High Cholinesterase Activity Research Articles

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Plasma Cholinesterase Activity in Patients With Rheumatoid Arthritis and Toxoplasmosis.

Background and objective Rheumatoid arthritis (RA) is a chronic autoimmune disease causing synovium inflammation and functional impairment. Toxoplasmosis is an intracellular zoonotic parasitic infection anda risk factor in immunosuppressed diseases including RA. The involvement of the cholinergic mechanism is not clear when both diseases exist in combination. This study aimed to examine plasma cholinesterase (ChE) activity in patients suffering from RA with concomitant toxoplasmosis, taking into account the enzyme susceptibility to in vitro inhibitory challenge with the organophosphate dichlorvos in RA patients. Methods This was a case-control study involving 88 RA patients and 61 healthy controls of both genders.The RA patients were allocated into three groups. The first group received no therapy (n=14), the second group received conventional anti-arthritis therapy (n=49), and the third group received conventional+biologic therapy(n=25). Plasma ChE activity was determined by an electrometric method. Plasma samples were screened for Toxoplasmagondii (T. gondii)infection, using ELISAT. gondii antibodies IgG andIgM.In vitro inhibition of plasma ChE activitywas assessed by incubating the samples with dichlorvos at0.25 and 0.5 μM.The time-dependent dichlorvos (0.25 μM)-induced plasma ChE inhibition and its kinetics weredetermined. Results The RA patients comprised 76 (86.4%) females and 12 males (13.6%), whereas healthy controls included 22 (36.1%) females and 39 (63.9%) males. The rates of toxoplasmosis IgG positivity in controls and RA patients were 26.2% and 39.8%, respectively. Plasma ChE activity in patients with RA was significantly higher than that in the control group, by 16%. Plasma ChE values of RA patients with conventional therapy and conventional + biologic therapy were higher than that of the control group, by 18% and 27%, respectively. Odds and risk ratios of elevated plasma ChE activity (20%) in RA patients with therapyindicated that high plasma ChE activity among RA patients with therapyis a risk factor. The plasma ChE activity of T. gondii IgG-positive RA patients was not significantly different from that of theIgG-negative ones. Dichlorvos at 0.25 and 0.5 μM significantly inhibited in vitro plasma ChE activity incontrols and RA patients. The rates of plasma ChE inhibition by dichlorvos were lower in the RA groups with conventional therapyin comparison with those in the control group (77% vs. 91%). Examining the dichlorvos time-dependent ChE inhibition kinetics, RA groups showed increases in the half-life of inhibition by 23.6% to 32.7% and the total inhibition time by 23.5% to 32.5%, together with decreases in the inhibition rate constant by 19% to 24.5%, an indication of reduced inhibition rate of plasma ChE activity compared to that of the control group. Conclusions The autoimmune nature of RA and its chronicity might have contributed to the increase in plasma ChE activity among the patients. This increase in enzyme activity could be a risk factor in RA patients undergoing conventional therapy alone or in combination with biologic therapy; however, the clinical significance of such a condition remains obscure at present. The in vitro inhibition of plasma ChE activity in RA patients suggests reduced susceptibility of the enzyme to ChE inhibition by dichlorvos. Toxoplasmosis was not a risk factor when plasma ChE activity was taken into account among RA patients.

The mechanistic basis for the toxicity difference between juvenile rats and mice following exposure to the agricultural insecticide chlorpyrifos

At high exposure levels, organophosphorus insecticides (OPs) exert their toxicity in mammals through the inhibition of brain acetylcholinesterase (AChE) leading to the accumulation of acetylcholine in cholinergic synapses and hyperactivity of the nervous system. Currently, there is a concern that low-level exposure to OPs induces negative impacts in developing children and the chemical most linked to these issues is chlorpyrifos (CPF). Our laboratory has observed that a difference in the susceptibility to repeated exposure to CPF exists between juvenile mice and rats with respect to the inhibition of brain AChE. The basis for this difference is unknown but differences in the levels of the detoxification mechanisms could play a role. To investigate this, 10-day old rat and mice pups were exposed daily for 7 days to either corn oil or a range of dosages of CPF via oral gavage. Four hours following the last administration of CPF on day 16, brain, blood, and liver were collected. The inhibition of brain AChE activity was higher in juvenile rats as compared to juvenile mice. The levels of activity of the detoxification enzymes and the impact of CPF exposure on their activity were determined in the two species at this age. In blood and liver, the enzyme paraoxonase-1 (PON1) hydrolyzes the active metabolite of CPF (CPF-oxon), and the enzymes carboxylesterase (CES) and cholinesterase (ChE) act as alternative binding sites for CPF-oxon removing it from circulation and providing protection. Both species had similar levels of PON1 activity in the liver and serum. Mice had higher ChE activity in liver and serum than rats but, following CPF exposure, the percentage inhibition was similar between species at an equivalent dosage. Even though rats had slightly higher liver CES activity than mice, the level of inhibition following exposure was higher in rats. In serum, juvenile mice had an 8-fold higher CES activity than rats, and exposure to a CPF dosage that almost eliminated CES activity in rats only resulted in 22% inhibition in mice suggesting that the high serum CES activity in mice as compared to rats is a key component in this species difference. In addition, there was a species difference in the sensitivity of CES to inhibition by CPF-oxon with rats having a lower IC50 in both liver and serum as compared to mice. This greater enzyme sensitivity suggests that saturation of CES would occur more rapidly in juvenile rats than in mice, resulting in more CPF reaching the brain to inhibit AChE in rats.

Isoquinoline Alkaloid Contents in Macleaya cordata Extracts and Their Acetylcholinesterase and Butyrylcholinesterase Inhibition.

An important strategy for treating neurodegenerative disorders is to maintain the levels of acetylcholine in the synaptic cleft by blocking the cholinesterases. Searching for new effective compounds with inhibited acetylcholinesterase and butyrylcholinesterase activity is one of the most significant challenges of the modern scientific research. The aim of this study was the optimization of the condition for cholinesterase activity determination by high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) in terms of concentrations of enzymatic reaction mixture components, temperature of incubation, and incubation time. In vitro investigation of acetylcholinesterase and butyrylcholinesterase activity inhibition by some isoquinoline alkaloids and extracts obtained from the aerial part and roots of Macleaya cordata collected in May, July, and September. Acetylcholinesterase and butyrylcholinesterase activity inhibition of the extracts obtained from the plant had not been tested previously. The application of the HPLC method allowed eliminating absorption of interfering components, for example, alkaloids such as sanguinarine and berberine. The HPLC method was successfully applied for the evaluation of the acetylcholinesterase inhibitory activity in samples such as plant extracts, especially those containing colored components adsorbing at the same wavelength as the adsorption wavelength of 5-thio-2-nitro-benzoic acid, which is the product of the reaction between thiocholine (product of the hydrolysis of acetyl/butyrylthiocholine reaction) with Ellman’s reagent. Moreover, liquid chromatography coupled with a triple quadrupole mass spectrometer (LC–QqQ–ESI–MS/MS) analysis allowed evaluating the identification of relevant bioactive compounds in the obtained plant extracts. The investigated alkaloids, especially sanguinarine and chelerythrine, and all the Macleaya cordata extracts, especially the extract obtained from the aerial part collected in May, exhibited very high cholinesterase activity inhibition. HPLC-DAD was also applied for the kinetics study of the most active alkaloids sanguinarine and chelerythrine. Our investigations demonstrated that these plant extracts can be recommended for further in vivo experiments to confirm their cholinesterase inhibition activity.

Characterisation of cholinesterases in mucous secretions and their localisation in epidermis of Labeo rohita and Cirrhinus mrigala.

Cholinesterases are multifunctional enzymes and have been associated with diverse physiological functions in addition to their classical role at synapses. In the present study, cholinesterase (ChE) isozymes have been characterised in mucous secretions and their activity has been localised in the epidermis of Labeo rohita and Cirrhinus mrigala. Zymography using specific substrates and inhibitors revealed the presence of two ChE isozymes-ChE-1 and ChE-2. The isozyme ChE-1 was characterised as an atypical butyrylcholinesterase and ChE-2 as a typical acetylcholinesterase in skin mucous secretions of both the fish species. Enzyme histochemical analysis demonstrated the presence of ChE activity in the epidermis of the fish species investigated. In both the fish species, strong ChE activity was observed in the outer-layer epithelial cells, taste buds and neuromasts. The middle and basal layer epithelial cells showed moderate to weak ChE activity. Club cells and mucous goblet cells showed the absence of ChE activity. Characterisation with specific inhibitors indicates that acetylcholinesterase (AChE) was the major cholinesterase type expressed in the epidermis of the two fish species investigated. Immunohistochemical localisation of apoptotic and cell proliferation markers, in addition, revealed high expression of active caspase 3 in the outer-layer epithelial cells, and proliferating cell nuclear antigen (PCNA) in the middle and basal layer epithelial cells. High ChE activity in caspase 3-positive cells in the outer layer of the epidermis and low in PCNA-positive cells in middle and basal layers could point towards the possible involvement of ChEs in cell death and their final extrusion from skin surface.

Interplay of ROS and behavioral pattern in fluoride exposed Drosophila melanogaster

Reactive oxygen species (ROS) is known to be associated with the process of aging and other health hazards. Organisms are compelled to compromise with body homeostasis when exposed to toxic substances. In the present study sodium fluoride (NaF) exposure (10–100 μgmL−1) to Drosophila melanogaster in the parental (P) generation leads to increase in adult mortality and alteration in male-female ratio in the P and F1 (1st Filial) generation. Post-treatment alterations in selected behavioral traits (crawling, embedding and climbing) were observed in larvae and adults. Altered behavioral pattern was found to be associated with reduced mitochondrial activity and decreased number of viable brain cells in treated individuals. Interestingly, higher cholinesterase activities in treated males in comparison to females demonstrate a definite sex bias in NaF-induced response. Hyper-activation of antioxidant enzyme like catalase and reduced superoxide dismutase (SOD) and glutathione-s-transferase (GST) activity indicate a shift in the oxidative status after fluoride exposure. Additionally, increase in lipid peroxidation suggests enhancement in ROS which is further validated through increment in protein carbonyl content. Hence, the observations of the present study propose behavioral alterations resulting from increased ROS after chronic exposure to sub-lethal concentrations of NaF in D. melanogaster.

Aluminium sulfate exposure: A set of effects on hydrolases from brain, muscle and digestive tract of juvenile Nile tilapia (Oreochromis niloticus)

Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1μg·mL−1 (G1) and 3μg·mL−1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.

Neural influences on human intestinal epithelium in vitro.

We present the first systematic and, up to now, most comprehensive evaluation of the basic features of epithelial functions, such as basal and nerve-evoked secretion, as well as tissue resistance, in over 2200 surgical specimens of human small and large intestine. We found no evidence for impaired nerve-evoked epithelial secretion or tissue resistance with age or disease pathologies (stomach, pancreas or colon cancer, polyps, diverticulitis, stoma reversal). This indicates the validity of future studies on epithelial secretion or resistance that are based on data from a variety of surgical specimens. ACh mainly mediated nerve-evoked and basal secretion in the small intestine, whereas vasoactive intestinal peptide and nitric oxide were the primary pro-secretory transmitters in the large intestine. The results of the present study revealed novel insights into regional differences in nerve-mediated secretion in the human intestine and comprise the basis by which to more specifically target impaired epithelial functions in the diseased gut. Knowledge on basic features of epithelial functions in the human intestine is scarce. We used Ussing chamber techniques to record basal tissue resistance (R-basal) and short circuit currents (ISC; secretion) under basal conditions (ISC-basal) and after electrical field stimulation (ISC-EFS) of nerves in 2221 resectates from 435 patients. ISC-EFS was TTX-sensitive and of comparable magnitude in the small and large intestine. ISC-EFS or R-basal were not influenced by the patients' age, sex or disease pathologies (cancer, polyps, diverticulitis). Ion substitution, bumetanide or adenylate cyclase inhibition studies suggested that ISC-EFS depended on epithelial cAMP-driven chloride and bicarbonate secretion but not on amiloride-sensitive sodium absorption. Although atropine-sensitive cholinergic components prevailed for ISC-EFS of the duodenum, jejunum and ileum, PG97-269-sensitive [vasoactive intestinal peptide (VIP) receptor 1 antagonist] VIPergic together with L-NAME-sensitive nitrergic components dominated the ISC-EFS in colonic preparations. Differences in numbers of cholinergic or VIPergic neurons, sensitivity of epithelial muscarinic or VIP receptors, or stimulus frequency-dependent transmitter release were not responsible for the region-specific transmitter contribution to ISC-EFS. Instead, the low atropine-sensitivity of ISC-EFS in the colon was the result of high cholinesterase activity because neostigmine revealed cholinergic components. Colonic ISC-EFS remained unchanged after tachykinin, P2X, P2Y or A1 and A2 receptor blockade. R-basal was smaller and ISC-basal was higher in the small intestine. TTX and bumetanide decreased ISC-basal in all regions, suggesting nerve-dependent secretory tone. ISC-basal was atropine-sensitive in the small intestine and PG97-269-sensitive in the large intestine. This comprehensive study reveals novel insights into region-specific nerve-mediated secretion in the human small and large intestine.

Serum Total Cholinesterase Activity on Admission Is Associated with Disease Severity and Outcome in Patients with Traumatic Brain Injury.

BackgroundTraumatic brain injury (TBI) is one of the leading causes of neurological disability. In this retrospective study, serum total cholinesterase (ChE) activities were analyzed in 188 patients for diagnostic as well as predictive values for mortality.Methods and FindingsWithin 72 hours after injury, serum ChE activities including both acetylcholinesterase and butyrylcholinesterase were measured. Disease severity was evaluated with Acute Physiology and Chronic Health Evaluation (APACHE) II score, Glasgow Coma Score, length of coma, post-traumatic amnesia and injury feature. Neurocognitive and functional scores were assessed using clinical records. Of 188 patients, 146 (77.7%) survived and 42 (22.3%) died within 90 days. Lower ChE activities were noted in the non-survivors vs. survivors (5.94±2.19 vs. 7.04±2.16 kU/L, p=0.023), in septic vs. non-infected patients (5.93±1.89 vs. 7.31±2.45 kU/L, p=0.0005) and in patients with extremely severe injury vs. mild injury (6.3±1.98 vs. 7.57±2.48 kU/L, p=0.049). The trajectories of serum ChE levels were also different between non-survivors and survivors, septic and non-infected patients, mild and severely injured patients, respectively. Admission ChE activities were closely correlated with blood cell counts, neurocognitive and functional scores both on admission and at discharge. Receiver operating characteristic analysis showed that the area under the curve for ChE was inferior to that for either APACHE II or white blood cell (WBC) count. However, at the optimal cutoff value of 5 kU/L, the sensitivity of ChE for correct prediction of 90-day mortality was 65.5% and the specificity was 86.4%. Kaplan-Meier analysis showed that lower ChE activity (<5 kU/L) was more closely correlated with poor survival than higher ChE activity (>5 kU/L) (p=0.04). After adjusting for other variables, ChE was identified as a borderline independent predictor for mortality as analyzed by Binary logistic regression (P=0.078).ConclusionsLowered ChE activity measured on admission appears to be associated with disease severity and outcome for TBI patients.

Composites of silica with immobilized cholinesterase incorporated into polymeric shell.

Synthetic approaches for new nanocomposite materials with relatively high cholinesterase activity have been developed. The peculiarity of the formation of such systems is the introduction of cholinesterase into polymer with subsequent incorporation on the ready-made silica particles and into the polysiloxane matrixes during sol-gel synthesis. Evaluation of the cholinesterase activity has been fulfilled through the imitation of the acetylcholine chloride decomposition reaction. Values of activity for cholinesterase nanocomposites demonstrated in this work are higher than those for the native cholinesterase. The higher activity of cholinesterase contained in nanocomposites was found for those prepared using highly dispersed silica.

English

To investigate the incidence of pesticide poisoning using serum cholinesterase activity patterns in a horticultural farm, 616 people comprising of 496 pesticide handlers (test group) and a control group of 120 persons participated in the study. A semi-structured questionnaire was used to obtain demographic information, while the activities of serum cholinesterase, serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase and bilirubin were estimated using standard commercial kits and absorbance measured using kinetic colorimetric tests. All the pesticide handlers (100%) were males, with majority (80.3%) aged 20 to 35 years old. Of the test population, 6% had significant cholinesterase enzyme depressions with no symptoms of exposure recorded. Significant difference (p 0.05) between the test and control groups based on years of handling pesticides. Use of pesticides in successive spray seasons significantly inhibited cholinesterase activity among the spray team, supervisors and harvesters (p<0.05). Higher cholinesterase activity was observed in the 31 to 40 age group with significant changes in cholinesterase activity (p<0.05) observed among those aged below 40 years. The study indicates that cholinesterase activity can be used effectively as an indicator of exposure to pesticides. Key words: Cholinesterase activity, pesticide handlers, Naivasha, monitoring program.

Effect of soil contamination with petroleum products on esterase activity in earthworm Lumbricus terrestris

Among biomarkers of exposure the suitability of esterases especially carboxylesterases (CarE), metabolizing wide array of xenobiotics being important for organismal functioning, was very often underlined. Moreover, neurotoxic effects of constituents of petroleum products may be exert by inhibition of cholinesterase (ChE) isozymes in animals. Here, we examined esterase activity toward acetylthiocholine iodide and p-nitrophenyl acetate in earthworms Lumbricus terrestris reared through four weeks on loamy sand soil contaminated with petroleum products (unleaded petrol, used engine oil), in two weeks intervals. Such contamination was simulated in one m volume soil containers, at a dose of 6 g kg of dry soil (experimental field in Mydlniki, Krakow suburb, Poland; 50.0815N, 19.84730E). We compared the effects of these contaminants action in soil collected in the year of treatment and one year later. We evaluated also effects of animals rearing on soil unremediated and remediated with “cocktail” of microorganism, and also checked the enzymatic activities response in two weeks intervals. We found lower ChE activity in animals reared on petroleum derivatives contaminated soil when compared with control animals, but only in the case of four weeks rearing on soil collected in the year of treatment. Similar response of ChE in animals reared on soil collected a year after the treatment, but estimated in earlier period of time – after two weeks exposure to petrol unleaded contamination was observed. CarE activity characterized higher variability with time than ChE activity. Generally, the activity of CarE lowered from the beginning to the end of the rearing (in all experimental groups). Animals reared on remediated soil, in most cases, had higher ChE activity than those reared on unremediated soil (control and diesel oil exposed animals).

In vivo studies on inhibition and recovery of B-esterase activities in Biomphalaria glabrata exposed to azinphos-methyl: Analysis of enzyme, substrate and tissue dependence

Cholinesterases and carboxylesterases belong to the group of B-esterases, the serine superfamily of esterases that are inhibited by organophosphorus compounds. It is now generally accepted that before using the B-esterases as biomarkers of exposure to organophosphorus and carbamates in a given species, the biochemical characteristics of these enzymes should be carefully studied. In this study, the enzyme/s and the tissue/s to be selected as sensitive biomarkers of organophosphorus exposition in the freshwater gastropod Biomphalaria glabrata were investigated. Firstly, the substrate dependence of cholinesterase and carboxylesterase activities in whole organism soft tissue and in different tissues of the snail (head-foot, pulmonary region, digestive gland, and gonads) was analyzed. Measurements of cholinesterase activity were performed using three substrates: acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), and butyrylthiocholine (BuSCh). Carboxylesterase activity was determined using four different substrates: 1-naphthyl acetate (1-NA), 2-naphthyl acetate (2-NA), p-nitrophenyl acetate (p-NPA), and p-nitrophenyl butyrate (p-NPB). Regardless of the tissue analyzed, the highest specific activity was obtained when using AcSCh, followed by PrSCh. Cholinesterase activity measured with BuSCh was very low in all cases. On the other hand, the highest cholinesterase activity was measured in head-foot and in pulmonary region, representing in the case of AcSCh hydrolysis 196% and 180% of the activity measured in whole organism soft tissue, respectively. In contrast, AcSCh hydrolysis in digestive gland and gonads was 28% and 50% of that measured in whole organism soft tissue. Regarding carboxylesterase activity, although all tissues hydrolyzed the four substrates assayed, substrate preferences varied among tissues. In particular, digestive glands showed higher carboxylesterase activity than the other tissues (299%, 359% and 137% of whole organism soft tissue activity) when measured with 1-NA, 2-NA and p-NPA as substrates, respectively. In contrast, with p-NPB as substrate, the highest carboxylesterase activity was observed in pulmonary region. Exposure of the snails for 48h to azinphos-methyl concentrations in the range of 0.05–2.5mgL−1 resulted in different degrees of inhibition of cholinesterase and carboxylesterase activities, depending on the enzyme, pesticide concentration, the substrate, and the tissue analyzed. In general, carboxylesterase activity measured with p-NPA and p-NPB was much more sensitive to azinphos-methyl inhibition than cholinesterase activity. The results also showed that while B-esterase activities in whole organism soft tissue and pulmonary region recovered completely within 14 days, carboxylesterase activity in digestive glands remained highly inhibited. On the whole, the results of the present study emphasize how important it is to characterize and measure cholinesterase and carboxylesterase activities jointly to make a proper assessment of the impact of organophosphorus pesticides in non-target species.

Health Indices of the Green Turtle (Chelonia mydas) Along the Pacific Coast of Baja California Sur, Mexico. I. Blood Biochemistry Values

Abstract The green sea turtle (also known locally as black turtle; Chelonia mydas) is 1 of 5 species of sea turtles found along the coast of the state of Baja California Sur, Mexico, where several bays and lagoon systems provide an important growth and feeding habitat. We characterized blood biochemistry values of 67 green turtles captured at 2 mangrove estuaries along the Pacific coast of the Baja California Peninsula, Mexico, from 2005 to 2007. Blood samples were collected from live turtles for biochemical analysis of 18 parameters and analyzed by physical state (healthy, injured), size classes, season, and geographic location. Green turtles showed differences in the variability of the biochemical parameters between the 2 sites. In Punta Abreojos, injured sea turtles had lower calcium (28%), potassium (28%), and inorganic phosphorus (34.5%) levels and higher cholinesterase activity (16%) compared to healthy turtles. Juvenile turtles collected in Bahia Magdalena had higher glucose levels (34%) than subad...

Muscular cholinesterase activities and lipid peroxidation levels as biomarkers in several Mediterranean marine fish species and their relationship with ecological variables

Muscular cholinesterase activities, as potential markers of neurotoxic exposure, and lipid peroxidation levels, indicative of oxidative stress damage, both currently used in early-warning pollution monitoring, were characterised in eighteen fish species of ecologic and/or economic importance. These species comprise five orders and eleven families of teleosts and two species of elasmobranchs, feed using different strategies (benthic, epibenthic, endobenthic and pelagic), belong to different trophic levels and express different swimming behaviour. Their habitat ranges from 50 to 60 m (shallow or continental shelf) and 600 to 850 m (middle continental slope). Sampling took place in front of the Barcelona coast (NW Mediterranean) during four seasonal cruises in 2007. In the summer sampling, another site potentially exposed to a different pollution load (Vilanova) was included for comparison. Species, seasonal and site differences were tested and discussed in relation to chemical analysis of the local sediment, systematic position, habitat depth, feeding strategy, trophic level and swimming activity. Greater inter species differences rather than seasonal or site trends were seen in accordance to little pollution fluctuations. Higher cholinesterase activities were recorded in suprabenthos feeders, regardless of depth habitat, whereas LP levels were similar in all species except for the shark Scyliorhinus canicula in which they were consistently elevated. This study confirms and broadens former observations carried out with a more reduced number of fish species (Solé et al., 2008a).

In vitro characterization of cholinesterases in the earthworm Eisenia andrei

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min − 1 mg protein − 1 , respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min − 1 mg protein − 1 , but the time course did not reach a plateau under our experimental conditions. K m values were 0.142 ± 0.006 and 0.183 ± 0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC 50 values were 1.44 × 10 − 8 and 1.20 × 10 − 8 M, respectively) and by carbaryl (IC 50 values of 5.75 × 10 − 9 and 4.79 × 10 − 9 M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE ( iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.

Mixed Nerve Suture Facilitated by Enzyme-Staining Techniques

Identification of motor and sensory fascicles to correctly match the motor and sensory fascicles helps surgeons to correctly align nerve stumps. Motor fibers, with their high cholinesterase activity, can be differentiated from sensory fibers. I developed a new modification that requires less than 1 hour and shows clearer differentiation compared with other histochemical techniques. In my clinical research, proximal stumps can be stained as long as 16 months after injury, however, distal stumps cannot be stained after 5 days. After 5 days, this staining could provide useful proximal information and distal fascicles may be identified by anatomic or topographical knowledge. I think this staining is especially useful in proximal nerve lesions and nerve injuries that require nerve graft within 4 days after injury. Also, this technique is useful in functioning muscle transfer to find motor fascicles and, in a sensate flap, to identify the sensory fascicle.

Effects of Diazinon Exposure on Cholinesterase Activity in Different Tissues of European Eel (Anguilla anguilla)

Cholinesterase (ChE) activity was measured in brain, plasma, and whole eye ofAnguilla anguillaexperimentally exposed to a sublethal concentration of 0.042 mg/liter (0.50 of the 96-hr LC50) of the organophosphorous pesticide diazinon. Whole eye was the tissue which revealed higher values of ChE activity (8.17 μmol/min/g) in nonexposed animals. Brain, plasma, and whole eye ChE activity ofA. anguillawas inhibited at 6, 24, 48, 72, and 96 hr of diazinon exposure. Pesticide induced significant inhibitory effects on the ChE activity of this species ranging from >70% inhibition in brain tissue to >90% in plasma samples. Brain and plasma presented technical difficulties in their collection. Due to its ease of collection, high ChE activity levels, and sensitivity to ChE pesticide inhibition, the use of whole eye is recommended for post-mortem ChE analysis inA. anguilla.

Brain regional distribution of physostigmine and its relation to cerebral blood flow following intravenous administration in rats

3H-labeled physostigmine (50 micrograms.kg-1) was administered intravenously to rats, and its concentration in brain tissue and spinal cord was assessed by quantitative autoradiography. Regional cerebral blood flow (rCBF) was measured with iodo-14C-antipyrine autoradiography in control rats and in animals injected i.v. with a dose of physostigmine similar to that used for the distribution studies. Tissue concentration of 3H-physostigmine was correlated with rCBF for 37 brain regions. A high degree of correlation was found at 0.5 min after drug injection, r (correlation coefficient) = 0.87. This association decreased at later times (5 min r = 0.73, 12 min r = 0.24). Structures with high cholinesterase activity (caudate-putamen, amygdala, hippocampus) showed greater retention of physostigmine over time. The highest initial physostigmine concentrations were found in regions lacking a blood-brain barrier (pineal bland, median eminence, choroid plexus) (range = 10.4-23.8 nCi/mg) and the lowest in white matter (corpus callosum, internal capsule, hippocampus commisure, spinal cord dorsal column) (range = 1.2-2.6 nCi/mg). Initial concentrations of the drug in the areas in which physostigmine induced vasodilatation (motor, sensory, temporal and occipital cortex, claustrum, and superior collicullus) were not different from concentrations in areas of comparable basal rCBF in which no such effect was observed. Variations in drug access to brain regions, then, do not explain the topographical variations of the cerebrovascular action of physostigmine.

Acetylcholine in neurons andf paraneurons: A histochemical study.

Several years ago we proposed a method for the localization of acetylcholine in the cholinergic nerve terminals. The method is based on a rapid precipitation of quarternary ammonium cations (such as acetylcholine or choline) by molybdic or tungstic heteropolyanions (such as phosphomolybdic acid, phosphotungstic acid and silicotungstic acid). The insoluble salts formed can be directly visualized under the electron microscope. In the synaptic vesicles, acetylcholine was localized as point-like precipitates, while the membranes were well preserved. Since these fixations are based on a rapid ionic interaction, the term "ionic fixation" was proposed. The ionic fixation performed on motor end-plates in various physiological conditions, provided different forms of cytochemical precipitates of acetylcholine (point-like, spot-like, diffuse or laminar). In contrast with the numerous physiological and neurochemical investigations into the acetylcholine mechanism of neurons, very little is known about paraneurons. The historical evolution of paraneuron research, more or less bound to that of the sympathetic paraganglion and of the APUD cells may explain this lack of results. In certain physiological conditions, acetylcholine can be revealed in paraneurons and seems to reflect a plasticity of the cells, as already observed in sympathetic ganglion cells. This paper further overviews the cholinergic mechanisms of exocrine secretory cells. The presence of a high cholinesterase activity exist in most of these exocrine cells, as far as we could verify. In addition to cholinesterase activity, the presence of acetylcholine, probably related to exocrine cell metabolism, was investigated with our histochemical method.