Publicaciones por año
Publicaciones del Departamento de Histología y Embriología
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.
An improved method combining two electrophoretic procedures: application to the separation of lens alpha-crystallin isoforms
1991 Jul-Aug;12(7-8):588-91
C Arruti 1 3 , S Chifflet 2 3
1 Departamento de Histología y Embriología, Facultad de Medicina, Montevideo, Uruguay. 2 Departamento de Bioquimica, Facultad de Medicina, Montevideo 3 Departamento de Biologia Celular, Faculted de Ciencias, Montevideo
DOI: 10.1002/elps.1150120720.
PMID: 1915250
Pubmed: https://pubmed.ncbi.nlm.nih.gov/1915250
Texto completo: https://doi.org/10.1002/elps.1150120720
Abstract:
Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS-PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat-bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of alpha-crystallins of young and embryonic quail lenses.
Localization of basic fibroblast growth factor binding sites in the chick embryonic neural retina
Differentiation 1990 Dec;45(3):161-7
A Cirillo 1 , C Arruti 1 2, Y Courtois 3, J C Jeanny 3
1 Laboratorio de Cultivo de Tejidos, Depto de Histología y Embriología, Facultad de Medicina, Avda General Flores 2125, 11800 Montevideo, Uruguay 2 Depto de Biologia Celular, Facultad de Humanidades y Ciencias, Tristin Narvaja 1464, 11200 Montevideo, Uruguay 3 Unité de Recherches Gérontologiques, INSERM U. 118, Unité Affilié CNRS, 29, rue Wilhem, F-75016 Paris, France
DOI: 10.1111/j.1432-0436.1990.tb00469.x
PMID:
Pubmed: https://pubmed.ncbi.nlm.nih.gov/
Texto completo: https://linkinghub.elsevier.com/retrieve/pii/S0301-4681(11)60217-X
Abstract:
We have investigated the localization of basic fibroblast growth factor (bFGF) binding sites during the development of the neural retina in the chick embryo. The specificity of the affinity of bFGF for its receptors was assessed by competition experiments with unlabelled growth factor or with heparin, as well as by heparitinase treatment of the samples. Two different types of binding sites were observed in the neural retina by light-microscopic autoradiography. The first type, localized mainly to basement membranes, was highly sensitive to heparitinase digestion and to competition with heparin. It was not developmentally regulated. The second type of binding site, resistant to heparin competition, appeared to be associated with retinal cells from the earliest stages studied (3-day-old embryo, stages 21-22 of Hamburger and Hamilton). Its distribution was found to vary during embryonic development, paralleling layering of the neural retina. Binding of bFGF to the latter sites was observed throughout the retinal neuroepithelium at early stages but displayed a distinct pattern at the time when the inner and outer plexiform layers were formed. During the development of the inner plexiform layer, a banded pattern of bFGF binding was observed. These bands, lying parallel to the vitreal surface, seemed to codistribute with the synaptic bands existing in the inner plexiform layer. The presence of intra-retinal bFGF binding sites whose distribution varies with embryonic development suggests a regulatory mechanism involving differential actions of bFGF on neural retinal cells.
Depletion of the Ca(++)-dependent releasable pool of glutamate in striatal synaptosomes associated with dendrotoxin-induced potassium channel blockade
J Neural Transm Gen Sect 1990 80(3):167-79
L Barbeito, J Siciliano, F Dajas
Neurochemistry Division, Faculty of Medicine, Montevideo, Uruguay, and Histology Department, Faculty of Medicine, Montevideo, Uruguay
DOI: 10.1007/BF01245118
PMID: 1970482
Pubmed: https://pubmed.ncbi.nlm.nih.gov/1970482
Texto completo: https://link.springer.com/article/10.1007/BF01245118
Abstract:
The presynaptic actions of the potassium channel blocker Dendrotoxin (DTX) on the Ca+2-dependent release of endogenous glutamate (GLU) and aspartate (ASP) have been tested in synaptosome-enriched preparations from rat striatum. 24 hours after the intrastriatal administration of DTX the K(+)-evoked release of GLU and ASP from the striatal synaptosomes was decreased by 40-45%. No changes in the total synaptosomal content of the amino acids were observed. Superfusion of immobilized synaptosomes with DTX or 4-amino-pyridine resulted in a dose-dependent increase in the basal outflow of GLU and ASP. The release of GLU stimulated by DTX was Ca+2-dependent and was not abolished by superfusing the synaptosomes with 50 microM D-ASP. Moreover, continuous superfusion of DTX (7 microM) to synaptosomes almost completely dumped the subsequent release of GLU and ASP stimulated by 20 mM K+. It is concluded that blockade of presynaptic K+ channels by DTX leads to a massive release of the transmitter pool of GLU (and possible also ASP) from isolated nerve terminals and to a depletion of the amino acid releasable pool.
The effect of eye-derived-growth-factor (EDGFs) on methionine incorporation in the different cell populations of bovine adult lens in organ culture
Exp Eye Res 1989 48(2):177-86
F Mascarelli 1 , Y Courtois 1 , C Arruti 2
1 Unité de Recherches Gérontologiques. U. 118 INSERM, 29 rue Wilhem, 75016 Parix, France 2 Laboratorio de Cultivo de Tejidos, Departamento de Histologia y Embriologia, Facultad de Medicina, Avenida General Flores, 2125 Montevideo, Uruguay
DOI: 10.1016/s0014-4835(89)80068-5
PMID: 2924806
Pubmed: https://pubmed.ncbi.nlm.nih.gov/2924806
Texto completo: https://www.sciencedirect.com/science/article/abs/pii/S0014483589800685?via%3Dihub
Abstract:
When adult bovine lenses were cultured in vitro, the purified retina-derived growth factors EDGF I or EDGF II, as well as the soluble fraction of the retina RE, increased the rate of incorporation of [35S]methionine into protein in cells belonging to different populations in the anterior epithelium as well as in fibers from the most superficial region of the cortex. These fiber cells were the most sensitive to stimulation by the retinal factors as they exhibit a significant increase of total protein synthesis 24 hr after addition of the factors to the culture medium. The epithelial cells studied--central epithelial cells and germinative cells--appeared stimulated only 1 day later. The stimulation of incorporation was not directed towards a particular subset of proteins but to all major polypeptides constituting the electrophoretic pattern of each cell population. It is suggested that this type of ocular signal, which stimulates the expression of a definite program, may act as a permissive signal.
Intrastriatal dendrotoxin injection: behavioral and neurochemical effects
Toxicon 1988 26(11):1009-15
R Silveira, J Siciliano, V Abo, L Viera, F Dajas
Neurochemistry Division, Instituto de Investigaciones Biologicas Clemente Estable, Montevideo, Uruguay, and Histology Department, Faculty ofMedicine, Montevideo, Uruguay
DOI: 10.1016/0041-0101(88)90199-7
PMID: 3245048
Pubmed: https://pubmed.ncbi.nlm.nih.gov/3245048
Texto completo: https://www.sciencedirect.com/science/article/abs/pii/0041010188901997
Abstract:
Unilateral striatal injection of dendrotoxin (DTX), a polypeptide isolated from the venom of the snake Dendroaspis angusticeps, in rats provoked a complex behavioral syndrome characterized by spontaneous circling towards the contralateral side, stereotypic like chewing movements and gnawing, abnormal postures and convulsions. All these symptoms achieved their maximum on the first day, disappearing during the first week after injection. Neurochemical analyses of striatal monoamines and monoamine metabolites showed a significant increase of dopamine and serotonin metabolites 20 hr after DTX injection. A group of animals sacrificed 15 days after toxin administration showed normal levels of monoamines and their metabolites, except for homovanillic acid levels which were still significantly increased. These data indicate that monoamines are involved in the behavioral syndrome elicited by DTX and are possibly related to its excitatory effect upon brain structures in vivo.
An eye-derived growth factor regulates epithelial cell proliferation in the cultured lens
Differentiation 1985 28(3):286-90
C Arruti 1 , A Cirillo 1 , Y Courtois 2
1 Laboratorio de Cultivo de Tejidos, Depto. de Histologia y Embriologia, Facultad de Medicina de Montevideo, Avda. Gral. Flores 2125, Montevideo, Uruguay 2 Unité de Recherches Gérontologiques, INSERM U. 118, CNRS ERA 842, Association Claude-Bernard, 29 rue Wilhem, Paris, France
DOI: 10.1111/j.1432-0436.1985.tb00837.x
PMID: 3996799
Pubmed: https://pubmed.ncbi.nlm.nih.gov/3996799
Texto completo: https://www.sciencedirect.com/science/article/abs/pii/S0301468111606762?via%3Dihub
Abstract:
Lenses in organ culture permit an analysis of factors acting on epithelial cell growth, while keeping the normal steric constraints of the cell population. By employing this technique with radioautography of epithelial whole mounts, we showed that the DNA synthesis found in the epithelia of cultured bovine lenses follows an organized spatial and temporal pattern during culture. Within the first 48 h, active cells were located at the preequatorial region ("germinative zone"), a distribution consistent with the in vivo spatial organization of multiplying cells. Starting at about 48 h, cells from the central region of the epithelium--a nonproliferating population--were triggered to synthesize DNA in the presence of eye-derived growth factor (EDGF). When cultured in serum-free medium, only a small fraction of the cells was labeled, but when a low serum concentration was present, this fraction reached 50% of the cell population. The stimulatory effect of EDGF required a lag period, but its effect reached a maximum exceeding that found for serum. However, the cells from the germinative region, having a cell density three- to four-fold higher than the central region, were not stimulated to proliferate. This occurred irrespective of the presence of EDGF or serum. If this growth-stimulatory activity derived from the retina were the actual factor controlling cell proliferation in the lens in vivo, then the results presented here would point to the presence of a regulatory mechanism similar to that known for some other hormones.
Monolayer organization by serially cultured bovine corneal endothelial cells: effects of a retina-derived growth-promoting activity
Exp Eye Res 1982 34(5):735-47
C Arruti 1 , Y Courtois 2
1 Laboratorio de Cultivo de Tejidos, Departmento de Histología y Embriología, Facultad de Medicina, Av. Gral Flores 2125, Montevideo, Uruguay 2 Unité de Recherches Gérontologiques U118, INSERM Développement et Sénescence Cellulaire, ERA 842, CNRS, 29 rue Wilhem, 75016 Paris, France
DOI: 10.1016/s0014-4835(82)80034-1
PMID: 7084337
Pubmed: https://pubmed.ncbi.nlm.nih.gov/7084337
Texto completo: https://www.sciencedirect.com/science/article/abs/pii/S0014483582800341?via%3Dihub
Abstract:
This study describes the ability of bovine corneal endothelial cells to proliferate and organize monolayers when cultured in the presence of a saline-soluble fraction of the retina (RE). This strongly stimulates cell proliferation by increasing the growth rate. In the continuous presence of RE, the cells organize a confluent monolayer composed of closely apposed polygonal cells lying on a basement membrane. Thus, over a large number of generations, these cells express most of their differentiated traits: contact inhibition of movement, proliferation arrest at monolayer formation, and the extracellular deposition of a material organized as a typical basement membrane. The presence of RE increases by a factor of 10 the number of cellular generations expressing these differentiated traits, and subcultured cells maintained in a medium lacking Re are unable to build up a similar organization at confluency, even if this is extended over several months. If these cultures are transferred to a medium containing RE, they behave like endothelial cells which have been always cultured with RE. We suggest that the expression of the endothelial differentiated state is related to the raised mitotic activity induced by the retinal factor.
Hysterectomy of the newborn guinea-pig, subsequent effects on the oestrous cycle and life span of the corpora lutea
J Endocrinol 1975 66(2):233-7
W Buño, E Carlevaro, L Riboni, H D'Albora, L de los Reyes, D Zipitria, R Domínguez
DOI: 10.1677/joe.0.0660233
PMID: 1172521
Pubmed: https://pubmed.ncbi.nlm.nih.gov/1172521
Texto completo: https://joe.bioscientifica.com/doi/10.1677/joe.0.0660233
Abstract:
Longer oestrous cycles result from neonatal hysterectomy than from hysterectomy in adult life. Section and cauterization of the utero-vaginal union also prolonged the vaginal closure period up to an average of 55 days. The destruction of the mesometrium did not lengthen the oestrous cycle. Uterine autografts in hysterectomized newoborn guinea-pigs did not prevent the long cycles.