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1.
IET Syst Biol ; 2(5): 222-33, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19045818

RESUMEN

One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors' experience may provide a productive template for systems biology investigations of other cellular systems.


Asunto(s)
Modelos Biológicos , Feromonas/metabolismo , Proteoma/metabolismo , Investigación/tendencias , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Simulación por Computador , Mapeo de Interacción de Proteínas/métodos
2.
Mol Reprod Dev ; 61(2): 135-41, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11803547

RESUMEN

We analyzed the presence of 3 beta-Hydroxysteroid Dehydrogenase/Delta(5-->4)-isomerase enzyme (3 beta-HSD) activity, a key enzyme of the steroid metabolic pathway, the mRNA of this enzyme, and the steroid metabolism in in vitro produced bovine embryos. 3 beta-HSD activity was detected in in vitro matured oocytes (74.4 +/- 1.4%), 1-cell (72.9 +/- 6.1%), 2-cell (61.8 +/- 7.4%), 8-cell (50 +/- 5%), morulae (50.8 +/- 2.6%), blastocysts (94.4 +/- 3%), and hatched blastocysts (100 +/- 0%) meanwhile the 4-cell stage showed a significant reduction (16.7 +/- 4.7%). When total embryonic RNA of different stages was subjected to RT-PCR assays, the mRNA of 3 beta-HSD was found to be present in all developmental stages of in vitro produced bovine embryos, from the oocyte to the blastocyst, with a marked decrease at the 4-cell stage. To determine whether the temporal pattern of enzyme activity was dependent on the maternal to zygotic transition, embryos were incubated in the presence of a transcription inhibitor, alpha-amanitin. The reappearance of the enzyme activity after the 4-cell stage was blocked in alpha-amanitin treated embryos, indicating the requirement of embryonic transcription. On the other hand, the embryonic steroid metabolism was tested by incubating blastocyst with tritiated pregnenolone. Analysis of the metabolites by TLC indicated the production of a compound with a mobility identical to progesterone. These results described the expression of the 3 beta-HSD and the activity of this metabolic enzyme in bovine oocytes and preimplantation embryos, suggesting that steroids may act as autocrine effectors on preimplantation embryo development.


Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Blastocisto/enzimología , Blastocisto/fisiología , 3-Hidroxiesteroide Deshidrogenasas/genética , Amanitinas/farmacología , Animales , Blastocisto/efectos de los fármacos , Bovinos , Células Cultivadas , Deshidroepiandrosterona/farmacología , Desarrollo Embrionario y Fetal , Femenino , Fertilización In Vitro , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oocitos/fisiología , Embarazo , Pregnenolona/metabolismo , Progesterona/metabolismo , ARN Mensajero/metabolismo
3.
Cell ; 107(6): 739-50, 2001 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-11747810

RESUMEN

In Saccharomyces cerevisiae, mothers and daughters have distinct fates. We show that Cbk1 kinase and its interacting protein Mob2 regulate this asymmetry by inducing daughter-specific genetic programs. Daughter-specific expression is due to Cbk1/Mob2-dependent activation and localization of the Ace2 transcription factor to the daughter nucleus. Ectopic localization of active Ace2 to mother nuclei is sufficient to activate daughter-specific genes in mothers. Eight genes are daughter-specific under the tested conditions, while two are daughter-specific only in saturated cultures. Some daughter-specific gene products contribute to cell separation by degrading the cell wall. These experiments define programs of gene expression specific to daughters and describe how those programs are controlled.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Fosfoproteínas/metabolismo , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas de Ciclo Celular/genética , Quitinasas/genética , Quitinasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas/genética , Filogenia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
4.
Reprod Fertil Dev ; 13(5-6): 411-20, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11833938

RESUMEN

The process of embryo implantation requires extensive remodelling of the endometrial extracellular matrix, a function largely performed by matrix-degrading metalloproteinases (MMPs). In the present study, we used trophoblast cells isolated from human term placentas to study the regulation of MMPs by nitric oxide (NO). Using a combination of zymography, Western blot and indirect immunofluorescence, we showed that MMP-2 and MMP-9 are increased during the conversion from low-motile cytotrophoblast cells to the highly motile and differentiated syncytiotrophoblast multinucleated cells. We also observed an increase in NO production and NO synthase (NOS) expression during this cellular differentiation process. In addition, we demonstrated a positive regulatory role of NO on the activity and protein expression of MMP-2 and MMP-9, because NO donors (NOC-18 and spermine-NONOate) or the NOS substrate (L-arginine) stimulate, whereas NOS inhibitors (N(G)-nitro-L-arginine methyl ester and N(G)-monomethyl-L-arginine) reduce the expression and gelatinolytic activity of MMP-2 and MMP-9 in isolated trophoblast cells. Taken together, these results suggest that, in differentiating trophoblasts, NO regulates the induction of matrix-degrading proteases required for invasion during embryo implantation.


Asunto(s)
Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Óxido Nítrico/farmacología , Trofoblastos/enzimología , Arginina/farmacología , Western Blotting , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Implantación del Embrión , Inhibidores Enzimáticos/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Embarazo , Trofoblastos/citología
5.
Brain Res Dev Brain Res ; 120(1): 83-6, 2000 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-10727733

RESUMEN

The aim of the present study was to determine the developmental pattern of progesterone metabolism in rat brain and spinal cord from embryonic day 13 (E13) to the perinatal period. A marked decrease in the 5alpha-reduction of progesterone in brain cortex was observed between E13 and postnatal day 5 (P5). Isopregnanolone was the predominant isomer in E13 in both cortex and spinal cord and its synthesis diminished gradually, while the concentration of allopregnanolone did not change significantly during development. The placental tissue was able to synthesize the 3alpha and 3beta isomers in E13, E16 and E19 embryos with allopregnanolone being the major metabolite in all the samples. We conclude that embryonic central nervous system tissues are able to synthesize neurosteroids at least from stage E13 and that they are developmentally regulated.


Asunto(s)
Sistema Nervioso Central/embriología , Sistema Nervioso Central/enzimología , Placenta/enzimología , Pregnanolona/biosíntesis , Progesterona/biosíntesis , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Animales , Radioisótopos de Carbono , Cromatografía en Capa Delgada , Femenino , Masculino , Embarazo , Progesterona/análisis , Progesterona/metabolismo , Ratas , Ratas Sprague-Dawley
6.
Proc Natl Acad Sci U S A ; 96(15): 8567-72, 1999 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-10411916

RESUMEN

We selected peptide aptamers from combinatorial libraries that disrupted cell-cycle arrest caused by mating pheromone in yeast. We used these aptamers as baits in two-hybrid hunts to identify genes involved in cell-cycle arrest. These experiments identified genes known to function in the pathway, as well as a protein kinase, the CBK1 product, whose function was not known. We used a modified two-hybrid system to identify specific interactions disrupted by these aptamers. These experiments demonstrate a means to perform "genetics" on the protein complement of a cell without altering its genetic material. Peptide aptamers can be identified that disrupt a process. These aptamers can then be used as affinity reagents to identify individual proteins and protein interactions needed for the process. Forward genetic analysis with peptide aptamer "mutagens" should be particularly useful in elucidating genetic networks in organisms and processes for which classical genetics is not feasible.


Asunto(s)
Ciclo Celular/efectos de los fármacos , Mutagénesis , Péptidos/farmacología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Ciclo Celular/genética , División Celular/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Factor de Apareamiento , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Eliminación de Secuencia
7.
Endocrinology ; 140(6): 2541-8, 1999 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10342840

RESUMEN

This study was aimed at testing the hypothesis that different forms of fibronectin (FN), produced as a consequence of the alternative splicing of the precursor messenger RNA, play specific roles during development of the ovarian follicle. In particular, we were interested in determining the effect of the ED-I (also termed ED-A) type III repeat, which is absent in the plasma form. Analysis of FN levels in follicular fluids corresponding to different stages of development of bovine follicles revealed marked changes in the concentrations of ED-I+ FN, whereas total FN levels remained relatively constant. ED-I+ FN levels were higher in small follicles, corresponding to the phase of granulosa cell proliferation. The hypothesis of a physiological role for ED-I+ FN was further supported by the finding of a regulation of the alternative splicing of FN in primary cultures of bovine granulosa cells by factors known to control ovarian follicular development. cAMP produced a 10-fold decrease in the relative proportion of the ED-I region. In contrast, transforming growth factor-beta elicited a 2-fold stimulation of overall FN synthesis and a 4-fold increase in the synthesis of ED-I containing FN. This effect was evident at the protein (Western blots) and messenger RNA (Northern blots) levels. Although a negative correlation (P < 0.001) was detected between ED-I+ FN and estradiol levels in follicular fluid, this steroid was unable to modulate in vitro the alternative splicing of FN. A possible mitogenic effect of ED-I+ FN was suggested by the observation that a recombinant peptide corresponding to the ED-I domain stimulated DNA synthesis in a bovine granulosa cell line (BGC-1), whereas a peptide corresponding to the flanking type III sequences had no effect. The hypothesis of ED-I+ FN as a growth regulatory factor was further strengthened by the fact that depletion of FN from BGC-1-conditioned medium, which contained ED-I+ FN, abrogated its mitogenic activity, whereas plasma FN was without effect. We propose that changes in the primary structure of FN may mediate some of the effects of gonadotropin and intraovarian factors during follicular development.


Asunto(s)
Empalme Alternativo , Fibronectinas/fisiología , Folículo Ovárico/crecimiento & desarrollo , Animales , Bovinos , Células Cultivadas , AMP Cíclico/fisiología , ADN/biosíntesis , Femenino , Fibronectinas/genética , Regulación de la Expresión Génica
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