible role of acetylcholinesterase in cell adhesion, we showed that the enzyme promoted neurite outgrowth in neuroblastoma cell lines, and conversely, that certain antiacetylcolinesterase antibodies abrogated cell-substrate adhesion. Interestingly, the antibodies most effective in this regard were catalytic. Preliminary epitope analysis indicated a conformational epitope in the N-terminal domain. This domain contains the active site within a deep gorge and the peripheral anionic site at the rim of the gorge. Peripheral-site inhibitors, but not active-site inhibitors, also interfered with adhesion, and competed with the catalytic monoclonal binding to acetylcholinesterase, indicating that the epitope recognized is associated with the peripheral anionic site. The inhibitor data also support the supposition that catalysis in these antibodies may have arisen from stable complexation of acetylcholinesterase with an inhibitor. We conclude that the catalytic antiacetylcholinesterase antibody interacts with structures associated with the peripheral anionic site, thus defining a novel site on the molecule involved in cell adhesion. This finding has implications for our understanding of the potential importance of this peripheral site in a variety of congenital, neoplastic, and degenerative conditions.

Cholinesterase-like catalytic antibodies: reaction with substrates and inhibitors.

We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.

Dimeric forms of cholinesterase in Sipunculus nudus.

In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low-salt (LSS), detergent (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium-Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130,000 and are likely G2 globular dimers of a 67,000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine. However, they also show a fairly high catalytic efficiency with other thiocholine esters as substrates, thus suggesting a wide and little-specialized conformation of the active site. Based on immunological cross-reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting forms and an enzyme-anchoring collagenous structure.

Characterization of cholinesterases from the parasitic nematode Trichinella spiralis.

1. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors. 2. The 5.3 and 13S forms identified in Trichinella extracts are also found in C. elegans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident. 3. The level of cholinesterases in excretory/secretory products is low. 4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.

A variant serum cholinesterase and a confirmed point mutation at Gly-365 to Arg found in a patient with liver cirrhosis.

A 64-year-old man was admitted to our hospital because of possible liver cirrhosis. His serum cholinesterase was anomalously low with a delta pH of 0.1 (normal range; 0.8-1.1). His enzyme was more heat-labile than the normal controls. Km value of his enzyme for benzoylcholine was 1.1 x 10(-5) mol/l, while that for normal controls was 2.3 x 10(-6) mol/l. In addition, isozymic alteration of his enzyme was observed. Sequencing of the white blood cell DNA of the patient showed a point mutation at nucleotide 1093 (GGA to CGA), which changes codon 365 from glycine to arginine.

Quantification and phenotyping of serum cholinesterase by enzyme antigen immunoassay: methodological aspects and clinical applicability.

An enzyme antigen immunoassay for a specific determination of serum cholinesterase is described. Polyclonal and monoclonal antibodies against cholinesterase have been used. Hydrophobic binding of the specific antibody to a microtitre plate was followed by incubation with the samples, and the activity of the bound cholinesterase was assayed by the Ellman method. The procedure has been optimized and characterized, with respect to antigen specificity, and the applicability of the assay for cholinesterase phenotyping is demonstrated. The cholinesterase activities, dibucaine-, scoline-, fluoride- and urea numbers were comparable with established reference values. The high sensitivity and specificity of the assay has been used for determination of cholinesterase in amniotic and cerebrospinal fluids, and its applicability in clinical medicine is indicated.

Immunological studies of families segregating the silent gene for plasma cholinesterase.

Seven families segregating apparently silent gene homozygotes have been investigated by enzyme-linked immunosorbent assays. These families confirm not only the heterogeneity of the silent gene in the form of E1s and E1t but in 3 families there is support for the segregation of the new gene E1x.

Immunoreactive plasma cholinesterase (EC 3.1.1.8) substance concentration, compared with cholinesterase activity concentration and albumin: inter- and intra-individual variations in a healthy population group.

Substance concentrations of plasma cholinesterase (EC 3.1.1.8) were measured in 94 healthy individuals without occupational exposure to known inhibitors (six samples from each individual). Immunoreactive cholinesterase substance concentrations showed an inter-individual variation corresponding to CVtotal = 22% (mean: 5.01 mg/l, SD: 1.11 mg/l). Intra-individual variations of immunoreactive cholinesterase substance concentration were correlated (r = 0.36) to intra-individual variation of albumin. Estimated by a repeated-measures analysis of variance, the observed intra-individual variation of cholinesterase substance concentration corresponded to CV = 8.8% (SD: 0.44 mg/l), which together with a CVerror = 6% (within and between runs), implies a biological intra-individual variation of cholinesterase substance concentration corresponding to CVintra = 6.4%. Specific catalytic activity (kU/mg immunoreactive cholinesterase) was influenced by the ChE-1 phenotype (phenotype U: 1.58 kU/mg, phenotype UA: 1.22 kU/mg), but not by body weight, height, age, and sex. Observed intra-individual variation of specific catalytic activity corresponded to 6.4% (SD: 0.10 kU/mg), which together with an estimated CVerror = 6.2% implies the biological intra-individual variations of specific catalytic cholinesterase activity to be insignificant. The insignificant CVintra makes specific catalytic cholinesterase activity a rational quantity for evaluation of unexpected fluctuations of cholinesterase activity concentrations.

Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases.

Monoclonal antibodies have served to characterize neurotactin, a novel Drosophila protein for which a role in cell adhesion is postulated. Neurotactin is a transmembrane protein, as indicated by epitope mapping and amino acid sequence. Similarly to other cell adhesion molecules, neurotactin accumulates in parts of the membrane where neurotactin-expressing cells contact each other. The protein is only detected during cell proliferation and differentiation, and it is found mainly in neural tissue and also in mesoderm and imaginal discs. Neurotactin has a large cytoplasmic domain rich in charged residues and an extracellular domain similar to cholinesterase that lacks the active site serine required for esterase activity. The extracellular domain also contains three copies of the tripeptide leucine-arginine-glutamate, a motif that forms the primary sequence of the adhesive site of vertebrate s-laminin.

Enzyme immunoassay of human cholinesterase (EC 3.1.1.8). Comparison of immunoreactive substance concentration with catalytic activity concentration in randomly selected serum samples from healthy individuals.

We developed an enzyme immunoassay (ELISA) for quantitation of plasma cholinesterase substance concentrations in native plasma or serum samples. The ELISA assay is based on polyclonal (rabbit) antihuman cholinesterase and a highly specific monoclonal (mouse) antibody, with a commercially available peroxidase-conjugated (rabbit) antibody directed against mouse immunoglobulins as the signal carrier. The detected serum cholinesterase substance concentrations (mean: 4.51 mg/l, SD: 0.90 mg/l) in randomly selected serum samples from 33 healthy individuals were closely and linearly related to the corresponding catalytic activity concentrations.