RESUMO
Myelination is a concerted mechanism tightly regulated in the brain. Although several factors are known to participate during this process, the complete sequence of events is far from being fully elucidated. Separate effects of apotransferrin (aTf) and thyroid hormone (TH) are well documented on rat myelin formation. TH promotes the maturation of oligodendrocyte progenitors (OPCs) into myelinating oligodendrocytes (OLGs), while aTf is able to induce the commitment of neural stem cells (NSCs) toward the oligodendroglial linage and favors OLG maturation. We have also demonstrated that Tf mRNA exhibited a seven-fold increase in hyperthyroid animals. These observations have led us to hypothesize that both factors may interplay during oligodendrogenesis. To assess the combined effects of aTf and TH on proper myelination in the rat brain, Tf expression and oligodendroglial maturation were evaluated at postnatal days 10 (P10) and 20 (P20) in several experimental groups. At P10, an up-regulation of both Tf mRNA and protein, as well as myelination, was found in hyperthyroid animals, while a decrease in Tf mRNA levels and myelin formation was detected in the hypothyroid group. At P20, no differences were found either in Tf mRNA or protein levels between hyperthyroid and control (Ctrol) rats, although differences in OLG differentiation remained. Also at P20, hypothyroid animals showed decreased Tf mRNA and protein levels accompanied with a less mature myelinating phenotype. Moreover, TH and aTf differentially regulate the expression of KLF9 transcription factor as well as TRα and TRß at P10 and P20. Our results suggest that TH is necessary early in OLG development for aTf action, as exogenous aTf administration was unable to counteract the effect of low TH levels in the hypothyroid state in all the time points analyzed. Furthermore, the fact that hyperthyroidism induced an increase in Tf expression and aTf-dependent regulation of TRα strongly suggests that Tf could be involved in some of TH later effects on OLG maturation. Here we describe the possible relationship between TH and aTf and its implication in oligodendrogenesis.
Assuntos
Apoproteínas/biossíntese , Bainha de Mielina/metabolismo , Fibras Nervosas Mielinizadas/metabolismo , Oligodendroglia/metabolismo , Hormônios Tireóideos/biossíntese , Transferrina/biossíntese , Animais , Animais Recém-Nascidos , Masculino , Camundongos , Ratos , Ratos WistarRESUMO
The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.
Assuntos
Animais , Bovinos , Malha Trabecular , Transferrina/biossíntese , Transferrina , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/biossíntese , Análise de Sequência de RNA , Malha Trabecular/metabolismo , Transferrina/genéticaRESUMO
The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.
Assuntos
Malha Trabecular/metabolismo , Transferrina/biossíntese , Transferrina/metabolismo , Animais , Bovinos , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Imunoprecipitação , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transferrina/genéticaRESUMO
Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.
Assuntos
Abelhas/genética , Ecdisteroides/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes de Insetos/genética , Hormônios Juvenis/fisiologia , Transferrina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Abelhas/crescimento & desenvolvimento , Abelhas/metabolismo , Northern Blotting , DNA Complementar/genética , Regulação para Baixo , Ecdisterona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônios Juvenis/farmacologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Filogenia , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transferrina/biossínteseRESUMO
It has been shown that the vitreous contains several intrinsic glycoproteins whose origin remains to be clarified. Isolated ciliary epithelium (CE) was assayed to verify its role in the synthesis and secretion of transferrin for the vitreous body. It was cultured in the presence of [35S]methionine and the incubation medium was processed for immunoprecipitation. Total RNA from CE was processed for RT-PCR and the amplification products were sequenced. Also, whole preparations of isolated CE were processed for immunolocalization of transferrin. From the incubation assays, a labeled peptide of about 80 kDa was immunopurified that is the expected size of transferrin. The RT-PCR and sequencing experiments detected the presence of transferrin mRNA. Both layers of the CE exhibited transferrin reactivity, following immunohistochemical processing. Taken altogether, these results indicate the CE as one of the possible sources of vitreous intrinsic transferrin.
Assuntos
Corpo Ciliar/metabolismo , Transferrina/biossíntese , Transferrina/metabolismo , Animais , Corpo Ciliar/anatomia & histologia , Técnicas de Cultura , Epitélio/anatomia & histologia , Epitélio/metabolismo , Imuno-Histoquímica , Testes de Precipitina , RNA Mensageiro/biossíntese , Coelhos , Transferrina/genéticaRESUMO
The FSH molecular mechanism of action is best recognized for its stimulation of the adenylyl cyclase/cAMP pathway via activation of a G protein. Recently, links between cAMP, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) signaling pathways in thyroid and granulosa cells have been observed. The aim of this study was to investigate the possible role of the PI3K/PKB pathway in FSH regulation of Sertoli cell function. Twenty-day-old rat Sertoli cell cultures were used. An increase in phosphorylated PKB (P-PKB) levels in response to FSH and dibutyryl-cAMP was observed. These increments in P-PKB levels were not observed in the presence of two PI3K inhibitors, wortmannin and Ly 294002. Inhibition of protein kinase A (PKA) by H89 did not decrease FSH stimulation of P-PKB levels. Taken together, these results indicate that FSH increases P-PKB levels in a PI3K-dependent and PKA-independent manner in rat Sertoli cells. In addition, wortmannin partially inhibited the ability of FSH to stimulate two well-known parameters of Sertoli cell function - transferrin secretion and lactate production - at doses equal to or lower than 0.1 microM. Related to lactate production, a decrease in FSH stimulation of lactate dehydrogenase activity and of basal and FSH-stimulated glucose uptake was observed in the presence of wortmannin. These metabolic changes were in most cases accompanied by changes in the levels of P-PKB. Altogether, these results suggest a meaningful role of the PI3K/PKB pathway in the mechanism of action of FSH in rat Sertoli cells.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Células de Sertoli/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Sobrevivência Celular , Células Cultivadas , AMP Cíclico/biossíntese , Glucose/metabolismo , Guanosina Trifosfato/metabolismo , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/biossíntese , Masculino , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Sprague-Dawley , Células de Sertoli/efeitos dos fármacos , Transferrina/biossínteseRESUMO
Iron overload is a major concern in hepatitis C virus (HCV) infection because excess iron can promote hepatocyte damage by activating iron-mediated lipid peroxidation. This may also facilitate viral replication. The objective of this pilot study was to test the hypothesis that Puerto Rican HCV patients have an altered serum iron (SIR) profile. Twenty-three HCV patients and 38 non-HCV controls were compared in terms of their serum iron, iron-binding capacity, percent saturation of transferrin, available binding capacity, and ferritin. Statistically significant differences (p < 0.01, Student's t-test) were found between the HCV patients and the non-HCV controls for SIR, transferrin saturation, and ferritin. The mean SIR concentration and transferrin saturation were 25% higher in HCV patients relative to controls. HCV patients had a mean ferritin value 48% higher than controls. These pilot study data indicate that Puerto Rican HCV patients have an altered iron balance and may be more susceptible to iron-induced oxidative stress.
Assuntos
Hepatite C/metabolismo , Ferro/sangue , Estudos de Casos e Controles , Ferritinas/sangue , Hepatócitos/metabolismo , Humanos , Estresse Oxidativo , Porto Rico , Transferrina/biossínteseRESUMO
PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous. The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin. METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver. The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris. Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin. RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry. CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.
Assuntos
Corpo Ciliar/metabolismo , Transferrina/biossíntese , Animais , Northern Blotting , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Fluorometria , Imuno-Histoquímica , Técnicas In Vitro , Iris/metabolismo , Masculino , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Coelhos , Transcrição Gênica , Transferrina/genéticaRESUMO
Transferrin occurs in the vitreous at a higher relative concentration than found in the plasma or in the aqueous humor. This has been related to a possible local synthesis of transferrin by some component of the eye, although convincing evidence for this has not been available. Recently, the intraocular synthesis of several vitreous glycoproteins, possibly by the ciliary body, was demonstrated by our group. This paper reports experiments that characterize vitreous transferrin as one of these glycoproteins. Vitreous of rabbits injected intravitreally either with 3H-fucose or 3H-tyrosine and killed 40 hr after the injection was processed for 1D and 2D-electrophoresis, followed by immunoblot techniques or fluorography. It was possible to detect a labeled polypeptide with the same molecular weight and pI of rabbit plasma transferrin. Furthermore, this labeled polypeptide could be specifically eluted from columns of Sepharose conjugated with antibody against rabbit plasma transferrin. Thus, these results demonstrate that at least part of the transferrin of the vitreous is synthesized within the eye.