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1.
FASEB J ; 31(2): 544-555, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28148778

RESUMO

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.


Assuntos
Acetilcolinesterase/classificação , Acetilcolinesterase/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Variação Genética , Microdomínios da Membrana/fisiologia , Receptor Muscarínico M3/metabolismo , Animais , Encéfalo/enzimologia , Fígado/enzimologia , Camundongos , Miocárdio/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Muscarínico M3/genética
2.
J Mol Neurosci ; 58(4): 411-5, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26697857

RESUMO

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.


Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Movimento , RNA Mensageiro/genética , Tauopatias/genética , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Hipocampo/crescimento & desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Especificidade da Espécie , Tauopatias/metabolismo , Tauopatias/fisiopatologia , Proteínas tau/genética
3.
Chem Biol Interact ; 229: 36-43, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25636489

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Anisóis/farmacologia , Inibidores da Colinesterase/farmacologia , Electrophorus/metabolismo , Ativação Enzimática/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Monoterpenos/farmacologia , Animais , Anisóis/química , Inibidores da Colinesterase/química , Humanos , Cinética , Monoterpenos/química , Óleos Voláteis/química , Óleos Voláteis/farmacologia
4.
Chem Biol Interact ; 216: 1-8, 2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-24680793

RESUMO

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.


Assuntos
Fosfatase Alcalina/metabolismo , Laminina/metabolismo , Fígado/enzimologia , Microdomínios da Membrana/enzimologia , Fosfatase Alcalina/genética , Animais , Regulação Enzimológica da Expressão Gênica , Laminina/genética , Fígado/metabolismo , Camundongos , Camundongos Knockout , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
5.
J Mol Neurosci ; 53(3): 429-35, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24242952

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , RNA Mensageiro/metabolismo , Acetilcolinesterase/genética , Animais , Encéfalo/enzimologia , Glicosilfosfatidilinositóis/metabolismo , Humanos , Intestinos/enzimologia , Rim/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Músculos/enzimologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , RNA Mensageiro/genética , Medula Espinal/enzimologia
6.
Int J Cancer ; 132(2): 297-307, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22696308

RESUMO

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.


Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Transcriptoma , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Análise Mutacional de DNA , Feminino , Expressão Gênica , Hipocalcina/genética , Hipocalcina/metabolismo , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Análise Multivariada , Proteína Inibidora de Apoptose Neuronal/genética , Proteína Inibidora de Apoptose Neuronal/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes
7.
FEBS J ; 277(21): 4519-29, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20883446

RESUMO

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg(-1), P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg(-1), P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg(-1)). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg(-1), P = 0.031). Glycosylphosphatidylinositol-linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3'-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.


Assuntos
Carcinoma de Células Renais/genética , Colinesterases/genética , Neoplasias Renais/genética , Rim/metabolismo , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Butirilcolinesterase/genética , Butirilcolinesterase/metabolismo , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/patologia , Colinesterases/sangue , Colinesterases/metabolismo , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/enzimologia , Rim/patologia , Neoplasias Renais/enzimologia , Neoplasias Renais/patologia , Lectinas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Biochim Biophys Acta ; 1802(9): 754-64, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20670915

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Laminina/genética , Microdomínios da Membrana/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/patologia , 5'-Nucleotidase/metabolismo , 5'-Nucleotidase/fisiologia , Acetilcolinesterase/genética , Acetilcolinesterase/fisiologia , Animais , Caveolina 3/genética , Caveolina 3/metabolismo , Laminina/deficiência , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/patologia , Distrofia Muscular Animal/metabolismo , Multimerização Proteica
9.
J Neurosci Res ; 88(1): 155-66, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19610099

RESUMO

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.


Assuntos
Envelhecimento/metabolismo , Cérebro/metabolismo , Colinesterases/metabolismo , Envelhecimento/genética , Animais , Astrócitos/metabolismo , Western Blotting , Colinesterases/genética , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Processamento de Imagem Assistida por Computador , Lectinas/metabolismo , Camundongos , Camundongos Endogâmicos , Microscopia Confocal , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
FEBS J ; 276(7): 2074-83, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19292875

RESUMO

Besides esterase activity, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze o-nitroacetanilides through aryl acylamidase activity. We have reported that BuChE tetramers and monomers of human blood plasma differ in o-nitroacetanilide (ONA) hydrolysis. The homology in quaternary structure and folding of subunits in the prevalent BuChE species (G4(H)) of human plasma and AChE forms of fetal bovine serum prompted us to study the esterase and amidase activities of fetal bovine serum AChE. The k(cat)/K(m) values for acetylthiocholine (ATCh), ONA and its trifluoro derivative N-(2-nitrophenyl)-trifluoroacetamide (F-ONA) were 398 x 10(6) M(-1) min(-1), 0.8 x 10(6) M(-1) min(-1), and 17.5 x 10(6) M(-1) min(-1), respectively. The lack of inhibition of amidase activity at high F-ONA concentrations makes it unlikely that there is a role for the peripheral anionic site (PAS) in F-ONA degradation, but the inhibition of ATCh, ONA and F-ONA hydrolysis by the PAS ligand fasciculin-2 points to the transit of o-nitroacetalinides near the PAS on their way to the active site. Sedimentation analysis confirmed substrate hydrolysis by tetrameric 10.9S AChE. As compared with esterase activity, amidase activity was less sensitive to guanidine hydrochloride. This reagent led to the formation of 9.3S tetramers with partially unfolded subunits. Their capacity to hydrolyze ATCh and F-ONA revealed that, despite the conformational change, the active site architecture and functionality of AChE were partially retained.


Assuntos
Acetanilidas/metabolismo , Acetilcolinesterase/metabolismo , Acetiltiocolina/metabolismo , Acetanilidas/química , Acetilcolinesterase/sangue , Acetiltiocolina/química , Animais , Bovinos , Guanidina/farmacologia , Hidrólise , Cinética
11.
Chem Biol Interact ; 175(1-3): 332-5, 2008 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-18571151

RESUMO

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.


Assuntos
Envelhecimento/metabolismo , Encéfalo/enzimologia , Butirilcolinesterase/metabolismo , RNA Mensageiro/genética , Animais , Camundongos
12.
Chem Biol Interact ; 175(1-3): 340-2, 2008 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-18482720

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Neoplasias Renais/enzimologia , Humanos , Neoplasias Renais/classificação , Neoplasias Renais/patologia
13.
Biol Chem ; 389(4): 425-32, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18208347

RESUMO

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.


Assuntos
Amidoidrolases/metabolismo , Butirilcolinesterase/metabolismo , Acetanilidas/química , Acetanilidas/metabolismo , Amidoidrolases/sangue , Butirilcolinesterase/sangue , Colinesterases/sangue , Colinesterases/metabolismo , Colo/metabolismo , Humanos , Rim/metabolismo , Estrutura Molecular , Especificidade por Substrato
14.
Neurochem Int ; 50(3): 531-9, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17178175

RESUMO

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.


Assuntos
Butirilcolinesterase/metabolismo , Laminina/fisiologia , Timo/enzimologia , Animais , Butirilcolinesterase/isolamento & purificação , Laminina/genética , Camundongos
15.
J Mol Neurosci ; 30(1-2): 51-4, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17192624

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Adenocarcinoma/enzimologia , Butirilcolinesterase/metabolismo , Neoplasias do Colo/enzimologia , Colo/enzimologia , Humanos , Cinética , Reto/enzimologia , Valores de Referência
16.
Int J Biochem Cell Biol ; 38(7): 1092-101, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16412684

RESUMO

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.


Assuntos
5'-Nucleotidase/metabolismo , Ativação Enzimática/genética , Laminina/deficiência , Fígado/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/metabolismo , 5'-Nucleotidase/análise , Animais , Laminina/química , Laminina/genética , Fígado/química , Camundongos , Camundongos Mutantes/genética , Músculo Esquelético/química , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Miocárdio/química , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise
17.
Anal Biochem ; 348(1): 64-8, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16289445

RESUMO

An electrothermal atomic absorption spectrometric procedure for zinc determination in animal tissues is first optimized and then used to measure the metal content of different tissues from normal and dystrophic mice. The procedure involves minimal manipulation of the sample to overcome the severe contamination problems normally associated with the measurement of low zinc levels. The samples (recommended amount 10 mg) are slurried in 2 ml of a 10-mM tetramethylammonium hydroxide solution containing 0.1 g/100 ml silicone antifoam. After mild heating at 60 degrees C for 10 min and homogenization for 1 min, the suspensions are submitted to a 10-fold dilution and then injected into the electrothermal atomizer. Calibration is carried out using aqueous standard solutions of zinc prepared in the same suspension media. The reliability of the whole procedure is verified using three certified reference materials.


Assuntos
Zinco/análise , Animais , Encéfalo/metabolismo , Eletroquímica , Rim/química , Fígado/química , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/química , Distrofia Muscular Animal , Sensibilidade e Especificidade , Espectrofotometria Atômica/métodos , Fatores de Tempo
18.
J Neurochem ; 95(4): 1035-46, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16135075

RESUMO

Half of congenital muscular dystrophy cases arise from laminin alpha2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean +/- SD 1.42 +/- 0.28 micromol acetylthiocholine/h/mg protein, U/mg) was decreased by approximately 50% in dystrophic thymus (0.77 +/- 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT-PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (G2A, 64%) and monomers (G1A, 19%), as well as hydrophilic tetramers (G4H, 9%) and monomers (G1H, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.


Assuntos
Acetilcolinesterase/metabolismo , Laminina/deficiência , Distrofias Musculares/metabolismo , Timo/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/genética , Animais , Northern Blotting/métodos , Western Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Eletroquímica/métodos , Imunoprecipitação/métodos , Lectinas/metabolismo , Camundongos , Distrofia Muscular Animal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Timo/enzimologia
19.
Neurochem Int ; 45(1): 129-39, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15082230

RESUMO

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.


Assuntos
Acetilcolinesterase/metabolismo , Baço/enzimologia , Acetilcolinesterase/análise , Animais , Ativação Enzimática/fisiologia , Lectinas/metabolismo , Camundongos , Ligação Proteica/fisiologia , Baço/química , Baço/citologia
20.
Int J Biochem Cell Biol ; 36(3): 422-33, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14687921

RESUMO

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.


Assuntos
5'-Nucleotidase/metabolismo , Laminina/deficiência , Fígado/enzimologia , Sefarose/análogos & derivados , 5'-Nucleotidase/química , 5'-Nucleotidase/isolamento & purificação , Animais , Western Blotting , Laminina/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Músculo Esquelético/enzimologia , Nucleotidases/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Tripsina
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