RESUMO
In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.
Assuntos
Estrogênios/farmacologia , Fator de Crescimento Neural/metabolismo , Plasticidade Neuronal , Neurônios Aferentes/fisiologia , Receptor trkA/metabolismo , Útero/inervação , Adrenérgicos/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Ensaio de Imunoadsorção Enzimática , Estrogênios/metabolismo , Feminino , Gânglios Espinais/anatomia & histologia , Gânglios Espinais/citologia , Guanetidina/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Simpatectomia Química , Útero/crescimento & desenvolvimento , Útero/metabolismoRESUMO
Local protein synthesis within axons has been studied on a limited scale. In the present study, several techniques were used to investigate this synthesis in sciatic nerve, and to show that it increases after damage to the axon. Neurofilament (NF) mRNAs were probed by RT-PCR, Northern blot and in situ hybridization in axons of intact rat sciatic nerve, and in proximal or distal stumps after sciatic nerve transection. RT-PCR demonstrated the presence of NF-L, NF-M and NF-H mRNAs in intact sciatic nerve, as well as in proximal and distal stumps of severed nerves. Northern blot analysis of severed nerve detected NF-L and NF-M, but not NF-H. This technique did not detect the three NFs mRNAs in intact nerve. Detection of NF-L and NF-M mRNA in injured nerve, however, indicated that there was an up-regulation in response to nerve injury. In situ hybridization showed that NF-L mRNA was localized in the Schwann cell perinuclear area, in the myelin sheath, and at the boundary between myelin sheath and cortical axoplasm. RNA and protein synthesizing activities were always greater in proximal as compared to distal stumps. NF triplet proteins were also shown to be synthesized de novo in the proximal stump. The detection of neurofilament mRNAs in nerves, their possible upregulation during injury and the synthesis of neurofilament protein triplet in the proximal stumps, suggest that these mRNAs may be involved in nerve regeneration, providing a novel point of view of this phenomenon.
Assuntos
Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Nervo Isquiático/metabolismo , Animais , Autorradiografia , Axotomia , Northern Blotting , Hibridização In Situ , Masculino , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Nervo Isquiático/cirurgia , Regulação para CimaRESUMO
The insert of a clone from a lambdagt11 Echinococcus granulosus (Platyhelminth, Cestoda) protoscolex cDNA library, showed an open reading frame whose deduced protein sequence presents a high homology with all described thioredoxins (TRX). The TRX active site (Trp-Cys-Gly-Pro-Cys) is completely conserved. With a monospecific antibody, selected from a total anti-protoscolex sera by the isolated clone, a 12 kDa polypeptide was immunoprecipitated from a protoscolex total protein extract. Furthermore, an antiserum raised against a recombinant EgTRX also recognizes a 12 kDa band in these extracts. The recombinant protein presents TRX activity, using the insulin reduction assay. Finally, a TRX activity was characterized in protoscolex extracts. In all organisms where TRXs were studied, they participate in a cascade of redox exchanges, contributing to the maintaining of cell homeostasis. Considering that the parasitic flatworm E. granulosus is probably submitted to an important oxidative stress due to host defences, EgTRX protein could be involved in the survival strategies of this parasite.
Assuntos
Echinococcus/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Southern Blotting , Western Blotting , Clonagem Molecular , Echinococcus/crescimento & desenvolvimento , Echinococcus/metabolismo , Expressão Gênica , Proteínas de Helminto/química , Proteínas de Helminto/classificação , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Soros Imunes , Insulina/metabolismo , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Oxirredução , Estrutura Secundária de Proteína , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tiorredoxinas/química , Tiorredoxinas/classificaçãoRESUMO
The homeobox-containing gene EgHbx3 from Echinococcus granulosus is expressed in the stalk of protoscoleces. The expression of the homeobox-containing gene EgHbx3 from E. granulosus was studied in protoscoleces by in situ hybridisation techniques. A method for performing in situ hybridisations on whole mounted hydatic material is described. Our results show a clear positive signal at the protoscolex stalk, when using the antisense EgHbx3 probe. This signal is particularly strong in the proximal end of the stalk (with respect to the protoscolex).