Grubic Z

General

Full name : Grubic Zoran

First name : Zoran

Mail : Institute for Pathophysiology, School of Medicine, University of Ljubljana, Zaloska cesta 4, P.O. Box 2211

Zip Code : 1001 Ljubljana

City : Ljubljana

Country : Slovenia

Email : Zoran.Grubic@mf.uni-lj.si

Phone : +38615437038

Fax : (386-61)302272

Website :

Directory :

References (52)

Title : In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle - Mis_2017_Molecules_22_
Author(s) : Mis K , Grubic Z , Lorenzon P , Sciancalepore M , Mars T , Pirkmajer S
Ref : Molecules , 22 : , 2017
Abstract : Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and alpha-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.
ESTHER : Mis_2017_Molecules_22_
PubMedSearch : Mis_2017_Molecules_22_
PubMedID: 28846617

Title : Non-synaptic roles of acetylcholinesterase and agrin - Gros_2014_J.Mol.Neurosci_53_454
Author(s) : Gros K , Parato G , Pirkmajer S , Mis K , Podbregar M , Grubic Z , Lorenzon P , Mars T
Ref : Journal of Molecular Neuroscience , 53 :454 , 2014
Abstract : Proteins in living organisms have names that are usually derived from their function in the biochemical system their discoverer was investigating. Typical examples are acetylcholinesterase and agrin; however, for both of these, various other functions that are not related to the cholinergic system have been revealed. Our investigations have been focused on the alternative roles of acetylcholinesterase and agrin in the processes of muscle development and regeneration. Previously, we described a role for agrin in the development of excitability in muscle contraction. In this study, we report the effects of agrin on secretion of interleukin 6 in developing human muscle. At the myoblast stage, agrin increases interleukin 6 secretion. This effect seems to be general as it was observed in all of the cell models analysed (human, mouse, cell lines). After fusion of myoblasts into myotubes, the effects of agrin are no longer evident, although agrin has further effects at the innervation stage, at least in in vitro innervated human muscle. These effects of agrin are another demonstration of its non-synaptic roles that are apparently developmental-stage specific. Our data support the view that acetylcholinesterase and agrin participate in various processes during development of skeletal muscle.
ESTHER : Gros_2014_J.Mol.Neurosci_53_454
PubMedSearch : Gros_2014_J.Mol.Neurosci_53_454
PubMedID: 24326956

Title : Acetylcholinesterase and agrin: Different functions, similar expression patterns, multiple roles - Mis_2013_Chem.Biol.Interact_203_297
Author(s) : Mis K , Matkovic U , Pirkmajer S , Sciancalepore M , Lorenzon P , Mars T , Grubic Z
Ref : Chemico-Biological Interactions , 203 :297 , 2013
Abstract : Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.
ESTHER : Mis_2013_Chem.Biol.Interact_203_297
PubMedSearch : Mis_2013_Chem.Biol.Interact_203_297
PubMedID: 23117006

Title : The cholinergic and non-cholinergic effects of organophosphates and oximes in cultured human myoblasts - Katalinic_2013_Chem.Biol.Interact_203_144
Author(s) : Katalinic M , Mis K , Pirkmajer S , Grubic Z , Kovarik Z , Mars T
Ref : Chemico-Biological Interactions , 203 :144 , 2013
Abstract : Organophosphorus compounds (OPs) and oximes may interfere with other molecules than AChE in the living systems, affecting in this way various cellular processes and underlying mechanisms. These non-cholinergic effects may contribute to the clinical status in OP poisoning and therefore deserve equal scientific attention. Here, we investigated the effects of tabun and oxime K048 on the processes known to be involved in muscle response to the environmental factors, like IL-6 release and the regulation of the heat shock proteins (HSPs). While IL-6 stimulates muscle regeneration, which follows well known OP-induced myopathy, HSPs have cytoprotective effect against various stress factors including xenobiotics. All our experiments were carried out on cultured human myoblasts, as the precursors of muscle regeneration. We found unchanged AChE mRNA level after tabun/K048 treatment meaning that tabun and K048 did not interfere with the transcription or stability of this mRNA in the time period tested, even if AChE catalytic activity was significantly affected. On the other hand, after myoblast exposure to tabun, we observed significant changes in the protein levels of HSP 27 and in the secretion of IL-6. Namely, secretion of IL-6 decreased to 53% and the level of HSP 27 increased by 34% compared to the control level. Both effects were attenuated if myoblasts were pretreated with oxime K048, but not if they were treated with K048 after exposure to tabun. The molecular mechanism underlying these effects remains to be elucidated. However, it seems that these effects could be associated with OPs and oximes as a specific group of compounds rather than as a specific compound itself. Overall, the effects of OPs and oximes demonstrated here might play an important role in muscle regeneration which importantly determines the final outcome of OP myotoxicity.
ESTHER : Katalinic_2013_Chem.Biol.Interact_203_144
PubMedSearch : Katalinic_2013_Chem.Biol.Interact_203_144
PubMedID: 23047024

Title : Acetylcholinesterase is involved in apoptosis in the precursors of human muscle regeneration - Pegan_2010_Chem.Biol.Interact_187_96
Author(s) : Pegan K , Matkovic U , Mars T , Mis K , Pirkmajer S , Brecelj J , Grubic Z
Ref : Chemico-Biological Interactions , 187 :96 , 2010
Abstract : The best established role of acetylcholinesterase (EC 3.1.1.7, AChE) is termination of neurotransmission at cholinergic synapses. However, AChE is also located at sites, where no other cholinergic components are present and there is accumulating evidence for non-cholinergic functions of this protein. In the process of skeletal muscle formation, AChE is expressed already at the stage of mononuclear myoblast, which is long before other cholinergic components can be demonstrated in this tissue. Myoblast proliferation is an essential step in muscle regeneration and is always accompanied by apoptosis. Since there are several reports demonstrating AChE participation in apoptosis one can hypothesize that early AChE expression in myoblasts reflects the development of the apoptotic apparatus in these cells. Here we tested this hypothesis by following the effect of siRNA AChE silencing on apoptotic markers and by determination of AChE level after staurosporine-induced apoptosis in cultured human myoblasts. Decreased apoptosis in siRNA AChE silenced myoblasts and increased AChE expression in staurosporine-treated myoblasts confirmed AChE involvement in apoptosis. The three AChE splice variants were differently affected by staurosporine-induced apoptosis. The hydrophobic (H) variant appeared unaffected, a tendency towards increase of tailed (T) variant was detected, however the highest, 8-fold increase was observed for readthrough (R) variant. In the light of these findings AChE appears to be a potential therapeutic target at muscle injuries including organophosphate myopathy.
ESTHER : Pegan_2010_Chem.Biol.Interact_187_96
PubMedSearch : Pegan_2010_Chem.Biol.Interact_187_96
PubMedID: 20338155

Title : Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures - Gajsek_2008_Chem.Biol.Interact_175_50
Author(s) : Gajsek N , Jevsek M , Mars T , Mis K , Pirkmajer S , Brecelj J , Grubic Z
Ref : Chemico-Biological Interactions , 175 :50 , 2008
Abstract : Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest "fractionation of movement" capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.
ESTHER : Gajsek_2008_Chem.Biol.Interact_175_50
PubMedSearch : Gajsek_2008_Chem.Biol.Interact_175_50
PubMedID: 18691702

Title : Effects of acetylcholinesterase gene silencing on its activity in cultured human skeletal muscle - Mis_2006_J.Mol.Neurosci_30_31
Author(s) : Mis K , Mars T , Golicnik M , Jevsek M , Grubic Z
Ref : Journal of Molecular Neuroscience , 30 :31 , 2006
Abstract : In spite of several reports demonstrating that acetylcholinesterase (AChE [EC 3.1.1.7]) expression is importantly regulated at the level of its mRNA, we still know little about the relationship between AChE mRNA level and the level of mature, catalytically active enzyme in the cell. Better insight into this relationship is, however, essential for our understanding of the molecular pathways underlying AChE synthesis in living cells. We have approached this problem previously (Grubic et al., 1995; Brank et al., 1998; Mis et al., 2003; Jevsek et al., 2004); however, recently introduced small interfering RNA (siRNA) methodology, which allows blockade of gene expression at the mRNA level, opens new possibilities in approaching the AChE mRNA-AChE activity relationship. With this technique one can eliminate AChE mRNA in the cell, specifically and at selected times, and follow the effects of such treatment at the mature enzyme level. In this study we followed AChE activity in siRNA-treated cultured human myoblasts. Our aim was to find out how the temporal profile of the AChE mRNA decrease is reflected at the level of AChE activity under normal conditions and after inhibition of preexisting AChE by diisopropyl phosphorofluoridate (DFP).AChE activity was determined at selected time intervals after siRNA treatment in both myoblast homogenates and in culture medium to follow the effects of siRNA treatment at the level of intracellular AChE synthesis and at the level of AChE secreted from the cell.
ESTHER : Mis_2006_J.Mol.Neurosci_30_31
PubMedSearch : Mis_2006_J.Mol.Neurosci_30_31
PubMedID: 17192616

Title : Expression of MuSK in in vitro-innervated human muscle - Gajsek_2006_J.Mol.Neurosci_30_27
Author(s) : Gajsek N , Jevsek M , Grubic Z
Ref : Journal of Molecular Neuroscience , 30 :27 , 2006
Abstract : Unlike rodent or avian muscle, which forms clusters of acetylcholine receptors (AChRs) on its surface, exhibits cross striations, and contracts spontaneously even if cultured in the absence of the nerve, human muscle must be innervated to reach such differentiation level under in vitro conditions (Kobayashi and Askanas, 1985; Mars et al., 2001). Because it is known that AChR clustering and other aspects of neuromuscular junction (NMJ) formation necessitate the activation of muscle-specific kinase (MuSK), one explanation of this inability of human muscle is that it has no MuSK or that it cannot be activated in the absence of the nerve. To test this hypothesis we analyzed cultured human muscle for the expression of MuSK at two stages of differentiation: postfusion myotube and innervated, contracting myotube. Analyses were carried out at the mRNA level, as no reliable anti-MuSK antibodies are available for the immunocytochemical demonstration of MuSK in cultured human muscle. The presence of MuSK, however, can be tested indirectly, as it can be activated in the absence of the nerve simply by growing muscle culture on laminin coating (Kummer et al., 2004). In the second part of our study, we therefore tested human myotubes for the presence and activation of MuSK by exposing them to laminin coating and by analyzing them afterwards for the areas of postsynaptic differentiation typical for NMJ formation.
ESTHER : Gajsek_2006_J.Mol.Neurosci_30_27
PubMedSearch : Gajsek_2006_J.Mol.Neurosci_30_27
PubMedID: 17192614

Title : Expression and distribution of acetylcholinesterase among the cellular components of the neuromuscular junction formed in human myotube in vitro - Mis_2005_Chem.Biol.Interact_157-158_29
Author(s) : Mis K , Mars T , Jevsek M , Strasek H , Golicnik M , Brecelj J , Komel R , King MP , Miranda AF , Grubic Z
Ref : Chemico-Biological Interactions , 157-158 :29 , 2005
Abstract : The results of our recent investigations on the expression and distribution of acetylcholinesterase (EC. 3.1.1.7, AChE) in the experimental model of the in vitro innervated human muscle are summarized and discussed here. This is the only model allowing studies on AChE expression at all stages of the neuromuscular junction (NMJ) formation in the human muscle. Since it consists not only of the motor neurons and myotubes but also of glial cells, which are essential for the normal development of the motor neurons, NMJs become functional and differentiated in this system. We followed AChE expression at various stages of the NMJ formation and in the context of other events characteristic for this process. Neuronal and muscular part were analysed at both, mRNA and mature enzyme level. AChE is expressed in motor neurons and skeletal muscle at the earliest stages of their development, long before NMJ starts to form and AChE begins to act as a cholinergic component. Temporal pattern of AChE mRNA expression in motor neurons is similar to the pattern of mRNA encoding synaptogenetic variant of agrin. There are no AChE accummulations at the NMJ at the early stage of its formation, when immature clusters of nicotinic receptors are formed at the neuromuscular contacts and when occasional NMJ-mediated contractions are already observed. The transformation from immature, bouton-like neuromuscular contacts into differentiated NMJs with mature, compact receptor clusters, myonuclear accumulations and dense AChE patches begins at the time when basal lamina starts to form in the synaptic cleft. Our observations support the concept that basal lamina formation is the essential event in the transformation of immature neuromuscular contact into differentiated NMJ, with the accumulation of not only muscular but also neuronal AChE in the synaptic cleft.
ESTHER : Mis_2005_Chem.Biol.Interact_157-158_29
PubMedSearch : Mis_2005_Chem.Biol.Interact_157-158_29
PubMedID: 16256091

Title : Expression of acetylcholinesterase in the developing neuromuscular junction in the in vitro innervated human muscle -
Author(s) : Jevsek M , Mars T , Mis K , King MP , Davidson E , Park H , Miranda AF , Grubic Z
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :7 , 2004
PubMedID:

Title : Formation of neuromuscular junctions in co-cultures of human muscle and rat embryonic spinal cord is accompanied by differentiation of neuronal and glial cells. -
Author(s) : Mars T , Yu KJ , Tang XM , Miranda AF , Cambi F , Grubic Z , King MP
Ref : Cholinergic Mechanisms, CRC Press :635 , 2004
PubMedID:

Title : Poster (86) Human muscle forms functional neuromuscular junctions in cocultures with the embryonic spinal cord explants of rat, mouse and human -
Author(s) : Jevsek M , Mars T , Mis K , King MP , Yu KJ , Grubic Z
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :366 , 2004
PubMedID:

Title : Poster (4) Expression of AChE at the developing neuromuscular junction in human skeletal muscle in vitro. -
Author(s) : Mis K , Jevsek M , Mars T , King MP , Davidson E , Park H , Miranda AF , Grubic Z
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :323 , 2004
PubMedID:

Title : Control of expression of nicotinic receptor and acetylcholinesterase in the developing neuromuscular junction in the human muscle. -
Author(s) : Grubic Z , Mis K , Jevsek M , Brank M , King MP , Miranda AF , Brecelj J , Mars T
Ref : Cholinergic Mechanisms, CRC Press :113 , 2004
PubMedID:

Title : mRNAs encoding acetylcholinesterase, butyrylcholinesterase and agrin 19 share the same temporal pattern of expression during development of rat spinal cord. -
Author(s) : Mis K , Davidson E , Park H , King MP , Mars T , Jevsek M , Brank M , Grubic Z
Ref : Cholinergic Mechanisms, CRC Press :641 , 2004
PubMedID:

Title : Origin of acetylcholinesterase in the in vitro formed neuromuscular junction. -
Author(s) : Jevsek M , Mars T , Mis K , Grubic Z
Ref : Cholinergic Mechanisms, CRC Press :597 , 2004
PubMedID:

Title : Origin of acetylcholinesterase in the neuromuscular junction formed in the in vitro innervated human muscle - Jevsek_2004_Eur.J.Neurosci_20_2865
Author(s) : Jevsek M , Mars T , Mis K , Grubic Z
Ref : European Journal of Neuroscience , 20 :2865 , 2004
Abstract : Synaptic basal lamina is interposed between the pre- and postsynaptic membrane of the neuromuscular junction (NMJ). This position permits deposition of basal lamina-bound NMJ components of both neuronal and muscle fibre origin. One such molecule is acetylcholinesterase (AChE). The origin of NMJ AChE has been investigated previously as the answer would elucidate the relative contributions of muscle fibers and motor neurons to NMJ formation. However, in the experimental models used in prior investigations either the neuronal or muscular components of the NMJs were removed, or the NMJs were poorly differentiated. Therefore, the question of AChE origin in the intact and functional NMJ remains open. Here, we have approached this question using an in vitro model in which motor neurons, growing from embryonic rat spinal cord explants, form well differentiated NMJs with cultured human myotubes. By immunocytochemical staining with species-specific anti-AChE antibodies, we are able to differentiate between human (muscular) and rat (neuronal) AChE at the NMJ. We observed strong signal at the NMJ after staining with human AChE antibodies, which suggests a significant muscular AChE contribution. However, a weaker, but still clearly recognizable signal is observed after staining with rat AChE antibodies, suggesting a smaller fraction of AChE was derived from motor neurons. This is the first report demonstrating that both motor neuron and myotube contribute synaptic AChE under conditions where they interact with each other in the formation of an intact and functional NMJ.
ESTHER : Jevsek_2004_Eur.J.Neurosci_20_2865
PubMedSearch : Jevsek_2004_Eur.J.Neurosci_20_2865
PubMedID: 15579140

Title : Localization of mRNAs encoding acetylcholinesterase and butyrylcholinesterase in the rat spinal cord by nonradioactive in situ hybridization - Mis_2003_J.Histochem.Cytochem_51_1633
Author(s) : Mis K , Mars T , Jevsek M , Brank M , Zajc-Kreft K , Grubic Z
Ref : Journal of Histochemistry & Cytochemistry , 51 :1633 , 2003
Abstract : In spite of intensive investigations, the roles of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8) in the central nervous system (CNS) remain unclear. A role recently proposed for BuChE as an explanation for survival of AChE knockout mice is compensation for AChE activity if it becomes insufficient. Neuronal contribution of both enzymes to the cholinesterase pool in the neuromuscular junction has also been suggested. These proposals imply that BuChE expression follows that of AChE and that, in addition to AChE, BuChE is also expressed in alpha-motor neurons. However, these assumptions have not yet been properly tested. Histochemical approaches to these problems have been hampered by a number of problems that prevent unambiguous interpretation of results. In situ hybridization (ISH) of mRNAs encoding AChE and BuChE, which is the state-of-the-art approach, has not yet been done. Here we describe rapid nonradioactive ISH for the localization of mRNAs encoding AChE and BuChE. Various probes and experimental conditions had been tested to obtain reliable localization. In combination with RT-PCR, ISH revealed that, in rat spinal cord, cells expressing AChE mRNA also express BuChE mRNA but in smaller quantities. alpha-Motor neurons had the highest levels of both mRNAs. Virtual absence of transcripts encoding AChE and BuChE in glia might reflect a discrepancy between mRNA and enzyme levels previously reported for cholinesterases.
ESTHER : Mis_2003_J.Histochem.Cytochem_51_1633
PubMedSearch : Mis_2003_J.Histochem.Cytochem_51_1633
PubMedID: 14623931

Title : Kinetic Model of Ethopropazine Interaction with Horse Serum Butyrylcholinesterase and Its Docking into the Active Site - Golicnik_2002_Arch.Biochem.Biophys_398_23
Author(s) : Golicnik M , Sinko G , Simeon-Rudolf V , Grubic Z , Stojan J
Ref : Archives of Biochemistry & Biophysics , 398 :23 , 2002
Abstract : The action of a potent tricyclic cholinesterase inhibitor ethopropazine on the hydrolysis of acetylthiocholine and butyrylthiocholine by purified horse serum butyrylcholinesterase EC 3.1.1.8 was investigated at 25 and 37 C The enzyme activities were measured on a stopped-flow apparatus and the analysis of experimental data was done by applying a six-parameter model for substrate hydrolysis The model which was introduced to explain the kinetics of Drosophila melanogaster acetylcholinesterase Stojan et al 1998 FEBS Lett 440 85?88 is defined with two dissociation constants and four rate constants and can describe both cooperative phenomena apparent activation at low substrate concentrations and substrate inhibition by excess of substrate For the analysis of the data in the presence of ethopropazine at two temperatures we have enlarged the reaction scheme to allow primarily its competition with the substrate at the peripheral site but the competition at the acylation site was not excluded The proposed reaction scheme revealed upon analysis competitive effects of ethopropazine at both sites at 25 C three enzyme inhibitor dissociation constants could be evaluated at 37 C only two constants could be evaluated Although the model considers both cooperative phenomena it appears that decreased enzyme sensitivity at higher temperature predominantly for the ligands at the peripheral binding site makes the determination of some expected enzyme substrate and/or inhibitor complexes technically impossible The same reason might also account for one of the paradoxes in cholinesterases activities at 25 C at low substrate concentrations are higher than at 37 C Positioning of ethopropazine in the active-site gorge by molecular dynamics simulations shows that A328 W82 D70 and Y332 amino acid residues stabilize binding of the inhibitor 2002 Elsevier Science
ESTHER : Golicnik_2002_Arch.Biochem.Biophys_398_23
PubMedSearch : Golicnik_2002_Arch.Biochem.Biophys_398_23
PubMedID: 11811945

Title : Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Comparison to the radioactive technique - Zajc_2000_Pflugers.Arch_439_R66
Author(s) : Zajc Kreft K , Kreft S , Komel R , Grubic Z
Ref : Pflugers Arch , 439 :R66 , 2000
Abstract : A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.
ESTHER : Zajc_2000_Pflugers.Arch_439_R66
PubMedSearch : Zajc_2000_Pflugers.Arch_439_R66
PubMedID: 10653146

Title : Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Comparison to the radioactive technique - Kreft_2000_Pflugers.Arch_439_r066
Author(s) : Kreft KZ , Kreft S , Komel R , Grubic Z
Ref : Pflugers Arch , 439 :r066 , 2000
Abstract : A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.
ESTHER : Kreft_2000_Pflugers.Arch_439_r066
PubMedSearch : Kreft_2000_Pflugers.Arch_439_r066
PubMedID: 28176077

Title : Combined effects of glucocorticoids and electromechanical activity on the acetylcholinesterase expression in the fast rat muscle - Vodiskar_1999_Chem.Biol.Interact_119-120_333
Author(s) : Vodiskar J , Zgonc V , Ribaric S , Grubic Z
Ref : Chemico-Biological Interactions , 119-120 :333 , 1999
Abstract : Protein synthesis is impaired in the glucocorticoid (GC)-treated fast mammalian muscle. Electromechanical activity was reported to alleviate this effect. Acetylcholinesterase (AChE; EC 3.1.1.7) synthesis in the skeletal muscle is regulated by both, GCs and electromechanical activity. In light of the above reports, one would expect that electrical stimulation will prevent GC-mediated fall of AChE synthesis in the muscle. On the other hand, a substantial body of evidence suggests that electromechanical activity exerts its effect at the AChE mRNA level, while GCs most probably act at the translational or early posttranslational level. Different levels of action would be more consistent with the independent and therefore additive influences of the two regulatory factors. In order to ascertain whether glucocorticoid and electromechanical effects interact in the control of AChE activity, we compared the effects of GCs on normal, nonstimulated fast rat skeletal muscle, with those of GC-treated and simultaneously electrically stimulated (tonic pattern, 10 Hz) muscle. Untreated and stimulated-only muscles were used as respective controls. The effects on the fast extensor digitorum longus muscle and slow soleus muscle, treated similarly were compared. As expected, chronic GC treatment and electrical stimulation of fast rat muscles with slow activity patterns both downregulated AChE activity. However, no additional decrease in AChE activity was observed, if stimulated fast muscle was simultaneously treated with GCs, suggesting that slow pattern of electromechanical activity prevents GC-mediated downregulation of AChE. The most plausible explanation of this observation is, that muscle activity blocks expression of some generally acting factors, which are induced by GCs and are responsible for the impaired synthesis of several proteins including AChE.
ESTHER : Vodiskar_1999_Chem.Biol.Interact_119-120_333
PubMedSearch : Vodiskar_1999_Chem.Biol.Interact_119-120_333
PubMedID: 10421469

Title : Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site - Sentjurc_1999_Biochim.Biophys.Acta_1430_349
Author(s) : Sentjurc M , Pecar S , Stojan J , Marchot P , Radic Z , Grubic Z
Ref : Biochimica & Biophysica Acta , 1430 :349 , 1999
Abstract : Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.
ESTHER : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedSearch : Sentjurc_1999_Biochim.Biophys.Acta_1430_349
PubMedID: 10082962

Title : Morphometric characteristics of myonuclear distribution in the normal and denervated fast rat muscle fiber - Milanic_1999_Chem.Biol.Interact_119-120_321
Author(s) : Milanic T , Kunstelj A , Mars T , Grubic Z
Ref : Chemico-Biological Interactions , 119-120 :321 , 1999
Abstract : Unlike the majority of mammalian tissues, which have mononuclear cells as a basic unit of the tissue composition, skeletal muscle fiber is a polynuclear syncytium. Polynuclear organization offers an additional possibility for the regulation of protein expression: it can also be controlled at the level of myonuclear distribution and specialization. Distribution of myonuclei can be considered as the distribution of genes. Variability in gene concentration may have important impact on the regional differentiation of the muscle fiber, since it leads to different regional concentrations of various gene products including factors controlling their expression. The aim of the present study was: 1) to provide morphometrical data on the myonuclear distribution in the junctional and extrajunctional regions of the normal fast rat muscle fiber, and 2) to analyze, whether morphometrical parameters of nuclear distribution change after mechanical denervation of this muscle. Single muscle fibers were isolated from the normal and denervated fast rat m. sternomastoideus. Their neuromuscular junctions were stained by thiocholine histochemical procedure and myonuclei were fluorescently labeled by Hoechst 33342. Myonuclear distribution in each individual muscle fiber was morphometrically analyzed on the image analyzer. Synaptic concentrations of myonuclei were found to exceed extrasynaptic concentrations by a factor of 17. The number of myonuclei accumulated at the endplates did not change after one or two weeks of denervation, neither did the morphometric parameters of these nuclei. A higher concentration of myonuclei due to muscle atrophy was observed in the extrasynaptic region and the longitudinal axis of these nuclei also became significantly shorter. Unchanged morphometric parameters of the junctional myonuclei after denervation are indicative of either irreversibility of the nerve-induced formation of nuclear clusters in this region or persistence of the factors responsible for their formation and maintenance.
ESTHER : Milanic_1999_Chem.Biol.Interact_119-120_321
PubMedSearch : Milanic_1999_Chem.Biol.Interact_119-120_321
PubMedID: 10421467

Title : Localization of cells expressing AChE mRNA in rat striatum using nonradioactive in situ hybridization - Mis_1999_Chem.Biol.Interact_119-120_327
Author(s) : Mis K , Zajc-Kreft K , Grubic Z
Ref : Chemico-Biological Interactions , 119-120 :327 , 1999
Abstract : A better understanding of the role of AChE in mammalian brain requires knowledge of the distribution of AChE synthesizing cells in this tissue. The aim of the present study is to test a nonradioactive approach for the localization of AChE mRNA positive cells in rat striatum. Nonradioactive in situ hybridization has not been used before for the localization of this mRNA in mammalian brain. In order to find optimal conditions for localization, we employed both RNA and oligonucleotide probes. We also examined various prehybridization protocols and approaches. The total number of cells in brain sections was determined by subsequent fluorescent staining of the nuclei. Optimal AChE mRNA localization was obtained with a digoxigenine-labeled RNA probe. We were not able to localize AChE mRNA with nonradioactively 3' end-labeled oligonucleotides. An acetylation step prior to hybridization was found to be essential for optimal signal/background ratios; high nonspecific staining was observed, if this step was omitted. In accordance with reports of other authors, who used radioactive in situ hybridization, we found very low percentages of AChE mRNA-positive cells in striatum, although this area exhibits very high AChE staining. In comparison to radioactive techniques, the nonradioactive approach avoids the risks of radioactivity, and is much less time consuming. In our experiments AChE mRNA localization in striatum was practically the same as that demonstrated previously by radioactive approaches.
ESTHER : Mis_1999_Chem.Biol.Interact_119-120_327
PubMedSearch : Mis_1999_Chem.Biol.Interact_119-120_327
PubMedID: 10421468

Title : Glucocorticoids differentially control synthesis of acetylcholinesterase and butyrylcholinesterase in rat liver and brain - Weber_1999_Chem.Biol.Interact_119-120_341
Author(s) : Weber U , Brank M , Grubic Z
Ref : Chemico-Biological Interactions , 119-120 :341 , 1999
Abstract : Mammalian organisms possess two cholinesterases: acetylcholinesterase (AChE, EC 3.1.1.7.) and butyrylcholinesterase (BuChE, EC 3.1.1.8.). A clear explanation for this dual expression of acetylcholine-hydrolyzing enzymes is still missing. Better knowledge on how these two enzymes respond to various physiological or pharmacological factors would importantly contribute to the understanding of their function. The aim of the present study is to elucidate glucocorticoid (GC) influences on the synthesis of AChE and BuChE in rat liver and brain. Female Wistar rats were treated with dexamethasone until body weight loss was greater than 15%, signaling full expression of a GC response. At this stage, liver and brain were isolated and AChE and BuChE activities were determined in their homogenates. A new approach, based on precise radiometric measurements of AChE and BuChE activities in the polysomal fractions, prepared under non-denaturing conditions, was used to study GC influences on the early stages of biosynthesis of both enzymes. We found a differential GC influence on AChE and BuChE. In brain, only BuChE activity was affected (-30%), while AChE remained practically unchanged. In liver, BuChE activity fell by 60%, while AChE lost only 18% of its control activity. In case of BuChE, decreased activities in the whole homogenates correlated with decreased activities in the polysomal fractions, suggesting that early stages of enzyme biosynthesis were primarily affected. On the other hand, decreased AChE activity in liver homogenates was not paralleled by a significant change at the level of polysomal AChE activity in this organ, suggesting that higher AChE turn-over is primarily responsible for the decreased activity in homogenate. These results, together with the GC-mediated elimination of the correlation between brain and liver BuChE activities, strongly support the proposal of Edwards and Brimijoin (J.A. Edwards, S. Brimijoin, Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat, Biochem. Pharmacol. 32 (1983) 1183-1189) that BuChE is regulated by systemically acting factors, including various hormones, while regulation of AChE is primarily tissue-specific.
ESTHER : Weber_1999_Chem.Biol.Interact_119-120_341
PubMedSearch : Weber_1999_Chem.Biol.Interact_119-120_341
PubMedID: 10421470

Title : Control levels of acetylcholinesterase expression in the mammalian skeletal muscle - Grubic_1999_Chem.Biol.Interact_119-120_309
Author(s) : Grubic Z , Zajc-Kreft K , Brank M , Mars T , Komel R , Miranda AF
Ref : Chemico-Biological Interactions , 119-120 :309 , 1999
Abstract : Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.
ESTHER : Grubic_1999_Chem.Biol.Interact_119-120_309
PubMedSearch : Grubic_1999_Chem.Biol.Interact_119-120_309
PubMedID: 10421466

Title : 3.5 kb AChE mRNA is more sensitive to degradation than 2.3 kb AChE mRNA in the denervated fast rat skeletal muscle -
Author(s) : Grubic Z , Kreft S , Zajc-Kreft K
Ref : Journal de Physiologie (Paris) , 92 :435 , 1998
PubMedID:

Title : Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle - Brank_1998_Eur.J.Biochem_251_374
Author(s) : Brank M , Zajc-Kreft K , Kreft S , Komel R , Grubic Z
Ref : European Journal of Biochemistry , 251 :374 , 1998
Abstract : Acetylcholinesterase (AChE) is responsible for the hydrolysis of acetylcholine in the neuromuscular junction and other cholinergic synapses. Insight into the mechanisms controlling AChE expression in skeletal muscle is important for understanding formation, plasticity, and various dysfunctions of the neuromuscular junction. We have investigated the mechanisms responsible for the decreased AChE activity in the fast rat sternomastoideus muscle after chronic glucocorticoid treatment. Under such conditions fast skeletal muscles become atrophic and loose 30-40% of their AChE activity. In order to establish at which level synthesis of AChE is affected by glucocorticoids, we studied the effects of chronic dexamethasone treatment at both AChE mRNA and mature enzyme levels. Reduced rate of AChE recovery after subtotal irreversible AChE inhibition was observed during the first week of dexamethasone treatment, but not later. Statistical analyses of four independent northern blots revealed unchanged AChE mRNA levels. At the same time, we observed more than 60% decrease in the (G1+G2)/A12 ratio of molecular forms at the expense of G forms. It has been generally accepted that globular G1 and G2 molecular forms are synthesized in the rough endoplasmic reticulum as precursors of asymmetric (A) AChE forms, assembled in the Golgi apparatus. Reduced levels of G1 and G2 AChE forms, in combination with unchanged AChE mRNA, are therefore consistent with the reports demonstrating that glucocorticoids downregulate muscle protein synthesis at the translational level. Our findings support but not entirely prove the concept that impaired translation and/or posttranslational control are the primary cause of decreased AChE activity in the glucocorticoid-treated muscle.
ESTHER : Brank_1998_Eur.J.Biochem_251_374
PubMedSearch : Brank_1998_Eur.J.Biochem_251_374
PubMedID: 9492307

Title : Glucocorticoid Control of Acetylcholinesterase and Butyrylcholinesterase Expression in the Mammalian Organism -
Author(s) : Zajc-Kreft K , Brank M , Weber U , Grubic Z
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :79 , 1998
PubMedID:

Title : Degradation of AChE mRNA in the Normal and Denervated Rat Skeletal Muscle -
Author(s) : Zajc-Kreft K , Kreft S , Grubic Z
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :125 , 1998
PubMedID:

Title : Application of the nonradioactive in situ hybridization for the localization of acetylcholinesterase mRNA in the central nervous system of the rat\; comparison to the radioactive technique - Kreft_1996_Pflugers.Arch_431_R309
Author(s) : Kreft S , Zajc-Kreft K , Zivin M , Sket D , Grubic Z
Ref : Pflugers Arch , 431 :R309 , 1996
Abstract : In this preliminary report nonradioactive digoxigenine-based and radioactive in situ hybridization procedures for the localization of acetylcholinesterase mRNA were tested and compared in rat brain. General patterns of Ache mRNA localization observed by both techniques did not differ significantly and were practically the same as reported in previous in situ studies on the mammalian brain. Shorter procedure time and avoidance of precautions necessary at work with radioactive materials are major advantages of nonradioactive technique. Under- and over- staining can be prevented by direct examination of coloring reaction. Faint staining in the control experiment with heterologous DNA suggests that proper stringency is essential for the specificity of staining.
ESTHER : Kreft_1996_Pflugers.Arch_431_R309
PubMedSearch : Kreft_1996_Pflugers.Arch_431_R309
PubMedID: 8739388

Title : Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance - Grubic_1995_Biochim.Biophys.Acta_1249_155
Author(s) : Grubic Z , Stalc A , Sentjurc M , Pecar S , Gentry MK , Doctor BP
Ref : Biochimica & Biophysica Acta , 1249 :155 , 1995
Abstract : Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.
ESTHER : Grubic_1995_Biochim.Biophys.Acta_1249_155
PubMedSearch : Grubic_1995_Biochim.Biophys.Acta_1249_155
PubMedID: 7599168

Title : Myoblast fusion and innervation with rat motor nerve alter distribution of acetylcholinesterase and its mRNA in cultures of human muscle - Grubic_1995_Neuron_14_317
Author(s) : Grubic Z , Komel R , Walker WF , Miranda AF
Ref : Neuron , 14 :317 , 1995
Abstract : To elucidate the mechanisms underlying acetylcholinesterase (AChE) localization, we analyzed the distribution of AChE and Ache mRNA during myogenesis in cocultures of human muscle and fetal rat spinal cord. We observed a temporal coincidence in alterations of AChE localization and nuclei expressing the message, suggesting developmental regulation at the mRNA level. Nonuniform mRNA staining among nuclei suggests asynchronous regulation, also supporting an earlier proposal that transcription proceeds intermittently. Asynchrony seems to be overridden by generally acting factors during myoblast fusion, when message is up-regulated, and at the onset of muscle contractions, when it becomes restricted to some nuclei in the junctional region and focal patches of AChE appear near nerve contacts. Coincidence of mRNA down-regulation and synthesis of stable basal lamina-bound AChE suggests coordinated adaptation, so that sufficient enzyme may be derived from low message levels.
ESTHER : Grubic_1995_Neuron_14_317
PubMedSearch : Grubic_1995_Neuron_14_317
PubMedID: 7857641

Title : Developmental Expression of Acetylcholinesterase in Skeletal Muscle -
Author(s) : Grubic Z , Miranda AF
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :37 , 1995
PubMedID:

Title : Alterations in the Topography of Acetylcholinesterase Active Site Gorge after Binding of Peripheral Anionic Site Ligands -
Author(s) : Stalc A , Grubic Z , Sentjurc M , Pecar S , Gentry MK , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :125 , 1995
PubMedID:

Title : The influence of chronic treatment with dexamethasone on the acetylcholinesterase activity in rat skeletal muscle - Brank_1993_Chem.Biol.Interact_87_249
Author(s) : Brank M , Grubic Z
Ref : Chemico-Biological Interactions , 87 :249 , 1993
Abstract : In the conditions of chronically elevated glucocorticoid agents in plasma, a drop in AChE activity of about 45% was reported. This data suggests the possibility that among other factors glucocorticoids also control AChE activity in the skeletal muscles. The question addressed in the present investigation was if AChE activity was reduced uniformly or selectively in the rat skeletal muscles after chronic application of dexamethasone? Selective effects of glucocorticoids on the AChE activity in different muscles and/or different types or regions of muscles would suggest the potential of these agents to regulate AChE metabolism in the skeletal muscle according to the environmental demands. Specific activity of skeletal muscle AChE was reduced in sternomastoideus (SM), extensor digitorum longus (EDL) and diaphragm (D) but not in soleus (SOL) after chronic dexamethasone treatment. Axial SM (white part) was more affected than distal white muscle EDL. AChE was better preserved in red rather than in white parts of muscles. The endplate-free region lost twice as much of specific AChE activity than the endplate-rich region. Our results suggest, but do not prove that glucocorticoid agents act in a selective way on the AChE metabolism of the skeletal muscles.
ESTHER : Brank_1993_Chem.Biol.Interact_87_249
PubMedSearch : Brank_1993_Chem.Biol.Interact_87_249
PubMedID: 8343982

Title : Synthesis of the DNA probe for the determination of rat AChE mRNA - Grubic_1993_Chem.Biol.Interact_87_245
Author(s) : Grubic Z , Komel R
Ref : Chemico-Biological Interactions , 87 :245 , 1993
Abstract : This is a preliminary report on our attempts of synthesis by polymerase chain reaction (PCR), the cDNA probe for the determination of mRNA of the AChE catalytic subunit. As our strategy we took the advantage of the fact that sequence identity of AChE gene increases with phylogenetic proximity. Single codon usage could therefore be applied. Two non-degenerate PCR primers were synthesised corresponding to AChE regions which were highly conservative among species analyzed until now. The sequence amplified by these two primers should be 339 base pairs long as concluded from mouse AChE sequence. By determining the nucleotide sequence of the PCR product and by comparison of this sequence with the corresponding mouse AChE region, we would be able to verify the correspondence of our PCR product to the rat AChE gene fragment. Only the first four amino acids of our PCR product flanking Phe 200, which is the first amino acid from the A2 primer, are 100% homologous with the mouse AChE. However, from the next 18 amino acids towards the N-terminal, only 4 are homologous with the mouse AChE. Since we expected more than 90% homology between the phylogenetically closely related species of mouse and rat, we doubt that the DNA sequence obtained belongs to the rat AChE gene.
ESTHER : Grubic_1993_Chem.Biol.Interact_87_245
PubMedSearch : Grubic_1993_Chem.Biol.Interact_87_245
PubMedID: 8343981

Title : The effects of pretreatment with soman simulator in the skeletal muscle: direct interactions with acetylcholinesterase - Grubic_1993_Chem.Biol.Interact_87_253
Author(s) : Grubic Z , Brank M , Brzin M
Ref : Chemico-Biological Interactions , 87 :253 , 1993
Abstract : Soman simulator PDP is a compound that has a chemical structure identical to soman, except that the fluorine atom is replaced by a methyl group which makes PDP unable to bind covalently to the AChE active center. In rats, late mortality observed after treatment with high doses of soman could be prevented by PDP pretreatment. Such pretreatment has been much less efficient in primates. The effect of PDP in rats has been explained by blocking the deposition of soman in so-called soman depots in which soman is stored intact and subsequently released. In this paper we demonstrate that in the presence of PDP, inhibition of rat muscle AChE by soman is reduced in rat but not in human muscle homogenates. This result suggests that at least part of the beneficial effects of PDP pretreatment in rat might be due to the direct interaction of PDP with AChE resulting in reduced AChE phosphorylation by soman.
ESTHER : Grubic_1993_Chem.Biol.Interact_87_253
PubMedSearch : Grubic_1993_Chem.Biol.Interact_87_253
PubMedID: 8343983

Title : The influence of soman simulator on reactivation by HI-6 of soman-inhibited acetylcholinesterase in preparations of rat and human skeletal muscle - Brank_1993_Biochem.Pharmacol_45_499
Author(s) : Brank M , Sentjurc M , Stalc A , Grubic Z
Ref : Biochemical Pharmacology , 45 :499 , 1993
Abstract : The aim of our study was to elucidate the phenomenon called "soman depot". Our investigations were focused on the depot formed in the skeletal muscle and on the effects of 1,2,2-trimethylpropyl dimethylphosphonate (PDP), a reported blocker of soman depot formation. The following questions were addressed: (1) how much of acetylcholinesterase (EC 3.1.1.7, AChE) activity can additionally be recovered by Hagedorn bispiridinium oxime reactivator 2-hydroxyimino-methylpyridinium-1-methyl-4'-carbamoyl-pyridinium-1 '-methylether dichloride monohydrate (HI-6) in the skeletal muscle preparations if they are pretreated by PDP prior to incubation in soman (1,2,2-trimethylpropyl methylphosphonofluoridate)? (2) Is this effect uniform along the muscle fibre or different in the endplate in comparison to the endplate-free region? (3) Is the effect of PDP species specific, i.e. does it differ between rat and human muscle? (4) What are the molecular mechanisms of the effects of PDP? PDP pretreatment increased the reactivation of soman-inhibited AChE by HI-6 in both regions of rat skeletal muscle. This increase was smaller in human skeletal muscle. The PDP-mediated increase in HI-6 reactivation was most efficient in the endplate-rich region of rat diaphragm as demonstrated biochemically and histochemically, but it could not be explained by the blockade of soman depot alone since it was also observed at low soman concentrations, at which soman depot is not supposed to form. This PDP effect could be better explained by the direct interactions of PDP with AChE resulting in decreased AChE phosphorylation. Soman concentration-dependent increase in HI-6 reactivation by PDP, which was more efficient at a high than a low soman concentration and could therefore originate from blockade of soman depot, was observed in the endplate-free region of rat diaphragm. It was also found in human muscle but was again smaller in this species. According to our EPR study, solubilization of soman in the lipophilic cell membrane compartment can be excluded as a mechanism producing significant soman depot. In general, our results suggest a more complex mechanism of PDP action than reported previously.
ESTHER : Brank_1993_Biochem.Pharmacol_45_499
PubMedSearch : Brank_1993_Biochem.Pharmacol_45_499
PubMedID: 8382066

Title : Poster: Pretreatment with atropine retards aging and improves reactivatabilty of soman inhibited AChE in rat and human skeletal muscle preparations in vitro -
Author(s) : Grubic Z , Serko A
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :300 , 1991
PubMedID:

Title : Poster: Propidium, a peripheral anionic site binding ligand, retards aging and increases reactivatability of soman inhibited AChE in rat diaphragm in vitro -
Author(s) : Grubic Z
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :299 , 1991
PubMedID:

Title : Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle\; comparison to SAD-128 and PAM-2 - Grubic_1989_Arch.Toxicol_63_68
Author(s) : Grubic Z , Tomazic A
Ref : Archives of Toxicology , 63 :68 , 1989
Abstract : HI-6 is presently considered the most potent oxime antidote against soman poisoning in mice, rats, dogs and monkeys. However, it is still an open question whether efficiency of HI-6, observed in experimental animals, can be extrapolated to soman intoxicated humans. In this paper efficiency of HI-6 and possible mechanisms of action were compared in rat and human fresh muscle preparations. In rat muscle, about 50% of control AChE activity could be recovered by both therapeutic (5 min after soman) and prophylactic (5 min before soman) application of HI-6. On the other hand, in human muscle therapeutic treatment restored only 5%, while prophylactic application of HI-6 again resulted in about 50% recovery of control AChE activity. As revealed by comparison of the prophylactic effects of HI-6 and the non-oxime bispyridinium compound SAD-128, competitive inhibition of AChE plays a minor role as a protective mechanism. Immediate reactivation of rapidly aging human AChE must therefore be instituted for successful protective treatment by HI-6. Retardation of aging, a direct effect of SAD-128, was roughly estimated to improve reactivation by HI-6 for about 10% of control AChE activity of the human muscle. PAM-2 proved completely inefficient as a therapeutic and as a prophylactic agent on both rat and human muscle preparations.
ESTHER : Grubic_1989_Arch.Toxicol_63_68
PubMedSearch : Grubic_1989_Arch.Toxicol_63_68
PubMedID: 2742504

Title : Iso-OMPA-induced potentiation of soman toxicity in rat correlates with the inhibition of plasma carboxylesterases - Grubic_1988_Arch.Toxicol_62_398
Author(s) : Grubic Z , Sket D , Brzin M
Ref : Archives of Toxicology , 62 :398 , 1988
Abstract : Recently, the question was raised as to why iso-OMPA, generally known as a selective irreversible inhibitor of butyrylcholinesterase (BuChE), potentiates soman toxicity in rats but not in mice. Mice are known to have higher carboxylesterase (CarbE) and lower BuChE activity in plasma than rat. It could be hypothesized that it is the iso-OMPA inhibition of plasma CarbE, and not of BuChE, which is responsible for potentiation of soman toxicity in iso-OMPA-pretreated rats. In order to test this hypothesis two doses of iso-OMPA were administered to rats prior to soman. The two doses were selected in such a way that both were high enough to inhibit more than 90% of plasma BuChE activity; plasma CarbE activity, however, was only slightly inhibited by the lower and substantially by the higher dose of iso-OMPA. Our results demonstrate that iso-OMPA-induced potentiation of soman toxicity correlates with the inhibition of CarbE and not with the inhibition of BuChE activity in rat plasma. Relative resistance of mice to iso-OMPA-induced potentiation of soman toxicity could therefore be explained by a higher proportion of CarbE activity remaining uninhibited after iso-OMPA pretreatment. By having their active centers unoccupied, CarbE molecules can bind soman and reduce its concentration in neuronal tissue and motor end-plates.
ESTHER : Grubic_1988_Arch.Toxicol_62_398
PubMedSearch : Grubic_1988_Arch.Toxicol_62_398
PubMedID: 3242452

Title : Alpha-bungarotoxin binding to the myotome and choline acetyltransferase activity in the rabbit embryo - Grubic_1984_J.Comp.Neurol_222_452
Author(s) : Grubic Z , Tennyson VM , Chang HW , Kremzner LT , Penn AS
Ref : Journal of Comparative Neurology , 222 :452 , 1984
Abstract : We have previously found incomplete sarcomeres and acetylcholinesterase activity in the myoblasts of the myotome of the rabbit at day 13 of gestation. We now report that an acetylcholine (ACh)-synthesizing enzyme and the nicotinic receptor are present at this stage as well. A study of the myotome using [125I]alpha-bungarotoxin shows that the mononucleated myoblasts have alpha-bungarotoxin binding sites before they migrate away to form multinucleated myotubes. Choline acetylcholinesterase activity and/or a different ACh-synthesizing enzyme are found at early stages of development, even before the spinal nerve has formed. An ACh-synthesizing enzyme is present in the notochord, a neural tube-dorsal root ganglion preparation, as well as in rows of myotomes separated from the latter preparation. Assays of isolated myotomes with very little adherent mesenchyme indicate that the enzyme is located either within the myotome or in its immediate vicinity. Cholinergic components, therefore, are associated with the mononucleated myoblasts of the myotome before they fuse to form myotubes and before they receive their permanent innervation.
ESTHER : Grubic_1984_J.Comp.Neurol_222_452
PubMedSearch : Grubic_1984_J.Comp.Neurol_222_452
PubMedID: 6699213

Title : Cholinesterases in skeletal muscle and sympathetic ganglia -
Author(s) : Brzin M , Klinar B , Sketelj J , Kiauta T , Grubic Z , Sket D
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :259 , 1984
PubMedID:

Title : Poster 69. Tbe detoxication capacity of rat liver at soman concentrations present in the tissue after application of sublethal doses -
Author(s) : Grubic Z , Sketelj J , Brzin M
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984
PubMedID:

Title : Recovery of acetylcholinesterase in the diaphragm, brain, and plasma of the rat after irreversible inhibition by soman: a study of cytochemical localization and molecular forms of the enzyme in the motor end plate - Grubic_1981_J.Neurochem_37_909
Author(s) : Grubic Z , Sketelj J , Klinar B , Brzin M
Ref : Journal of Neurochemistry , 37 :909 , 1981
Abstract : Recovery of AChE activity in the motor end plate region and end plate free region of the rat diaphragm was studied after irreversible inhibition by soman. Recovery was slow during the first 2 days and only 4 S and 10 S molecular forms of AChE were present in the end plate region. However, cytochemical evidence indicates that synaptic AChE has already started to accumulate and that the synthesis of AChE in muscle and Schwann cell might even be enhanced. Tubular structures, observed underneath the motor end plate, may serve to transport the enzyme from its sites of synthesis in the sarcoplasmic reticulum. Asymmetric molecular forms of AChE in he end plate region appeared later during recovery and, one week after poisoning, their activity was only about 50% of normal value. The limited ability of newly synthesized AChE to attach to the subcellular structures and, therefore, be retained in the muscle, may explain the phase of slow recovery. In accordance with this view, AChE activity in brain recovered in a similar way as in muscle, whereas soluble plasma cholinesterases recovered faster, apparently without a slow initial phase.
ESTHER : Grubic_1981_J.Neurochem_37_909
PubMedSearch : Grubic_1981_J.Neurochem_37_909
PubMedID: 7320730

Title : Quantitative evaluation of the trapping reaction of copperthiocholine histochemical procedures for localization of cholinesterases - Grubic_1978_Histochem_56_213
Author(s) : Grubic Z , Brzin M
Ref : Histochemistry , 56 :213 , 1978
Abstract : The aim of the present study was to investigate whether the trapping reaction of the histochemical procedure for the localization of ChE of Koelle and Friedenwald (1949) and its modification by Brzin and Pucihar (1976) proceeds quantitatively. The weight of the precipitate formed in the tissue sample during the histochemical procedure was compared with enzyme activity of an equal sample. The differential magnetic microbalance was used for measurements of reduced weight and for previous determination of density of the precipitate. The evidence for the composition of the final product was drawn from the quantitative analysis of copper and iodine and from the infrared spectra. Tsuji's statement (1974) that cuprous copper thiocholine iodide is the final product of histochemical procedures investigated was confirmed. It was found that the trapping reaction of the original as well as of the modified procedure under our experimental conditions in tissue sections proceeds quantitatively which means that one of the basic conditions for reliable localization is fulfilled.
ESTHER : Grubic_1978_Histochem_56_213
PubMedSearch : Grubic_1978_Histochem_56_213
PubMedID: 689917

Title : Automation of a magnetic diver balance for reduced weight and microgasometric measurements -
Author(s) : Oman S , Grubic Z , Brzin M
Ref : Analytical Biochemistry , 83 :211 , 1977
PubMedID: 920939

Title : A radiometric method for the determination of choline acetylase activity based on thin-layer chromatography -
Author(s) : Grubic Z , Kiauta T , Brzin M
Ref : Analytical Biochemistry , 74 :354 , 1976
PubMedID: 962095

Title : Notes on the thin-layer chromatography radiometric assay of cholinesterase activity -
Author(s) : Grubic Z , Sketelj J , Brzin M
Ref : Analytical Biochemistry , 69 :306 , 1975
PubMedID: 1211634