RESUMO
BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.
Assuntos
Citoglobina/metabolismo , Fígado Gorduroso , Células Estreladas do Fígado , Cirrose Hepática , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Colestase/tratamento farmacológico , Colestase/metabolismo , Descoberta de Drogas , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Camundongos , Camundongos Knockout , Substâncias Protetoras/farmacologia , Proteínas Recombinantes/farmacologia , Resultado do TratamentoRESUMO
Xenopus vasa-like gene 1 (XVLG1), a DEAD-Box Helicase 4 (DDX4) gene identified as a vertebrate vasa homologue, is required for the formation of primordial germ cells (PGCs). However, it remains to be clarified when and how XVLG1 functions in the formation of the germ cells. To gain a better understanding of the molecular mechanisms underlying XVLG1 during PGC development, we injected XVLG1 morpholino oligos into germ-plasm containing blastomeres of 32-cell stage of Xenopus embryos, and traced cell fates of the injected blastomere-derived PGCs. As a result of this procedure, migration of the PGCs was impaired and the number of PGCs derived from the blastomeres was significantly decreased. In addition, TUNEL staining in combination with in situ hybridization revealed that the loss of PGCs peaked at stage 27 was caused by apoptosis. This data strongly suggests an essential role for XVLG1 in migration and survival of the germ cells.
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Células Germinativas/fisiologia , Xenopus/metabolismo , Animais , Movimento Celular , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Morfolinos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.
Assuntos
Biomarcadores/metabolismo , Globinas/imunologia , Globinas/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatite C/metabolismo , Cirrose Hepática/metabolismo , Actinas/imunologia , Animais , Anticorpos/imunologia , Compostos Azo , Proteínas de Ligação ao Cálcio/imunologia , Células Cultivadas , Citoglobina , Proteínas da Matriz Extracelular/imunologia , Humanos , Imuno-Histoquímica , Camundongos , Miofibroblastos/metabolismo , Antígenos Thy-1/imunologiaRESUMO
As a result of a high-throughput in situ hybridization screening for adult mouse testes, we found that the mRNA for Tmco5 is expressed in round and elongating spermatids. Tmco5 belongs to the Tmco (Transmembrane and coiled-coil domains) gene family and has a coiled-coil domain in the N-terminal and a transmembrane domain in the C-terminal region. A monoclonal antibody raised against recombinant TMCO5 revealed that the protein is expressed exclusively in the elongating spermatids of step 9 to 12 and is localized to the manchette, a transiently emerging construction, which predominantly consists of cytoskeleton microtubules and actin filaments. This structure serves in the transport of Golgi-derived non-acrosomal vesicles. Moreover, induced expression of TMCO5 in CHO cells resulted in the co-localization of TMCO5 with ß-tubulin besides the reorganization of the Golgi apparatus. Judging from the results and considering the domain structure of TMCO5, we assume that Tmco5 may have a role in vesicle transport along the manchette.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Espermátides/metabolismo , Vesículas Transportadoras/metabolismo , Envelhecimento/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas do Citoesqueleto/genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espermatogênese , Testículo/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.
Assuntos
Mitocôndrias/enzimologia , Isomerases de Dissulfetos de Proteínas/análise , Animais , Bovinos , Fracionamento Celular , Masculino , Camundongos , Microscopia Imunoeletrônica , Microssomos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/imunologia , Isomerases de Dissulfetos de Proteínas/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
In order to know when the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) first appears in germ line cells and whether the protein is also present in somatic cells as is vasa protein in Drosophila, the spatio-temporal distribution of the protein in Xenopus embryos was carefully investigated by fluorescent microscopy. Part of the observation was performed by whole-mount immunocytochemistry and immunoblotting. A distinct fluorescence of XVLG1 protein was first recognized in a juxta-nuclear location of germ line cells or presumptive primordial germ cells (pPGC) at stage 12 (late gastrula) and remained associated with the pPGC or primordial germ cells (PGC) throughout the following stages until stage 46 (feeding tadpole). In contrast, weak fluorescence was seen in the animal hemisphere rather than in the vegetal hemisphere of cleaving embryos and in the perinuclear region of somatic cells at stages 10-42 (early gastrula to young tadpole), respectively. Nearly the same pattern as revealed by fluorescence was seen by whole-mount immunocytochemistry, except that a small amount of XVLG1 protein seemed to be present in the germ plasm and pPGC of embryos earlier than stage 12. The presence of the protein in the somatic cells and the PGC was also shown by immunoblotting.
RESUMO
The African clawed frog, Xenopus laevis, has a ZZ/ZW-type sex-determination system. We previously reported that a W-linked gene, Dm-W, can determine development as a female. However, the mechanisms of early sex differentiation remain unclear. We used microarrays to screen for genes with sexually dimorphic expression in ZZ and ZW gonads during early sex differentiation in X laevis and found several steroidogenic genes. Importantly, the steroid 17α-hydroxylase gene Cyp17a1 and the aromatase gene Cyp19a1 were highly expressed in ZZ and ZW gonads, respectively, just after sex determination. At this stage, we found that Cyp17a1, Cyp19a1, or both were expressed in the ZZ and ZW gonads in a unique mass-in-line structure, in which several masses of cells, each surrounded by a basement membrane, were aligned along the anteroposterior axis. In fact, during sex differentiation, ovarian cavities formed inside each mass of Cyp17a1- and Cyp19a1-positive cells in the ZW gonads. However, the mass-in-line structure disappeared during testicular development in the ZZ testes. These results suggested that the mass-in-line structure found in both ZZ and ZW gonads just after sex determination might be formed in advance to produce ovarian cavities and then oocytes. Consequently, we propose a view that the default sex may be female in the morphological aspect of gonads in X laevis.
Assuntos
Aromatase/genética , Gônadas/citologia , Gônadas/embriologia , Ovário/embriologia , Diferenciação Sexual/genética , Proteínas de Xenopus/genética , Xenopus laevis , Animais , Embrião não Mamífero , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Gônadas/metabolismo , Masculino , Análise em Microsséries , Ovário/citologia , Ovário/metabolismo , Processos de Determinação Sexual/genética , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological characteristics of Alzheimer's disease (AD). NFTs are composed of tubular filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature of AD is excessive generation of nitric oxide (NO). Protein disulfide isomerase (PDI) is a chaperon protein located in the endoplasmic reticulum (ER). It was recently reported that NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. In addition, we previously reported the presence of PDI-immunopositive NFTs in AD. Here, we found that protein disulfide isomerase P5 (P5), which is a member of the PDI protein family, was co-localized with tau in NFTs. To our knowledge, this is the first report of P5-immunopositive inclusion in AD. Furthermore, we showed that S-nitrosylated P5 was present and the expression level of P5 was decreased in AD brains compared with that of control brains. We also demonstrated that the knock-down of PDI or P5 by siRNA could affect the viability of SH-SY5Y cells under ER stress. Previously, the observation of S-nitrosylated PDI in AD was reported. NO may inhibit P5 by inducing S-nitrosylation in the same manner as PDI, which inhibits its enzymatic activity allowing protein misfolding to occur in AD. The accumulation of misfolded proteins induces ER stress and may cause apoptosis of neuronal cells through S-nitrosylation and down-regulation of PDI and P5 in AD.
Assuntos
Doença de Alzheimer/patologia , Lobo Frontal/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuroblastoma/patologia , Emaranhados Neurofibrilares/enzimologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Isomerases de Dissulfetos de Proteínas/genética , Fatores de Tempo , Proteínas tau/metabolismoRESUMO
Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin-binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.
Assuntos
Regulação para Baixo , Histona Desacetilases/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Desacetilase 6 de Histona , Histona Desacetilases/deficiência , Homeostase , Humanos , Camundongos , Mitocôndrias/enzimologiaRESUMO
P5, one of the protein disulphide isomerase (PDI) family members, catalyses disulphide bond formation in proteins and exhibits molecular chaperone and calcium binding activities in vitro, whereas its physiological significance remains controversial. Recently, we have reported that P5 localizes not only in the ER but also in mitochondria, although it remains unclear so far about its physiological significance(s) of its dual localization. Here we report that H(2)O(2)- or rotenone-induced cell death is suppressed in MTS-P5 cells, which stably express P5 in mitochondria. H(2)O(2)-induced cell death in Saos-2 cells occurred, in large part, through caspase-independent and poly(ADP-ribose) polymerase (PARP)-dependent manner. In MTS-P5 cells challenged with H(2)O(2) treatment, PARP was still activated, whereas release of cytochrome c or apoptosis-inducing factor and intramitochondrial superoxide generation were suppressed. We also found that mitochondrial P5 was in close contact with citrate synthase and maintained large parts of its activity under H(2)O(2) exposure. These results suggest that mitochondrial P5 may upregulate tricarboxylic acid cycle and possibly, other intramitochondrial metabolism.
Assuntos
Morte Celular , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Estresse Oxidativo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Apoptose , Fator de Indução de Apoptose/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
We have reported elsewhere that offspring from the No. 65 female of Xenopus laevis cleaved normally, but their development was arrested at the onset of gastrulation, like the Ambystoma ova-deficient (o) mutant, irrespective of mating with different wild-type males, and that an acidic, 38 kDa protein present in wild-type eggs was lacking in eggs of the female. In the current study, we first determined the partial amino acid sequence (VANLE) of one of the well-separated tryptic peptides from the protein, which was found in elongation factor 1 delta (Ef1delta) in Xenopus, and finally identified the protein as one of the Ef1delta isoforms, Ef1delta2, by peptide mass spectrometry. RT-PCR analyses for Ef1delta2 and its close homolog Ef1delta1 in wild-type oocytes and embryos demonstrated that both transcripts are maternal and Ef1delta1 is present more abundantly than Ef1delta2 throughout the stages examined. Importantly, the amount of the Ef1delta2 transcript per embryo decreased gradually after gastrulation, in accordance with the gradual decrease of the 38 kDa protein per embryo reported in our earlier study. Because pharmacological inhibition of translation induces gastrulation arrest in wild-type embryos, it is reasonable to conclude that the mutant embryos arrest in development due to the lack of Ef1delta2 that is indispensable for translation. Thus, the present study provides the first molecular information on the cause of the gastrulation-defective mutation in Amphibia.
Assuntos
Gástrula/metabolismo , Mutação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Gástrula/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Immunoblotting , Masculino , Espectrometria de Massas , Peso Molecular , Oócitos/citologia , Oócitos/metabolismo , Fator 1 de Elongação de Peptídeos/química , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de Proteína , Fatores de Tempo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologiaRESUMO
Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.
Assuntos
Quimiocina CXCL12/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Receptores CXCR4/metabolismo , Transdução de Sinais , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Sequência de Bases , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Clonagem Molecular , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fenótipo , RNA Mensageiro/metabolismoRESUMO
We have developed a novel method for microinjection into Xenopus eggs and embryos. Microinjection was performed into eggs or embryos that were placed in wells (ca. 2.5 mm x 2.5 mm x 0.8 mm for each well) at the bottom of a commercially available hybridoma dish, which was filled with 1.5% methylcellulose solution. Eggs or embryos, rotated to a desired orientation in the viscous methylcellulose solution with a hair loop, could remain in the orientation for more than twenty minutes. Accordingly, samples such as mRNAs, DNAs, proteins and antisense morpholino oligonucleotides could be easily and efficiently microinjected into any part (animal, vegetal, dorsal, lateral or ventral) of more than five hundred eggs and embryos in one day. In addition, methylcellulose did not interfere with the development of the injected eggs and embryos.
Assuntos
Microinjeções/métodos , Xenopus laevis/embriologia , Animais , Corantes/administração & dosagem , Dextranos/administração & dosagem , Desenvolvimento Embrionário , Feminino , Fluoresceínas/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Metilcelulose , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Óvulo/citologia , Soluções , Viscosidade , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Using a large-scale in situ hybridization screening system, we found that mRNA coding for ME1, a basic helix-loop-helix (bHLH) transcription factor, was abundantly expressed in Paneth cells of adult small intestinal crypts. Other functionally related E-protein mRNAs, ME2, and E2A, however, could not be detected in the cells. ME1 mRNA was first detected in the jejunum and ileum two weeks after birth when the number of Paneth cells starts to increase. ME1 is the first identified bHLH transcription factor expressed in the Paneth cells and may be used as a molecular marker and a key molecule for analyzing transcriptional regulation in the Paneth cell.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Mucosa Intestinal/fisiologia , Camundongos/genética , Celulas de Paneth/fisiologia , RNA Mensageiro/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica , Hibridização In Situ , Jejuno/crescimento & desenvolvimento , Jejuno/fisiologia , Camundongos/fisiologiaRESUMO
Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23-48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44-47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43-48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages.
Assuntos
Células Germinativas , Xenopus laevis/embriologia , Animais , Apoptose , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Sondas Moleculares , Fatores de Transcrição/genética , Proteínas de Xenopus/genéticaRESUMO
Using a large-scale in situ hybridization screening, we found that the mRNA coding for Xenopus glutamate receptor interacting protein 2 (XGRIP2) was localized to the germ plasm of Xenopus laevis. The mRNA is maternally transcribed in oocytes and, during maturation, transported to the vegetal germ plasm through the late pathway where VegT and Vg1 mRNAs are transported. In the 3'-untranslated region (UTR) of the mRNA, there are clusters of E2 and VM1 localization motifs that were reported to exist in the mRNAs classified as the late pathway group. With in situ hybridization to the sections of embryos, the signal could be detected in the cytoplasm of migrating presumptive primordial germ cells (pPGCs) until stage 35. At stage 40, when the cells cease to migrate and reach the dorsal mesentery, the signal disappeared. A possible role of XGRIP2 in pPGCs of Xenopus will be discussed.
Assuntos
Proteínas de Transporte/genética , Citosol/metabolismo , Células Germinativas/metabolismo , Mães , Transdução de Sinais , Transcrição Gênica/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo , Animais , Movimento Celular , Clonagem Molecular , DNA Complementar/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Transporte Proteico , RNA Mensageiro/genética , Fatores de Tempo , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
The monoclonal antibody to the beta-subunit of H(+)/K(+)-ATPase (mAbHKbeta) cross-reacts with a protein that acts as a molecular chaperone for the structural maturation of sarcoplasmic reticulum (SR) Ca(2+)-ATPase. We partially purified a mAbHKbeta-reactive 65-kDa protein from Xenopus ovary. After in-gel digestion and peptide sequencing, the 65-kDa protein was identified as methionine aminopeptidase II (MetAP2). The effects of MetAP2 on SR Ca(2+)-ATPase expression were examined by injecting the cRNA for MetAP2 into Xenopus oocytes. Immunoprecipitation and pulse-chase experiments showed that MetAP2 was transiently associated with the nascent SR Ca(2+)-ATPase. Synthesis of functional SR Ca(2+)-ATPase was facilitated by MetAP2 and prevented by injecting an antibody specific for MetAP2. These results suggest that MetAP2 acts as a molecular chaperone for SR Ca(2+)-ATPase synthesis.
Assuntos
Aminopeptidases/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Animais , Feminino , Imunoprecipitação , Metionil Aminopeptidases , Microinjeções , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Ligação Proteica , RNA Complementar/administração & dosagem , RNA Complementar/genética , Coelhos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Xenopus laevisRESUMO
In order to know the role of the Xdsg gene in presumptive PGCs (pPGCs) of Xenopus, we attempted to inhibit the translation of Xdsg mRNA in pPGCs by injecting antisense morpholino oligo (asMO), together with Fluorescein Dextran-Lysine (FDL), into single germ plasm-bearing cells of 32-cell embryos. Among three types of asMOs complementary to different parts of the 5'-untranslated region of Xdsg mRNA tested, only one asMO, designated as Xdsg-3, inhibited the translation of the mRNA in FDL-labeled pPGCs, resulting in the absence of labeled PGCs in experimental tadpoles. On the other hand, two other asMOs, Xdsg-1 and -2, did not inhibit the translation, so that a similar number of labeled PGCs found in FDL-injected but asMO-uninjected control tadpoles were observed in experimental tadpoles derived from asMO-injected embryos. Surprisingly, use of Xdsg-3 asMO resulted in the disappearance of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) from FDL-labeled pPGCs by inhibiting the translation of XVLG1 mRNA. However, the effect of Xdsg-3 asMO on the translation of Xdsg and XVLG1 mRNAs and PGC formation could be canceled by the coinjection with Xdsg mRNA. Consequently, the Xdsg protein in pPGCs may play an important role in the formation of PGCs by regulating the production of XVLG1 protein.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Proteínas de Xenopus/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Linhagem da Célula , Feminino , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligonucleotídeos Antissenso/química , Xenopus , Proteínas de Xenopus/químicaRESUMO
We investigated the mode of migration of presumptive primordial germ cells (pPGC) in the endoderm cell mass of Xenopus embryos at stages 7-40. The molecules underlying the migration were also studied cytochemically and immunocytologically. By examining the relative positions of pPGC and somatic cells derived from the single, fluorescein-dextran lysine (FDL)-injected, germ plasm-bearing cells of stage 6 embryos, pPGC in embryos at stages 7-23 and those at stages later than 24 were assumed to passively and actively migrate in the endoderm cell mass, respectively. This assumption was supported by the observation that F-actin, essential for active cell migration, was recognized on pPGC of the latter stages, but never on those of the former ones. In addition, the molecule like CXC chemokine receptor 4 (CXCR4) found on directionally migrating PGC in mouse and zebrafish, probably Xenopus CXCR4 (xCXCR4), was detected on pPGC only at latter stages. Accordingly, F-actin and xCXCR4, and probably beta1-integrin and collagen type IV, which are indispensable for the formation of F-actin, are thought to be involved in the active migration of pPGC in the endoderm cell mass.
Assuntos
Movimento Celular/fisiologia , Endoderma/fisiologia , Células Germinativas/fisiologia , Xenopus/embriologia , Actinas/metabolismo , Animais , Cromatografia de Afinidade , Primers do DNA , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Células Germinativas/metabolismo , Imuno-Histoquímica , Receptores CXCR4/metabolismoRESUMO
We performed high-throughput in situ hybridization screening of sections of mouse epidermis using an equalized skin cDNA library as probes and identified a novel gene giving rise to two splicing variants, both of which are expressed in the spinous layer. This gene was mapped between two genes encoding keratinocyte-related peptides, suprabasin and keratinocyte differentiation-associated protein (Kdap), on human chromosome 19q13.1. These gene products appeared to carry functional signal sequences. We then designated these two splicing variants as dermokine-alpha and -beta. Northern blotting and quantitative RT-PCR revealed that dermokine-alpha/-beta, suprabasin, and Kdap were highly expressed in stratified epithelia. In mouse embryonic development, dermokine-alpha/-beta began to be expressed during the period of stratification. Also, in differentiating primary cultured human keratinocytes, transcription of dermokine-alpha/-beta, suprabasin, and Kdap was induced. These findings indicated that dermokine-alpha/-beta, suprabasin, and Kdap are secreted from the spinous layer of the stratified epithelia and that these genes form a novel gene complex on the chromosome.