Actin depolymerizing factor （ADF）, highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin fi...Actin depolymerizing factor （ADF）, highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin filaments. Four cDNAs （designated GhADF2, GhADF3, GhADF4, and GhADF5, respectively） encoding ADF proteins were isolated from cotton （Gossypium hirsutum） fiber cDNA library. GhADF2 cDNA is 705 bp in length and deduces a protein with 139 amino acids. GhADF3 cDNA is 819 bp in length and encodes a protein of 139 amino acids. GhADF4 cDNA is 804 bp in length and deduces a protein with 143 amino acids. GhADF5 cDNA is 644 bp in length and encodes a protein of 141 amino acids. The molecular evolutionary relationship of these genes was analyzed by means of bioinformatics. GhADF2 is closely related to GhADF3 （99% identity） and PetADF2 （89% identity）. GhADF4 is closely related to AtADF6 （78% identity）, and GhADF5 is closely related to AtADF5 （83% identity）. These results demonstrated that the plant ADF genes are highly conserved in structure. RT-PCR analysis showed that GhADF2 is predominantly expressed in fiber, whereas, GhADF5 is mainly expressed in cotyledons. On the other hand, it seems that GhADF3 and GhADF4 have no tissue specificity. Expression levels of different ADF genes may vary considerably in the same cell type, suggesting that they might be involved in regulating tissue development of cotton and the each ADF isoform may diverge to form the functional difference from the other ADFs during evolution.展开更多
Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephospho...Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin;however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent;gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.展开更多
Sea Island cotton(Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether
The plant actin depolymerizing factor （ADF） binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Ara...The plant actin depolymerizing factor （ADF） binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues （Arg98 and Lys100） located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 （K82/A） and the α-helix 4 （R135/A, R137/A） form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion （Allium cepa） peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization.展开更多
目的探讨辛伐他汀对慢性脑低灌注(CCH)大鼠认知功能及海马组织丝切蛋白表达的影响。方法选择40只SD大鼠随机分为假手术组(CCH模型不结扎颈总动脉)、CCH组(建立CCH模型)、溶剂组(CCH模型+0.5%羟甲基纤维素钠灌胃)和辛伐他汀组(CCH模型+0...目的探讨辛伐他汀对慢性脑低灌注(CCH)大鼠认知功能及海马组织丝切蛋白表达的影响。方法选择40只SD大鼠随机分为假手术组(CCH模型不结扎颈总动脉)、CCH组(建立CCH模型)、溶剂组(CCH模型+0.5%羟甲基纤维素钠灌胃)和辛伐他汀组(CCH模型+0.5%羧甲基纤维素钠+辛伐他汀灌胃),每组10只。检测大鼠海马组织丝切蛋白及突触后致密蛋白95(PSD-95)表达。结果与假手术组比较,CCH组和溶剂组总行程、站立次数、辨别指数、穿越平台次数及PSD-95表达明显降低,定位航行实验和第1~3天工作记忆实验逃避潜伏期明显延长,丝切蛋白表达明显升高(P<0.05)。与CCH组和溶剂组比较,辛伐他汀组的总行程、站立次数、相对辨别指数、穿越平台次数明显升高,定位航行实验和第1,3天工作记忆实验逃避潜伏期明显缩短(P<0.05)。辛伐他汀组丝切蛋白表达明显低于CCH组和溶栓组,PSD-95表达明显高于CCH组和溶栓组(1.09±0.96 vs 1.57±0.33和1.65±0.21,1.41±0.11 vs 1.14±0.16和1.01±0.02,P<0.05)。结论辛伐他汀通过下调大鼠海马丝切蛋白表达,增加PSD-95表达,改善CCH所致大鼠认知障碍。展开更多
基金This work was supported by the National Natural Sciences Foundation of China (No. 30470930)the Ministry of Education of China (No. 104130)the National Program for Basic Research and Development (973) of China (No. 2004CB117304).
文摘Actin depolymerizing factor （ADF）, highly conserved in all eukaryotic cells, is a low molecular mass of actin-binding protein, which plays a key role in modulating the polymerizing and depolymerizing of the actin filaments. Four cDNAs （designated GhADF2, GhADF3, GhADF4, and GhADF5, respectively） encoding ADF proteins were isolated from cotton （Gossypium hirsutum） fiber cDNA library. GhADF2 cDNA is 705 bp in length and deduces a protein with 139 amino acids. GhADF3 cDNA is 819 bp in length and encodes a protein of 139 amino acids. GhADF4 cDNA is 804 bp in length and deduces a protein with 143 amino acids. GhADF5 cDNA is 644 bp in length and encodes a protein of 141 amino acids. The molecular evolutionary relationship of these genes was analyzed by means of bioinformatics. GhADF2 is closely related to GhADF3 （99% identity） and PetADF2 （89% identity）. GhADF4 is closely related to AtADF6 （78% identity）, and GhADF5 is closely related to AtADF5 （83% identity）. These results demonstrated that the plant ADF genes are highly conserved in structure. RT-PCR analysis showed that GhADF2 is predominantly expressed in fiber, whereas, GhADF5 is mainly expressed in cotyledons. On the other hand, it seems that GhADF3 and GhADF4 have no tissue specificity. Expression levels of different ADF genes may vary considerably in the same cell type, suggesting that they might be involved in regulating tissue development of cotton and the each ADF isoform may diverge to form the functional difference from the other ADFs during evolution.
文摘Destrin, also called actin-depolymerizing factor (ADF), exists in resting parotid tissue as phosphorylated (inactive) and dephosphorylated (active) forms, and β-adrenergic stimulation of this tissue induces dephosphorylation of destrin. It is suggested that destrin dephosphorylation is involved in cortical F-actin disruption observed in parallel with β-agonist-induced amylase secretion. At present, the phosphorylation/dephosphorylation mechanism of destrin in parotid tissue is not known. We previously detected, in a crude rat parotid extract, a constitutively active protein kinase catalyzing phosphorylation of destrin;however, its identification has been hampered by difficulty in its enrichment. The purpose of this study was to explore a simple purification method(s) for this enzyme. To this end, we first developed a high-throughput dot-blot assay for the kinase with an anti-phosphodestrin antibody and then studied its purification by column chromatography on several media. We found that the kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite columns. In each chromatography, however, the kinase could be eluted, at the cost of resolution, only by sharp increases in the elution power of the eluent;gradual increases in the elution power resulted in unacceptably poor recovery. We confirmed that enzymatic properties of the kinase were not basically altered during the purification. Further purification of the kinase was achieved by native polyacrylamide gel electrophoresis (PAGE), which resolved the kinase activity into two bands and separated the activity from most proteins (the kinase activity after PAGE was detected with destrin-coated polyvinylidene difluoride membranes and the anti-phosphodestrin antibody). The two bands seem to constitute the major destrin-phosphorylating activity in the resting rat parotid gland. We here report its partial purification and characterization together with the detection methods.
文摘Sea Island cotton(Gossypium barbadense L.) has been highly valued in Verticillium wilt resistance and many fiber qualities including fiber length,strength,and fineness.To identify whether
基金supported by the Shandong Taishan Scholar programthe Shandong Natural Science Foundation(ZR2012CM022, ZRB019E7)the Laboratory of Biotechnology of Qingdao Agricultural University
文摘The plant actin depolymerizing factor （ADF） binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues （Arg98 and Lys100） located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 （K82/A） and the α-helix 4 （R135/A, R137/A） form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion （Allium cepa） peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization.
文摘目的探讨辛伐他汀对慢性脑低灌注(CCH)大鼠认知功能及海马组织丝切蛋白表达的影响。方法选择40只SD大鼠随机分为假手术组(CCH模型不结扎颈总动脉)、CCH组(建立CCH模型)、溶剂组(CCH模型+0.5%羟甲基纤维素钠灌胃)和辛伐他汀组(CCH模型+0.5%羧甲基纤维素钠+辛伐他汀灌胃),每组10只。检测大鼠海马组织丝切蛋白及突触后致密蛋白95(PSD-95)表达。结果与假手术组比较,CCH组和溶剂组总行程、站立次数、辨别指数、穿越平台次数及PSD-95表达明显降低,定位航行实验和第1~3天工作记忆实验逃避潜伏期明显延长,丝切蛋白表达明显升高(P<0.05)。与CCH组和溶剂组比较,辛伐他汀组的总行程、站立次数、相对辨别指数、穿越平台次数明显升高,定位航行实验和第1,3天工作记忆实验逃避潜伏期明显缩短(P<0.05)。辛伐他汀组丝切蛋白表达明显低于CCH组和溶栓组,PSD-95表达明显高于CCH组和溶栓组(1.09±0.96 vs 1.57±0.33和1.65±0.21,1.41±0.11 vs 1.14±0.16和1.01±0.02,P<0.05)。结论辛伐他汀通过下调大鼠海马丝切蛋白表达,增加PSD-95表达,改善CCH所致大鼠认知障碍。
