Production of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures in a pilot-scale bioreactor.

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low-cost production, although low yields remain the major challenge. The studies on scaling-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40-L pilot-scale STB. Employing cyclical operation together with a simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46-58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kL a) in the pilot-scale STB to be equivalent to the lab-scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L-1 day-1 , respectively, which are comparable to the lab-scale culture. Here, we demonstrate pilot-scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long-term, reproducible, and sustained production of rrBChE.

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor.

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)-1 or 7.5 ± 0.4 mg L-1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study.

Differential Expression of Cholinergic System Components in Human Induced Pluripotent Stem Cells, Bone Marrow-Derived Multipotent Stromal Cells, and Induced Pluripotent Stem Cell-Derived Multipotent Stromal Cells.

The components of the cholinergic system are evolutionary very old and conserved molecules that are expressed in typical spatiotemporal patterns. They are involved in signaling in the nervous system, whereas their functions in nonneuronal tissues are hardly understood. Stem cells present an attractive cellular system to address functional issues. This study therefore compared human induced pluripotent stem cells (iPSCs; from cord blood endothelial cells), mesenchymal stromal cells derived from iPSCs (iPSC-MSCs), and bone marrow-derived MSCs (BM-MSCs) from up to 33 different human donors with respect to gene expressions of components of the cholinergic system. The status of cells was identified and characterized by the detection of cell surface antigens using flow cytometry. Acetylcholinesterase expression in iPSCs declined during their differentiation into MSCs and was comparably low in BM-MSCs. Butyrylcholinesterase was present in iPSCs, increased upon transition from the three-dimensional embryoid body phase into monolayer culture, and declined upon further differentiation into iPSC-MSCs. In BM-MSCs a notable butyrylcholinesterase expression could be detected in only four donors, but was elusive in other patient-derived samples. Different nicotinic acetylcholine receptor subunits were preferentially expressed in iPSCs and during early differentiation into iPSC-MSCs, low expression was detected in iPS-MSCs and in BM-MSCs. The m2 and m3 variants of muscarinic acetylcholine receptors were detected in all stem cell populations. In BM-MSCs, these gene expressions varied between donors. Together, these data reveal the differential expression of cholinergic signaling system components in stem cells from specific sources and suggest the utility of our approach to establish informative biomarkers.

[Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).

Immunohistochemical analysis of hippocampal butyrylcholinesterase: Implications for regional vulnerability in Alzheimer's disease.

Studies of acetylcholine degrading enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in Alzheimer's disease (AD) have suggested their potential role in the development of fibrillar amyloid-ß (Aß) plaques (amyloid plaques). A recent genome-wide association study analysis identified a novel association between genetic variations in the BCHE locus and amyloid burden. We studied BChE immunoreactivity in hippocampal tissue sections from AD and control cases, and examined its relationship with amyloid plaques, neurofibrillary tangles (NFT), dystrophic neurites (DN) and neuropil threads (NT). Compared to controls, AD cases had greater BChE immunoreactivity in hippocampal neurons and neuropils in CA2/3, but not in the CA1, CA4 and dentate gyrus. The majority of amyloid plaques (> 80%, using a pan-amyloid marker X-34) contained discrete neuritic clusters which were dual-labeled with antibodies against BChE and phosphorylated tau (clone AT8). There was no association between overall regional BChE immunoreaction intensity and amyloid plaque burden. In contrast to previous reports, BChE was localized in only a fraction (~10%) of classic NFT (positive for X-34). A similar proportion of BChE-immunoreactive pyramidal cells were AT8 immunoreactive. Greater NFT and DN loads were associated with greater BChE immunoreaction intensity in CA2/3, but not in CA1, CA4 and dentate gyrus. Our results demonstrate that in AD hippocampus, BChE accumulates in neurons and plaque-associated neuritic clusters, but only in a small proportion of NFT. The association between greater neurofibrillary pathology burden and markedly increased BChE immunoreactivity, observed selectively in CA2/3 region, could reflect a novel compensatory mechanism. Since CA2/3 is generally considered more resistant to AD pathology, BChE upregulation could impact the cholinergic modulation of glutamate neurotransmission to prevent/reduce neuronal excitotoxicity in AD hippocampus.

Cholinesterase of rats experimentally infected by Cryptococcus neoformans: Relationship between inflammatory response and pathological findings.

The aim of this study was to assess the role of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) as biomarkers of inflammation and tissue injury on rats experimentally infected by Cryptococcus neoformans. For this purpose, 20 male rats were divided into two groups: 10 animals representing the uninfected control group (Group A) and 10 C. neoformans var. grubii infected animals (Group B). Blood and brain samples were collected on days 10 (A10 and B10), and 30 (A30 and B30) post-infection (PI) for hematological analyses; AChE (in lymphocytes and brain) and seric BChE activity; interleukins (IL-1, IL-6, and IL-10); nitrite/nitrate (NOx) levels; and markers of protein oxidation (AOPP) and lipid peroxidation (TBARS). As a result, when animals of Group A were compared to animals of Group B, it was observed leukocytosis (P<0.05) on day 10 PI; AChE activity increase (P<0.05) in lymphocytes (day 30 PI) and in brain (days 10 and 30 PI); BChE activity decrease (P<0.05) on day 10 PI; IL-1 and IL-6 increase (P<0.01) in both periods, while IL-10 had reduced levels (P<0.01) in the same periods; NOx levels increased (P<0.05) significantly on days 10 and 30 PI, while AOPP and TBARS levels increased significantly on day 30 PI; as well as pneumonia on infected rats. Therefore, based on the results obtained, it was possible to conclude that AChE and BChE behavior lead to a proinflammatory reaction evidenced by the enhancement of IL-1, IL-6, and NOx throughout the experiment associated with reduction on IL-10 levels, and cellular damage.

Japanese Huperzia serrata extract and the constituent, huperzine A, ameliorate the scopolamine-induced cognitive impairment in mice.

Huperzia serrata has been used as a Chinese folk medicine for many years. It contains huperzine A, which has a protective effect against memory deficits in animal models; however, it is unclear if H. serrata extract exerts any effects in Alzheimer's disease (AD) models. We used H. serrata collected in Japan and determined its huperzine A content using HPLC. We determined its inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. H. serrata extract (30 mg/kg/day) and donepezil (10 mg/kg/day) were orally administrated for 7 days. After repeated administration, we performed the Y-maze and passive avoidance tests. H. serrata extract contained 0.5% huperzine A; H. serrata extract inhibited AChE, but not BuChE. H. serrata extract ameliorated cognitive function in mice. These results indicate that Japanese H. serrata extract ameliorates cognitive function deficits by inhibiting AChE. Therefore, H. serrata extract may be valuable for the prevention or treatment of dementia in AD.

[The effect of hypoxia on cholinesterase activity in rat sensorimotor cortex].

This study reports the dynamics of changes in postnatal ontogenesis of the activity of soluble and membrane-bound forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in sensorimotor cortex of rats as well as the pattern of their changes after prenatal hypoxia (E14, 7% O2, 3 h) or acute hypoxia in adult animals (4 months, 7% O2, 3 h). In normally developing rats the activity of the membrane-bound AChE form in the sensorimotor cortex gradually increased up to the end of the first month after birth and remained at this high level during all further postnatal ontogenesis, while the activity of the soluble form of AChE reached its maximum on the 10th day after birth and decreased significantly by the end of the first month. In animals exposed to prenatal hypoxia the activity both of the soluble and membrane bound forms of AChE during the first two weeks after birth was 20-25% lower, as compared to controls but increased by the end of the first month and even exceeded the control values remaining increased up to old age (1.5 years). The activity of both BChE forms in rat sensorimotor cortex at all stages of postnatal ontogenesis was significantly lower than of AChE, although the dynamics of their changes was similar to that of AChE. Prenatal hypoxia led to a decrease in the activity of the membrane-bound form of BChE, as compared to controls, practically at all developmental stages studied, but was higher at the end of the first month after birth. At the same time, the activity of the soluble form of BChE was decreased only on the 20th day of development, as compared to the control, but increased from the end of the first month of life onwards. Acute hypoxia in adult rats also led to a decrease in the activity of both forms of AChE and BChE in the sensorimotor cortex but the dynamics of these changes was different for each enzyme. Thus, insufficient oxygen supply to the nervous tissue at different stages of ontogenesis has a significant effect on the activity and ratio of various forms of cholinesterases exhibiting either growth factor or signaling properties. This may lead to changes in brain development and formation of behavioural reactions, including learning and memory, and also increase the risk of development of the sporadic form of Alzheimer's disease (AD)--one of the most common neurodegenerative diseases of advanced age. This study expands our knowledge of the properties of brain cholinesterases under normal and pathological conditions and may be useful for developing new approaches towards prevention and treatment of AD.

Manufacturing economics of plant-made biologics: case studies in therapeutic and industrial enzymes.

Production of recombinant biologics in plants has received considerable attention as an alternative platform to traditional microbial and animal cell culture. Industrially relevant features of plant systems include proper eukaryotic protein processing, inherent safety due to lack of adventitious agents, more facile scalability, faster production (transient systems), and potentially lower costs. Lower manufacturing cost has been widely claimed as an intuitive feature of the platform by the plant-made biologics community, even though cost information resides within a few private companies and studies accurately documenting such an advantage have been lacking. We present two technoeconomic case studies representing plant-made enzymes for diverse applications: human butyrylcholinesterase produced indoors for use as a medical countermeasure and cellulases produced in the field for the conversion of cellulosic biomass into ethanol as a fuel extender. Production economics were modeled based on results reported with the latest-generation expression technologies on Nicotiana host plants. We evaluated process unit operations and calculated bulk active and per-dose or per-unit costs using SuperPro Designer modeling software. Our analyses indicate that substantial cost advantages over alternative platforms can be achieved with plant systems, but these advantages are molecule/product-specific and depend on the relative cost-efficiencies of alternative sources of the same product.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy.

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Butyrylcholinesterase is associated with ß-amyloid plaques in the transgenic APPSWE/PSEN1dE9 mouse model of Alzheimer disease.

Histochemical analysis of Alzheimer disease (AD) brain tissues indicates that butyrylcholinesterase (BuChE) is present in ß-amyloid (Aß) plaques. The role of BuChE in AD pathology is unknown, but an animal model developing similar BuChE-associated Aß plaques could provide insights. The APPSWE/PSEN1dE9 transgenic mouse (ADTg), which develops Aß plaques, was examined to determine if BuChE associates with these plaques, as in AD. We found that in mature ADTg mice, BuChE activity associated with Aß plaques. The Aß-, thioflavin-S- and BuChE-positive plaques mainly accumulated in the olfactory structures, cerebral cortex, hippocampal formation, amygdala, and cerebellum. No plaques were stained for acetylcholinesterase activity. The distribution and abundance of plaque staining in ADTg closely resembled many aspects of plaque staining in AD. Butyrylcholinesterase staining consistently showed fewer plaques than were detected with Aß immunostaining but a greater number of plaques than were visualized with thioflavin-S. Double-labeling experiments demonstrated that all BuChE-positive plaques were Aß positive, whereas only some BuChE-positive plaques were thioflavin-S positive. These observations suggest that BuChE is associated with a subpopulation of Aß plaques and may play a role in AD plaque maturation. A further study of this animal model could clarify the role of BuChE in AD pathology.

The apolipoprotein E ε4 allele plays pathological roles in AD through high protein expression and interaction with butyrylcholinesterase.

The apolipoprotein E (ApoE) ε4 allele has consistently been established as an Alzheimer's disease (AD) risk factor, but its pathological contribution to AD is obscure. Certain butyrylcholinesterase (BuChE) polymorphisms together with the ApoE ε4 allele synergistically increase the risk of AD. In addition, AD risk factors, i.e. advanced age, female gender and ApoE ε4 are associated with different levels of CSF BuChE in AD patients, and BuChE protein attenuates Aß fibrillization in vitro. Here we investigated the roles of ApoE and BuChE gene products as modulators of pathological features of AD in vivo. We found that AD risk factors were associated with different levels of ApoE protein in the CSF of AD patients (n=115). Women and ApoE ε4 carriers had the highest levels of ApoE protein (up by 50-120%, p<0.01-0.0001), which were increased with age (r=0.30, p<0.0006). The CSF surrogate markers of pathological features of AD, i.e. high tau and P-tau, low Aß(42) and high tau/Aß(42) ratio, were associated with high levels of ApoE protein. Intriguingly, high ApoE protein levels were not only associated with low amounts of BuChE, but they also altered the aging and activity of this enzyme in concentration- and isoform-dependent manners, particularly in the presence of Aß peptides. Both ApoE and BuChE levels were also differentially related to levels of the proinflammatory cytokine IL-1ß. In conclusion, ApoE ε4 might impart its pathological role through high protein expression and interaction with BuChE, which in turn might modulate central cholinergic activity and Aß load in the brain.

The PRiMA-linked cholinesterase tetramers are assembled from homodimers: hybrid molecules composed of acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during development of chicken brain.

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.

High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system.

Butyrylcholinesterase (BChE: EC 3.1.1.8) serves as a natural scavenger for a variety of drugs, poisons, and organophosphorous compounds by hydrolyzing their ester bonds. Large scale production of recombinant human BChE (rhBChE) has been reported in transgenic goat. Here we demonstrate high-level expression of rhBChE with biological activity comparable to that of natural and recombinant enzymes, through the Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. We constructed the full-length hBChE cDNA into the plasmid pFastBac. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP) was cloned downstream to the polyhedron (pH) promoter. Transfection was carried out by subcutaneous injection of 4-5th instar silkworm larvae. Approximately 4-7 days after infection, high-level expression of recombinant proteins was observed as indicated by the orange fluorescence of the larvae under blue light illumination. The hemolymph of the infected larvae was harvested, purified and assayed for BChE activity. The total units of BChE activity after purification were around 6.4 units per larvae. The K(m) and V(max) values of rhBChE were determined to be 17.7 microM and 2194 U/l hemolymph, respectively. By SDS-PAGE and Western analysis, the size of silkworm rhBChE was estimated to be 85 kDa. The results indicate that the silkworm larva is a good alternative system to produce bioactive rhBChE. Further optimization and modifications will be necessary for large-scale production of rhBChE. This should provide a rapid, low-cost, and high yield rhBChE for therapeutic applications.

Transgenic plants as a source for the bioscavenging enzyme, human butyrylcholinesterase.

Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high-level expression of human BChE in plants. Plant-derived BChE is shown to be biochemically similar to human plasma-derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant-derived bioscavenger to protect animals against an organophosphorous pesticide challenge.

Protein profile and alpha-lactalbumin concentration in the milk of standard and transgenic goats expressing recombinant human butyrylcholinesterase.

The expression of recombinant proteins of pharmaceutical interest in the milk of transgenic farm animals can result in phenotypes exhibiting compromised lactation performance, as a result of the extraordinary demand placed on the mammary gland. In this study, we investigated differences in the protein composition of milk from control and transgenic goats expressing recombinant human butyrylcholinesterase. In Experiment 1, the milk was characterized by gel electrophoresis and liquid chromatography/mass spectrometry in order to identify protein bands that were uniquely visible in the transgenic milk and/or at differing band densities compared with controls. Differences in protein content were additionally evaluated by computer assisted band densitometry. Proteins identified in the transgenic milk only included serum proteins (i.e. complement component 3b, ceruloplasmin), a cytoskeleton protein (i.e. actin) and a stress-induced protein (94 kDA glucose-regulated protein). Proteins exhibiting evident differences in band density between the transgenic and control groups included immunoglobulins, serum albumin, beta-lactoglobulin and alpha-lactalbumin. These results were found to be indicative of compromised epithelial tight junctions, premature mammary cell death, and protein synthesis stress resulting from transgene expression. In Experiment 2, the concentration of alpha-lactalbumin was determined using the IDRing assay and was found to be significantly reduced on day 1 of lactation in transgenic goats (4.33 +/- 0.97 vs. 2.24 +/- 0.25 mg/ml, P < 0.01), but was not different from non-transgenic controls by day 30 (0.99 +/- 0.46 vs. 0.90 +/- 0.11 mg/ml, P > 0.05). We concluded that a decreased/delayed expression of the alpha-lactalbumin gene may be the cause for the delayed start of milk production observed in this herd of transgenic goats.

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk.

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1-5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.

The butyrylcholinesterase knockout mouse as a model for human butyrylcholinesterase deficiency.

Butyrylcholinesterase (BChE) is an important enzyme for metabolism of ester drugs. Many humans have partial or complete BChE deficiency due to genetic variation. Our goal was to create a mouse model of BChE deficiency to allow testing of drug toxicity. For this purpose, we created the BChE knockout mouse by gene-targeted deletion of a portion of the BCHE gene (accession number M99492). The BChE(-/-) mouse had no BChE activity in plasma, but it had low residual butyrylthiocholine hydrolase activity in all other tissues attributed to carboxylesterase ES-10. The BChE(-/-) mouse had a normal phenotype except when challenged with drugs. Nicotinic receptor function as indicated by response to nicotine seemed to be normal in BChE(-/-) mice, but muscarinic receptor function as measured by response to oxotremorine and pilocarpine was altered. Heart rate, blood pressure, and respiration, measured in a Vevo imager, were similar in BChE(+/+) and BChE(-/-) mice. Like BChE(-/-) humans, the BChE(-/-) mouse responded to succinylcholine with prolonged respiratory arrest. Bambuterol was not toxic to BChE(-/-) mice, suggesting it is safe in BChE(-/-) humans. Challenge with 150 mg/kg pilocarpine i.p., a muscarinic agonist, or with 50 mg/kg butyrylcholine i.p., induced tonicclonic convulsions and death in BChE(-/-) mice. This suggests that butyrylcholine, like pilocarpine, binds to muscarinic receptors. In conclusion, the BChE(-/-) mouse is a suitable model for human BChE deficiency.

Elevated butyrylcholinesterase and acetylcholinesterase may predict the development of type 2 diabetes mellitus and Alzheimer's disease.

Plasma levels of C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and lipid peroxides are elevated and concentrations of endothelial nitric oxide (eNO) decreased in type 2 diabetes mellitus and Alzheimer's disease. This suggests that both these diseases are low-grade systemic inflammatory conditions and are closely associated with each other. Recent studies revealed that plasma and tissue concentrations of enzymes butyrylcholinesterase and acetylcholinesterase are elevated in type 2 diabetes and Alzheimer's disease. Acetylcholine has anti-inflammatory actions. Hence, elevated butyrylcholinesterase and acetylcholinesterase concentrations will lead to a decrease in the levels of acetylcholine that could trigger the onset of low-grade systemic inflammation seen in type 2 diabetes and Alzheimer's disease. In view of this, we propose that butyrylcholinesterase and acetylcholinesterase will not only serve as therapeutic targets but also may serve as markers to predict the development of type 2 diabetes mellitus and Alzheimer's disease.

Cholinesterase activity in human pulmonary arteries and veins: correlation with mRNA levels.

Isolated intact human pulmonary arteries and veins were used to determine the acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) activities in the absence or presence of two selective cholinesterase (ChE) inhibitors, iso-OMPA or BW284c51, respectively. These results were compared with the mRNA levels for each enzyme in human pulmonary vessels. Total ChE activities measured in presence of acetylthiocholine (ACTI, 1 mM) in intact vascular preparations were 45+/-04 and 114+/-07 mU/g tissue in human pulmonary arteries (n=14) and veins (n=14), respectively. These activities were completely abolished in presence of 10 microM neostigmine. In both types of vessels AChE and BChE activities were observed. These activities were at least 2-fold higher in human pulmonary veins when compared with arteries and were correlated with the accumulation of the corresponding transcripts (n=8). In each type of vessel, similar total ChE activities were detected in homogenized and intact preparations, while in human bronchial preparations this activity was 5-fold higher in homogenates than in intact preparations. Together these results provide evidence that the ChE activities in human pulmonary vessels may be extracellular and that the higher activity measured in veins as compared to arteries was associated with the differential accumulation of the corresponding transcripts.