Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

The observation of acetylcholinesterase (AChE) type H (AChEH), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChEH The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChEH, accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChEH, and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors.

Heat-inactivated mycobacteria activate the toll-like receptor 2 and 4 pathways in the zebrafish model of tuberculosis.

Based on previous evidence demonstrating the efficacy of inactivated mycobacteria for the control of fish mycobacteriosis, we explored the protective efficacy of two inactivated Mycobacterium bovis administered via parenteral and mucosal routes against Mycobacterium marinum infection mimicking natural conditions in the zebrafish model of tuberculosis. Although we did not observe a clear effect of any of the immunostimulants on mycobacterial burden, the results showed a significant increase in TLR2 and TLR4 gene expression levels in fishes parenterally immunized with inactivated Bacillus Calmette-Guérin (BCG). Our findings demonstrated that the TLR2 and the TLR4 signaling pathways are involved in the immune response elicited by inactivated mycobacteria in the zebrafish model of tuberculosis and support the use of inactivated mycobacteria in vaccine formulations for the control of mycobacteriosis.

Expression profiling shows differential molecular pathways and provides potential new diagnostic biomarkers for colorectal serrated adenocarcinoma.

Serrated adenocarcinoma (SAC) is a recently recognized colorectal cancer (CRC) subtype accounting for 7.5 to 8.7% of CRCs. It has been shown that SAC has a poorer prognosis and has different molecular and immunohistochemical features compared with conventional carcinoma (CC) but, to date, only one previous study has analyzed its mRNA expression profile by microarray. Using a different microarray platform, we have studied the molecular signature of 11 SACs and compared it with that of 15 matched CC with the aim of discerning the functions which characterize SAC biology and validating, at the mRNA and protein level, the most differentially expressed genes which were also tested using a validation set of 70 SACs and 70 CCs to assess their diagnostic and prognostic values. Microarray data showed a higher representation of morphogenesis-, hypoxia-, cytoskeleton- and vesicle transport-related functions and also an overexpression of fascin1 (actin-bundling protein associated with invasion) and the antiapoptotic gene hippocalcin in SAC all of which were validated both by quantitative real-time PCR (qPCR) and immunohistochemistry. Fascin1 expression was statistically associated with KRAS mutation with 88.6% sensitivity and 85.7% specificity for SAC diagnosis and the positivity of fascin1 or hippocalcin was highly suggestive of SAC diagnosis (sensitivity = 100%). Evaluation of these markers in CRCs showing histological and molecular characteristics of high-level microsatellite instability (MSI-H) also helped to distinguish SACs from MSI-H CRCs. Molecular profiling demonstrates that SAC shows activation of distinct signaling pathways and that immunohistochemical fascin1 and hippocalcin expression can be reliably used for its differentiation from other CRC subtypes.

Changes in the Acetylcholinesterase Enzymatic Activity in Tumor Development and Progression.

Acetylcholinesterase is a well-known protein because of the relevance of its enzymatic activity in the hydrolysis of acetylcholine in nerve transmission. In addition to the catalytic action, it exerts non-catalytic functions; one is associated with apoptosis, in which acetylcholinesterase could significantly impact the survival and aggressiveness observed in cancer. The participation of AChE as part of the apoptosome could explain the role in tumors, since a lower AChE content would increase cell survival due to poor apoptosome assembly. Likewise, the high Ach content caused by the reduction in enzymatic activity could induce cell survival mediated by the overactivation of acetylcholine receptors (AChR) that activate anti-apoptotic pathways. On the other hand, in tumors in which high enzymatic activity has been observed, AChE could be playing a different role in the aggressiveness of cancer; in this review, we propose that AChE could have a pro-inflammatory role, since the high enzyme content would cause a decrease in ACh, which has also been shown to have anti-inflammatory properties, as discussed in this review. In this review, we analyze the changes that the enzyme could display in different tumors and consider the different levels of regulation that the acetylcholinesterase undergoes in the control of epigenetic changes in the mRNA expression and changes in the enzymatic activity and its molecular forms. We focused on explaining the relationship between acetylcholinesterase expression and its activity in the biology of various tumors. We present up-to-date knowledge regarding this fascinating enzyme that is positioned as a remarkable target for cancer treatment.

The levels of both lipid rafts and raft-located acetylcholinesterase dimers increase in muscle of mice with muscular dystrophy by merosin deficiency.

Wild type and dystrophic (merosin-deficient) Lama2dy mice muscles were compared for their density of lipid rafts. The 5-fold higher level of caveolin-3 and the 2-3 times higher level of ecto-5'-nucleotidase activity in raft preparations (Triton X-100-resistant membranes) of dystrophic muscle supported expansion of caveolar and non-caveolar lipid rafts. The presence in rafts of glycosylphosphatidylinositol (GPI)-linked acetylcholinesterase (AChE) dimers, which did not arise from erythrocyte or nerve, not only revealed for the first time the capacity of the myofibre for translating the AChE-H mRNA but also an unrecognized pathway for targeting AChE-H to specialized membrane domains of the sarcolemma. Rafts of dystrophic muscle contained a 5-fold higher AChE activity/mg protein. RT-PCR for 3'-alternative mRNAs of AChE revealed AChE-T mRNA prevailing over AChE-R and AChE-H mRNAs in wild type mouse muscle. It also displayed principal 5'-alternative AChE mRNAs with exons E1c and E1e (the latter coding for N-terminally extended subunits) and fewer with E1d, E1a and E1b. The levels of AChE and butyrylcholinesterase mRNAs were unaffected by dystrophy. Finally, the decreased level of proline-rich membrane anchor (PRiMA) mRNA in Lama2dy muscle provided for a rational explanation to the loss of PRiMA-bearing AChE tetramers in dystrophic muscle.

Cholinesterase activity in brain of senescence-accelerated-resistant mouse SAMR1 and its variation in brain of senescence-accelerated-prone mouse SAMP8.

The early-onset, irreversible, severe deficits of learning and memory in the senescence-accelerated mouse (SAM)-prone/8 (SAMP8) support its use as an animal model for human dementias of early onset. Possible implication of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in cognitive dysfunction of SAMP8 mice was studied by comparing cholinesterase (ChE) expression in brains of SAMP8 mice and of their normal control, SAM-resistant/1 (SAMR1) mice. The level of ChE mRNAs was the same in SAMP8 and SAMR1 brains, which agreed with their equal AChE activity (3.09 +/- 1.45 vs. 3.07 +/- 1.44 mumol.hr(-1).mg protein(-1), U/mg), but not with a doubled BuChE activity in SAMP8 brain (0.14 +/- 0.05 vs. 0.07 +/- 0.02 U/mg; P < 0.01). This great increase in neural BuChE activity may contribute to cognitive deficit of SAMP8 mice. Hydrophilic (G(4) (H), 8%) and amphiphilic (G(4) (A), 74%) AChE tetramers, besides dimers and monomers (G(2) (A) + G(1) (A), 18%), were identified in SAMR1 brains. They also contained G(4) (H) BuChE forms (18%) as well as G(4) (A) (53%) and G(2) (A) + G(1) (A) (29%) species. Although SAMP8 brain displayed proportions of AChE and BuChE forms that were similar to those of SAMR1 brain, phenyl-agarose chromatography with detergent-free extracts showed a rise in the proportion of secretory G(4) (H) BuChE from 35% in SAMR1 to 44% in SAMP8 brain. The strong immunolabelling of glial fibrillary acidic protein (GFAP), a marker of reactive gliosis, in SAMP8 brain and the consideration of BuChE as a marker of glial cells suggest a relationship between phenotypic changes in neuroglial cells and the excess of BuChE activity in SAMP8 brain.

The level of butyrylcholinesterase activity increases and the content of the mRNA remains unaffected in brain of senescence-accelerated mouse SAMP8.

Looking at cholinesterases (ChEs) changes in age-related mental impairment, the expression of ChEs in brain of senescence accelerated-resistant (SAMR1) and senescence accelerated-prone (SAMP8) mice was studied. Acetylcholinesterase (AChE) activity was unmodified and BuChE activity increased twofold in SAMP8 brain. SAMR1 brain contained many AChE-T mRNAs, less BuChE and PRiMA mRNAs and scant AChE-R and AChE-H mRNAs. Their content unchanged in SAMP8 brain. Amphiphilic (G(4)(A)) and hydrophilic (G(4)(H)) AChE and BuChE tetramers, besides amphiphilic dimers (G(2)(A)) and monomers (G(1)(A)) were identified in SAMR1 brain and their distribution was little modified in SAMP8 brain. Blood plasma does not seem to provide the excess of BuChE activity in SAMP8 brain; it probably arises from glial cell changes owing to astrocytosis.

The expression of cholinesterases in human renal tumours varies according to their histological types.

The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.

Butyrylcholinesterase activity and molecular components in thymus of healthy and merosin-deficient Lama2dy mice.

The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.

The increased ecto-5'-nucleotidase activity in muscle, heart and liver of laminin alpha2-deficient mice is not caused by an elevation in the mRNA content.

We have previously shown that mouse muscle and liver contain catalytically active and inactive ecto-5'-nucleotidase (eNT) variants and that eNT activity in these tissues increases in laminin alpha2 (merosin)-deficient Lama2dy mice. These results prompted us to study whether: (1) the increase of eNT activity depends on the change in the content of merosin between healthy and dystrophic organs; (2) the active and inactive eNT variants arise from the same or distinct mRNAs; (3) the enhancement of the activity is caused by an increase in the eNT mRNA content. Compared to healthy organs, the activity in dystrophic organs increased four-fold in muscle, 1.7-fold in liver, 1.4-fold in heart and not at all in kidney and lung. The level of immunolabelled eNT protein per unit of activity suggested a similar ratio of inactive to active eNT in healthy liver, kidney, heart and muscle, which increased greatly in lung. The size of the eNT subunit in liver, kidney, heart and muscle (72 kDa) decreased to 66 kDa in lung. The identification of a single RT-PCR product suggested that active and inactive eNT arise from the same mRNA and are generated by a differential post-translational processing. Compared to the content in muscle, the amount of eNT mRNA was 12-fold higher in liver and kidney, eight-fold in heart and five-fold in lung. The relative content of eNT mRNA was unaffected by the deficiency of merosin.

Thymus acetylcholinesterase activity is reduced in mice with congenital muscular dystrophy.

Lama2dy mice constitute an animal model for congenital muscular dystrophy (CMD) by merosin (laminin alpha2-chain) deficiency. This pathology affects the properties of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of mouse skeletal muscle and nerves (Moral-Naranjo et al., 1999, 2002). AChE and BChE are involved through catalytic and noncatalytic actions in multiple processes, such as hydrolysis of acetylcholine (ACh), morphogenesis, hematopoiesis, and tumorigenesis (Soreq and Seidman, 2001). AChE and BChE molecules can be globular (G1, G2, and G4) or asymmetric forms (A4, A8, and A12) (Massoulié, 2002), and G molecules can show amphiphilic (detergent-interacting, GA) or hydrophilic (GH) behavior. AChE catalytic subunits are encoded by three mRNAs (T, H, or R) generated by alternative splicing. The presence of AChE in lymphoid tissues (Rossi et al., 1991; Nieto-Cerón et al., 2004), the role of immune responses in muscular dystrophy (Spencer and Tidball, 2001), the abnormalities of Lama2dy thymus (Magner et al., 2000), and the role of ACh in thymocyte function (Kawashima and Fujii, 2000) prompted us to investigate thymus AChE and the possible effect of merosin deficiency on it.

Acetyl- and butyrylcholinesterase activities decrease in human colon adenocarcinoma.

Apart from the hydrolysis of acetylcholine (ACh), acetyl- (AChE) and butyrylcholinesterase (BChE), through noncatalytic mechanisms, intervene in hematopoiesis, morphogenesis, and neurogenesis (Layer and Willbold, 1995; Soreq and Seidman, 2001). Cholinesterase (ChE) molecules occur as globular (G1, G2, and G4) and asymmetric (A4, A8, and A12) forms (Legay, 2000; Massoulié, 2002). The G species might display amphiphilic (GA) or hydrophilic (GH) properties (Perrier et al., 2002). The involvement of ChEs in tumorigenesis is supported by the measurement of ChE activity in tumors (García-Ayllón et al., 2001; Ruiz-Espejo et al., 2003), the amplification of ChE genes in leukemias and ovarian tumors, and the relationship between the expression of AChE and the aggressiveness of astrocytomas(Perry et al., 2002). This research was undertaken to determine whether ChE activity is altered in gut carcinomas.

[Utility of biomarkers in abdominal pain management]. / Utilidad de los biomarcadores en el manejo del dolor abdominal.

EN: Abdominal pain conditions that fall into the category of acute abdomen (AA) are the most important ones to identify quickly. Diagnostic delay can lead to death or significant complications. Biological markers have the potential to improve the diagnostic and prognostic capacity of clinical assessment and the conventional complement of tests. This review aims to explore the relevance of several markers to the management of AA in the emergency department. Creactive protein (CRP), procalcitonin, and lactate are the biomarkers most often used in the emergency department. CRP is often analyzed in the context of AA, but it is very difficult to establish a cutoff that gives good sensitivity and specificity. The kinetics of CRP make it the most sensitive biomarker and one that is appropriate for assessing severity before the onset of clinical signs of severe sepsis or altered hemodynamics. Lactate is a marker of poor tissue perfusion, a key element in the management of severe sepsis and septic shock in AA. Since lactate testing is easy and inexpensive, this important biomarker is useful in the emergency department.

ES: Las manifestaciones de las enfermedades que subyacen bajo el término de dolor abdominal agudo (DAA) como motivo de consulta en urgencias, pueden ser sutiles en su inicio y variables en el tiempo, lo que dificulta su reconocimiento precoz. Entre ellas son prioritarias las englobadas bajo el término de abdomen agudo (AA) o situación de DAA tiempo-dependiente. Los biomarcadores pueden mejorar el manejo de estos pacientes, añadiendo información adicional a la valoración clínica y a las exploraciones complementarias, e incrementando la capacidad diagnóstica y pronóstico. Los biomarcadores más utilizados en urgencias son la proteína C reactiva (PCR), la procalcitonina (PCT) y el lactato. La PCR ha sido el marcador más estudiado en el diagnóstico del DAA, y es muy difícil establecer un punto de corte que proporcione buena sensibilidad y especificidad. La PCT es el biomarcador más sensible y adecuado, gracias a su particular cinética, para valorar la gravedad antes de que los signos clínicos de sepsis grave o alteración hemodinámica hagan su aparición. El lactato es un marcador de hipoperfusión tisular y elemento clave en el manejo de la sepsis grave y del shock séptico en el abdomen agudo, lo que añadido a su fácil y rápida obtención y a su bajo coste, definen su importancia y utilidad en los servicios de urgencias.

MRNA Levels of ACh-Related Enzymes in the Hippocampus of THY-Tau22 Mouse: A Model of Human Tauopathy with No Signs of Motor Disturbance.

The microtubule-associated protein Tau tends to form aggregates in neurodegenerative disorders referred to as tauopathies. The tauopathy model transgenic (Tg) THY-Tau22 (Tau22) mouse shows disturbed septo-hippocampal transmission, memory deficits and no signs of motor dysfunction. The reports showing a hippocampal downregulation of choline acetyltransferase (ChAT) in SAMP8 mice, a model of aging, and an upregulation of acetylcholinesterase (AChE) in Tg-VLW mice, a model of FTDP17 tauopathy, may lead to think that the supply of ACh to the hippocampus can be threatened as aging or Tau pathology progress. The above was tested by comparing the mRNA levels for ACh-related enzymes in hippocampi of wild-type (wt) and Tau22 mice at ages when the neuropathological signs are debuting (3-4 months), moderate (6-7 months) and extensive (>9 months). Age-matched Tau22 and wt mice hippocampi displayed similar ChAT, AChE-T, butyrylcholinesterase (BChE) and a proline-rich membrane anchor (PRiMA) mRNA levels, any change most likely arising from ACh homeostasis. The unchanged hippocampal levels of AChE-T mRNA and enzyme activity observed in Tau22 mice, expressing G272V-P301S hTau, differed from the increase in AChE-T mRNA and activity observed in Tg-VLW mice, expressing G272V-P301L-R406W hTau. The difference supports the idea that AChE upregulation may proceed or not depending on the particular Tau mutation, which would dictate Tau folding, the accessibility/affinity to kinases and phosphatases, and P-Tau aggregation with itself and protein partners, transcription factors included.

Acetylcholinesterase activity of electric eel is increased or decreased by selected monoterpenoids and phenylpropanoids in a concentration-dependent manner.

The profitable insecticidal action of monoterpenoids prompted us to test their efficiency against stored-grain beetle species, via inhibition of acetylcholinesterase (AChE). For this, we first studied the ability of the monoterpenoids geraniol, linalool, camphor, fenchone, carvone and γ-terpinene, besides the phenylpropanoids trans-anethole and estragole to inhibit Electrophorus AChE. The results indicated that while AChE activity increased (15-35%) with 40 µM geraniol, camphor, γ-terpinene and linalool, the activity decreased (60-40%) with 5mM carvone, γ-terpinene, and fenchone. The Km for AChE was 0.52 ± 0.02 mM in control assays, which fell to 0.28 ± 0.01 mM or 0.32 ± 0.01 mM in assays with 20 µM linalool or γ-terpinene added. In the millimolar range, the terpenoids behaved as weak inhibitors. Unexpectedly, AChE inhibition by camphor, carvone, γ-terpinene, and fenchone gave Hill numbers ranging 2.04-1.57, supporting the idea that AChE was able to lodge more than one monoterpenoid molecule. The plots of 1/v vs. 1/S at varying monoterpenoid provided straight lines, fenchone and γ-terpinene acting as competitive inhibitors and carvone and camphor as non-competitive inhibitors. Moreover, the secondary plots of the slope KM(app)/Vmax(app) vs. [I] and of 1/Vmax(app) vs. [I] gave parabolic curves, which lent support to the proposed capacity of AChE to bind more than one monoterpenoid molecule. The fitting of the curves to a second-order polynomial equation allowed us to calculate the inhibition constants for the interaction of AChE with fenchone, γ-terpinene, carvone and camphor. The previously unnoticed increase in AChE activity with monoterpenoids should be considered as a reminder when advising the use of essential oils of plants or their constituents as anti-AChE agents to attenuate pathological signs of Alzheimer's disease.

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes.

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.

Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice.

The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.

Molecular properties of acetylcholinesterase in mouse spleen.

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.

The lipid raft-bound alkaline phosphatase activity increases and the level of transcripts remains unaffected in liver of merosin-deficient LAMA2dy mouse.

Alkaline phosphatase (AP) and other proteins add glycosylphosphatidylinositol (GPI) before addressing to raft domains of the cell membrane. Our previous report showing an increased density of lipid rafts in muscle of dystrophic Lama2dy mice prompted us to compare livers of normal (NL) and dystrophic mice (DL) for their levels of rafts. With this aim, hepatic rafts were isolated as Triton X-100 resistant membranes, and identified by their abundance of flotillin-2, alkaline phosphatase (AP) and other raft markers. The comparable abundance of cholesterol and flotillin-2 in rafts of NL and DL contrasted with the double AP activity both in rafts of DL and whole DL. The AP mRNA level was the same in NL and DL. Sedimentation analysis profiles revealed AP activity of NL distributed between dimeric (dAP) and monomeric AP (mAP), whose proportions and lectin-binding extent changed in DL. The increased AP activity and changed AP glycosylation in DL, the prevalence of mAP in NL and the enhanced stability of dAP in DL demonstrated the critical role that glycosylation and oligomerization play for AP catalysis. The higher AP activity of DL probably arises from dystrophy-associated changes in glycosyl transferases, which alter AP glycosylation and subunit folding with profitable effects for AP stability and catalysis.

Most acetylcholinesterase activity of non-nervous tissues and cells arises from the AChE-H transcript.

While the functional implications of AChE-T, PRiMA and ColQ have been firmly established, those of glypiated AChE remain uncertain. Insights into the physiological meaning of glycosylphosphatidylinositol (GPI)-linked AChE-H were gained by comparing nervous and non-nervous tissues for the amount of AChE mRNA variants they contained. PCR showed that AChE-T mRNA prevailed in the mouse brain, spinal cord, sciatic nerve and muscle, and AChE-H mRNA in the bone marrow and thymus, as well as in the human gut. The similar levels of AChE-T and AChE-H mRNAs in mouse liver and human kidney contrasted with the almost exclusive presence of catalytically active AChE-H in both organs. The absence of PRiMA mRNA in liver suggested that the tetramers made of AChE-T fail to bind to the cell membrane and are secreted due to the lack of PRiMA in non-nervous organs. In contrast, glypiated AChE-H is largely and lastingly bound to the cell membrane. Thus, non-synaptic glypiated AChE-H seems to be the counterpart of synaptic PRiMA-linked AChE-T, the former designed for clearing ACh waves, the latter for confronting ACh bursts, and both for helping to protect cells against the harmful effects of durable nicotinic and muscarinic activation.