Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inw...Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa展开更多
Microtransplantation of rat brain neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological appro...Microtransplantation of rat brain neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological approaches.In this review,we show that oocytes injected with adult rat brain neurolemma elicited tetrodotoxin-sensitive inward ion currents upon membrane depolarization,which were increased in a concentration-dependent manner by treatment with the pyrethroid insecticides permethrin and deltamethrin.Under our initial protocols,oocyte health was reduced over time and neurolemma incorporation varied between batches of oocytes from different frogs,limiting the usefulness of the assay for regulatory issues.A collection of changes to the assay procedure,data acceptance criteria,and analysis method yield substantially improved precision and,hence,assay performance.These changes established this ex vivo approach as a toxicologically relevant assay to study the toxicodynamic action of pyrethroids on ion channels in their native state using neurolemma fragments prepared from juvenile and adult rat brains.展开更多
Calpains are a superfamily of Ca2+-dependent cysteine proteases, implicated in various cellular processes and thus probably necessary in all the stages of cell life. The first extended report of quantification of tota...Calpains are a superfamily of Ca2+-dependent cysteine proteases, implicated in various cellular processes and thus probably necessary in all the stages of cell life. The first extended report of quantification of total RNAs within the developmental stages of Xenopus laevis was described in this study. Decreases of total RNAs were positively associated with waves of apoptotic cell death (onset of gastrulation, and morphogenesis). Using qPCR, the temporal expression pattern of CAPN1 and CPAN8b (XCL-2) were characterized during the Xenopus laevis embryogenesis. Transcripts of the CAPN1 and CAPN8 genes were detectable from gastrula stage and their levels oscillated throughout development. The expression of the CAPN1 (mu/I) gene was observed in earliest stage, indicating a maternal origin, while expression of the CAPN8b gene was detectable after midblastula transition. The levels of the two transcripts then started to rise again obviously as a result of zygotic expression (stage 11). The CAPN1 gene expression was particularly expressed at tailbud stage, while the CAPN8 transcripts were found at gastrula, neurula and tailbud stages. This is the first report of quantification of mRNAs CAPN8b and CAPN1 (mu/I) within the developmental stages of Xenopus laevis by qPCR.展开更多
To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized ...To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.展开更多
Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 3...Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.展开更多
Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nucl...Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.展开更多
The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orches...The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.展开更多
We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, ...We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.展开更多
The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incuba...The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.展开更多
We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA f...We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.展开更多
Kruppel-like factor 4(Klf4) is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key down...Kruppel-like factor 4(Klf4) is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711(dnTcf711), which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.展开更多
Decabromodiphenyl ether (decaBDE),as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE m...Decabromodiphenyl ether (decaBDE),as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R (1-1000 ng/L) in water from stage 46/47 (free swimming larvae,system of Nieuwkoop and Faber) to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis (presented by forelimb emergence,FLE).Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers (PBDEs) (sum of 39 congeners) in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.展开更多
Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sort...Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.展开更多
A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the rea...A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only eggderived lamin LIII.展开更多
Inositol requiring enzyme-1(IRE1)is highly conserved from yeasts to humans.Upon endoplasmic reticulum(ER)stress,IRE1 activates X-box-binding protein 1(XBP1)by unconventional splicing of XBP1 mRNA,which activates unfol...Inositol requiring enzyme-1(IRE1)is highly conserved from yeasts to humans.Upon endoplasmic reticulum(ER)stress,IRE1 activates X-box-binding protein 1(XBP1)by unconventional splicing of XBP1 mRNA,which activates unfolded protein response(UPR)to restore ER homeostasis.In mice,IRElαplays an essential role in extraembryonic tissues.However,its precise action during the early stage of development is unknown.In this study,the gain and loss-of-function analyses were used to investigate the function of Xenopus IRElα(xIRElα).The effects of xIRElαduring embryo development were detected with RT-PCR and whole mount in situ hybridization.ER stress was induced by tunicamycin.The apoptotic cells were measured by TUNNEL assays.Although both gain and loss of xIRElαfunction had no significant effect on Xenopus embryogenesis,knockdown of xIRElαcould rescue tunicamycin-induced developmental defects and apoptosis.The finding indicates that xIRElαis not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.展开更多
The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyi...The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.展开更多
文摘Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa
基金supported by the Council of the Advancement of Pyrethroid Human Risk Assessment(CAPHRA)(#S17110000000004).
文摘Microtransplantation of rat brain neurolemma into the plasma membrane of Xenopus laevis oocytes is an ex vivo method used to study channels and receptors in their native state using standard electrophysiological approaches.In this review,we show that oocytes injected with adult rat brain neurolemma elicited tetrodotoxin-sensitive inward ion currents upon membrane depolarization,which were increased in a concentration-dependent manner by treatment with the pyrethroid insecticides permethrin and deltamethrin.Under our initial protocols,oocyte health was reduced over time and neurolemma incorporation varied between batches of oocytes from different frogs,limiting the usefulness of the assay for regulatory issues.A collection of changes to the assay procedure,data acceptance criteria,and analysis method yield substantially improved precision and,hence,assay performance.These changes established this ex vivo approach as a toxicologically relevant assay to study the toxicodynamic action of pyrethroids on ion channels in their native state using neurolemma fragments prepared from juvenile and adult rat brains.
文摘Calpains are a superfamily of Ca2+-dependent cysteine proteases, implicated in various cellular processes and thus probably necessary in all the stages of cell life. The first extended report of quantification of total RNAs within the developmental stages of Xenopus laevis was described in this study. Decreases of total RNAs were positively associated with waves of apoptotic cell death (onset of gastrulation, and morphogenesis). Using qPCR, the temporal expression pattern of CAPN1 and CPAN8b (XCL-2) were characterized during the Xenopus laevis embryogenesis. Transcripts of the CAPN1 and CAPN8 genes were detectable from gastrula stage and their levels oscillated throughout development. The expression of the CAPN1 (mu/I) gene was observed in earliest stage, indicating a maternal origin, while expression of the CAPN8b gene was detectable after midblastula transition. The levels of the two transcripts then started to rise again obviously as a result of zygotic expression (stage 11). The CAPN1 gene expression was particularly expressed at tailbud stage, while the CAPN8 transcripts were found at gastrula, neurula and tailbud stages. This is the first report of quantification of mRNAs CAPN8b and CAPN1 (mu/I) within the developmental stages of Xenopus laevis by qPCR.
基金Project supported by the National Key Basic Research Program of China(No. 2003CB415005)the National Natural Science Foundation of China(No. 20377044)the Hi-Tech Research and Development Program(863) of China (No. 2003AA646010)
文摘To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.
文摘Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis.,and its effects on embryonic development were studied.Survival rate of the antikeratin-injected embryos was much lower(only 35.67% at gastrula)than that of the control(74.85% at gastrula),in which embryos were injected with mouse IgG.Most of survivors in the experimental series showed aberrant external appearance.On the other hand,in cleavage stage,ie 2-7h after fertilization,immunohistochemical staining of embryos showed that the expermental embryos were mostly keratin negative,while embryos of the control ones were keratin positive.When introducing this antikeratin into one cell of a 2-cell embryo,only the uninjected half of the embryo continued its development while the other half could not develop at all.These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.
文摘Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.
文摘The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.
文摘We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.
文摘The plasmid p^(SV-Luc20)or the mRNA of luciferase gene transcribed from p^(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.
文摘We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments. The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.
基金supported by the Start-up Funding of Henan University of Science and Technology(13480027) to Q. C.the Key Science Foundation of Nanjing Medical University(2015NJMUZD002)+2 种基金the Natural Science Foundation of Higher Education Institutions of Jiangsu Province(16KJB-180020)Natural Science Foundation of Jiangsu Province (BK20171053)National Natural Science Funds of China (81702747) to C.L
文摘Kruppel-like factor 4(Klf4) is a zinc finger transcription factor and plays crucial roles in Xenopus embryogenesis.However, its regulation during embryogenesis is still unclear. Here, we report that Tcf711, a key downstream transducer of the Wnt signaling pathway, could promote Klf4 transcription and stimulate Klf4 promoter activity in early Xenopus embryos. Furthermore, cycloheximide treatment showed a direct effect on Klf4 transcription facilitated by Tcf711. Moreover, the dominant negative form of Tcf711(dnTcf711), which lacks N-terminus of the β-catenin binding motif, could still activate Klf4 transcription, suggesting that this regulation is Wnt/β-catenin independent.Taken together, our results demonstrate that Tcf711 lies upstream of Klf4 to maintain its expression level during Xenopus embryogenesis.
基金supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No. KZCX2-YW-420-3,KZCX2-YW-Q-02-05)the National Natural Science Foundation of China (No.20437020,20677074)
文摘Decabromodiphenyl ether (decaBDE),as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R (1-1000 ng/L) in water from stage 46/47 (free swimming larvae,system of Nieuwkoop and Faber) to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis (presented by forelimb emergence,FLE).Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers (PBDEs) (sum of 39 congeners) in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.
文摘Animal pole cells (AC) and vegetal pole cells (VC) dissociated from early Xenopus gastrulae were intermingled, and the cell sorting process occurring within the aggregate was analyzed. The overall process of cell sorting was found to morphologically consist of two steps, “concentrification” and “polarization”, as designated here. First, AC and VC clusters emerged at random positions in the aggregate, and the individual clusters gradually assembled themselves by 5 hours in culture (5 hC), forming a concentric arrangement, in which the AC cluster was enveloped by the VC cluster. This concentrification step is essentially consistent with the descriptions in earlier studies. As the next step, the AC and VC clusters moved up and down from 7.5 to 12 hC, resulting in the vertical polarization, namely, a serial array just like in vivo. Immunohistochemical analyses showed that AC expressed both C- and E-cadherins, while VC only expressed C-cadherin, as in vivo, suggesting the normal participation of cadherin system. On the other hand, the actin localization showed that the actin bundles accumulated at the edge of the AC cluster until the concentrification was completed, and gradually decreased during the polarization step. Another important finding was that AC cluster could generate cartilage tissues during the long-term (7 days) culture, evidence for a healthy inductive interaction between the AC and VC. Taken together, the present experimental system allows the AC and VC to be viable and grow into an embryo-like organization.
文摘A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei. Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear membrane, nuclear pore complexes, and decondensed chromatin etc. Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei. Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only eggderived lamin LIII.
文摘Inositol requiring enzyme-1(IRE1)is highly conserved from yeasts to humans.Upon endoplasmic reticulum(ER)stress,IRE1 activates X-box-binding protein 1(XBP1)by unconventional splicing of XBP1 mRNA,which activates unfolded protein response(UPR)to restore ER homeostasis.In mice,IRElαplays an essential role in extraembryonic tissues.However,its precise action during the early stage of development is unknown.In this study,the gain and loss-of-function analyses were used to investigate the function of Xenopus IRElα(xIRElα).The effects of xIRElαduring embryo development were detected with RT-PCR and whole mount in situ hybridization.ER stress was induced by tunicamycin.The apoptotic cells were measured by TUNNEL assays.Although both gain and loss of xIRElαfunction had no significant effect on Xenopus embryogenesis,knockdown of xIRElαcould rescue tunicamycin-induced developmental defects and apoptosis.The finding indicates that xIRElαis not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.
文摘The complex transformation of a tadpole to a frogduring amphibian development is under the control of thyroid hormone (T3). T3 is known to regulate gene transcription through its nuclear receptors. We have previouslyisolated many genes which are up-regulated by T3 in theintestine of Xenopus laevis tadpoles. We have now cloneda full- length cDNA for one such gene (IU12). Sequenceanalysis shows that the IU12 cDNA encodes a plasmamembrane protein with 12 transmembrane domains andhomologous to a mammalian gene associated with cell activation and organ development. Similarly, we have foundthat IU12 is activated during intestinal remodeling whenboth cell death and proliferation take place. Furthermore,IU12 is an early T3-response gene and its expression in theintestine during T3-induced metamorphosis mimics thatduring normal development. These results argue for a roleof IU12 in the signal transduction pathways leading to intestinal metamorphosis.
