EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mo...EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.展开更多
One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to e...One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.展开更多
The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA st...The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted： ct39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.展开更多
A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and ad...A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.展开更多
Retroviral mediated gene transfer of humanglobin gene into hematopoietic stem cells is apromising approach for thalassemia gene therapy.Major problem of the transferred globin gene was lowlevel expression of the gene ...Retroviral mediated gene transfer of humanglobin gene into hematopoietic stem cells is apromising approach for thalassemia gene therapy.Major problem of the transferred globin gene was lowlevel expression of the gene with its proximal cis-acting sequence. The locus control region (LCR) ofthe human β-globin gene cluster consists of four majorDNase I hypersensitive sites (HS). When linked展开更多
Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant ...Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.展开更多
Seventy-four members from 9 adult polycystic kidney disease(APKD)familieswere analysed with Southern blot and 3′hypervariable region/Pvu Ⅱ,3′HVR/Bgl ⅡRFLPs.The results showed that 3′ HVR fragments were highly pol...Seventy-four members from 9 adult polycystic kidney disease(APKD)familieswere analysed with Southern blot and 3′hypervariable region/Pvu Ⅱ,3′HVR/Bgl ⅡRFLPs.The results showed that 3′ HVR fragments were highly polymorphic.Theheterozygote frequencies of Pvu Ⅱ,Bgl Ⅱ and PvuⅡ+Bg1Ⅱ fragments were 92%,84.7%and 98.6%,respectively.The maximum lod score for linkage between 3′HVR and APKDwas 9.71 at a recombination fraction 0.045.We successfully applied 3′HVR probe to thegene diagnosis of 17 symptomatic patients and 7 patients in the presymptomatic stage.展开更多
920363 Characterization and purificationof a glucose-6-phosphate dehydrogenasevariant Gd(-) Zhanjiang in human erythro-cytes.CAI Wangwei (蔡望伟),et al.ZhanjiangMed Coll.Chin J Hematol 1991;12(11):575-577.
OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and ant...OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. RESULTS: It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. CONCLUSION: The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.展开更多
Mycophenolic acid(MPA,1)and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases.Despite chemical synthetic achievements,the biosynthetic formation of a ...Mycophenolic acid(MPA,1)and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases.Despite chemical synthetic achievements,the biosynthetic formation of a seven-carbon carboxylic acid pharmacophore side chain of 1,especially the processes involving the cleavage of the prenyl side chain between DHMP(4)and DMMPA(5),remains unknown.In this work,we identified a membrane-bound prenyltransferase,PgMpaA,that transfers FPP to 4 to yield FDHMP(6).Compound 6 undergoes the first cleavage step via a new globin-like enzyme PgMpaB to form a cryptic intermediate 12.Heterologous expression of PgMpa genes in Aspergillus nidulans demonstrates that the second cleavage step(from 12 to 5)of 1 is a PgMpa clusterindependent process in vivo.Our results,especially the discovery of the broad tolerance of substrates recognized by PgMpaB,set up a strategy for the formation of"pseudo-isopentenyl"natural products using fungal globin-like enzymes.展开更多
A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by rev...A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.展开更多
Objective：To investigate the potential efficacy of panaxadiol saponins component（PDS-C）,a biologically active fraction isolated from total ginsenosides,to reverse chemotherapy-induced myelosuppression and pancytope...Objective：To investigate the potential efficacy of panaxadiol saponins component（PDS-C）,a biologically active fraction isolated from total ginsenosides,to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide（CTX）.Methods：Mice with myelosuppression induced by CTX were treated with PDS-C at a low-（20 mg/kg）,moderate-（40 mg/kg）,or high-dose（80 mg/kg） for 7 consecutive days.The level of peripheral white blood cell（WBC）,neutrophil（NEU） and platelet（PLT） were measured,the histopathology and colony formation were observed,the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot.Results：In response to PDS-C therapy,the peripheral WBC,NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner.Similarly,bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells（P〈0.01）.PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice,as evidenced by significantly increase in colony formation units-granulocytes/monocytes and-megakaryocytes（P〈0.01）.The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway,this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase（p-MEK） and extracellular signal-regulated kinases（p-ERK）,and receptor tyrosine kinase（C-kit） and globin transcription factor 1（GATA-1） in hematopoietic cells of CTX-treated mice（P〈0.05）.Conclusions：PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice,probably mediated by a mechanism involving MEK and ERK protein kinases,and C-kit and GATA-1 transcription factors.PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.展开更多
A newly developed method of RT-PCR/competitive PCR for measuring the relative and ab-solute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study thealterations of globin gen...A newly developed method of RT-PCR/competitive PCR for measuring the relative and ab-solute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study thealterations of globin gene expressions in the patients with β-thalassemia pre-and post-hydroxyurea(HU)treatment.It was found for the first time that HU had the effect of enhancing β-globin gene expression insome patients.Two cases with β-thalassemia who were subjected to HU treatment for over two years showeda marked increase in β-globin mRNA level and β-globin chain synthesis,resulting in more effective erythro-poiesis and the alleviation of clinical symptoms.展开更多
The transcription of essentially the entire eukaryotic genome produces a huge amount of non-coding RNAs.Among them,long noncoding RNAs(lncRNAs)consist of a significant portion that widely exists across mammal genome,g...The transcription of essentially the entire eukaryotic genome produces a huge amount of non-coding RNAs.Among them,long noncoding RNAs(lncRNAs)consist of a significant portion that widely exists across mammal genome,generating from high-throughput transcriptomic studies in the last decade.Although the functions of most lncRNAs remain to be further investigated,many of them have already been shown to play critical roles during normal development and disease conditions.Increasing evidence indicates that lncRNAs involve in versatile biological processes during erythroid proliferation and differentiation,including erythroid cell survival,heme metabolism,globin switching and regulation,erythroid enucleation,etc,via cis-or trans-mediated molecular mechanisms.In this review,we focus on recent advances regarding the functions and mechanisms of lncRNAs in normal erythropoiesis.展开更多
文摘EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.
基金The National Natural Science Foundation of China under contract Nos 30640015 and 30771644the Major Program of Zhejiang Province of China under contract No. 2005C23085
文摘One adult α-globin gene and one β-globin gene have been cloned from the large yellow croaker Pseudosciaena crocea. Linkage analysis indicated that the α- and β-globin genes were oriented head-to-head relative to each other. To identify the regulatory elements present in the intergenic and intragenic regions of the globin complex, the intergenic region alone or together with the β-globin gene first intron was cloned into the luciferase-reporter vector pGL3-Basic respectively, and the chimeric constructs were tran- siently transfected into Vero cells and primary fish erythrocytes. The intergenic region cannot support the high-level expression of luciferase. However, the promoter activity of the intergenic region was strongly stimulated by the positive regulatory elements （PRE） located in the β-globin gene intron 1. Thus, it is proposed that the intergenic promoters and intragenic PRE were necessary for the effective expression of the linked α- and β-globin genes.
基金supported by a grant from a major program of Zhejiang Province Commission of Science and Technologythe National Natural Science Foundation of China under contract No.2005C23085.
文摘The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted： ct39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.
文摘A cosmid construct μLCRAγψβδβ were induced into mouse erythroleukemia cell lines 585 that expresses murine adult globin only and MEL GM 979 that expresses both murine embryonic and adult globins.Similar patterns of human globin gene expression were displayed in the two MEL cell lines transfected with the construct.Inducible expression of the Aγ and β gene was observed during induced cell differentiation.However,the expression level of the Aγ globin gene is much higher than that of the β globin gene in either uninduced or induced MEL transformants.No γ to β switching happened in the stable MEL transformants following a continuous culture.The much more effective enhance of the μLCR on the Aγ globin gene than that on the β globin gene is resulted probably from the fact that the distance between the LCR and the β globin gene is much longer than that between the LCR and the Aγ globin gene in the construct,in comparison with other constructs containing HS2 or μLCR linked to both of γ and β globin genes in different order.Two suggestions can be derived from these results:1) A competition between the γ and β globin gene for interaction with the LCR may indeed present,but only an enough long distance difference between the LCR to the γ and to the β gene can effectively influence the competition;2) Unlike transgenic mice,MEL cells are incapable of reconstructing the regulatory information involved in developmental control when it is provided by a fragment of the β globin gene cluster with limited length.
文摘Retroviral mediated gene transfer of humanglobin gene into hematopoietic stem cells is apromising approach for thalassemia gene therapy.Major problem of the transferred globin gene was lowlevel expression of the gene with its proximal cis-acting sequence. The locus control region (LCR) ofthe human β-globin gene cluster consists of four majorDNase I hypersensitive sites (HS). When linked
文摘Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.
文摘Seventy-four members from 9 adult polycystic kidney disease(APKD)familieswere analysed with Southern blot and 3′hypervariable region/Pvu Ⅱ,3′HVR/Bgl ⅡRFLPs.The results showed that 3′ HVR fragments were highly polymorphic.Theheterozygote frequencies of Pvu Ⅱ,Bgl Ⅱ and PvuⅡ+Bg1Ⅱ fragments were 92%,84.7%and 98.6%,respectively.The maximum lod score for linkage between 3′HVR and APKDwas 9.71 at a recombination fraction 0.045.We successfully applied 3′HVR probe to thegene diagnosis of 17 symptomatic patients and 7 patients in the presymptomatic stage.
文摘920363 Characterization and purificationof a glucose-6-phosphate dehydrogenasevariant Gd(-) Zhanjiang in human erythro-cytes.CAI Wangwei (蔡望伟),et al.ZhanjiangMed Coll.Chin J Hematol 1991;12(11):575-577.
文摘OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. RESULTS: It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. CONCLUSION: The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.
基金supported by the National Natural Science Foundation of China(31870022)the Fundamental Research Funds for the Central Universities(XDJK2018B035 and 201941001,China)+3 种基金the Scientific Research Starting Foundation of Southwest University(SWU117034,China)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao,China)(No.2018SDKJ0401-2)Taishan Scholar Youth Expert Program in Shandong Province(tsqn201812021,China)supported by the Venture&Innovation Support Program for Chongqing Overseas Returnees and the Thousand Young Talents Program of China.
文摘Mycophenolic acid(MPA,1)and its derivatives are first-line immunosuppressants used in organ transplantation and for treating autoimmune diseases.Despite chemical synthetic achievements,the biosynthetic formation of a seven-carbon carboxylic acid pharmacophore side chain of 1,especially the processes involving the cleavage of the prenyl side chain between DHMP(4)and DMMPA(5),remains unknown.In this work,we identified a membrane-bound prenyltransferase,PgMpaA,that transfers FPP to 4 to yield FDHMP(6).Compound 6 undergoes the first cleavage step via a new globin-like enzyme PgMpaB to form a cryptic intermediate 12.Heterologous expression of PgMpa genes in Aspergillus nidulans demonstrates that the second cleavage step(from 12 to 5)of 1 is a PgMpa clusterindependent process in vivo.Our results,especially the discovery of the broad tolerance of substrates recognized by PgMpaB,set up a strategy for the formation of"pseudo-isopentenyl"natural products using fungal globin-like enzymes.
基金Financial support from the National Natural Science Foundation of China (Grant Nos. 20175029 and 20375040) is gratefully acknowledged.
文摘A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.
基金Supported by the Science and Technology Program Project of Zhejiang Province(No.2015C33173)Chinese Medicine Foundation of Young Talents in Zhejiang Province(No.2014ZQ006)Australia-China Institutional Links Research Program sponsored by the International Development Program of Education Australia(No.IDP 2-8)
文摘Objective：To investigate the potential efficacy of panaxadiol saponins component（PDS-C）,a biologically active fraction isolated from total ginsenosides,to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide（CTX）.Methods：Mice with myelosuppression induced by CTX were treated with PDS-C at a low-（20 mg/kg）,moderate-（40 mg/kg）,or high-dose（80 mg/kg） for 7 consecutive days.The level of peripheral white blood cell（WBC）,neutrophil（NEU） and platelet（PLT） were measured,the histopathology and colony formation were observed,the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot.Results：In response to PDS-C therapy,the peripheral WBC,NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner.Similarly,bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells（P〈0.01）.PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice,as evidenced by significantly increase in colony formation units-granulocytes/monocytes and-megakaryocytes（P〈0.01）.The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway,this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase（p-MEK） and extracellular signal-regulated kinases（p-ERK）,and receptor tyrosine kinase（C-kit） and globin transcription factor 1（GATA-1） in hematopoietic cells of CTX-treated mice（P〈0.05）.Conclusions：PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice,probably mediated by a mechanism involving MEK and ERK protein kinases,and C-kit and GATA-1 transcription factors.PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.
基金the Chinese"863"Research Grant and NIH Grant HLB-29623
文摘A newly developed method of RT-PCR/competitive PCR for measuring the relative and ab-solute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study thealterations of globin gene expressions in the patients with β-thalassemia pre-and post-hydroxyurea(HU)treatment.It was found for the first time that HU had the effect of enhancing β-globin gene expression insome patients.Two cases with β-thalassemia who were subjected to HU treatment for over two years showeda marked increase in β-globin mRNA level and β-globin chain synthesis,resulting in more effective erythro-poiesis and the alleviation of clinical symptoms.
基金supported by the National Key Research and Development Program of China(2016YFA0102300 and 2017YFA0103102)CAMS Innovation Fund for Medical Sciences(2016-I2M-3-002,2016-I2M-1-018 and 2017-I2M-1-015)+3 种基金National Natural Science Foundation of China(81870089 and 81700105)CAMS Medical Epigenetics Research Center(2018PT31033)Supported by the Fundamental Research Funds for the Central Universities(3332018157)State Key Laboratory of Experimental Hematology Research Grant(157-z18-07).
文摘The transcription of essentially the entire eukaryotic genome produces a huge amount of non-coding RNAs.Among them,long noncoding RNAs(lncRNAs)consist of a significant portion that widely exists across mammal genome,generating from high-throughput transcriptomic studies in the last decade.Although the functions of most lncRNAs remain to be further investigated,many of them have already been shown to play critical roles during normal development and disease conditions.Increasing evidence indicates that lncRNAs involve in versatile biological processes during erythroid proliferation and differentiation,including erythroid cell survival,heme metabolism,globin switching and regulation,erythroid enucleation,etc,via cis-or trans-mediated molecular mechanisms.In this review,we focus on recent advances regarding the functions and mechanisms of lncRNAs in normal erythropoiesis.
