Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
Differential regulation of FAK+ and PYK2/Cakbeta, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity
Eur J Neurosci 1998 10(5):1667-75
P Derkinderen, J Siciliano 1 , M Toutant, J A Girault
INSERM U 114, Chaire de Neuropharmacologie, Collège de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France 1 Departamento de Histología, Facultad de Medicina, 11800 Montevideo, Uruguay
DOI: 10.1046/j.1460-9568.1998.00174.x
PMID: 9751139
Pubmed: https://pubmed.ncbi.nlm.nih.gov/9751139
Texto completo: https://onlinelibrary.wiley.com/doi/10.1046/j.1460-9568.1998.00174.x
Abstract:
FAK+, an isoform of focal adhesion kinase preferentially expressed in brain and PYK2/Cakbeta (proline-rich tyrosine kinase 2/cell adhesion kinasebeta) are two related cytoplasmic tyrosine kinases. They are candidates for coupling electrical activity and stimulation of neurotransmitter receptors to short and long-term changes in synaptic properties, cytoskeletal organization and gene expression in neurons. As the same set of stimuli appear capable of stimulating FAK and/or PYK2 in non-neuronal cells and in cell lines with neuronal characteristics, we investigated the selectivity of regulation of these two kinases in mature nervous tissue. Using rat hippocampal slices, we compared the regulation of FAK+ and PYK2 by stimuli known to be active on one or the other of these two kinases in other cell types: lysophosphatidic acid (LPA), carbachol, depolarization, and hyperosmolarity. Phosphorylation of FAK+ was markedly increased by carbachol and LPA. Carbachol effects occurred via activation of M1 muscarinic receptors and nicotinic receptors. The effects of carbachol and LPA were prevented by protein kinase C inhibitors, whereas 8-Br-cAMP attenuated the effects of carbachol but not of LPA. Tyrosine phosphorylation of PYK2 but not of FAK+ was very strongly enhanced by depolarization and hyperosmolarity. This study and our previous results show that FAK+ and PYK2 are regulated differentially in hippocampal slices: FAK+ is phosphorylated on tyrosine in response to stimulation of G protein-coupled receptors, whereas PYK2 is mainly sensitive to depolarization and hyperosmolarity. Thus, FAK+ and PYK2 may provide specific and separate links between activation of neurotransmitters receptors, depolarization and tyrosine phosphorylation in mature hippocampus.
Analysis of nuclear degradation during lens cell differentiation
Cell Death Differ 1998 5(4):251-61
M F Counis 1 , E Chaudun 1 , C Arruti 2 , L Oliver 3 , M Sanwal 4 , Y Courtois 1 , A Torriglia 1
1 U450 INSERM, développement, vieillissement et pathologie de la rétine, Unité associée CNRS, Association Claude Bernard, 29 rue Wilhem, Paris, 75016, France 2 Laboratorio de Cultivo de Tejidos, Facultad de Medicina, Av General Flores 2125, Montevideo, 11800, Uruguay 3 INSERM U 419, Institut de Biologie, 9 quai Moncousu, Nantes, Cédex 01, 44035, France 4 Department of Biochemistry, University of Western Ontario, London, NGA 5C1, Canada
DOI: 10.1038/sj.cdd.4400351
PMID: 10200471
Pubmed: https://pubmed.ncbi.nlm.nih.gov/10200471
Texto completo: https://doi.org/10.1038/sj.cdd.4400351
Abstract:
Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.
Dephosphorylation of Ser-137 in DARPP-32 by protein phosphatases 2A and 2C: different roles in vitro and in striatonigral neurons
Biochem J 1998 330 ( Pt 1):211-6
F Desdouits 1 , J C Siciliano 1 2 , A C Nairn 3 , P Greengard 3 , J A Girault 1
1 INSERM U114, Chaire de Neuropharmacologie, Collège de France, Paris, France. 2 Departamento de Histologia, Facultad de Medicina, Montevideo, Uruguay 3 Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, U.S.A
DOI: 10.1042/bj3300211
PMID: 9461512
Pubmed: https://pubmed.ncbi.nlm.nih.gov/9461512
Texto completo: https://portlandpress.com/biochemj/article-abstract/330/1/211/36870/Dephosphorylation-of-Ser-137-in-DARPP-32-by?redirectedFrom=fulltext
Abstract:
DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr=32000) is highly expressed in striatonigral neurons in which its phosphorylation is regulated by several neurotransmitters including dopamine and glutamate. DARPP-32 becomes a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr-34 by cAMP- or cGMP-dependent protein kinases. DARPP-32 is also phosphorylated on Ser-137 by protein kinase CK1 (CK1), in vitro and in vivo. This phosphorylation has an important regulatory role since it inhibits the dephosphorylation of Thr-34 by calcineurin in vitro and in striatonigral neurons. Here, we show that DARPP-32 phosphorylated by CK1 is a substrate in vitro for protein phosphatases 2A and 2C, but not protein phosphatase 1 or calcineurin. However, in substantia nigra slices, dephosphorylation of Ser-137 was markedly sensitive to decreased temperature, and not detectably affected by the presence of okadaic acid under conditions in which dephosphorylation of Thr-34 by protein phosphatase 2A was inhibited. These results suggest that, in neurons, phospho-Ser-137-DARPP-32 is dephosphorylated by protein phosphatase 2C, but not 2A. Thus, DARPP-32 appears to be a component of a regulatory cascade of phosphatases in which dephosphorylation of Ser-136 by protein phosphatase 2C facilitates dephosphorylation of Thr-34 by calcineurin, removing the cyclic nucleotide-induced inhibition of protein phosphatase 1.
Differential regulation of proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) and pp125(FAK) by glutamate and depolarization in rat hippocampus
J Biol Chem 1996 271(46):28942-6
J C Siciliano 1 2 , M Toutant 1 , P Derkinderen 1 , T Sasaki 3 , J A Girault 1
1 INSERM U 114, Chaire de Neuropharmacologie, Collège de France, 11 place Marcelin Berthelot, 75231 Paris Cedex 05, France 2 Departamento de Histología, Facultad de Medicina, 11800 Montevideo, Uruguay 3 Department of Biochemistry, Cancer Research Institute, Sapporo Medical University, S-1 W-17, Sapporo 060, Japan
DOI: 10.1074/jbc.271.46.28942
PMID: 8910543
Pubmed: https://pubmed.ncbi.nlm.nih.gov/8910543
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(18)35018-X
Abstract:
The mechanisms by which stimuli that raise cytosolic free Ca2+ concentrations in neurons can increase protein tyrosine phosphorylation are not known. Using rat hippocampal slices and cortical synaptosomes, we have examined the regulation of two highly related cytoplasmic tyrosine kinases, pp125 focal adhesion kinase (pp125(FAK)) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta). Membrane depolarization increased tyrosine phosphorylation of PYK2/CAKbeta and pp125(FAK). These effects were blocked by EGTA or by protein kinase C inhibitors (RO31-8220; GF109203X) and mimicked by ionomycin or phorbol 12-myristate 13-acetate, in the case of pp125(FAK), or their combination in the case of PYK2/CAKbeta. Glutamate and specific agonists of ionotropic (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate) or metabotropic (trans-1-aminocyclopentane-1,3, -dicarboxylate) glutamate receptors stimulated the phosphorylation of pp125(FAK), but not of PYK2/CAKbeta. Glutamate effects were prevented by GF109203X. Thus, in hippocampal slices, tyrosine phosphorylation of pp125(FAK) and PYK2/CAKbeta are regulated differentially by pathways involving Ca2+ and protein kinase C. pp125(FAK) and PYK2/CAKbeta may provide specific links between neuronal activity, increases in cytosolic Ca2+ and protein tyrosine phosphorylation, which may be important for neuronal survival, and synaptic plasticity.
Regulation of a neuronal form of focal adhesion kinase by anandamide
Science 1996 273(5282):1719-22
P Derkinderen 1 , M Toutant 1 , F Burgaya 1 , M Le Bert 1 , J C Siciliano 1 2 , V de Franciscis 1 3 , M Gelman 1 , J A Girault 1
1 INSERM U 114, Chaire de Neuropharmacologie, Collège de France, 11 place Marcelin Berthelot, 75231 Paris cedex 05, France. 2 Present address: Departamento de Histología, Facultad de Medicina, Montevideo, Uruguay 3 On leave of absence from Centro di Endocrinologia ed Oncologia Sperimentale del CNR, Università di Napoli “Federico II,” Naples, Italy.
DOI: 10.1126/science.273.5282.1719
PMID: 8781236
Pubmed: https://pubmed.ncbi.nlm.nih.gov/8781236
Texto completo: https://www.science.org/doi/10.1126/science.273.5282.1719
Abstract:
Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinase is a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.
Intrinsic neuronal cell bodies in the rat ovary
Neurosci Lett 1996 205(1):65-7
The present study describes ganglia and isolated neurones in the ovary of the Wistar rat, employing histological and histochemical techniques. Four kinds of ganglia in the postpubertal and young adult rat were identified: the mesovarial, hilar, medullary and cortical ganglia. Isolated neurones were also found, being dispersed along blood vessels in the ovary medulla and near the follicles. The soma diameters of these neuronal cells ranged from 25 to 50 microns. In the prepubertal rat, only the mesovarial and hilar ganglia were observed. They contained small neurones with soma diameters ranging from 10 to 15 microns. NADPH-diaphorase activity was detected in some isolated neurones and in the cortical and hilar ganglia in all rats examined.
Laboratory of Biology of Reproduction, Department of Histology and Embryology, School of Medicine, Gral. Flores 2125, CP 11800, Montevideo, Uruguay
DOI: 10.1016/0304-3940(96)12361-2
PMID: 8867022
Pubmed: https://pubmed.ncbi.nlm.nih.gov/8867022
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/0304394096123612
Abstract:
The present study describes ganglia and isolated neurones in the ovary of the Wistar rat, employing histological and histochemical techniques. Four kinds of ganglia in the postpubertal and young adult rat were identified: the mesovarial, hilar, medullary and cortical ganglia. Isolated neurones were also found, being dispersed along blood vessels in the ovary medulla and near the follicles. The soma diameters of these neuronal cells ranged from 25 to 50 microns. In the prepubertal rat, only the mesovarial and hilar ganglia were observed. They contained small neurones with soma diameters ranging from 10 to 15 microns. NADPH-diaphorase activity was detected in some isolated neurones and in the cortical and hilar ganglia in all rats examined.
alpha-Crystallin polypeptides in developing chicken lens cells
Exp Eye Res 1995 61(2):181-7
A de Maria , C Arruti
Departamento de Biología Celular, Facultad de Ciencias and Laboratorio de Cultivo de Tejidos, Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800 Montevideo, Uruguay
DOI: 10.1016/s0014-4835(05)80038-7
PMID: 7556482
Pubmed: https://pubmed.ncbi.nlm.nih.gov/7556482
Texto completo: https://www.sciencedirect.com/science/article/abs/pii/S0014483505800387?via%3Dihub
Abstract:
We provide evidence that the different cells that form the chicken lens have isoelectric variants of alpha-crystallins at early and late developmental stages. We separated the alpha A and alpha B-crystallin subclasses by sodium dodecylsulphate polyacrylamide gel electrophoresis and then further resolved each by isoelectric focusing and assays with specific anti alpha-crystallin antibodies. We found that the annular pad, cortical and nuclear fibers, as well as the epithelial cells, contain alpha A and alpha B native chains and their respective isoelectric variants. These results on adult and embryonic lenses obtained a short time after the onset of alpha-crystallin expression suggest that lens cells, having different phenotypes, are able to produce post-translational modifications of the alpha A and alpha B chains as a part of their developmental program.
Neonatal acetylcholinesterase inhibition by fasciculin 2 in rats: a model for the study of central nervous system development?
Toxicon 1995 Jul;33(7):909-16
B Bolioli 1 , F Blasina 1, R Silveira 2, F Dajas 3
1 Histology Department, Faculty of Medicine, Institute de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, Montevideo, Uruguay 2 Cell Biology Division, Institute de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, Montevideo, Uruguay 3 Neurochemistry Division, Institute de Investigaciones Biológicas Clemente Estable, Avda. Italia 3318, Montevideo, Uruguay
DOI: 10.1016/0041-0101(95)00025-h
PMID: 8588215
Pubmed: https://pubmed.ncbi.nlm.nih.gov/8588215
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/0041-0101(95)00025-H
Abstract:
Fasciculin 2 (FAS), a potent acetylcholinesterase (AChE, EC 3.1.1.7) inhibitory peptide with affinity for the enzyme in the nanomolar range was utilized together with two other AChE inhibitors (Paroxon and BW284c51) to study the role of AChE in central nervous system development. When drugs were intracisternally injected at postnatal days 3 and 5, only FAS showed a significant inhibition of hippocampus and striatum AChE (39% and 77% inhibition, respectively). After FAS treatment, animals showed convulsive behaviour which was blocked by subcutaneous pretreatment with atropine sulfate (10 mg/kg). An assessment of developmental indices showed no alteration in neurological reflex maturation, motor behaviour or cell morphology. Body weight gain was significantly lower only in FAS-treated animals compared to controls during the preweaning period. To investigate the specificity of this effect a synthetic loop of FAS (showing no activity in vitro or in vivo) and oxidized FAS (showing a weak inhibition in vitro and no activity in vivo) were also intracisternally injected. Animals injected with the loop showed normal body weight development while those treated with oxidized FAS showed impairment in body weight. In conclusion, FAS was the most potent drug at inhibiting neonatal AChE in vivo without nonspecific brain damage. Impairment in body weight seems to be dependent on AChE involvement, although the possibility of a direct FAS effect is discussed. These results point to FAS intracisternal treatment as a useful in vivo model to study the role of AChE in the critical period of early postnatal central nervous system development.
Characterisation of eye-lens DNases: long term persistence of activity in post apoptotic lens fibre cells
Cell Death Differ 1995 2(1):47-56
C Arruti 1 , E Chaudun 2 , A De Maria 1 , Y Courtois 2 , M F Counis 3
1 Departamento de Biologia Celular, Facultad de Ciencias y Laboratorio de Cultivo de Tejidos, Facultad de Medicina, Avda, Gral, Flores 2125, Montevideo, Uruguay 2 Unité de Recherches Gérontologique, CNRS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France 3 Unité 118 INSERM, 29 rue Wilhem, 75016, Paris, France
DOI:
PMID: 17180015
Pubmed: https://pubmed.ncbi.nlm.nih.gov/17180015
Texto completo:
Abstract:
Fibre cells in the ocular lens exhibit a constitutive apoptotic process of nuclear degradation that includes chromatin breakage, generating a ladder pattern of DNA fragments. This process is intrinsic to the normal terminal differentiation program. Despite the loss of nucleus and cytoplasmic organelles, the terminal differentiated fibre cells remain in the lens during the whole life span of the individual. The lens cells thus provide a unique system in which to determine the presence and fate of endonucleases once the chromatin has been cleaved. We report here on the presence of DNase activity in nucleated and anucleated lens cells. Using a nuclease gel assay and double-stranded DNA as substrate, we found active 30 and 60 kDa DNases. The enzymatic activities were Ca(2+), Mg(2+) dependent, and active at neutral pH. The relative amount of these forms changed during development and aging of the lens fibre cells. Both forms were inhibited by Zn(2+), aurintricarboxylic acid, and G-actin. The proteins were also separated by SDS-PAGE, renatured after removing SDS and incubated in the presence of native DNA adsorbed to a membrane. Therefore it was possible to demonstrate, by means of a nick translation reaction, that the enzymes produced single strand cuts. Based on these findings we propose that these chick lens nucleases are probably related to DNase I.
DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease
J Cell Physiol 1994 158(2):354-64
E Chaudun 1 , C Arruti 2 , Y Courtois 1 , F Ferrag 1 , J C Jeanny 1 , B N Patel 3 , C Skidmore 3 , A Torriglia 1 , M F Counis 1
1 Unité de Recherches Cerontologiques 118 INSERM, UA 630 CRNS, Association Claude Bernard, 29 rue Wilhem, 75016 Paris, France (E.C., Y.C., F.F., I.C.)., A.T., M.F.C.); 2 Laboratorio de Cultivo de Tejidos, Departarnento de Histologia y Embriologia, Facultad de Medicina, Montevideo, Uruguay, (C.A.); 3 Department of Biochemistry and Physiology, University of Reading, Reading RC6 LA), England (B.N.P., C,S)
DOI: 10.1002/jcp.1041580218
PMID: 8106572
Pubmed: https://pubmed.ncbi.nlm.nih.gov/8106572
Texto completo: https://doi.org/10.1002/jcp.1041580218
Abstract:
Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.