Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects: antioxidant, enzyme inhibitory, anti-inflammatory properties and LC-ESI-MS profile.

Brachychiton populneus is one of the unexploited Tunisian plants, traditionally eaten as food and used for medicinal purposes. The present study aimed to investigate the phytochemical components of the seeds, leaves and flowers from B. populneus using three different solvents and to explore their antioxidant, anti-inflammatory and neuroprotective effects. Further, this study was focused on the identification of phenolic compounds from the most active extract. In vitro, all extracts showed strong antioxidant property by DPPH, ferrous ion chelating and lipid peroxidation-inhibiting assays, noticeable anti-inflammatory activity by protein denaturation and membrane stabilization methods and important neuroprotective effects by acetylcholinesterase inhibitory test. In vivo, B. populneus (50, 100 and 200 mg/kg, i.p.) showed significant dose-response anti-inflammatory effects against carrageenan-induced paw edema. With respect to the phenolic profile, the leaf methanol extract presented eight phenolic acids, one flavone and four flavonoids, with salvianolic acid B (820.3 mg/kg), caffeic acid (224.03 mg/kg), syringic acid (100.2 mg/kg) and trans-ferulic acid (60.02 mg/kg) as the major compounds. The results of the current study suggested that B. populneus could be a precious source of health-benefitting biomolecules and may be developed as new antioxidant, anti-inflammatory and AChE inhibitors.

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R.

PRX-105 is a plant-derived recombinant version of the human 'read-through' acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50nmol/kg PRX-105, 2min before being exposed to 1.33×LD50 and 1.5×LD50 of toxin and 10min after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t½) in mice was 994 (±173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t½ in humans was substantially longer than in mice (average 26.7h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation.

Cell membrane enveloped polymeric nanosponge for detoxification of chlorpyrifos poison: In vitro and in vivo studies.

Organophosphates are highly toxic compounds as they are involved in irreversible inhibition of acetylcholinesterase, causing various neurotoxic effects via acetylcholine accumulation throughout the nervous system. Traditional treatments for organophosphate poisoning are not effective enough to overcome all the toxic effects. There is a need for alternate treatment of life threatening poisoning of organophosphates. For this purpose a biomimetic nanosponge of poly (lactic-co-glycolic acid) is prepared, characterized and analysed as an antidote for organophosphate poisoning. In this nanosponge red blood cell membranes are used for coating poly lactic co-glycolic acid nanoparticles. In vitro studies are conducted to investigate the retention of acetylcholinesterase activity on the prepared nanosponge as well as to assess the scavenging ability of prepared nanosponge for model organophosphate, chlorpyrifos. In vivo studies are conducted to evaluate the detoxification potential of nanosponge in rabbit model, poisoned with chlorpyrifos. Hepatotoxicity and renal toxicity of nanosponge/chlorpyrifos complex is also studied in survived rabbits and the data is analysed statistically.

(II) Physiological profiling of an endogenous peptide in the basal forebrain: Age-related bioactivity and blockade with a novel modulator.

Previous studies have suggested that neurodegeneration is an aberrant form of development, mediated by a novel peptide from the C-terminus of acetylcholinesterase (AChE). Using voltage-sensitive dye imaging we have investigated the effects of a synthetic version of this peptide in the in vitro rat basal forebrain, a key site of degeneration in Alzheimer's disease. The brain slice preparation enables direct visualisation in real-time of sub-second meso-scale neuronal coalitions ('Neuronal Assemblies') that serve as a powerful index of brain functional activity. Here we show that (1) assemblies are site-specific in their activity profile with the cortex displaying a significantly more extensive network activity than the sub-cortical basal forebrain; (2) there is an age-dependency, in both cortical and sub-cortical sites, with the younger brain (p14 rats) exhibiting more conspicuous assemblies over space and time compared to their older counterparts (p35-40 rats). (3) AChE-derived peptide significantly modulates the dynamics of neuronal assemblies in the basal forebrain of the p14 rat with the degree of modulation negatively correlated with age, (4) the differential in assembly size with age parallels the level of endogenous peptide in the brain, which also declines with maturity, and (5) this effect is completely reversed by a cyclised variant of AChE-peptide, 'NBP14'. These observations are attributed to an enhanced calcium entry that, according to developmental stage, could be either trophic or toxic, and as such may provide insight into the basic neurodegenerative process as well as an eventual therapeutic intervention.

Liposomes are effective carriers for the ocular delivery of prophylactics.

Liposomes containing acetylcholinesterase were prepared by the freeze-drying method. The multilamellar morphology of the vesicles was revealed by freeze-fracture electron microscopy and their size distribution was determined by quasi-elastic light scattering. The vesicle diameters were in the range of about 0.2-4.0 micron. The liposome preparations were tested for their ocular delivery of an entrapped cholinesterase enzyme in counteracting the miotic effect of diisopropylfluorophosphate (DFP), a prototype of a family of organophosphate poisons. The topical application of the enzyme-containing liposomes to the rabbit eye was found to confer a significant level of protection against DFP-induced miosis. In comparing the prophylactic effectiveness of different enzyme-bearing liposomes, positively charged vesicles were found to be more effective than either neutral or negatively charged vesicles. Although the precise protective mechanism is not clear, our in vitro studies indicate that DFP molecules freely associate with liposomes and tear fluid promotes the release of liposome-entrapped enzymes. Thus, it is conceivable that the enzyme-liposome complex may act somewhat like a sponge by sequestering DFP molecules which diffuse into the vesicle, and also by releasing the entrapped enzyme to combine with DFP, thereby neutralizing its in vivo toxic effect.

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice.

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

Comparison of the behavioural effects of infusion of carbachol and acetylcholinesterase into the rat substantia nigra.

It has been postulated for many years that acetylcholinesterase (AChE) may play a nonclassical role in the substantia nigra, unrelated to its ability to hydrolyse acetylcholine. In this study the behavioural effects of unilateral infusion of AChE and a cholinergic agonist, carbachol, were compared. Carbachol induced ipsiversive circling over a very short time scale (minutes), whereas AChE induced contraversive circling, but over a longer time course-10 days. Both agents showed selectivity of response within the substantia nigra: acetylcholinesterase was only effective when infused into the most rostral region of the substantia nigra and its effects were limited to the pars compacta. In contrast, carbachol had effects in both the pars compacta and reticulata, with a graded sensitivity to carbachol in the rostral/caudal plane; infusions into rostral regions induced high rates of circling compared to more caudal areas, suggesting that the cholinergic input to the substantia nigra is not homogenous, but greater in rostral regions. This disparity between the effects of carbachol and AChE would, therefore, suggest that AChE is not exerting its long-term behavioural actions via a cholinergic mechanism, both in terms of time course of the response and the areas within the substantia nigra sensitive to these agents.

Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.

Vaccination against gastrointestinal nematodes of sheep using purified secretory acetylcholinesterase from Trichostrongylus colubriformis--an initial pilot study.

Purified secretory acetylcholinesterase (sAChE) from Trichostrongylus colubriformis was used as a candidate vaccine against mixed T. colubriformis, Haemonchus contortus and C. oncophora challenge infections of sheep. Cross species protection was achieved with an average reduction in worm burden of all species of 31%, rising to 58% in individual cases. There was no consistent reduction in faecal egg counts and increases in anti-T. colubriformis sAChE IgG antibody levels following vaccination were modest. We suggest that improved antigen delivery systems will result in increased host protection.

[Methods of treating experimental Neguvon poisoning and a comparison of their effects]. / Zpusoby lécení experimentální otravy Neguvonem a srovnání jejich úcinku.

The effect of conventional antidotal therapy of oral intoxication due to organophosphorus insecticide Neguvon in male mice is compared to the effect of peritoneal dialysis. As a dialysate, the combination of ACHE reactivator with atropine, distilled water and ACHE preparation has been used. In order to evaluate treatment results, the clinical course of intoxication and the increase of blood ACHE activity inhibited with perorally Neguvon were investigated. The highest increase in activity of inhibited blood ACHE has been stated by the peritoneal dialysis with ACHE preparation. Furthermore, the effects of two dialysates were compared. The first one consisted in ACHE reactivator with atropine, and the second one in ACHE preparation, respectively. The ACHE containing dialysate showed better therapeutical results. The mentioned acetylcholinesterase is stated to be inhibited through the treatment with 50% of Neguvon dosage applied to the experimental animals. Thus the actual dose of Neguvon inducing proper intoxication was reduced to its half amount.

Encapsulation of enzymes in liposomes: high encapsulation efficiency and control of substrate permeability.

Enzyme encapsulation into liposomes is a promising technique to stabilize and prevent them from denaturation and proteolysis. We demonstrate this using acetylcholinesterase which is the main target for pesticides. In order to achieve a reasonable encapsulation yield, we analyzed the parameters involved in each step of various encapsulation procedures. The only encapsulation method which did not denature the protein was the lipid film hydration technique, however the encapsulation efficiency was usually low. The efficiency could be increased up to more than 40% by induction of a specific interaction between the enzyme and the lipid surface. Once encapsulated, the enzyme encountered another problem: the permeability barrier of the lipid membrane drastically diminished the activity of the enzyme entrapped in the liposome by reducing the entrance rate of the substrate molecules and then reducing the substrate concentration inside the liposome. To solve this problem, we controlled the permeability of the liposome wall by reconstituting a porin from Escherichia coli. We succeeded to recover the full functionality of the enzyme, while retaining the protection against denaturation and proteolytic enzymes.

Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase.

The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.

Acetylcholinesterase inhibition: a novel approach in the treatment of neurogenic orthostatic hypotension.

BACKGROUND: Pharmacological treatment of orthostatic hypotension is often limited because of troublesome supine hypertension. OBJECTIVE: To investigate a novel approach to treatment using acetylcholinesterase inhibition, based on the theory that enhanced sympathetic ganglion transmission increases systemic resistance in proportion to orthostatic needs. DESIGN: Prospective open label single dose trial. MATERIAL: 15 patients with neurogenic orthostatic hypotension caused by: multiple system atrophy (n = 7), Parkinson's disease (n = 3), diabetic neuropathy (n = 1), amyloid neuropathy (n = 1), and idiopathic autonomic neuropathy (n = 3). METHODS: Heart rate, blood pressure, peripheral resistance index (PRI), cardiac index, stroke index, and end diastolic index were monitored continuously during supine rest and head up tilt before and one hour after an oral dose of 60 mg pyridostigmine. RESULTS: There was only a modest non-significant increase in supine blood pressure and PRI. In contrast, acetylcholinesterase inhibition significantly increased orthostatic blood pressure and PRI and reduced the fall in blood pressure during head up tilt. Orthostatic heart rate was reduced after the treatment. The improvement in orthostatic blood pressure was associated with a significant improvement in orthostatic symptoms. CONCLUSIONS: Acetylcholinesterase inhibition appears effective in the treatment of neurogenic orthostatic hypotension. Orthostatic symptoms and orthostatic blood pressure are improved, with only modest effects in the supine position. This novel approach may form an alternative or supplemental tool in the treatment of orthostatic hypotension, specially for patients with a high supine blood pressure.

Bromoacetylcholine and acetylcholinesterase introduced via liposomes into motor nerve endings block increases in quantal size.

We incorporated bromoacetylcholine (an inhibitor of choline acetyltransferase), acetylcholinesterase, or both into liposomes made of phosphatidylcholine. Frog sartorius muscles were exposed to these liposomes for 30-60 min. The liposome treatment itself did not decrease the size of the quanta compared to untreated controls. Then the preparations were exposed for 10-20 min to a hypertonic solution, which increases the rate of spontaneous quantal release and elicits an increase in the amount of acetylcholine released per quantum. Following the hypertonic treatment the quanta were significantly smaller in the liposome-treated preparations. Most of the difference occurred because in the preparations not exposed to the liposomes quantal size increased following the hypertonic treatment. This increase is thought to be due to the incorporation of more acetylcholine into each quantum. Our conclusions are that the treatments decreased the acetylcholine concentration in the cytoplasm, and that the increase in size occurs because additional acetylcholine is added to the vesicles containing the quanta from the cytoplasm.

Effect of diplodiatoxin (Stenocarpella maydis) on some enzymatic profiles in male and female rats.

Acute and subacute effects of diplodiatoxin were monitored with special reference to biochemical target enzymes like acid phosphatase (AcP), alkaline phosphatase (AkP), and acetylcholinesterase (AChE) in male and female rats. For acute toxicity study the rats were treated with single oral dose of 5.7 mg/kg of diplodiatoxin, whereas for subacute toxicity study the rats were orally treated with 0.27 mg/kg/day for 21 days. Diplodiatoxin caused loss in body weight and feed intake with other clinical symptoms. Due to the acute and subacute treatment of diplodiatoxin significant decreases were observed in serum AcP and AkP and also in liver AkP, whereas liver AcP increased in both male and female treated rats. Further, significant inhibition of brain AChE was observed in acute and subacute treated animals, indicating its effect on nerve synapsis. Sexual dimorphism was recorded when the activity of male rats was compared with female rats. The values were near those of controls on Day 7 (posttreatment), indicating recovery in the altered enzymes once the treatment was ceased. These results suggest that diplodiatoxin is toxic and has potential to affect the normal functioning of individuals and can cause changes in vital tissues such as liver.

Filogenia da sensibilidade da acetilcolinesterase cerebral de peixes ao metil-paraoxon como um possível marcador ambiental / Phylogeny of the sensitivity of fish brain acetylcholinesterase to methyl-paraoxon as a possible marker environmental

Todos os animais com células nervosas e musculares possuem a enzima acetilcolinesterase (EC 3.1.1.7, AChE) e essa seqüência de aminoácidos está presente em muitas outras proteínas, com ou sem atividade catalítica, fato que permite que essa proteína seja utilizada em estudos de filogenia e de evolução. A grande diferença de afinidade entre substratos e inibidores é utilizada como um biomarcador para estudos genéticos de vários grupos de animais, especialmente insetos. Peixes possuem uma grande diferença na sensibilidade da AChE ao metil-paraoxon (MP) em relação aos animais terrestres e essa diferença pode estar relacionada à evolução. As constantes cinéticas de inibição (CCI) para o mecanismo de inibição progressivamente irreversível da AChE cerebral ao MP foram determinadas em duas fontes de AChE como um modelo para avaliação do potencial de uso das CCI como biomarcador para estudos evolucionários e filogenéticos. As CCI da AChE cerebral de seis exemplares de tainha (Mugil liza), coletados em duas lagoas da costa do Estado do Rio de Janeiro (Araruama e Saquarema), em tempos diferentes (2005 e 2007, respectivamente), foram determinadas em dois laboratórios distintos (CESTEH - FIOCRUZ e Dept. Bioquímica - UERJ). As CCI, medidas separadamente em cada exemplar, indicaram que essas constantes são preservadas em todos os exemplares de uma mesma espécie e que a metodologia empregada pode ser conduzida em laboratórios distintos sem grandes variações. A AChE cerebral de tainha foi tomada como um exemplo de enzima menos sensível (IC50 = 2118nM) e a de galinha comercial (Gallus gallus domesticus) como um exemplo de uma enzima muito mais sensível ao MP (IC50 = 26nM)...

The enzyme acetylcholinesterase (EC 3.1.1.7, AChE) is present in all animalswith neurons and muscle cells, and many other proteins have the same sequence, with or without catalytic activity, allowing them to be used for phylogenetic and evolutionary studies. The great difference between substrates and inhibitor affinities makes it useful as a biomarker for genetic studies of various animals groups, especially insects. There are great differences among fish in AChE sensitivity to methyl-paraoxon (MP) in relation to terrestrial animals, and these differences can be related to evolution. The inhibition kinetic constants (IKC) for progressive irreversible inhibition of brain AChE with MP were determined in these two AChE sources as models for evaluating IKC as potential biomarkers in evolutionary and phylogenetic studies, especially among fish. IKC of brain AChE from six specimens of Mugil liza, a very common coastal fish, was collected from two of Brazil's lagoons in Rio de Janeiro State during 2005 at Araruama and 2007 at Saquarema. First samples were assayed at CESTEH - Fundação Oswaldo Cruz and the latter at Dept. Bioquímica - Universidade do Estado do Rio de Janeiro. The IKC was measured separately for each fish showing that these constants weremaintained for all animals of the same species and that this methodology can beused in different laboratories without variations. The cerebral AChE of tainha was used as an example of a less sensitive enzyme (Concentration which inhibits 50% of enzyme activity after 30 minutes of incubation, or IC50, = 2118nM). The commercial hen (Gallus gallus domesticus) was used an example of a very highly sensitiveenzyme to MP (IC50 = 26nM)...