Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C...Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.展开更多
The present results demonstrated that in an adult rat model of permanent middle cerebral artery occlusion （pMCAO）, pretreatment with bilobalide reduced brain water content and infarct area, down-regulated aquaporin ...The present results demonstrated that in an adult rat model of permanent middle cerebral artery occlusion （pMCAO）, pretreatment with bilobalide reduced brain water content and infarct area, down-regulated aquaporin 1, 4 mRNA expression in brain edema tissue, then inhibited their synthesis in the striatum, in particular at the early stage of ischemia （at 8 hours after pMCAO）, inhibited glial fibrillary acidic protein expression, and lightened reactive gliosis. These data sug-gest that bilobalide attenuates brain edema formation due to reduced expression of aquaporins.展开更多
Ammonia induces astrocyte swelling, which is strongly associated with overexpression of aquaporin-4. However, the mechanisms by which ammonia induces astrocyte swelling, and subsequently upregulating aquaporin-4 expre...Ammonia induces astrocyte swelling, which is strongly associated with overexpression of aquaporin-4. However, the mechanisms by which ammonia induces astrocyte swelling, and subsequently upregulating aquaporin-4 expression, remain unknown. In the present study, astrocytes were cultured in vitro and exposed to ammonium chloride （NH4CI）, followed by propofol protein kinase C agonist, or antagonist, respectively. Astrocyte morphology was observed by light microscopy, and aquaporin-4 expression was detected by western blot analysis. Results showed that propofol or protein kinase C agonist significantly attenuated the degree of NH4CI-induced astrocyte swelling and inhibited increased aquaporin-4 expression. Propofol treatment inhibited aquaporin-4 overexpression in cultured astrocyte induced by NH4CI; protein kinase C pathway activation is potentially involved.展开更多
As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most imp...As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most important factor limiting rubber production. On the basis of an EST down-regulated in TPD-affected rubber trees, a 903 bp cDNA denoted HbPIP2;2 was isolated from the bark tissue with a combination of in silico cloning and RT-PCR. The cDNA contains an 867 hp ORF, 13 bp 5＇UTR and 23 bp 3＇ UTR. Sequence analysis indicated that HbPIP2;2 encodes 288 amino acids with a theoretical molecular weight （Mw） of 30. 71 kDa and isolectric point （Pi） of 8.20. Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the plasma membrane and harbor one conserved MIP domain that can be grouped into plasma membrane intrinsic proteins （PIPs） of aquaporin （AQP） family. Homolo- gy search revealed that the protein shares a similarity of more than 90% with the homologues in Ricinus communis, Popttlus trichocarpa, Juglans regia and Theobro- ma cacao, suooorting a hie.hly conserved evolution. This study provided basis for further uncovering the regulatory role of AOPs in TPD occurrence.展开更多
基金Supported by the National Natural Science Foundation of China(Nos.30700827 and 30871301)Jilin Provincial Science & Technology Department of China(Nos.20070719 and 20080731)Northeast Normal University,China(Nos.20070401,NENU-STC07005)
文摘Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.
基金a Research Subject of General Hospital of Shenyang Military Area Command of Chinese PLA
文摘The present results demonstrated that in an adult rat model of permanent middle cerebral artery occlusion （pMCAO）, pretreatment with bilobalide reduced brain water content and infarct area, down-regulated aquaporin 1, 4 mRNA expression in brain edema tissue, then inhibited their synthesis in the striatum, in particular at the early stage of ischemia （at 8 hours after pMCAO）, inhibited glial fibrillary acidic protein expression, and lightened reactive gliosis. These data sug-gest that bilobalide attenuates brain edema formation due to reduced expression of aquaporins.
基金Supported by a Grant from Zhejiang Provincial Health Department, No. 2007A057
文摘Ammonia induces astrocyte swelling, which is strongly associated with overexpression of aquaporin-4. However, the mechanisms by which ammonia induces astrocyte swelling, and subsequently upregulating aquaporin-4 expression, remain unknown. In the present study, astrocytes were cultured in vitro and exposed to ammonium chloride （NH4CI）, followed by propofol protein kinase C agonist, or antagonist, respectively. Astrocyte morphology was observed by light microscopy, and aquaporin-4 expression was detected by western blot analysis. Results showed that propofol or protein kinase C agonist significantly attenuated the degree of NH4CI-induced astrocyte swelling and inhibited increased aquaporin-4 expression. Propofol treatment inhibited aquaporin-4 overexpression in cultured astrocyte induced by NH4CI; protein kinase C pathway activation is potentially involved.
基金Supported by National Natural Science Foundation of China(31371556)Natural Science Foundation of Hainan Province(312026)Fundamental Research Fund for the Rubber Research Institute in Chinese Academy of Tropical Agricultural Sciences(1630022011014)
文摘As a syndrome with tapping incision blocked partly or entirely during latex exploiting in rubber tree （Hevea brasiliensis ）, ＂Tapping panel dryness （TPD）＂ causes great yield losses, thereby becoming the most important factor limiting rubber production. On the basis of an EST down-regulated in TPD-affected rubber trees, a 903 bp cDNA denoted HbPIP2;2 was isolated from the bark tissue with a combination of in silico cloning and RT-PCR. The cDNA contains an 867 hp ORF, 13 bp 5＇UTR and 23 bp 3＇ UTR. Sequence analysis indicated that HbPIP2;2 encodes 288 amino acids with a theoretical molecular weight （Mw） of 30. 71 kDa and isolectric point （Pi） of 8.20. Bioinformatics analysis suggested that the deduced protein is predicted to have six transmembrane helices located to the plasma membrane and harbor one conserved MIP domain that can be grouped into plasma membrane intrinsic proteins （PIPs） of aquaporin （AQP） family. Homolo- gy search revealed that the protein shares a similarity of more than 90% with the homologues in Ricinus communis, Popttlus trichocarpa, Juglans regia and Theobro- ma cacao, suooorting a hie.hly conserved evolution. This study provided basis for further uncovering the regulatory role of AOPs in TPD occurrence.
