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1.
Mol Hum Reprod ; 10(4): 215-21, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14985476

RESUMO

Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by approximately 2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This "FAAH activator" was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A(2), C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B(4) or prostaglandins E(2) and F(2)alpha had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated.


Assuntos
Blastocisto/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Metabolismo dos Lipídeos , Oxigenases de Função Mista/metabolismo , Útero/metabolismo , Amidoidrolases , Animais , Meios de Cultivo Condicionados , Sistema Enzimático do Citocromo P-450/genética , Ativação Enzimática , Feminino , Camundongos , Camundongos Knockout , Oxigenases de Função Mista/genética , Fosfolipase D/metabolismo , Gravidez , Receptores de Canabinoides/metabolismo
2.
Anat Embryol (Berl) ; 199(5): 459-69, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10221457

RESUMO

The organization of the cytoskeleton has been studied during mouse differentiation in cells of the myenteric neuronal lineage. The entire gut was examined starting from day 12.5 of embryonic life (E12.5) until birth (P0). Immunocytochemistry was performed to evaluate the expression of five of the most represented neurofilaments proteins (the low, NF-L, medium, NF-M, and heavy, NF-H, molecular weight subunits, alpha-internexin and peripherin) and of two of the microtubule-associated proteins (MAPI and MAP2a+2b). In parallel, the appearance in the differentiating myenteric neurons of filamentous and microtubular structures and their intracytoplasmatic distribution were observed under the electron microscope. A differential immunohistochemical expression of the structural proteins was found. Immature cells expressed alpha-internexin, peripherin, NF-M and MAP1 by day E12.5; alpha-internexin expression was strong in these cells, but gradually decreased with age and was practically absent in adulthood. Conversely, the expression of the other three proteins increased with cell differentiation and was still present in adulthood. NF-L and NF-H expression appeared later, by day E16.5, and was weak for the entire pre- and postnatal life. MAP2a+2b was never expressed. Under the electron microscope, at day E12.5 the cytoskeleton was already organized in filamentous and microtubular structures. At this age neurofilaments were few and mainly located in the cell processes, and microtubules were numerous and mainly assembled in the neuritic growth cones, together with synaptic vesicles. With ageing, neurofilaments and microtubules were ubiquitous in the neuron. Data obtained demonstrate that cytoskeletal proteins gradually accumulate in the cells of the neuronal lineage in parallel with the organization of the cytoskeletal structures, which in turn mediate important neural events by the earliest stages of murine embryonic life, including growth of nerve processes and initiation of axonal transport.


Assuntos
Microtúbulos , Plexo Mientérico/embriologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular , Desenvolvimento Embrionário e Fetal , Feminino , Técnicas Imunoenzimáticas , Mucosa Intestinal/metabolismo , Intestinos/inervação , Camundongos , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Plexo Mientérico/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Neurônios/ultraestrutura , Gravidez
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